Biomedical Engineering Encyclopedia Vol-1-3

ENCYCLOPEDIA OF
BIOMEDICAL ENGINEERING
This page intentionally left blank
ENCYCLOPEDIA OF
EDITOR IN CHIEF
Roger Narayan
University of North Carolina at Chapel Hill, Chapel Hill,
NC, United States
VOLUME 1
Section Editors
Min Wang
The University of Hong Kong, Pokfulam, Hong Kong
Cato Laurencin
University of Connecticut Health Center, Farmington, CT, United States
Xiaojun Yu
Stevens Institute of Technology, Hoboken, NJ, United States
Amsterdam • Boston • Heidelberg • London • New York • Oxford

Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright Ó 2019 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photo-
copying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to
seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the
Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted
herein).
Notice
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in
research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods,
compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the
safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or
damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.
Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
ISBN 978-0-12-804829-0
For information on all publications visit our website

at http://store.elsevier.com
Publisher: Oliver Walter

Acquisition Editor: Blerina Osmanaj
Publishing Services Manager: Beckie Brand
Associate Content Project Manager: Kshitija Iyer
Cover Designer: Greg Harris
Printed and bound in the United States

EDITORIAL BOARD
EDITOR IN CHIEF
Roger Narayan
University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
SECTION EDITORS
Levi Hargrove
Rehabilitation Institute of Chicago, Chicago, IL, United States
Christian Hellmich
TU Wien, Vienna University of Technology, Vienna, Austria
Sri Krishnan
Ryerson University, Toronto, ON, Canada
Cato Laurencin
Diego Mantovani
Laval University, Quebec City, QC, Canada
William Z Rymer
Pankaj Vadgama
Queen Mary University of London, London, United Kingdom
Min Wang
Alexander Wong
University of Waterloo, Waterloo, ON, Canada
Xiaojun Yu
v
EDITOR IN CHIEF
Roger Narayan
Dr. Roger Narayan is a professor in the Joint Department of Biomedical Engineering at the
University of North Carolina and North Carolina State University. He is an author of over 200
publications as well as several book chapters on processing, characterization, and modeling of bio-
logical and biomedical materials. Dr. Narayan has edited several books, including Biomedical Mate-
rials, Printed Biomaterials, Computer Aided Biomanufacturing, Diamond-Based Materials for Biomedical
Applications, Medical Biosensors for Point of Care (POC) Applications, Monitoring and Evaluation of Bioma-
terials and their Performance In Vivo, Nanobiomaterials: Nanostructured Materials for Biomedical Applica-
tions, and the ASM Handbook on Materials for Medical Devices. He has previously served as chair of the
Functional Materials Division of The Minerals, Metals & Materials Society and is currently chair-elect
of the Bioceramics Division of American Ceramics Society. Dr. Narayan has received several honors
for his research activities, including the North Carolina State University Alcoa Foundation Engi-
neering Research Achievement Award, the North Carolina State University Sigma Xi Faculty
Research Award, the University of North Carolina Jefferson-Pilot Fellowship in Academic Medicine,
the National Science Faculty Early Career Development Award, the Office of Naval Research Young
Investigator Award, the American Ceramic Society Richard M. Fulrath Award, the Royal Academy of Engineering Distinguished Visiting
Fellowship, and TMS Brimacombe Medal. He has served as Fulbright Scholar at the University of Otago, the National Polytechnic Institute
(Mexico City), and the University of Sao Paulo. He has been elected as Fellow of ASM International, the American Association for the
Advancement of Science, the American Ceramic Society, and the American Institute for Medical and Biological Engineering.
vii
SECTION EDITORS
Levi Hargrove
Dr. Hargrove is currently the Director of the Center for Bionic Medicine and of the Neural Engi-
neering for Prosthetic and Orthotics Laboratory at the Shirley Ryan AbilityLab. He is also an
Associate Professor in the Departments of Physical Medicine and Rehabilitation and the
McCormick School of Engineering at Northwestern University.
A major goal of his research is to develop clinically realizable myoelectric control systems
that can be made available to persons with limb loss in the near future. His research addresses
all levels of amputation and has been published in the Journal of the American Medical Associ-
ation and the New England Journal of Medicine, and multiple patents. Key projects include the
development of advanced and adaptive control systems for prosthetic legs, improving control
of robotic hand prostheses, and intramuscular EMG signal processing. In 2012, Dr. Hargrove
cofounded Coapt, a company to transition advanced rehabilitation technologies from the
research laboratory to patients’ homes.
Christian Hellmich
Dr. Christian Hellmich, Full Professor at the Department of Civil Engineering of the Vienna
University of Technology (TU Wien), is the director of the Institute for Mechanics of Materials
and Structures. At TU Wien, he received his engineering, Ph.D., and habilitation degrees (in
1995, 1999, and 2004, respectively). From 2000 to 2002, he was a Max Kade Postdoctoral
Fellow in the Department of Civil and Environmental Engineering at the Massachusetts Insti-
tute of Technology. His work is strongly focused on well-validated material and (micro)struc-
tural models, in terms of theoretical foundations and applications to concrete, soil, rock,
wood, bone, and biomedical implants, up the structural level (tunnels, pipelines, bridges, bio-
logical organs such as the skeleton)dwith complementary experimental activities if necessary.
He has led several projects for the tunnel, railway, and pipeline industries, as well as interna-
tional research activities sponsored by the European Commission, including the coordination
of the mixed industry-academia consortium “BIO-CT-EXPLOIT” at the crossroads of numer-
ical simulation and computer tomography, or the cross-domain COST action NAMABIO inte-
grating engineers, physicists, (stem) cell biologists, and medical doctors across the European
continent and beyond. He has published more than 130 papers in international refereed
scientific journals in the fields of engineering mechanics, materials science, and theoretical biology, more than 20 book chapters,
and more than 120 papers in refereed conference proceedings. Dr. Hellmich has served as the Chairman of both the Properties of
Materials Committee of the Engineering Mechanics Division of the American Society of Civil Engineers (ASCE), and the Porome-
chanics and Biomechanics Committees of the Engineering Mechanics Institute (EMI), as associate editor of the Journal of Engineering
Mechanics (ASCE), and as Coeditor in Chief of the Journal of Nanomechanics and Micromechanics (ASCE). As community service, he
has (co)chaired and/or supported more than 50 international conferences (including chairmanship of the 2013 Biot Conference on
Poromechanics and the 2015 CONCREEP conference; both EMI-ASCE supported), and he has reviewed for 128 different scientific
journals and 15 science foundations. He was awarded the Kardinal Innitzer Science Award of the Archbishopry of Vienna in 2004
(for his habilitation thesis), the Science Award of the State of Lower Austria in 2005 (for his achievements in the micromechanics of
hierarchical composites), and he was the recipient of the 2008 Zienkiewicz Award for Young Scientists in Computational Engi-
neering Sciences, sponsored by the European Community on Computational Methods in Applied Sciences (ECCOMAS). For further
activities in the multiscale poromicromechanics of bone materials, he received one of the highly prestigious ERC Grants of the
ix
x Section Editors
European Research Council in 2010; and he was elected member of the Young Academy of the Austrian Academy of Sciences in
2011. In 2012, he was rewarded the prestigious Walter L. Huber Research Prize of the ASCE, for his contributions to the micropor-
omechanics of hierarchical geomaterials and biomaterials; he was elected Fellow of EMI in 2014 and was corecipient of the 2017
Kajal Mallick Memorial Award of the Institution of Civil Engineers (United Kingdom).
Sri Krishnan
Sridhar (Sri) Krishnan received B.E. degree in Electronics and Communication Engineering
from the College of Engineering, Guindy, Anna University, Chennai, India, in 1993, and
M.Sc. and Ph.D. degrees (with student fellowship from Alberta Heritage Foundation for
Medical Research) in Electrical and Computer Engineering from The University of Calgary,
Calgary, Alberta, Canada, in 1996 and 1999, respectively. Sri Krishnan joined Ryerson Univer-
sity in July 1999 and is currently a Professor in the Department of Electrical and Computer
Engineering. Since July 2011, he is an Associate Dean (Research, Development and External
Partnerships) for the Faculty of Engineering and Architectural Science. He is also the Codi-
rector of the Institute for Biomedical Engineering, Science and Technology (iBEST) and an
affiliate scientist at the Keenan Research Centre in St. Michael’s Hospital, Toronto.
Since January 2002 Sri Krishnan held various administrative leadership positions in the
Department of Electrical and Computer Engineering and the Faculty of Engineering and
Architectural Science. In 2010–2011, Sri Krishnan held Visiting Appointments in University
of Rennes 1 (France), Grenoble Institute of Technology (France) and Indian Institute of Tech-
nology (Madras). Sri Krishnan is a registered professional engineer in the Province of Ontario and is a senior member of IEEE (EMBS
and SP societies). He was the Founding Chair (2005–2015) of IEEE Signal Processing Society, Toronto Section and Region 7 (Can-
ada), and a Founding Member of the IEEE Engineering in Medicine and Biology Society, Toronto Section. He currently serves as
a Technical Committee Member (Biomedical Signal Processing) of IEEE EMBS.
Sri Krishnan held the Canada Research Chair position (2007–2017) in Biomedical Signal Analysis. Sri Krishnan has successfully
supervised/trained 10 postdoc fellows, 10 Ph.D., 30 Masters (thesis), 9 Masters (project), 42 RAs, and 20 Visiting RAs. Sri Krishnan’s
research interests include adaptive signal representations and analysis and their applications in biomedicine, multimedia (audio),
and biometrics. He has published 295 papers in refereed journals and conferences, filed 10 invention disclosures, and has one US
patent. He has presented keynote/plenary/invited talks in more than 35 international conferences and workshops. Sri Krishnan also
serves as a reviewer, committee member, and chair for many international conferences, journals, and granting bodies. Sri Krishnan’s
academic interests include (interdisciplinary) curriculum design, experiential learning, and innovation. Sri Krishnan serves in the
advisory boards of research institutes, innovation centers, incubator zones, and business organizations.
Sri Krishnan is a recipient/awarded Outstanding Canadian Biomedical Engineer Award 2016; Certificate of Appreciation from
PEO York Chapter 2016; Fellow of Canadian Academy of Engineering in 2014; 2014 Exemplary Service Award from IEEE Toronto
Section; 2014 Certificate of Merit from IEEE Signal Processing Society; 2013 Achievement in Innovation Award from Innovate Cal-
gary; 2011 Sarwan Sahota Distinguished Scholar Award; 2011 Certificate of Appreciation from IEEE Signal Processing Society; 2010
Shastri Visiting Professorship; 2010 French Embassy Visiting Researcher; 2008 Ontario Research Innovation Award from Bio-
discovery Toronto; 2007 Canadian Engineers’ Young Engineer Achievement Award from Engineers’ Canada; 2006 New Pioneers
Award in Science and Technology; 2006 South Asian Community Achiever Award; 2006 IEEE Toronto Section Best Chapter Chair
Award; 2005 IEEE AESS Best Chapter Chair Award; 2005 IEEE Certificate of Appreciation from Six Societies; Six Best Research Paper
Awards coauthored with his graduate students in International Conferences; and 2005 FEAS Research Excellence Award.
Cato Laurencin
Cato T. Laurencin, M.D., Ph.D. is the University Professor at UCONN. He is the eighth desig-
nated in UCONN’s history. He is Professor of Chemical Engineering, Professor of Materials
Science and Engineering, and Professor of Biomedical Engineering, and the Van Dusen Distin-
guished Endowed Professor of Orthopaedic Surgery. He directs the Institute for Regenerative
Engineering and the Raymond and Beverly Sackler Center at the University of Connecticut.
Dr. Laurencin earned his B.S.E. degree in Chemical Engineering from Princeton University.
He earned his Ph.D. in Biochemical Engineering/Biotechnology from the Massachusetts Insti-
tute of Technology where he was named a Hugh Hampton Young Fellow. At the same time, he
earned his M.D., Magna Cum Laude from the Harvard Medical School where he received the
Robinson Award for Surgery.
Dr. Laurencin is an expert in biomaterials, nanotechnology, stem cell science, and, the new
field he has pioneered, Regenerative Engineering. He is a fellow of American Institute of Chemical
Section Editors xi
Engineers and was named one of the 100 Engineers of the Modern Era by the AICHE. He received the Percy Julian Medal from National
Organization of Black Chemists and Chemical Engineers, and the Pierre Galletti Award from the American Institute of Medical and
Biological Engineering. He has received the NIH Director’s Pioneer Award and the National Science Foundation Emerging Frontiers
in Research and Innovation Award for his research in Regenerative Engineering.
Dr. Laurencin is an elected member of the National Academy of Engineering, the National Academy of Medicine, the Indian
National Academy of Engineering, the Indian National Academy of Sciences, and the African Academy of Sciences. He is an acade-
mician and foreign member of the Chinese Academy of Engineering.
Dr. Laurencin has two awards named in his honor. The W. Montague Cobb Institute and the National Medical Association estab-
lished the Cato T. Laurencin Lifetime Research Achievement Award, while the Society for Biomaterials established The Cato T. Lau-
rencin, M.D., Ph.D. Travel Fellowship Award.
Dr. Laurencin received the Presidential Faculty Fellow Award from President Bill Clinton and the Presidential Award for Excel-
lence in Science, Mathematics, and Engineering Mentoring from President Barack Obama. He is the recipient of the National Medal
of Technology and Innovation, America’s highest award for technological achievement from President Barack Obama in ceremonies
at the White House.
Diego Mantovani, Ph.D., FBSE.

Prof. Diego Mantovani is the director of Laboratory for Biomaterials and Bioengineering at
Laval University, in Canada, and senior scientist of the Regenerative Medicine Division of
the Quebec University Hospital Research Centre. He received his doctoral degree jointly
from University of Technology of Compiègne, France, and Laval University in 1999 and his
joint Diploma in Engineering from Politecnico di Milano and the University of Technology
of Compiegne, France, in 1993. After an industrial postdoc (1999), he becomes professor
at Laval University School of Science and Engineering in 2000. Since the beginning he estab-
lished is Laboratory at the University Hospital Research Center in Quebec City. Within his
team, researches focus on surface modifications by plasma, thin polymer functional films,
cell–materials interactions, degradable metals, scaffolds, and bioreactors for the replacement
and regeneration of cardiovascular tissue. He has authored more than 260 original articles,
holds 5 patents, and presented more than 185 keynotes, invited and seminar lectures world-
wide. His H-index is 43 (June 2018), and his works were cited more than 7000 times. He was
President of the Canadian Society for Biomaterials (2008–2009), and Executive Cochair of
the World Biomaterials Congress in 2016 in Montreal, Canada. In 2012, he was elected Fellow of the World Biomaterials Science
and Engineering Society. Since 2012, he is the holder of the Canada Research Chair 1 in Biomaterials and Bioengineering for the
Innovation in Surgery. He was member of ad hoc panels at FDA, ISO, and Health Canada and member of a number of funding,
regulatory and scientific committees worldwide. He is Adjunct Professor at Politecnico di Milano and Universita del Piemonte Ori-
entale in Italy, as well as at the Vellore Institute of Technology, in India. He was invited professor in several universities worldwide,
including Campinas, Brasil (2012–2015), Bologna (2015), Bordeaux (2014), Siao Tong West, China (2012), Cergy-Pontoise
(2012), ParisTech (2011), Buenos Aires (2010), Namur, Belgium (2008), Tor Vergata, Italy (2007), Ankara, Turkey (2006), and
others. He is member of the editorial board of five scientific journals in the field and of the advisory board of three medical devices
consortia worldwide.
William Z Rymer
Professor William Z Rymer is Professor of Physical Medicine and Rehabilitation and Physi-
ology at the Rehabilitation Institute of Chicago, Chicago, IL, United States. His focus of
work includes pathophysiology, stroke, spinal cord injury, spinal circuits, biomedical engi-
neering, and neural signal processing.
xii Section Editors
Pankaj Vadgama
Pankaj Vadgama qualified in Degree in Medicine and then in Chemistry at the University of
Newcastle upon Tyne, United Kingdom, with a First Class Honors BSc. He is a chemical
pathologist, becoming a Fellow of the Royal College of Pathology. He completed his Ph.D.
on medical biosensors as an MRC Fellow at Newcastle, and while there, he was made Director
of the Biosensors Group and later appointed as Professor of Clinical Biochemistry at the
University of Manchester, subsequently becoming Research Dean for the Faculty of Medicine.
He was appointed Director of the Interdisciplinary Research Centre in Biomedical Materials at
Queen Mary, University of London and was, until recently, Head of the Department of Clin-
ical Biochemistry, Barts Health NHS Trust. His main interests are variously biosensors,
applied bioelectrochemistry, point-of-care testing, and membrane technology. He has pub-
lished over 200 papers. He is also Fellow of the Royal Society of Medicine, Institute of Physics,
Royal Society of Chemistry, the Institute of Materials Minerals and Mining, and the Royal
Society of Biology. He was given the Foundation Award of the Association of Clinical
Biochemistry and Laboratory Medicine, has been a Sandoz Lecturer of the British Geriatric
Society, and delivered the Latner lecture at the University of Newcastle. He has served on
various UK Research Council grants award committees and is at present member of the Insti-
tute of Materials Minerals and Mining Smart Materials and Nano Committees and the
Biomedical Materials Application Division. He sits on various BSI committees and was Chair of the ISO subpanel on nanomedicine
nomenclature. He sits on various editorial boards and is Editor in Chief of Bioelectrochemistry. He is Deputy Chair of the Council for
the Frontiers of Science based in Uganda directed at research training in East Africa.
Min Wang
Min Wang is a Full Professor at The University of Hong Kong (HKU), and as Programme
Director (2013–2018), he has led HKU’s Medical Engineering Programme (which is retitled
to “Biomedical Engineering Programme” in 2018). He has worked in universities in the
United Kingdom (1991–1997), Singapore (1997–2002), and Hong Kong (2002–Present)
and has been a Guest Professor or Adjunct Professor of several universities in mainland China
(Shanghai Jiao Tong University, Zhejiang University, Tianjin University, Southwest Jiao tong
University, etc.). He was awarded BSc (1985) and Ph.D. (1991), both in Materials Science and
Engineering, by Shanghai Jiao Tong University and University of London, respectively. He is
a chartered engineer (CEng, 1995; UK) and chartered scientist (CSci, 2005; UK). He is an elec-
ted fellow of professional societies in the United Kingdom, Hong Kong, United States, and
internationally (FIMMM, 2001; FIMechE, 2007; FHKIE, 2010; FBSE, 2011; FAIMBE, 2012;
WAC Academician, 2013). Since 1991, he has been conducting research in biomaterials and
tissue engineering and developing new biomaterials using the composite/hybridization
approach. He was a founding member of UK’s Interdisciplinary Research Centre (IRC) in
Biomedical Materials at the University of London. His biomaterials research has covered
metals, polymers, ceramics, and composites and includes surface modification of materials or scaffolds. In recent years, he has
focused on nanobiomaterials, electrospinning, and 3D printing. He and his research staff/students have won many awards at inter-
national conferences. He has authored a large number of research papers as well as many book chapters. His research has been
widely cited by other researchers around the world. He has given many conference presentations, including more than 150 invited
talks at international conferences. He has also given more than 110 seminars in universities, research institutes, and hospitals in
Europe, North America, Asia, and Australia. He has been Chairman/Organizer of many conferences and has served in committees
of more than 70 international conferences. He is the Founding Series Editor of Springer Series in Biomaterials Science and Engineering
books and has been Editor, Associate Editor, or member of the Editorial Board of 20 international, printed journals, including Inter-
national Materials Reviews, Composites Science and Technology, Surface and Coatings Technology, Journal of Materials Science: Materials in
Medicine, and Journal of the Royal Society Interface. He has acted as a referee for more than 110 international journals in the fields of
materials science and engineering, biomaterials and tissue engineering, physics, chemistry, medicine, dentistry, medical devices,
biofabrication, nanoscience, nanotechnology, and 3D printing. He has been active in professional society activities and has served
in various roles in these societies. He was Chairman of the Biomedical Division of Hong Kong Institution of Engineers (HKIE). He
serves/has served in the Nomination Committee of World Academy of Ceramics (WAC) and the ICF-BSE Steering Committee of the
International College of Fellows of the International Union of Societies for Biomaterials Science and Engineering (IUS-BSE). He has
been an elected Council Member of Chinese Society for Biomaterials, Hong Kong Institution of Engineers, Asian Biomaterials Feder-
ation, World Association for Chinese Biomedical Engineers (WACBE), and Administrative Council of International Federation for
Medical and Biological Engineering (IFMBE). (http://web.hku.hk/memwang/).
Section Editors xiii
Alexander Wong
Alexander Wong, P.Eng., is currently the Canada Research Chair in Artificial Intelligence and
Medical Imaging, Codirector of the Vision and Image Processing Research Group, and an
Associate Professor in the Department of Systems Design Engineering at the University of
Waterloo. He had previously received the B.A.Sc. degree in Computer Engineering from
the University of Waterloo, Waterloo, ON, Canada, in 2005, the M.A.Sc. degree in Electrical
and Computer Engineering from the University of Waterloo, Waterloo, ON, Canada, in
2007, and Ph.D. degree in Systems Design Engineering from the University of Waterloo,
ON, Canada, in 2010. He was also an NSERC postdoctoral research fellow at Sunnybrook
Health Sciences Centre. He has published over 400 refereed journal and conference papers,
as well as patents, in various fields such as computational imaging, artificial intelligence,
computer vision, and medical imaging, and has received numerous awards such as 13 paper
awards at international conference and an Early Researcher Award from the Ministry of
Economic Development and Innovation.
Xiaojun Yu
Dr. Yu is Associate Professor, Biomedical Engineering at Stevens Institute of Technology,
Hoboken, NJ, United States. Dr. Yu’s primary research interests focus on tissue engineering,
polymeric biomaterials and drug delivery. His current research activities include nano- and
microscale functionalization of biomimic three-dimensional scaffolds for neural and
musculoskeletal tissue repair and regeneration, investigation of cell and material interac-
tions in bioreactors, development of controlled release systems for the delivery of growth
factors and drugs, and manipulation of microenvironment for stem cell proliferation and
differentiation.
CONTRIBUTORS TO VOLUME 1
Tyler Ackley M Bohner

UConn Health, Farmington, CT, United States RMS Foundation, Bettlach, Switzerland
Rafiq Ahmad M E Bronner
Chonbuk National University, Jeonju-si, Jeollabuk-do, California Institute of Technology, Pasadena, CA, USA
Republic of Korea
Ashley C Brown
Song Ih Ahn North Carolina State University and University of North
George W. Woodruff School of Mechanical Engineering, Carolina at Chapel Hill, Raleigh, NC, United States;
Wallace H. Coulter Department of Biomedical and North Carolina State University, Raleigh, NC,
Engineering, Institute for Electronics and United States
Nanotechnology, Parker H. Petit Institute for
Arnaud Bruyas
Bioengineering and Bioscience, Georgia Institute of
Stanford University, Stanford, CA, United States
Technology, Atlanta, GA, United States
S A Busch
Khiam Aik Khor
Athersys, Inc., Cleveland, OH, USA
Nanyang Technological University,
Singapore Ruth Cameron
University of Cambridge, Cambridge, United Kingdom
James M Anderson
Case Western Reserve University, Cleveland, OH, Sophie Cazalbou
United States UMR 5085 UPS-INPT-CNRS, Toulouse, France
Lavinia Cosmina Ardelean Paul Z Chen
“Victor Babes” University of Medicine and Pharmacy University of Waterloo, Waterloo, ON, Canada
Timisoara, Timisoara, Romania
Shiyu Cheng
A Atala Beijing Engineering Research Center for
Wake Forest Baptist Medical Center, WinstoneSalem, BioNanotechnology and CAS Key Laboratory for
NC, USA Biomedical Effects of Nanomaterials and Nanosafety,
CAS Center for Excellence in Nanoscience, National
S T Avecilla
Center for NanoScience and Technology, Beijing, P. R.
New York Presbyterian Hospital, Weill Cornell Medical
China
College, New York, NY, USA
Daniel Chester
Hani A Awad
North Carolina State University and University of North
University of Rochester, Rochester, NY, United States
Carolina at Chapel Hill, Raleigh, NC, United States;
Besim Ben-Nissan and North Carolina State University, Raleigh, NC,
University of Technology Sydney, Sydney, NSW, United States
Australia
Andy H Choi
Serena Best University of Technology Sydney, Sydney, NSW,
University of Cambridge, Cambridge, United Kingdom Australia
Aldo R Boccaccini Ezharul Hoque Chowdhury
University of Erlangen-Nuremberg, Erlangen, Germany Monash University, Clayton, VIC, Australia
xv
xvi Contributors to Volume 1
Tzahi Cohen-Karni Margaret A T Freeberg

Carnegie Mellon University, Pittsburgh, PA, United University of Rochester, Rochester, NY, United States
States
Katie Gailiunas
M Csete University of Connecticut, Storrs, CT, United States
Huntington Medical Research Institutes, Pasadena, CA,
Emmanuel Gibon
USA
M M Cushing Stuart B Goodman
New York Presbyterian Hospital, Weill Cornell Medical Stanford University, Stanford, CA, United States
College, New York, NY, USA
Paul Frank Gratzer
Michael A Daniele School of Biomedical Engineering, Dalhousie University,
North Carolina State University and University of North Halifax, NS, Canada
and North Carolina State University, Raleigh, NC, Frank X Gu
United States University of Waterloo, Waterloo, ON, Canada
Natalia Davidenko Vincenzo Guarino

University of Cambridge, Cambridge, United Kingdom National Research Council of Italy, Naples, Italy
Lin Guo
Caroline N Dealy
UConn Health, Farmington, CT, United States The University of Hong Kong, Pokfulam, Hong Kong
Babak Hassan Beygi
Jinqi Deng
The Hong Kong Polytechnic University of Hong Kong,
Beijing Engineering Research Center for
Hung Hom, Hong Kong
BioNanotechnology and CAS Key Laboratory for
Biomedical Effects of Nanomaterials and Nanosafety, Y Hayashi
CAS Center for Excellence in Nanoscience, National Department of Dental Regenerative Medicine, Center of
Center for NanoScience and Technology, Beijing, P. R. Advanced Medicine for Dental and Oral Diseases,
China; and Sino-Danish College, University of Chinese National Center for Geriatrics and Gerontology,
Academy of Sciences, Beijing, P. R. China Research Institute, Obu, Japan
Kyle G Doherty J S Hayes
University of Liverpool, Liverpool, United Kingdom NUI Galway, Galway, Ireland
Dionysios Douroumis Michelle Hobert
University of Greenwich, Greenwich, United Kingdom University of Connecticut, Storrs, CT, United States
Bin Duan T Hochgreb-Hägele
University of Nebraska Medical Center, Omaha, NE, California Institute of Technology, Pasadena, CA, USA
United States
Rong Huang
Felipe Eltit Queensland University of Technology, Brisbane, QLD,
The University of British Columbia, Vancouver, BC, Australia
Canada
K Ishida
Jorge Luis Escobar Ivirico Tokyo University of Science, Noda, Chiba, Japan
University of Connecticut, Storrs, CT, United States;
Manisha Jassal
and University of Connecticut Health Center,
Stevens Institute of Technology, Hoboken, NJ, United
Farmington, CT, United States
States
Bing Fang
Wenkai Jia
University of Delaware, Newark, DE, United States
Michigan Technological University, Houghton, MI,
Melanie Fisher United States
UConn Health, Farmington, CT, United States
Sirui Jiang
Kate Fox Case Western Reserve University, Cleveland, OH,
RMIT University, Melbourne, VIC, Australia United States
Contributors to Volume 1 xvii
Xingyu Jiang Dimitrios A Lamprou

Beijing Engineering Research Center for University of Kent, Canterbury, United Kingdom
BioNanotechnology and CAS Key Laboratory for
Cato T Laurencin
Biomedical Effects of Nanomaterials and Nanosafety,
University of Connecticut Health Center, Farmington,
CAS Center for Excellence in Nanoscience, National
CT, United States; and University of Connecticut,
Center for NanoScience and Technology, Beijing, P. R.
Storrs, CT, United States
China; and Sino-Danish College, University of Chinese
Academy of Sciences, Beijing, P. R. China Yunki Lee
Radoslaw Junka George W. Woodruff School of Mechanical Engineering,
Stevens Institute of Technology, Hoboken, NJ, United Wallace H. Coulter Department of Biomedical
States Engineering, Institute for Electronics and
Jacob G Kallenbach Bioengineering and Bioscience, Georgia Institute of
University of Rochester, Rochester, NY, United States Technology, Atlanta, GA, United States
Victoria R Kearns Andreas Lendlein
University of Liverpool, Liverpool, United Kingdom Institute of Biomaterial Science and Berlin Brandenburg
Stephnie M Kennedy Center for Regenerative Therapies, Teltow, Germany
University of Liverpool, Liverpool, United Kingdom Hannah J Levis
Yusuf Khan University of Liverpool, Liverpool, United Kingdom
University of Connecticut Health Center, Farmington,
Jiao Jiao Li
CT, United States
University of Sydney, Sydney, NSW, Australia; Kolling
Gilson Khang Institute, Northern Sydney Local Health District, St
Chonbuk National University, Jeonju-si, Jeollabuk-do, Leonards, NSW, Australia; and Sydney Medical School
Republic of Korea Northern, University of Sydney, St Leonards, NSW,
Australia
Kristi L Kiick
University of Delaware, Newark, DE, United States Zhong Li
Nanyang Technological University, Singapore
Sungwoo Kim
Stanford University, Stanford, CA, United States Liliana Liverani
University of Erlangen-Nuremberg, Erlangen,
YongTae Kim
Germany
George W. Woodruff School of Mechanical Engineering,
Wallace H. Coulter Department of Biomedical Thilanga Liyanage
Engineering, Institute for Electronics and University of Kentucky, Lexington, KY, United States
Tianzhi Luo
Bioengineering and Bioscience, Georgia Institute of
Technology, Atlanta, GA, United States
Christopher Mancuso
Nicholas J Kohrs
University of Connecticut, Storrs, CT, United States
University of Kentucky, Lexington, KY, United States
Ohan S Manoukian
D S Koslov
University of Connecticut Health, Farmington, CT,
Wake Forest Baptist Medical Center, WinstoneSalem,
United States; and University of Connecticut, Storrs,
NC, USA
CT, United States
Sangamesh G Kumbar
Ethan A Marrow
University of Connecticut Health, Farmington, CT,
North Carolina State University and University of North
United States; and University of Connecticut, Storrs,
CT, United States
and North Carolina State University, Raleigh, NC,
Vincenzo La Carrubba United States
University of Palermo, Palermo, Italy
Z Master
Rebecca Lace Albany Medical College, Albany, NY, USA; and
University of Liverpool, Liverpool, United Kingdom University of Alberta, Edmonton, AB, Canada
xviii Contributors to Volume 1
Stefani Mazzitelli David A Puleo

University of Ferrara, Ferrara, Italy University of Kentucky, Lexington, KY, United States
R Morita Zichen Qian
Tokyo University of Science, Noda, Chiba, Japan Michigan Technological University, Houghton, MI,
United States
Mahboob Morshed
Independent University, Dhaka, Bangladesh Ru Qing Yu
The University of Hong Kong, Hong Kong
Amir Najarzadeh
University of Kentucky, Lexington, KY, United States Daniel Radke
Michigan Technological University, Houghton, MI,
M Nakashima
United States
Department of Dental Regenerative Medicine, Center of
Advanced Medicine for Dental and Oral Diseases, P Rajan
National Center for Geriatrics and Gerontology, The Scripps Research Institute, La Jolla, CA, USA
Research Institute, Obu, Japan
Sahil Kumar Rastogi
Claudio Nastruzzi Carnegie Mellon University, Pittsburgh, PA, United
University of Ferrara, Ferrara, Italy States
Inn Chuan Ng Muhammad Y Razzaq
National University of Singapore, Singapore Institute of Biomaterial Science and Berlin Brandenburg
Center for Regenerative Therapies, Teltow, Germany
D H Nguyen
Shiley Eye Center and Institute for Genomic Medicine, Lucien Reclaru
University of California at San Diego, La Jolla, CA, USA VVSA, branch of Richemont International SA Varinor
Innovation, Delémont, Switzerland
Mitsuo Niinomi
Tohoku University, Sendai, Japan; Osaka University, Markus Reinthaler
Osaka, Japan; Meijo University, Nagoya, Japan; and Institute of Biomaterial Science and Berlin Brandenburg
Nagoya University, Nagoya, Japan Center for Regenerative Therapies, Teltow, Germany
Bridget Oei Nicholas P Rhodes
UConn Health, Farmington, CT, United States University of Liverpool, Liverpool, United Kingdom
Anurag Ojha Kelsey Richard
University of Connecticut, Storrs, CT, United States UConn Health, Farmington, CT, United States
M Oshima R G Richards
Tokyo University of Science, Noda, Chiba, Japan AO Research Institute, Davos, Switzerland
H Ouyang Aaqil Rifai
Shiley Eye Center and Institute for Genomic Medicine, RMIT University, Melbourne, VIC, Australia
University of California at San Diego, La Jolla, CA, USA
Steven A Ross
Chi-Chun Pan University of Greenwich, Greenwich, United Kingdom
M Saito
Pornteera Pawijit Tokyo University of Science, Noda, Chiba, Japan
National University of Singapore, Singapore
Naseem Sardashti
Aura Penalosa University of Connecticut Health, Farmington, CT,
University of Connecticut, Storrs, CT, United States United States; and University of Connecticut, Storrs,
CT, United States
D Pergament
Case Western Reserve University School of Law, Mark Schröder
Cleveland, OH, USA; and Children’s Law Group, LLC, Institute of Biomaterial Science and Berlin Brandenburg
Chicago, IL, USA Center for Regenerative Therapies, Teltow, Germany
Elena Pirogova Nikolaos Scoutaris
RMIT University, Melbourne, VIC, Australia University of Greenwich, Greenwich, United Kingdom
Contributors to Volume 1 xix
Swaminathan Sethuraman Mian Wang

SASTRA Deemed University, Thanjavur, India Northeastern University, Boston, MA, United States
Jeong Eun Song Min Wang
Chonbuk National University, Jeonju-si, Jeollabuk-do, The University of Hong Kong, Pokfulam, Hong Kong
Republic of Korea
Qiong Wang
Alexander Martin Stahl The University of British Columbia, Vancouver, BC,
Stanford University, Stanford, CA, United States Canada
Teagen Stedman Rizhi Wang
University of Connecticut Health, Farmington, CT, The University of British Columbia, Vancouver, BC,
United States Canada
Anuradha Subramanian Thomas J Webster
SASTRA Deemed University, Thanjavur, India Northeastern University, Boston, MA, United States;
Millicent O Sullivan and Wenzhou Medical University, Wenzhou, China
Rachel L Williams
Haoran Sun University of Liverpool, Liverpool, United Kingdom
Christian Wischke
Mitch Tahtinen Institute of Biomaterial Science and Berlin Brandenburg
Michigan Technological University, Houghton, MI, Center for Regenerative Therapies, Teltow, Germany
United States
Man Sang Wong
Jordon Tan The Hong Kong Polytechnic University of Hong Kong,
Temasek Polytechnic, Singapore Hung Hom, Hong Kong
A E Ting Yin Xiao
Athersys, Inc., Cleveland, OH, USA Queensland University of Technology, Brisbane, QLD,
Nirmalya Tripathy Australia
University of Washington, Seattle, WA, United States Yunzhi Yang
T Tsuji Stanford University, Stanford, CA, United States
Tokyo University of Science, Noda, Chiba, Japan; and Hanry Yu
Organ Technologies Inc., Tokyo, Japan National University of Singapore, Singapore; Agency for
N Turovets Science, Technology and Research (A*STAR),
University of California, Irvine, CA, USA Singapore; BioSyM, Singapore-MIT Alliance for
Research and Technology, Singapore; and Nanfang
Morgan A Urello Hospital, Southern Medical University, Guangzhou,
University of Delaware, Newark, DE, United States China
Antonio Valdevit
Xiaojun Yu
Stevens Institute of Technology, Hoboken, NJ, United Stevens Institute of Technology, Hoboken, NJ, United
States; and SEA Limited, Columbus, OH, United States
States
Nandakumar Venkatesan
K Zhang
University of Kentucky, Lexington, KY, United States
Shiley Eye Center and Institute for Genomic Medicine,
Chong Wang University of California at San Diego, La Jolla, CA, USA
Dongguan University of Technology, Dongguan, China
Feng Zhao
Guifang Wang Michigan Technological University, Houghton, MI,
Michigan Technological University, Houghton, MI, United States
United States
Qilong Zhao
Jing Yi Wang Shenzhen Institutes of Advanced Technology, Chinese
The University of Hong Kong, Hong Kong Academy of Sciences, Shenzhen, China
xx Contributors to Volume 1
Li Wu Zheng Yu Zheng
Prince Philip Dental Hospital, The University of Hong The Hong Kong Polytechnic University of Hong Kong,
Kong, Hong Kong Hung Hom, Hong Kong
Wenfu Zheng Yinghong Zhou
Beijing Engineering Research Center for Queensland University of Technology, Brisbane, QLD,
BioNanotechnology and CAS Key Laboratory for Australia
Biomedical Effects of Nanomaterials and Nanosafety,
CAS Center for Excellence in Nanoscience, National Hala Zreiqat
Center for NanoScience and Technology, Beijing, P. R. University of Sydney, Sydney, NSW, Australia
China
CONTENTS OF VOLUME 1
Editorial Board v
Editor in Chief vii
Section Editors ix
Contents of All Volumes xxv
Preface xxxv
Biomaterials: Science and Engineering

Alternative Processing Techniques for CoCr Dental Alloys 1
Lucien Reclaru and Lavinia Cosmina Ardelean
Bioceramics 16
Besim Ben-Nissan, Sophie Cazalbou, and Andy H Choi
Biomedical Composites 34
Min Wang and Qilong Zhao
Bulk Properties of Biomaterials and Testing Techniques 53
Min Wang and Chong Wang
Corrosion of Orthopedic Implants 65
Qiong Wang, Felipe Eltit, and Rizhi Wang
Decellularized Extracellular Matrix 86
Paul Frank Gratzer
Diamond, Carbon Nanotubes and Graphene for Biomedical Applications 97
Aaqil Rifai, Elena Pirogova, and Kate Fox
Gold Nanoparticles for Colorimetric Detection of Pathogens 108
Paul Z Chen and Frank X Gu
Manufacture of Biomaterials 116
Min Wang, Lin Guo, and Haoran Sun
Materials and Their Biomedical Applications 135
Min Wang and Bin Duan
Nano-Biomaterials and their Applications 153
Mian Wang and Thomas J Webster
xxi
xxii Contents of Volume 1
Natural Biopolymers for Biomedical Applications 162

Natalia Davidenko, Ruth Cameron, and Serena Best
Polymeric Coatings and Their Fabrication for Medical Devices 177
Dimitrios A Lamprou, Nikolaos Scoutaris, Steven A Ross, and Dionysios Douroumis
Porous Biomaterials and Scaffolds for Tissue Engineering 188
Liliana Liverani, Vincenzo Guarino, Vincenzo La Carrubba, and Aldo R Boccaccini
Preparation and Properties of Coatings and Thin Films on Metal Implants 203
Zhong Li and Khiam Aik Khor
Titanium Alloys 213
Mitsuo Niinomi
Biomaterials: In Vitro and in Vivo Studies of Biomaterials

Anatomy and Physiology for Biomaterials Research and Development 225
Inn Chuan Ng, Pornteera Pawijit, Jordon Tan, and Hanry Yu
Animal Models in Biomaterial Development 237
James M Anderson and Sirui Jiang
Blood–Biomaterial Interactions 242
Nicholas P Rhodes
Interaction Between Mesenchymal Stem Cells and Immune Cells in Tissue Engineering 249
Rong Huang, Yinghong Zhou, and Yin Xiao
Osseointegration of Permanent and Temporary Orthopedic Implants 257
J S Hayes and R G Richards
Tissue Response to Biomaterials 270
Jiao Jiao Li and Hala Zreiqat
Biomaterials: Biomaterial Applications and Advanced Medical Technologies

Biomaterials in Dentistry 278
Li Wu Zheng, Jing Yi Wang, and Ru Qing Yu
Biomaterials in Ophthalmology 289
Rachel L Williams, Hannah J Levis, Rebecca Lace, Kyle G Doherty, Stephnie M Kennedy, and Victoria R Kearns
Biomaterials in Orthopaedics 301
Emmanuel Gibon and Stuart B Goodman
Cell Encapsulation and Delivery 308
Stefani Mazzitelli and Claudio Nastruzzi
Drug Delivery Systems and Controlled Release 316
Nicholas J Kohrs, Thilanga Liyanage, Nandakumar Venkatesan, Amir Najarzadeh, and David A Puleo
Electrospinning and Electrospray for Biomedical Applications 330
Gene Delivery and Clinical Applications 345
Mahboob Morshed and Ezharul Hoque Chowdhury
Materials for Exoskeletal Orthotic and Prosthetic Systems 352
Man Sang Wong, Babak Hassan Beygi, and Yu Zheng
Contents of Volume 1 xxiii
Microfluidics for Biomedical Applications 368

Shiyu Cheng, Jinqi Deng, Wenfu Zheng, and Xingyu Jiang
Organs-on-Chips 384
Yunki Lee, Song Ih Ahn, and YongTae Kim
Shape-Memory Polymer Medical Devices 394
Muhammad Y Razzaq, Markus Reinthaler, Mark Schröder, Christian Wischke, and Andreas Lendlein
Regenerative Engineering
Adult Bone Marrow-Derived Stem Cells: Immunomodulation in the Context of Disease and Injury 406
A E Ting and S A Busch
Assessment of Cellular Responses of Tissue Constructs in vitro in Regenerative Engineering 414
Margaret A T Freeberg, Jacob G Kallenbach, and Hani A Awad
Assessment of Tissue Constructs In Vivo in Regenerative Engineering 427
Anuradha Subramanian and Swaminathan Sethuraman
Bioengineered Kidney and Bladder 432
D S Koslov and A Atala
Bioengineering Scaffolds for Regenerative Engineering 444
Zichen Qian, Daniel Radke, Wenkai Jia, Mitch Tahtinen, Guifang Wang, and Feng Zhao
Biomaterials for Tissue Engineering and Regenerative Medicine 462
Ohan S Manoukian, Naseem Sardashti, Teagen Stedman, Katie Gailiunas, Anurag Ojha,
Aura Penalosa, Christopher Mancuso, Michelle Hobert, and Sangamesh G Kumbar
Biomimetic Approaches for Regenerative Engineering 483
Nirmalya Tripathy, Rafiq Ahmad, Jeong Eun Song, and Gilson Khang
Bioreactors: System Design and Application for Regenerative Engineering 496
Antonio Valdevit
Bone Substitute Materials 513
M Bohner
Case Studies for Soft Tissue Regenerative Engineering 530
Jorge Luis Escobar Ivirico and Cato T Laurencin
Characterizing the Properties of Tissue Constructs for Regenerative Engineering 537
Yusuf Khan
Clinical and Laboratory Aspects of Hematopoietic Stem Cell Transplantation 546
S T Avecilla and M M Cushing
Dental Stem Cells 554
M Nakashima and Y Hayashi
Drug and Gene Delivery for Regenerative Engineering 565
Morgan A Urello, Tianzhi Luo, Bing Fang, Kristi L Kiick, and Millicent O Sullivan
Ethics of Issues and Stem Cell Research: the Unresolved Issues 584
Z Master
Eye Diseases and Stem Cells 598
H Ouyang, D H Nguyen, and K Zhang
xxiv Contents of Volume 1
Human Parthenogenetic Pluripotent Stem Cells 608

N Turovets and M Csete
Human Pluripotent Stem Cells 618
P Rajan
Introduction to Regenerative Engineering 624
Manisha Jassal, Radoslaw Junka, Cato T Laurencin, and Xiaojun Yu
Nanoelectronics for Neuroscience 631
Sahil Kumar Rastogi and Tzahi Cohen-Karni
Neural Crest Stem Cells 650
T Hochgreb-Hägele and M E Bronner
Osteoarthritis at the Cellular Level: Mechanisms, Clinical Perspectives, and Insights From Development 660
Melanie Fisher, Tyler Ackley, Kelsey Richard, Bridget Oei, and Caroline N Dealy
Reproductive Technologies, Assisted 677
D Pergament
Tooth Regenerative Therapy: Tooth Tissue Repair and Whole Tooth Replacement 686
M Oshima, K Ishida, R Morita, M Saito, and T Tsuji
Vascularized Tissue Regenerative Engineering Using 3D Bioprinting Technology 696
Sungwoo Kim, Arnaud Bruyas, Chi-Chun Pan, Alexander Martin Stahl, and Yunzhi Yang
Wound Healing and the Host Response in Regenerative Engineering 707
Daniel Chester, Ethan A Marrow, Michael A Daniele, and Ashley C Brown
CONTENTS OF ALL VOLUMES
VOLUME 1
Bioceramics 16
Paul Frank Gratzer
xxv
xxvi Contents of All Volumes

Titanium Alloys 213
Mitsuo Niinomi

Nicholas P Rhodes

Organs-on-Chips 384
Contents of All Volumes xxvii

Antonio Valdevit
M Bohner
Yusuf Khan
Z Master
P Rajan
xxviii Contents of All Volumes

D Pergament
VOLUME 2
Biomechanics
Biomechanics of Cells as Potential Biomarkers for Diseases: A New Tool in Mechanobiology 1
Dinesh R Katti, Kalpana S Katti, Shahjahan Molla, and Sumanta Kar
Bone Micro- and Nanomechanics 22
Caitlyn J Collins, Orestis G Andriotis, Vedran Nedelkovski, Martin Frank, Orestis L Katsamenis, and
Philipp J Thurner
Cell Adhesion: Basic Principles and Computational Modeling 45
Diego A Vargas and Hans Van Oosterwyck
Centrifugation and Hypergravity in the Bone 59
Carmelo Mastrandrea and Laurence Vico
Computational Modeling of Respiratory Biomechanics 70
Christian J Roth, Lena Yoshihara, and Wolfgang A Wall
Constitutive Modeling of Soft Tissues 81
Michele Marino
Continuum Mechanics and Its Practical Applications at the Level of Scaling Laws 111
Ko Okumura
CT-Based Bone and Muscle Assessment in Normal and Pathological Conditions 119
Paolo Gargiulo, Magnus K Gislason, Kyle J Edmunds, Jonathan Pitocchi, Ugo Carraro, Luca Esposito,
Massimiliano Fraldi, Paolo Bifulco, Mario Cesarelli, and Halldór Jónsson
Knowledge Extraction From Medical Imaging for Advanced Patient-Specific Musculoskeletal Models 135
Marie-Christine Ho Ba Tho and Tien Tuan Dao
Contents of All Volumes xxix
Mathematical Quantification of the Impact of Microstructure on the Various Effective

Properties of Bones 143
Miao-Jung Y Ou, Annalisa De Paolis, and Luis Cardoso
Multiphase Porous Media Models for Mechanics in Medicine: Applications to Transport Oncophysics
and Diabetic Foot 155
Pietro Mascheroni, Raffaella Santagiuliana, and Bernhard Schrefler
Multiscale Bone Mechanobiology 167
Stefan Scheiner, Maria-Ioana Pastrama, Peter Pivonka, and Christian Hellmich
Multiscale Mechanical Behavior of Large Arteries 180
Claire Morin, Witold Krasny, and Stéphane Avril
Nanoindentation-Based Characterization of Hard and Soft Tissues 203
Pasquale Vena and Dario Gastaldi
Nanomechanical Raman Spectroscopy in Biological Materials 215
Yang Zhang, Ming Gan, and Vikas Tomar
On the Use of Population-Based Statistical Models in Biomechanics 229
Justin Fernandez, Shasha Yeung, Alex Swee, Marco Schneider, Thor Besier, and Ju Zhang
Poroelasticity of Living Tissues 238
Andrea Malandrino and Emad Moeendarbary
Structural and Material Changes of Human Cortical Bone With Age: Lessons from the Melbourne
Femur Research Collection 246
Romane Blanchard, C David L Thomas, Rita Hardiman, John G Clement, David C Cooper, and
Peter Pivonka
Vascular Tissue Biomechanics: Constitutive Modeling of the Arterial Wall 265
Thomas Christian Gasser
Medical Devices
3D Printing in the Biomedical Field 275
Alexander K Nguyen, Roger J Narayan, and Ashkan Shafiee
Biocompatibility Evaluation of Orthopedic Biomaterials and Medical Devices: A Review of Safety
and Efficacy Models 281
Michel Assad and Nicolette Jackson
Biological Grafts: Surgical Use and Vascular Tissue Engineering Options for Peripheral Vascular Implants 310
Francesca Boccafoschi, Martina Ramella, Luca Fusaro, Marta C Catoira, and Francesco Casella
Current Advancements and Challenges in Stent-Mediated Gene Therapy 322
Shounak Ghosh, Katari Venkatesh, and Dwaipayan Sen
Dentistry: Restorative and Regenerative Approaches 332
Geetha Manivasagam, Aakash Reddy, Dwaipayan Sen, Sunita Nayak, Mathew T Mathew, and
Asokami Rajamanikam
Ephemeral Biogels: Potential Applications as Active Dressings and Drug Delivery Devices 348
Larreta-Garde Véronique, Picard Julien, and Giraudier Sébastien
Immunological Responses in Orthopedics and Transplantation 359
Caroline D Hoemann and Martin Guimond
xxx Contents of All Volumes
Iron-Based Degradable Implants 374

Sergio Loffredo, Carlo Paternoster, and Diego Mantovani
Medical Devices: Coronary Stents 386
Vanessa Montaño-Machado, Malgorzata Sikora-Jasinska, Carolina Catanio Bortolan, Pascale Chevallier,
and Diego Mantovani
Medical Devices in Otorhinolaryngology 399
Paolo Aluffi Valletti, Massimiliano Garzaro, and Valeria Dell’Era
Medical Devices in Neurology 409
Abbas Z Kouzani and Roy V Sillitoe
Obstetrics and Gynecology: Hysteroscopy 414
Antonio Santos-Paulo
Orthopedic Implants 425
Weihong Jin and Paul K Chu
Pharmacology: Drug Delivery 440
Frédéric Chaubet, Violeta Rodriguez-Ruiz, Michel Boissière, and Diego Velasquez
Prosthetic Aortic Valves 454
Anne-Sophie Zenses, Philippe Pibarot, Marie-Annick Clavel, Ezequiel Guzzetti, Nancy Cote, and Erwan Salaun
Urology and Nephrology: Regenerative Medicine Applications 467
Ingrid Saba, Stéphane Chabaud, Sophie Ramsay, Hazem Orabi, and Stéphane Bolduc
Zinc-Based Degradable Implants 478
Ehsan Mostaed, Malgorzata Sikora-Jasinska, and Maurizio Vedani
Medical Imaging
Biomechanics Imaging and Analysis 488
Reza Sharif Razavian, Sara Greenberg, and John McPhee
Breast Imaging: Mammography, Digital Tomosynthesis, Dynamic Contrast Enhancement 501
Mehran Ebrahimi
Diffusion Magnetic Resonance Imaging 505
Jennifer Shane Williamson Campbell and Gilbert Bruce Pike
Digital Holographic Microscopy 519
Farnoud Kazemzadeh and Alexander Wong
Digital Pathology 524
Matthew G Hanna and Liron Pantanowitz
Functional Magnetic Resonance Imaging 533
Jean Chen and Julien Cohen-Adad
Hemodynamic Imaging 545
Robert Amelard and Alexander Wong
Imaging Informatics 551
David A Koff and Thomas E Doyle
Macroscopic Pigmented Skin Lesion Prescreening 561
Eliezer Bernart, Eliezer Soares Flores, and Jacob Scharcanski
Contents of All Volumes xxxi
Magnetic Resonance Imaging 574

Rachel W Chan, Justin Y C Lau, Wilfred W Lam, and Angus Z Lau
Perceptual Quality Assessment of Medical Images 588
Hantao Liu and Zhou Wang
Radiomics 597
Farzad Khalvati, Yucheng Zhang, Alexander Wong, and Masoom A Haider
Ultrasound Elastography 604
Hyock Ju Kwon and Bonghun Shin
Rehabilitation Engineering and Integrative Technologies

Functional Electric Stimulation Therapy 614
Dejan B Popovic
ProsthesesdAssistive TechnologydSports 621
Bryce T J Dyer
ProsthesesdAssistive TechnologydUpper 632
Jonathon W Sensinger, Wendy Hill, and Michelle Sybring
Robotics: Exoskeletons 645
Daniel P Ferris, Bryan R Schlink, and Aaron J Young
RoboticsdSoft Robotics 652
Gursel Alici
VOLUME 3
Mathematical Techniques in Biomedical Engineering

Cardiac Modeling 1
Edward Vigmond and Gernot Plank
Mathematical Approaches for Medical Image Registration 21
Barbara Zitova
Mathematical Modeling of Gene Networks 33
Lakshmi Sugavaneswaran
Mathematical Modeling Tools and Software for BME Applications 56
Fred J Vermolen and Amit Gefen
Mathematical Techniques for Biomedical Image Segmentation 64
Roberto Rodríguez and Juan H Sossa
Mathematical Techniques for Circulatory Systems 79
Jason Carson, Raoul Van Loon, and Perumal Nithiarasu
Mathematical Techniques for Noninvasive Muscle Signal Analysis and Interpretation 95
Roberto Merletti, Ales Holobar, and Dario Farina
Optimization Techniques in BME 112
Jeevan Kumar Pant
xxxii Contents of All Volumes
Single-Cell-Based In Silico Models: A Tool for Dissecting Tumor Heterogeneity 130

Aleksandra Karolak, Saharsh Agrawal, Samantha Lee, and Katarzyna A Rejniak
Spectrotemporal Modeling of Biomedical Signals: Theoretical Foundation and Applications 144
Raymundo Cassani and Tiago H Falk
Statistical Modeling in Biomedical Engineering 164
Yunfeng Wu and Pinnan Chen
Time–Frequency Distributions in Biomedical Signal Processing 177
Yashodhan Athavale and Sridhar Krishnan
Wavelets in Biomedical Signal Processing and Analysis 193
Babak Azmoudeh and Dean Cvetkovic
Bioinstrumentation and Bioinformatics

A Systematic Workflow for Design and Computational Analysis of Protein Microarrays 213
Jonatan Fernández-García, Rodrigo García-Valiente, Javier Carabias-Sánchez, Alicia Landeira-Viñuela,
Rafael Góngora, María Gonzalez-Gonzalez, and Manuel Fuentes
Ambulatory EEG Monitoring 223
Bernard Grundlehner and Vojkan Mihajlovic
Automated EEG Analysis for Neonatal Intensive Care 240
Nathan Stevenson and Anton Tokariev
Big Data Calls for Machine Learning 258
Andreas Holzinger
Bioimpedance Spectroscopy Processing and Applications 265
Herschel Caytak, Alistair Boyle, Andy Adler, and Miodrag Bolic
Bioinformatics in Design of Antiviral Vaccines 280
Ashesh Nandy and Subhash C Basak
Bioinformatics in Disease Classification 291
Miguel Ángel Medina
Biopotential Monitoring 296
Julián Oreggioni, Angel A Caputi, and Fernando Silveira
Blood Gas Analysis and Instrumentation 305
Rebecca Symons, Robindro Chatterji, Kirsty Whenan, Rita Horvath, and Paul S Thomas
Computational Approaches in microRNA Biology 317
Ulf Schmitz, Shailendra K Gupta, Julio Vera, and Olaf Wolkenhauer
Detection and Classification of Breast Lesions Using Ultrasound-Based Imaging Modalities 331
Md Kamrul Hasan and Sharmin R Ara
DNA Microarrays: Fundamentals, Data Integration and Applications 349
Eduardo Valente and Miguel Rocha
ECG Monitoring: Present Status and Future Trend 363
Saurabh Pal
Genetic Algorithms for Breast Cancer Diagnostics 380
Florin Gorunescu and Smaranda Belciug
Contents of All Volumes xxxiii
Machine Learning in Biomedical Informatics 389

Carlos Fernandez-Lozano, Adrián Carballal, Cristian R Munteanu, Marcos Gestal, Víctor Maojo,
and Alejandro Pazos
Matrix Assisted Laser Desorption/Ionization as a New Cancer Diagnostic Tool 400
Bozena Hosnedlova, Marta Kepinska, Branislav Ruttkay-Nedecky, Carlos Fernandez, Tomas Parak,
Halina Milnerowicz, Jiri Sochor, Geir Bjørklund, and Rene Kizek
Medical Utility of NIR Monitoring 415
Zuzana Kovacsova, Gemma Bale, and Ilias Tachtsidis
Metabolomics in Biomaterial Research 432
Ana M Gil, Maria H Fernandes, and Iola F Duarte
Nucleic-Acid Sequencing 443
G Dorado, S Gálvez, H Budak, T Unver, and P Hernández
Optical Techniques for Blood and Tissue Oxygenation 461
Panayiotis Kyriacou, Karthik Budidha, and Tomas Y Abay
Polymerase Chain Reaction (PCR) 473
G Dorado, G Besnard, T Unver, and P Hernández
Single-Photon Emission Computed Tomography: Principles and Applications 493
Yong Du and Habib Zaidi
Translational Bioinformatics: Informatics, Medicine, and -Omics 507
Sergio Paraiso-Medina, David Perez-Rey, Raul Alonso-Calvo, Cristian R Munteanu, Alejandro Pazos,
Casimir A Kulikowski, and Victor Maojo
Ultrasonic Imaging in Biomedical Applications 515
Roman Gr Maev and Fedar M Severin
Index 523
PREFACE
The use by man of available technology to treat damaged or diseased tissue is older than the written historical
record. For example, the Mayan people created artificial teeth out of nacre, which were shown to be fused to the
bone (Bobbio, 1972; Westbroek & Marin, 1998). Giovanni Borelli’s studies of the cardiovascular system (e.g.,
the elasticity of arteries), which were published in De Motu Animalium (On Animal Motion), can be considered
as one of the foundations of the field of biomechanics (Parker, 2009). The hypothesis of an intrinsic ’animal
electricity’ by Luigi Galvani in the 18th century led to the development of the field of electrophysiology (Pic-
colino, 1997). In the 19th century, the development of the antiseptic approach to surgical procedures by Joseph
Lister made implantation of medical devices without certain postoperative infection possible; for example,
Lister described the use of silver wire for treatment of a fractured patella (Worboys, 2013). The discovery of X-
rays by Roentgen at the end of the 19th century was rapidly translated for medical imaging (Rowland, 1896;
Schuster, 1896). In our lifetime, the work by W. T. Green on generating new cartilage by seeding of chondrocytes
as well as by John Burke and Ioannis Yannas on generating skin substitutes is recognized as the birth of the field
of regenerative engineering (Vacanti, 2006).
At the beginning of the 21st century, the American Institute for Medical and Biological Engineering
identified several research areas for the field of biomedical engineering. These include: (a) functional geno-
mics and proteomics, (b) nanotechnology, (c) targeted drug delivery, (d) tissue engineering, and (e) the
development of new types of medical instrumentation (Hendee, Chien, Maynard, & Dean, 2002). As some of
these research areas have matured, others have become more prominent. Over the past few years, the use of
3D printing and bioprinting technologies to create medical devices has become more prominent. One benefit
of utilizing 3D printing and bioprinting for patient care is that medical imaging data (e.g., magnetic resonance
imaging and computed tomography data) may be employed to fashion prostheses or artificial tissues with
submicroscale features that conform with the requirements of the patient (Narayan, Doraiswamy, Chrisey, &
Chichkov, 2010; Skoog & Narayan, 2013). Another technology that will likely transform the field of
biomedical engineering over the coming decades involves the use of clustered regularly interspaced short
palindromic repeats (CRISPR)/Cas9 for engineering of the human genome. The interface between biomedical
engineering and the new field of genome engineering has already spawned research into new technologies for
delivery of genome editing tools into the body; the synergy between these fields will only grow over time
(Wright, Nuñez, & Doudna, 2016).
The goal of the Encyclopedia of Biomedical Engineering is to consider the principles and technologies that
underlie the field of biomedical engineering. The encyclopedia is divided into three volumes. The first volume
contains a section on biomaterials, which was edited by Min Wang at the University of Hong Kong, and
a section on regenerative engineering, which was edited by Cato Laurencin at the University of Connecticut and
Xiaojun Yu at the Stevens Institute of Technology. The second volume contains a section on rehabilitation
engineering and integrative technologies, which was edited by William Rymer and Levi Hargrove at North-
western University, a section on biomechanics, which was edited by Christian Hellmich at the Vienna University
of Technology, a section on medical devices, which was edited by Diego Mantovani at the University of Laval,
and a section on medical imaging, which was edited by Alexander Wong at the University of Waterloo. The third
volume contains a section on mathematical techniques in biomedical engineering, which was edited by Sri
Krishnan at Ryerson University, and a section on bioinstrumentation and bioinformatics, which was edited by
Pankaj Vadgama at Queen Mary University of London.
I would like express my sincere appreciation to the section editors and authors for all of their efforts on the
encyclopedia. I would also like thank Beckie Brand, Susan Dennis, Becky Gelson, Ginny Mills, Blerina Osmanaj,
xxxv
xxxvi Preface
Laura Escalante Santos, and Will Smaldon at Elsevier for their outstanding efforts to bring the encyclopedia to
publication. I hope that this work serves the biomedical engineering community by providing a resource that
considers topics at the interface of the biological sciences and engineering.
Roger J Narayan, M.D., Ph.D.

UNC/NCSU Joint Department of Biomedical Engineering.
References
Bobbio, A. (1972). The first endosseous alloplastic implant in the history of man. Bull. Hist. Dent, 20, 1–6.
Hendee, W. R., Chien, S., Maynard, C. D., & Dean, D. J. (2002). The National Institute of biomedical imaging and Bioengineering: history, status, and potential impact. Annals of
Biomedical Engineering, 30, 2–10.
Narayan, R. J., Doraiswamy, A., Chrisey, D. B., & Chichkov, B. N. (2010). Medical prototyping using two photon polymerization. Materials Today, 13, 44–50.
Parker, K. H. (February 2009). A brief history of arterial wave mechanics. Medical & Biological Engineering & Computing, 47(2), 111–118.
Piccolino, M. (October 1997). Luigi Galvani and animal electricity: two centuries after the foundation of electrophysiology. Trends in Neurosciences, 20(10), 443–448.
Rowland, S. (March 7, 1896). Report on the Application of the new Photography to medicine and surgery. Br Med J, 1(1836), 620–622.
Schuster, A. (January 18, 1896). On the new Kind of Radiation. Br Med J, 1(1829), 172–173.
Skoog, S. A., & Narayan, R. J. (2013). Stereolithography in medical device fabrication. Advanced Materials & Processes, 171, 32–36.
Vacanti, C. A. (July 2006). The history of tissue engineering. J Cell Mol Med, 10(3), 569–576.
Westbroek, P., & Marin, F. (1998). A marriage of bone and nacre. Nature, 392, 861–862.
Worboys, M. (September 20, 2013). Joseph Lister and the performance of antiseptic surgery. Notes and Records of the Royal Society of London, 67(3), 199–209.
Wright, A. V., Nuñez, J. K., & Doudna, J. A. (January 14, 2016). Biology and Applications of CRISPR systems: Harnessing Nature’s Toolbox for genome engineering. Cell, 164(1–2),
29–44.
PERMISSION ACKNOWLEDGMENTS
The following material is reproduced with kind permission of Taylor & Francis
Figure 1 On the Use of Population-Based Statistical Models in Biomechanics

www.taylorandfrancisgroup.com
The following material is reproduced with kind permission of American Association for the Advancement of
Science
Figure 8a Nanotechnology for Regenerative Engineering

www.aaas.org
The following material is reproduced with kind permission of Oxford University Press
Figure 1 Translational Bioinformatics for Personalised Medicine

www.oup.com
The following material is reproduced with kind permission of Nature Publishing Group
Figure 5 Continuum Mechanics and Its Practical Applications at the Level of Scaling Laws
Figure 2 Multi-scale Bone Mechanobiology
Figure 5 Functional Magnetic Resonance Imaging
Figure 4b Cell Mechanics and Cell Adhesion - Basic Principles and Computational Modeling
Figure 9 Diffusion Tensor Imaging
Figure 8 Corrosion of Biomaterials
Figure 8 Biomaterials for Tissue Engineering and Regenerative Medicine
Figure 3c Nanotechnology for Regenerative Engineering
Figure 4b Nanotechnology for Regenerative Engineering
Figure 6 Nanotechnology for Regenerative Engineering
i
ii Permission Acknowledgments

Figure 5 Drug and Gene Delivery for Regenerative Engineering
Figure 3 Holographic Microscopy
Figure 4 Radiomics
http://www.nature.com
BIOMATERIALS: SCIENCE AND ENGINEERING
Alternative Processing Techniques for CoCr Dental Alloys

Lucien Reclaru, VVSA, branch of Richemont International SA Varinor Innovation, Delémont, Switzerland
Lavinia Cosmina Ardelean, “Victor Babes” University of Medicine and Pharmacy Timisoara, Timisoara, Romania
© 2019 Elsevier Inc. All rights reserved.
Introduction 1
Manufacturing Technologies for CoCr Alloys 2
Evaluation of CoCr Alloys Manufactured by Different Technologies 5
Chemical Composition of the Alloys 5
Metallographical Evaluation 5
Corrosion Evaluation 8
Toxicological Aspects 13
Conclusions 15
References 15
Relevant Websites 15
Glossary
Castability The ease of forming a quality casting.
Electrochemical corrosion testing A process for studying various forms of metallic corrosion, which provides information
about the extent of corrosion activity.
Polarization curve Plot of current density versus electrode potential for a specific electrode-electrolyte combination.
Potentiodynamic polarization Probably the most commonly used polarization testing method for measuring corrosion
resistance; a technique where the potential of the electrode is varied at a selected rate by application of a current through the
electrolyte.
Sintering The process of compacting and forming a solid mass of material using heat or pressure without melting it.
Introduction
Since the early 1900s, a wide range of metals and their alloys are used in surgically implanted medical devices, prostheses and dental
materials, in order to provide improved physical and chemical properties, such as strength, durability and corrosion resistance
(Ardelean et al., 2015).
The classes of metals used in medical devices and dental materials include stainless steels, cobalt–chromium alloys, and titanium
(as alloys and unalloyed) (Wassell et al., 2002).
In addition, dental casting alloys are based on precious metals (gold, platinum, palladium, and silver), nickel, and copper and
may in some cases contain smaller amounts of many other elements, added to improve castability, handling, ceramic bond, or other
physical properties. Alloys used in dental applications classify as shown in Table 1.
Despite the wide range of dental alloys, each one of them has its shortcomings. Due to a number of factors, the use of CoCr based
alloys increased for the last years. Noble alloys became too expensive for the average population. Cobalt alloys are considered an
economic alternative to nonprecious nickel-based alloys, known as potential allergens. After a period of time when manufacturers
tried to produce better dental alloys, for example, CoCr based alloys doped with precious metals, which were quite a disappoint-
ment (Ardelean et al., 2015, 2010, 2016a), today the trend changed to improving the manufacturing process, as an alternative, and
not developing new compositions of the dental alloys. CoCr based alloys are known to have excellent corrosion resistance. Because
of their outstanding mechanical properties (e.g., high stiffness) these alloys are mainly used for the fabrication of removable partial
dentures, but also for metal-ceramic fixed dentures, where fine frameworks constructions are needed (Ardelean et al., 2015, 2016a).
Unfortunately, they are difficult to treat and to process for the dental technician and for the dentist in traditional casting technique
because of high hardness. Handling has considerably improved by using the new technologies which appeared in the last years, 3D
printing being considered as the “next industrial revolution.” In our “high-speed” and “low-cost” world these effortless and time
economically technologies, suitable for manufacturing the cheap CoCr alloys seem to be the best alternative available.
Encyclopedia of Biomedical Engineering, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.11100-6 1

2 Biomaterials: Science and Engineering j Alternative Processing Techniques for CoCr Dental Alloys
Table 1 Classification of dental alloys
Precious alloys Nonprecious alloys
Conventional crown and bridge alloys Nickel-based

High gold Type A-Base Ni–Cr, major secondary element: Be
Low gold Type B-Base Ni–Cr, major secondary element: Fe
Ag-based Type C-Base Ni–Cr, major secondary element: Mo
Alloys for the porcelain-fused-to metal technique Type D-Base Ni–Cr, major secondary element: Mn
High gold Type E-Base Ni–Cr–Fe
Low gold (NIOM Type B) CoCr-based, modified CoCr þ Pt, Ru, Nb, Au, In, Fe-CoCr
Pd-based: Pd Ag, Pd Sn, Pd Cu, Pd Cr, Pd Co Cu-based (bronze)
Titanium: Ti grade 1-4 , Ti-6Al-4V, Ti-6Al-7Nb, Ti- Mo, Ti-40Zr, Ti-Pd-Co, Ti-50Ni,
Ti-42.5Ni-7.5Pd, Ti-5Al-13Ta, Ti-5Ag, Ti-20Ag
Manufacturing Technologies for CoCr Alloys
Manufacturing technologies currently available for CoCr alloys are:

a. Traditional casting process (origin: bulk metal)
This traditional laborious and time consuming method implies castability problems and porosities which often appear when
specific flowing parameters of the casting machine are not respected.
b. Milling process (origin: bulk metal)
CAD/CAM technology implies obtaining a virtual image of the dental prosthesis based on an impression or a 3D image. The CAD
software virtually designs the prosthetic element and the milling unit physically manufactures it (Fig. 1). Milling CoCr blanks
(Fig. 2) may be difficult because its hardness and high demands are placed on the manufacturing unit (coolant delivery, rigidity
of the machine etc.) (Ardelean et al., 2016b).
A great progress was made by using milling units with four or five axes. In case of these milling units, the kinematics of the
machine is optimized, with large angulations of the fourth and fifth axes, more than 30 degrees. Thus it allows the milling and
the dry or wet grinding of very good quality dental prosthesis.
On the other hand, the elements obtained by milling do not show any structure defects such as porosities, cracks etc. (Fig. 3).
c. CAD/CAM sintering (origin: powder)
This is a new technology developed by Amann Girrbach, which uses nonprecious metal blanks with a wax-like texture and allows
them to be effortlessly dry milled in system’s benchtop machines. The milling is done in the green body state (unsintered metal
powder held together by a binder). These dry millable CrCr blanks are similar to partially sintered zirconia blanks and can be easily
processed in the preliminary state. After the required frameworks have been milled from the blank they are debinded and densely
sintered in a downstream process. The sintering process of the milled CoCr structures is easy carried out in a special furnace and
apparently gives a result with good structure quality: homogeneous, distortion-free frameworks without contraction cavities
(www.amanngirrbach.com).
d. 3D Printing Technologies (origin: powder)
In principle these technologies are very much alike, the differences come mainly in the process itself. 3D printers seamlessly inte-
grate with computer-assisted design (CAD) software and other digital files like magnetic resonance imaging.
Fig. 1 Milling of prosthetic elements according to CAD files.

Biomaterials: Science and Engineering j Alternative Processing Techniques for CoCr Dental Alloys 3
Fig. 2 CoCr blank for milling.
Fig. 3 CoCr prosthetic elements obtained by milling.
When talking about CoCr alloys manufacturing by 3D Printing, some alternatives are available:
• Selective laser sintering (SLS), developed by Dr. Carl Deckard in Austin USA.
In the mid-1980’s Deckard uses a moving laser beam to trace and selectively sinter powdered materials (including metals) into
successive cross-sections of a three-dimensional part. Selective laser sintering (SLS) technique uses a high power laser (CO2 laser)
to fuse small particles of metal powders into a mass representing a desired 3D object.
Based on a virtual image, the various powders (CoCr, NiCr or ceramic) are slowly built, layer by layer, as the 3D CAD software
measures thousands of cross-sections of each prosthetic element to determine exactly how each layer is to be constructed.
As in all rapid prototyping processes, the parts are built upon a platform that adjusts in height equal to the thickness of the layer
being built. After each cross-section is scanned, the powder bed is lowered by one layer thickness. Additional powder is deposited on
top of each solidified layer and sintered. The powder is maintained at an elevated temperature so that it fuses easily upon exposure
to the laser (www.popular3dprinters.com).
The laser selectively fuses metal powders by scanning cross-sections generated from a 3D digital description of the part, a CAD
file or scan data on the surface of a powder bed (Figs. 4 and 5) (Ardelean et al., 2016a; Reclaru et al., 2012a).
The laser sintering technique makes possible the manufacture of extremely accurate prosthetic elements with mechanical prop-
erties that correspond to any clinical requirement (Fig. 6).
• Direct metal laser sintering (DMLS), developed jointly by Rapid Product Innovations (RPI) and EOS GmbH in Munich,
Germany.
Starting in 1994, is the first commercial rapid prototyping method to produce metal parts in a single process. Metal powder, free of
binder or fluxing agent, is completely melted by the scanning of a high power laser beam to build the part with properties of the
original material. The absence of the polymer binder avoids the burn-off and infiltration steps, and results in a 95% dense steel part
compared to roughly 70% density in case of SLS. In addition DMLS has higher detail resolution than SLS, because of thinner layers,
enabled by a smaller powder diameter (www.popular3dprinters.com).
Fig. 4 3D digital description of the computer-based program for a fabrication tray.
Fig. 5 The laser selectively fuses the CoCr metal powder.
Fig. 6 Laser sintered prosthetic elements.
• Selective laser melting (SLM), started at the Fraunhofer Institute ILT in Aachen, Germany.
It is an additive manufacturing method that uses high powered laser to melt metallic powders together to shape the product from
a 3D CAD data. Renishaw uses a high powered ytterbium fiber laser to fuse metal powders (www.renishaw.com/en/metal-3d-
printing-for-healthcare). The recoater sweeps a layer of fine material powder and makes it ready for the laser to fuse them according
to the 2D cross section of each layer under a tightly controlled inert atmosphere. When the part is made completely, it goes for the
required heat treatment and postprocessing (www.popular3dprinters.com).
• Electron beam melting (EBM), developed by ARCAM AB in Sweden-General Electric-2016.

This is another type of additive manufacturing for metal parts, often classified as a rapid manufacturing method. The technology
manufactures parts by melting metal powder layer by layer with an electron beam in a high vacuum. Unlike some metal sintering
techniques, the parts are fully dense, void-free, and extremely strong (www.popular3dprinters.com).
Evaluation of CoCr Alloys Manufactured by Different Technologies

Chemical Composition of the Alloys
The chemical composition of the CoCr alloys is shown in Table 2 (compositions as given by the manufacturers).
When comparing chemical composition, CoCr alloys for casting, milling, CAD/CAM sinter process and SLS do not show great
differences.
In addition, the Ceramill Sintron alloy blanks contain an organic binder. Ceramill Sintron sinter alloy has comparable and, in
the case of some parameters, even superior strength properties than cast CoCr. Similar evaluations are available in case of laser-
melted alloys compared with cast CoCr alloys (Geis-Gerstorfer et al., 2013).
Metallographical Evaluation
The CoCr samples were embedded in a cold-curing resin on a methyl methacrylate basis (Technovit, Kulzer), then polished with SiC
paper (grit 320/500/1200/1400) and finally with diamond spray (6/3/1 mm) (Struers). Electrolytic etching has been done in a bath
of 100 mL H2O dest., 10 mL HCl conc. and 5 g chromium (VI)-oxide during 5 s under 0.4 V and 0.3 A. The microstructures of the
alloys were observed using a metallographical microscope (Polyvar Met, Reichert-Jung). Two scanning electron microscopes (JEOL
JSM 6300) equipped with an EDX system (Oxford, INCA) for local phase analysis and SEM Sigma Zeiss with an Oxford X-MAX EDX
Instrument) for microanalysis were used. The analyzed samples were covered with a gold flash.
a. Traditional casting process (origin: bulk metal)
The use of CoCr alloys is traditionally carried out by casting, which is quite an unwieldy process. The cast structure is a classical
dendritic one (Fig. 7).
b. Milling process (origin: bulk metal)
Figs. 8–10 show the micrographical structures of the CoCr alloy without and with chemical attack. These are specific CoCr alloy
gross flow structures, obtained either by continuous casting of molten metal, or by hot lamination and cut into a disc form. There
are no abnormalities to be noticed.
The chemical composition of the phases is given in Table 3.
c. CAD/CAM sintering process (origin: powder)
The micrographical structure without chemical attack shows micrometric porosities, homogeneously distributed in sections. No
significant defects may be seen (Fig. 11).
Table 2 Composition of the alloys (wt.%)
Element Cast Milled Ceramill Sintron SLS
Co 63.7 60 66 64.1
Cr 28.9 24 28 29.3
Mo 5.3 4.5 5 4.9
Mn 0.8 <1 <1
Si <1 <1
W 0.1 8.5
Fe 0.4 <1
Fig. 7 Metallographic observation of a cast CoCr alloy.

Fig. 8 Micrographical structure of the CoCr alloy without chemical attack.
Fig. 9 SEM observation of the CoCr alloy without chemical attack.
Fig. 10 Micrographical structure of the CoCr alloy after chemical attack.
Table 3 The chemical composition of the phases
Spectrum C O Al Si Cr Mn Fe Co Mo Total
Spectrum 1: matrix 0.85 28.45 0.85 64.46 5.38 100.00

Spectrum 2: white phase 1.07 38.61 0.38 35.48 24.46 100.00
Spectrum 3: inclusion 14.37 37.13 0.47 17.74 12.26 1.18 14.72 2.13 100.00
Fig. 11 Details without chemical attack.
After sintering, the microstructure is a fine (IG 6.5) and homogeneous one. Micrometric porosities, homogeneously distributed,
typical for sintered pieces, are noticeable (Fig. 12).
d. Selective laser sintering (origin: powder)
The structures are being examined in vertical and horizontal samples. In case of vertical samples (Fig. 13), pile of CrCo layers due to
laser action in order to construct the prosthetic element may be observed. In case of horizontal samples porosity in the structure of
CoCr layers may be observed (Fig. 14).
Previously existing tests made on Ceramill Sintron sinter alloy in comparison with a CoCr casting alloy with regard to their
biocompatibility properties and corrosion resistance state that both alloys are well below the maximum threshold limit value of
the corrosion standard. Similar to the casting and sinter alloys tested in this study, a low Co release was also established with
CoCr alloys which were processed using the SLM technique. The chemical solubility of cast CoCr alloy was also found to be
Fig. 12 Microstructure after sintering.
Fig. 13 Metallographic observation on a vertical sample.

Fig. 14 Metallographic observation on a horizontal sample.
very low with regard to the main components (Co, Cr, Mo) as well as with CoCr alloys which were processed using the SLM tech-
nique (Geis-Gerstorfer et al., 2012). The corrosion behavior of the sinter alloy is considered integrate in the existing range of CoCr
alloys that can be processed. Overall the chemical solubility of Ceramill Sintron was proven very low, due to the good biocompat-
ibility results that were also ascertained (Noack, 2012).
Corrosion Evaluation
Assessment of corrosion behavior has been based on electrochemical evaluation of general corrosion.
• Potentiodynamic Polarization
For the polarization tests, small discs of 1 cm2 surface were prepared. One millimetre of the disc surfaces were then removed using
a lathe, before the metallographic polishing with diamond paste of 1 mm was carried out. The polished discs were pressed into a pol-
ytetrafluoroethylene (PTFE) holder after having protected their lateral surfaces with a transparent, flexible silicon-polymer coating,
in order to prevent possible crevice corrosion during testing.
The test medium was an artificial saliva of the Fusayama type with composition NaCl 0.4 g/L; KCl 0.4 g/L; NaH2PO4* H2O
0.69 g/L; CaCl2*H2O 0.79 g/L and urea 1.0 g/L. Temperature was kept constant at 37 C and pH was 5.
An EG&G BY 273 potentiostat provided the potentiodynamic control of the working electrode. The counter-electrode was in
platinum and a saturated calomel electrode (SCE) served as the reference. The electrochemical polarization measurements were
carried out using the technique of a rotating electrode at a speed of 300 rpm.
All specimens were subjected to the following measurement technique: recording of the cathodic and anodic potentiodynamic
polarization curves from 1000 to þ 1200 mV versus SCE in de-aerated medium with a scanning rate of 0.03 mV/s.
In order to explore all the potential range, which an alloy can take in a given electrolyte, the potentiodynamic polarization curves
are plotted. Plots in a semilogarithmic version between 1000 and þ 1000 mV SCE of the CoCr alloys are displayed in Fig. 15.
Five CoCr samples obtained by four different techniques namely traditional casting (CoCr Flamarc alloy), two different SLS
devices (SLS1 and SLS2), a SLM process and a CAD/CAM milling process were evaluated. By comparison of the polarization curves,
the corrosion behavior is quite different. The comparison is made in the anodic domain between 0 and þ 500 mV SCE. Good
behavior is revealed by the casted Flamarc alloy and CoCr SLM. It is noted that the corrosion process is highly dependent on
Fig. 15 Potentiodynamic polarization curves in artificial Fusayama saliva, between 1000 and þ 1000 mV vs. SCE.
the manufacturing technology used, the specific operating parameters, and the physicochemical properties of the powders used in
process. Generally speaking, in this type of representation, as the polarization curves move to the right, the corrosion resistance
behavior is worse. It should be noted that the polarization curves for the SLS and SLM techniques, from a potential of 500 mV,
move in current values of milliampers. It is of interest to present the polarization curves in the linear axes.
Fig. 16 shows in linear representation the part of the polarization curves between 750 and þ 750 mV SCE in the scale of
cathodic–anodic currents comprising between 0.05 and 0.05 mA/cm2. This helps to visualize the breakdown potential Ebd,
another electrochemical quantity which characterizes the corrosion behavior of alloys. Ebd is the potential at which the anodic
current increases strongly. The potential range situated between “the corrosion potential” (E(i ¼ 0)) and the breakdown potential
represents the immunity zone in which corrosion is weak or even insignificant. The more this zone is extended, the less the alloy
risks being found in a situation where, by polarization or by galvanic effect resulting from the presence of another alloy, it can be led
to corrode rapidly.
According to the values of Ebd recorded, a very different corrosion behavior to of the CoCr studied alloys is observed.
The breakdown potential Ebd of CoCr SLS1 is around 370 mV. The immunity zone is very narrow, there will thus be the start of
corrosion from 100 mV without then having a passivity zone. A similar behavior is revealed by the Flamarc casted alloy with break-
down potential at 600 mV. As far as CoCr milling and CoCr SLM are concerned, the immunity zones extend up to 800 mV. Probably
CoCr SLM shows such behavior due the formation of chromium and cobalt oxides on the surface of the sample.
Scanning electron microscopy (SEM) examination of the surface after the corrosion test of the SLS1 sample shows cracks
(Fig. 17).
The spectral composition analysis by EDX (spectrum 1) clearly reveals the existence of sodium and chlorine oxygen, which are
corrosion products (Table 4).
The best resistance to corrosion is shown by the sample obtained by CAD/CAM milling and the worst by the sample obtained
from SLS1. The corrosion behavior of CoCr alloys strongly depends on the process of elaboration. In case of milling no heating/
sintering is involved and the bulk alloy has a compact structure, with no noticeable impurities. In case of CAD/CAM sintering,
Fig. 16 Polarization curves of the CoCr alloys presented in linear axes.
Fig. 17 SEM micrograph of the corroded surface after the polarization test for SLS1 sample.
Table 4 The spectral composition of the SLS1 sample
Spectrum O Na Si Cl Cr Co Mo Total
Spectrum 1 24.53 1.19 1.12 0.59 28.76 34.30 9.52 100.00
the presence of the binder and the final sintering of the prosthetic milled piece may potentially lead to corrosion problems. In case
of SLS, the manufacturing process itself and the powder nature of the alloy may lead to a higher corrosion risk.
• Extraction, Cations release

The release of cations has been measured in the Fusayama artificial saliva as well as in lactic acide 0.1N NaCl 0.1N according to
ISO 10271, 2001. 0.07N HCl medium is used in the evaluation of metals extracted from toys for children. Children are more
sensitive to the problems of toxicological reactions than adults. They have the tendency to put the toys in the mouth to suck
them, to the limit of swallowing small pieces which compose the toys. Today the laws for the protection of children are strict
(Annex XVII REACH, EN73-1 Safety of toys Migration of certain element), ASTM F 2923 (Standard Specification for Consumer
Product Safety for Children’s Jewelry) and California Proposition 65. On the other hand it is an excellent extraction medium
without special problems of preparation or quantitative analysis. The concentrations of 0.1% NaF and 0.1% KF are specific
for the composition of toothpastes. Thus it is interesting to make an evaluation of cations release of CoCr alloys in this type
of environment.
The solutions are filtered before use over a sterilized FalconÒ 0.22 mm cellulose acetate membrane; the release flasks used are of
FalconÒ sterile type made of polypropylene. The samples are in the rectangular shape of 14 35 mm and are firstly cleaned in
ethanol p.a. under ultrasound. The ratio of release solution volume/total sample surface is equal to 1. The extraction is carried
out at 37 C shielded from light for 168 h. Three samples of each CoCr alloy for each test environment are used. For each extraction
environment, two blank samples are measured as a reference. The solutions are analyzed by two cross technique ICP-OES Optima
4300 Perkin Elmer and ICP MS Perkin Elmer.
The matrices of the cations are measured according to the following diagram:
ICP MS/Ba, Be, Cd, Co, Hg, Li, Mo, Nb, Pb, Sb, Sn, Sr, Zn
ICP OES/Al, Cr, Cu, Fe, Ni, P, S, Ti, V, Zn.
ICP OES Hydrures/As, Hg, Sb, Se.
A statistical analysis of the released cations was performed for As, Ba, Cd, Co, Cr, Cu, Fe, Hf, Hg, Mo, Nb, Ni, Pb, Sb, Se, Te, Ti, V,
W, and Zr.
The maximum accepted risk for Type-I error (false rejection of the null hypothesis, leading to the erroneous conclusion that
differences between alloys exist) are:
Fe ( 22%), Cr, Cu, Mo, Ni, and Co ( 17%) in 0.07N HCl.
Fe ( 27%), Cr, Cu, Mo, Ni, and Co ( 22%) in 0.1%NaF þ 0.1%KF.
While demonstrating that the surfaces of the materials in contact with the biological medium degrade by a process of corrosion,
the preclinical evaluations do not make it possible to specify which constituent elements of the material dissolve preferentially, or in
what quantities. On the other hand, the negative effects that the materials may have on the cell medium, on the tissues or on the
body are precisely due to the presence of certain released components as degradation products, more particularly metal cations in
solution. The degradation of metal alloys in a biological medium is accompanied by release of cations such as Cr, Co, Ni, Ti etc. This
may lead to a situation with cumulative effects such as irritating, allergic, mutagenic, toxic are involved (Swiontkowski et al., 2001).
The extraction depends on a great number of parameters; on one hand the release environment (chemical composition, pH,
chemical stability in time and development of bacteria, temperature etc.), and on the other hand the sample parameters and the
extraction recipient (geometric shape, surface condition, volume/surface ratio, position of samples during release etc.). More infor-
mation for the choice of the environmental conditions are obtained by consulting potentialdpH equilibrium diagrams for the
system chemical element-water (Pourbaix, 1974). The choice of chemical elements to be analyzed is based on the composition
of the CoCr alloys and typical “impurities” in alloys making, and on certain toxic contaminants that might be present in the
material.
The release values for Co, Cr, Mo, Fe major elements in the composition of the powder used in the manufacture of the samples, is
shown in Fig. 18.
It is noted that the SLS1 sample releases large quantities of Cr: 1800 mg/L, Co: 1200 mg/L and Fe: 222 mg/L. from the 0.07N HCl
medium.
Further on the CoCr powder used in the SLS trial was examined and analyzed.
The composition is 64%–67% Co, 28%–30% Cr and 5%–6% Mo, and has at equilibrium of a monophasic structure (Fig. 19)
(the dot). The particle size distribution was also examined. The dimensions of the metal particles are distributed between 10 and
50 mm (Fig. 20).
Because there is nothing special regarding these measurements, probably the large quantities of Cr, Co, and Fe released are due to
the parameters of the machine that are not optimized, or to the performance of the machine.
Fig. 18 Release values of major elements Co, Cr, Mo, Fe.
Fig. 19 Composition of the CoCr powder used in SLS trial.
Fig. 20 Dimensions of the CoCr powder particles used in SLS trial.

Considering the extraction results, it should be noted that a mixture of cations at relatively high concentrations is found and
a cocktail effect cannot be ruled out, this concerns mainly the Co, Cr, Mo, Fe elements (Fig. 18), and, to a lesser extent, the elements
presented in Table 5.
Tables 5 and 6 show the values measured for each sample as well as those of blanks for taking into account any release from the
extraction recipients. The quantities measured in the releases are expressed in mg/L. For certain of the 20 analyzed elements the
concentrations have been found to be close to or below the detection limit. The elements that reveal concentrations at the limit
value detection, namely As, Ba, Cd, Hf, Hg, Nb, Sb, Se, and Zr are not shown in Tables 5 and 6.
The presence of Cu traces (Tables 5 and 6) in NiCr and CoCr alloys represents a problem in the world of iron and steel industry.
It is a permanent pollution of the process of elaboration that starts from metallic waste that comes from certain geographical areas.
Purification by oxidation in the cast iron tanks is not sufficient, and a second vacuum cast is quite expensive. Consequently, the
alloys based on Fe, Ni, Cr, Co, Mo found on the market have traces of copper.
It is well known that nickel causes contact allergy, considered a real threat to health.
In Europe 10%–15% for female adults and 1%–3% for male adults are allergic to nickel and in consequence the European
Union (UE) has accordingly decreed two directives:
a) the nickel release from parts in direct contact with the skin must be lower than 0.5 mg/cm2/week (European Parliament and
council directive 94/27/EC of June 30, 1994) and
b) all metallic parts that are inserted into pierced ears and other parts of the human body must not have a nickel release rate greater
than 0.2 mg/cm2/week (Commission directive 2004/96/EC of September 27, 2004).
In dentistry, allergies to metals are frequently reported. In particular, allergy to nickel in females is reported to vary from 9% to
20%, and in case of orthodontic patients with pierced ears, 30% are allergic to nickel, copper and chromium (Wiltshire and
Noble, 2007).
In certain countries, nickel-based, cheaper alloys have increasingly been subjected to more and more regulations or even banned
(Schnuch et al., 2011). However, piercing of other parts of the body has increased in the last years (Gutsche et al., 2008).
The authorities implementing regulatory rules (EU and national) were not aware of a persisting nickel problem, as they have
neglected to include epidemiological outcome control regarding morbidity as an integral part of the regulation.
Today we find ourselves with a Ni cocktail effect, in an allergic population, pierced, tattooed and with orthodontic devices made
of poor stainless steels. Probably the amount of nickel found (Tables 5 and 6) can generate a nickel cocktail effect.
• Crþ 6 release in Fusayama artificial saliva after electrochemical test

After electrochemical test, the solutions were analyzed quantitatively for Crþ 6 by the anodic stripping voltammetry (ASV) method
with PDV 6000 (Cogent Environmental Ltd. Cambridge UK/Gerber Instruments Ag Effretikon Switzerland) (Fig. 21).
The measurement is made on a polished graphite electrode. This is a very sensitive method, chromate (Crþ 6) can be measured in
the range 5–10 ppb.
The advantage of this technique is that chromate (Crþ 6) and not Crþ 3 or total chromium (Crþ 6 þ Crþ 3) is specifically measured.
For the measurement of total chromium we used the ICP method. As in the extracts, there is always a chemical equilibrium between
Crþ 3 and Crþ 6, the voltammetric technique makes it possible to measure only the chromate without having any interference with
the Crþ 3 cation. In addition, the coloring of the extracts hardly influences the measurement, as in spectrophotometric techniques.
The peak domain is: Chromate: 950 mV at 1250 mV versus Ag/AgCl. The basic parameters for electrochemical measurement are
shown in Table 7.
Table 5 Release values for each sample in the HCl medium
mg/L Ba Cu Hg Ni Pb Te Ti V W
Blank 0.2 18 0.2 1.0 0.3 7.1 9.5 0.8 0.6

SLS1 2.4 2.3 0.5 9.7 9.6 1.0 7.7 0.8 0.5
SLS2 0.2 1.0 0.5 1.0 24 1.0 4.2 0.5 0.5
Milling 0.2 42 0.5 3.5 3.0 6.2 11 0.8 0.6
Table 6 Release values for each sample in the NaF þ KF medium
mg/L Ba Cu Hg Ni Pb Te Ti V W
Blank 15 53 0.5 1.1 0.2 1.0 22 0.8 5.8

SLS1 17 120 0.2 3.3 0.3 2.9 24 61.7 3.1
SLS2 17 170 0.5 5.7 1.2 5.0 19 42.0 1.8
Milling 0.2 42 0.5 3.5 3.0 6.2 11 0.8 0.6
Fig. 21 Voltammetric analysis techniquedelectrochemical instrument and analytical cell assembly.
Table 7 The basic parameters for electrochemical measurement
Rest potential 1100 mV Mix time 30 s

Deposit potential 160 mV Deposit Time 60
Hold potential 700 mV Hold Time 15 s
Strip potential 700 mV Strip Datum Potential 700 mV
Low potential 700 mV Measure High Potential 1300 mV
Strip stop potential 1300 mV Strip Rate 500 mV/s
Clean potential 1400 mV Clean Time 20 s
Table 8 The execution programs
Area of concentration to measure (ppb) Concentration of the standard used (ppb) Deposit time (seconds)
5–50 30 150
5–200 100 100
200–800 500 60
The execution programs are designed as shown in Table 8.

Thus, if a chromate concentration between 5 and 50 ppb is to be measured in a sample, it will be necessary to use a reference
standard of 30 ppb with a time of 150 s. In Fig. 22, a measurement using a 500 ppb standard is presented. The unhatched peak is the
extract and the hatched peak represents the standard of 500 ppb in chromate.
Fig. 23 shows comparatively the quantities of Crþ 6 released in the two environments by the casting, milling and SLS technol-
ogies. The quantities of Cr migrated in the artificial saliva environment are greater than in the lactic acid. The detection limit is
200 mg/L. It is noteworthy that the SLS1 releases significantly higher amounts of Crþ 6, irrespective of the extraction medium.
The Cr release of alloys is significant because Crþ 6 has been shown to be a human carcinogen as well as a frequent allergen. Crþ 3
is not classifiable as to its carcinogenicity, but it is also described as an allergen.
According to EU Commission ECHA 130.000 people allergic to Crþ 6 are reported and their number increases annually.
During the degradation of the CoCr, CoCrMo or NiCr alloys, the Cr is released into Crþ 3, but it can also be oxidized at Crþ 6 at
the cellular level. Hexavalent chromium (Crþ 6) is mutagenic and carcinogenic; yet it is not the direct cause of these effects since it is
metabolized in the cytoplasm and especially in the nucleus of the cell in trivalent chromium (Crþ 3) which, in this form, produces
the lesions of the DNA.
On the other hand, Crþ 3 is neither mutagenic nor carcinogenic because its reactivity in this form. It reacts with the membrane
and the proteins of the cytoplasm before reaching the DNA within the cell nucleus, causing the cell death without mutations.
Toxicological Aspects
Biocompatibility becomes more and more a puzzle in the general picture of evaluating the effects of chemicals toxicity which are in
contact with us.
Fig. 22 Measurement using a 500 ppb standard.
Fig. 23 Quantities of Crþ 6 measured by anodic stripping voltammetry (ASV).
The different chemicals originating from different sources, released at different moments and from different places could
combine themselves to expose human beings to a cocktail of chemicals. Today one speaks of a burning scientific subject, the cocktail
effect.
European Chemical Agency (ECHA) is trying to understand how the chemicals are released from different sources and how they
combine with each other to give rise to a human exposure with adverse effects.
A factor of complication is that the individual chemicals could become more dangerous only because of other chemicals with
which they are mixed, the cocktail effect. Also, ECHA has developed a roadmap implementation plan on substances of very high
concern (SVHC), that is, the endocrine disrupters (ED), substances which are carcinogens, mutagens or toxic to reproduction (CMR)
and the sensitizers.
Conclusions
In the last 10 years the dental alloys market in Europe has undergone dramatic changes, mainly for reasons of economy and
biocompatibility, with important consequences (Wiltshire and Noble, 2007; Reclaru et al., 2012b; Rusu et al., 2014) such as
removing palladium from being an important part of dental alloys due to allergic reactions generated by PdCu alloys (Reclaru
et al., 2014; Durosaro and El-Azhary, 2008).
In particular many manufacturers of noble dental alloys dedicated themselves to the development of gold-based or platinum-
based noble alloys, containing almost exclusively noble metals (gold and platinum) without palladium, called “bio.”
To simplify the work of technicians, “universal” precious alloys are also proposed, which ensure reduction of the number of
alloys to be maintained in stock in the laboratory.
The price variations of precious metals on the rise and the world economic crisis that started in 2008 mean that even in countries
where precious alloys are traditionally used, nickel-based alloys are more frequently found. Nickel based alloys have become
a widely used substitute for the much more expensive precious metal alloys. But it is well known that nickel causes contact allergy,
considered a real threat to health (Setcos et al., 2006) so, the use of CoCr based alloys increased.
Nonprecious metal alloys are available in dental technology for the casting procedure, powders for 3D printing technologies and
as fully dense blanks for the milling technique. New CoCr blanks are now also available which are milled in the green body state
(unsintered metal powder held together by a binder) and then densely sintered.
Handling is considerably improved by the new technologies available for CoCr alloys: CAD/CAM milling and 3D Printing. The
labour-intensive and error-prone casting procedure and therefore time-consuming manual working stages are no longer required.
Because of the fast evolution of 3D Printing techniques, CoCr alloys in prosthetic dental applications have to be considered as
serious competitors in the next years. The mechanical and corrosion resistance, the price (low-cost) and the easiness of execution
from photographic imprints are in their advantage. It can be concluded that production procedures such as 3D Printing and milling
can successfully replace conventional casting procedures and represent a logical step towards the future.
References
Ardelean, L., Reclaru, L., Bortun, C., Ghiban, B., & Rusu, L. C. (2010). New aspects concerning co-Cr alloys. Metalurgia International, 15, 31–35.
Ardelean, L., Reclaru, L., Bortun, C., & Rusu, L. C. (2015). Assessment of dental alloys by different methods. In M. Aliofkhazraei (Ed.), Superalloys (pp. 141–170). Rjeka, Croatia:
InTech.
Ardelean, L., Reclaru, L., Bortun, C., & Rusu, L. C. (2016a). Investigations on dental alloys using metallographic observation, scanning electron microscopy, and energy-dispersive X-
ray spectroscopy. In S. G. Stanciu (Ed.), Micro and nanotechnologies for biotechnology (pp. 123–143). Rjeka, Croatia: InTech.
Ardelean, L., Reclaru, L., Bortun, C., & Rus, L. C. (2016b). Current technologies and materials for dental fixed prosthetic restorations. Solid State Phenomena, Advanced Materials
and Structures VI, 254, 132–137.
Commission directive 2004/96/EC of 27 September 2004. (2004). Official Journal of the European Community, L301, 51–52.
Durosaro, O., & El-Azhary, R. (2008). A 10-year retrospective study on palladium sensitivity. Dermatitis, 20, 208–213.
European Parliament and council directive 94/27/EC of 30 June 1994. (1994). Official Journal of the European Community, L188, 1–2.
Geis-Gerstorfer, J., Noack, F., & Reichert, A. (2012). As good as cast. Dental Dialogue, 13, 14–19.
Geis-Gerstorfer, J., Schille, C., & Schweizer, E. (2013). Comparison of the biocompatibility and corrosion properties of a CoCr sinter alloy with a casting alloy. Dental Dialogue, 14,
20–28.
Gutsche, P., Schmalz, G., & Landthaler, M. (2008). Prevalence of piercing in a German population. European Journal of Dermatology, 18, 26–28.
Noack, F. (2012). CoCr-revolution. Dental Dialogue, 13, 2–5.
Pourbaix, M. (1974). Atlas of electrochemical equilibrium in aqueous solution. Houston, Texas: NACE International Cebelcor.
Reclaru, L., Ardelean, L., Rusu, L. C., & Sinescu, C. (2012a). Co-Cr material selection in prosthetic restoration: Laser sintering technology. Solid State Phenomena, Advanced
Materials and Structures IV, 216, 412–415.
Reclaru, L., Unger, R. E., Kirkpatrick, C. J., et al. (2012b). Ni–Cr based dental alloys; Ni release, corrosion and biological evaluation. Materials Science and Engineering C, 32,
1452–1460.
Reclaru, L., Ardelean, L., Govor, I., Tigmeanu, C. V., & Rusu, L. C. (2014). In vitro toxicity testing of palladium using trifluorothymidine. Revista de Chimie, 65, 1149–1153.
Rusu, L. C., Bortun, C. M., Tanasie, G., et al. (2014). The cytotoxicity of dental alloys studied on cell culture. Romanian Journal of Morphology and Embriology, 55, 3–6.
Schnuch, A., Wolter, J., Geier, J., & Uter, W. (2011). Nickel allergy is still frequent in young German females – Probably because of insufficient protection from nickel-releasing
objects. Contact Dermatitis, 64, 142–150.
Setcos, J. C., Babaei-Mahamani, A., Di Silvio, L., Mijor, I. A., & Wilson, N. H. F. (2006). The safety of nickel containing dental alloys. Dental Materials, 22, 1163–1168.
Swiontkowski, M. F., Agel, J., Schwappach, J., McNair, P., & Welch, M. J. (2001). Cutaneous metal sensitivity in patients with orthopaedic injuries. Orthopaedics and Traumatology,
15, 86–89.
Wassell, R. W., Walls, A. W. G., & Steele, J. G. (2002). Crowns and extra-coronal restorations: Materials selection. British Dental Journal, 192, 199–211.
Wiltshire, W. A., & Noble, J. (2007). Allergies to dental materials. Vital Autumn, 27, 32.
Relevant Websites
http://www.amanngirrbach.com.
http://www.popular3dprinters.com.
http://www.renishaw.com/en/metal-3d-printing-for-healthcare.
Bioceramics
Besim Ben-Nissan, University of Technology Sydney, Sydney, NSW, Australia
Sophie Cazalbou, UMR 5085 UPS-INPT-CNRS, Toulouse, France
Andy H Choi, University of Technology Sydney, Sydney, NSW, Australia
Introduction 16
Biomaterials and Bioceramics 16
Bioceramic Production Methods 17
Aluminum Oxide (Al2O3) 19
Medical-Grade Alumina 21
AluminadBiolox Family 22
Zirconia-Toughened Alumina (ZTA) 23
Zirconia (ZrO2) and Partially Stabilized Zirconia (PSZ) 23
Bioglass 24
Glass and Glass Ceramics 24
Synthesis of Bioactive Glass 25
Laser spinning approach 26
Micro-emulsion approach 26
Solegel approach 26
Bioactive Glasses From Various Research Groups 26
Hench and co-workers: Bioglass® 27
Bioactive glass from Finland: Åbo Akademi 27
Bioactive glass from Japan: A-W glass 28
Calcium silicate bioactive glass 28
Bioactive Glass Composites 28
Silicon Nitride as Implant Material 29
Bioceramics for Drug Delivery 29
Bioceramics for Radiotherapy 29
Concluding Remarks 30
References 31
Introduction
Degeneration and diseases often result in the replacement of skeletal parts, such as the knees, hips, finger joints, elbows, vertebrae
and teeth, and repair of the mandible surgically. When a person suffers from a pain, the main concern for that individual is relieving
the pain and returning to a healthy and functional lifestyle. Biomaterials and specifically bioceramics are widely used for joint
repair, bone reconstruction and as dental implants.
Bioceramics prior to the 1970s were employed as implants to perform singular and biologically inert roles. The limitations with
these synthetic materials as tissue substitutes were emphasized with the growing realization that the cells and tissues of the body
perform many other vital regulatory and metabolic roles. Since then, the demands of bioceramics have changed, from maintaining
an essentially physical function without eliciting a host response to providing a more positive interaction with the host. This has
been accompanied by increasing demands on medical devices that they not only improve the quality of life but also extend its dura-
tion. More importantly, the exciting and potential opportunities associated with the use of new nanobioceramics and their compos-
ites with biogenic materials such as proteins, peptites, collagen and polysaccharides as body interactive materials, helping the body
to heal, or promoting the regeneration of tissues, thus restoring physiological functions. It is anticipated that the growth in these
areas will continue due to a number of factors, for instance, the need due to the aging population, improvements in technology
and lifestyle, a better understanding of body function, an increasing preference by younger to middle-aged candidates for under-
going surgery, improved aesthetics and the need for better functions (Ferrage et al., 2017).
Biomaterials and Bioceramics

By definition, a biomaterial is a nondrug substance which is ideal to be placed in a system that can replace or enhance the roles of
bodily organs or tissues. These materials are able to be in contact with bodily fluids and tissues while showing little or if any adverse
reactions for prolonged periods of time.
16 Encyclopedia of Biomedical Engineering, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.99867-2

Biomaterials: Science and Engineering j Bioceramics 17
The major key factors that are pertinent for the success of an implant are its biocompatibility and biofunctionality. Engineers and
surgeons have identified, even at the initial stages of this field, the problems related to the design and materials selection that
resulted in premature loss of implant function through mechanical failure, corrosion or inadequate biocompatibility of the compo-
nent. Depending on the applications, bioactive glass and glass ceramics in addition to ceramic materials are ideal candidates with
respect to the above functions, except for their brittle behavior under functional loading.
In this article, our aim is to examine the general definitions of bioceramics and glass as well as the preparation methods, prop-
erties and applications ceramics and bioactive glasses currently available and in use. We will also introduce the development and
progress of the commercially available and currently investigated bioceramics and bioglasses. Furthermore, their chemistry, bioac-
tivity and mechanisms of their bonding and interactions within a physiological environment, their preparation methods and their
applications in the biomedical field will also be covered.
Improvements of implant surfaces and tissue implant interfaces using surface modifications, micro- and nanoscale coatings have
been of interest worldwide during the past three decades. Bioceramics when used as body interactive material has the potential to
assist the body to heal, and/or promote the regeneration of tissues thereby restoring physiological functions. This approach is being
explored in the development of a new generation of bioceramics with a widened range of medical applications.
Porous materials specifically calcium phosphates are being used as bone grafts at an increasing pace due to the fact that they
permit the ingrowth of natural bone, supply of blood and nutrients, and thus providing a strong bond between the graft and
bone. On the other hand, “nanostructured materials” refer to certain materials which have intricate structures and sizes that fall
within the range of 10–100 nm. As a consequence of their size, an extensive development of technology has taken place in the fields
of ceramic and materials sciences and engineering during the last two decades. This is appropriate because most biological systems,
including protein complexes, viruses, and membranes exhibit natural nanostructures.
Recently, tissue engineering has taken a new direction by seizing the advantage of combining the use of living cells with three-
dimensional ceramic scaffolds to deliver vital cells to the damaged site of the patient. Extremely productive strategies have been
initiated during the last few decades to combine bioceramic implants with biogenic materials in the field of cell growth and differ-
entiation of osteogenic cells.
A stem cell is a cell from an embryo, fetus, or adult which has the capability to reproduce for long periods. It can also give rise to
specialized cells that make up the tissues and organs of the body. It has been demonstrated that these cells when implanted onto
immuno-deficient mice can combine with mineralized three-dimensional scaffolds to form a highly vascularized bone tissues.
Defects across the bone diaphysis can be treated with these cell cultured-bioceramic composites with good functional recovery
and excellent integration of the ceramic scaffold with bone. Excellent innovative works with bioceramics, biogenics and both
natural and synthetic composites are in progress for clinical applications (Ferrage et al., 2017). Different classes of these materials
are known based on their response to biological environment, for instance alumina (Al2O3) and zirconia (ZrO2) are classified as
bioinert; bioglass and glass ceramic are bioactive; while calcium phosphate ceramics are classified as bioactive and bio-resorbable.
Though bioceramics are widely used as implants in orthopedics, maxillofacial surgery and for dental implants, more developments
are in progress to extend their applications and achieving improvements in their performance and reliability. The method by which
these materials are synthesized or produced will govern their properties and applications as well as their performance within the
human body.
Bioceramic Production Methods

The production method as well as the processing route strongly determines the properties and microstructure of ceramic materials.
For these reasons, it is extremely vital to select the most suitable approach when synthesizing biomaterials with desired properties
and/or a combination of properties.
The most widely used production method for synthesizing advanced ceramics such as bioceramics include powder preparation
methods and wet chemical processing techniques such as sol–gel and co-precipitation in addition to traditional techniques such as
cold and hot isostatic pressing (Figs. 1–3). The abovementioned techniques have all been utilized for the fabrication of both mono-
lithic structures and coatings. Recently with the advent of the 3D printing bioceramics with complicated shapes are produced after
appropriate CT scans of the patients. The method applied in both maxillofacial and orthopedic surgery although the method for
ceramics in infancy and we will possibly utilize more this technique in the years to come (Ferrage et al., 2017).
Additive manufacturing of bioceramics, and more precisely stereolithography and selective laser sintering/melting, represent
promising techniques for their industrial applications. They can be considered not only as rapid prototyping methods, like they
were first designed for, but as real manufacturing processes. Already established for metals (SLM: Selective Laser Melting) and poly-
mers (SLS: Selective Laser Melting, SLA: Stereo Lithography), these 3PD methods allow investigations on the manufacturing of new
materials, such as bioceramics. Using such manufacturing processes combine several advantages, like a decrease in the fabrication
times and products costs, and a less constrained design of the parts.
Dissociation of ceramics and partial melting are serious material issues and has to be properly investigated for the 3D printing
process. New tools and methods need to be developed to investigate these multifaceted implications. Therefore, improvements
in the process and further scientific research still need to be achieved. A better understanding of the laser/materials interactions
would allow a better control through an easier adjustment of the processes’ parameters. This might also help controlling residual
thermal stress which is currently causing the formation of cracks in most of the produced ceramic parts, and sometimes their
crumbling.
18 Biomaterials: Science and Engineering j Bioceramics
Minerals
Powder
Powder Processing
Chemical
Processing “Green body”
Shaping ?
Sintering
Finished
Dense
Product Machining ? Compact
Fig. 1 A typical production process of monolithic bioceramics.
(A) (B) (C) Heating coils
Fill can Vacuum bakeout Hot isostatic press

Fig. 2 Schematic representation of hot isostatic pressing of bioceramics.
Fig. 3 Femoral and acetabular components of ceramic hip joint. Please note the cup has been macrotextured and hydroxyapatite coated.
Dimensional accuracy and surface finish are to be enhanced as well. Indeed, the post thermal treatments often required after SLS
and SLA can result in a dimensional shrinkage that is difficult to predict. The surface of the finished parts can suffer from the “stair-
case effect”: given that the parts are built layer by layer, each layer has to be as thin as possible so it cannot be distinguished from one
another. A bad surface finish results in the need of grinding or polishing the parts, which add an additional step to the
manufacturing procedure. Stereolithography is the 3DP method with the best resolution and thus the best surface finish. Still,
by optimizing raw materials, the robotic systems involved in the process and the process parameters, it is possible to decrease
the size of the minimal constitutive element of the printed part, hence the surface finish.
Unlike 3D-printed metal and polymer parts, the ceramic parts built by SLS or SLA are not “ready-to-use” as they require post-
treatments (thermal treatments for debinding and sintering) to reach acceptable mechanical properties. This eliminates one of the
main advantages mentioned above of 3DP for the processing of ceramics.
As they are well identified, these problems are being solved, step-wise. The technologies as well as the understanding of the
phenomena occurring keep improving. But the most difficult limitations to overcome are set by the materials used. Progress in
materials sciences and materials engineering could be the key to a better absorbance of a laser wavelength by a powder bed, or
to a better slurry viscosity.
Knowing its advantages on the ability to allow to produce biomedical parts based on patients anatomy (patient matched by CT
scan) and the use of complicated interconnected porous designs the 3DP process has a lot to offer to help surgeons to design
implants and help patients to reduce pain and suffering.
In comparison to modern techniques the traditional pressing method is accomplished by placing the powder into a die or mold
and applying pressure to achieve compaction. Hot pressing and hot isostatic pressing are the most widely used methods of
producing high pity pure bioceramics. Higher densities and small grain structures which are required by bioceramics can be fabri-
cated using hot isostatic pressing. The process involves the simultaneous application of heat and pressure being applied from all
directions using a pressurized gas such as argon or helium. On the other hand, hot pressing can be utilized to produce flat blocks
or plates and non-uniform components with relative ease (Fig. 1). Current monolithic ceramics for orthopedics applications use hot
isostatic pressing to achieve high densities.
A relatively new and promising method of bioceramic production for solid pieces and coatings is a solution-based synthesis
approach call sol–gel technique. Sol–gel processing is unique in that it can be used to produce different forms, such as powders,
platelets, coatings, fibers and monoliths of the same composition, merely by varying the chemistry, viscosity, and other factors
of a given solution (Ben-Nissan and Choi, 2006).
Composite can be described as a heterogeneous combination of two or more materials. It is possible through the use of the
composite approach and with the assistance of secondary substitutional phases to manipulate the required mechanical properties
of the composites such as strength and stiffness to a magnitude much close to the values of natural bone. Biocomposites can be
synthesized either by physically mixing or by introducing a new component into an existing ceramic material. Spray driers are
used in powder processing. This permits for property modifications of the ceramic materials and may even offer new material
functions.
The use of gel system such as hydrogels are another form of biocomposite which has been developed for biomedical applica-
tions. In essence, a gel can be thought of as a three-dimensional network immersed in a fluid. Both monolithic and coated materials
can be entrapped into a gel such that the properties of the materials can be improved and tailored to suit the specific needs of certain
biomedical devices.
The current focus is on the production of new bioceramics that are relevant to a broad range of applications, including: implant-
able surface-modified medical devices for better hard- and soft-tissue attachment; increased bioactivity for tissue regeneration and
engineering; cancer treatment; drug and gene delivery; treatment of bacterial and viral infections; delivery of oxygen to damaged
tissues; imaging; and materials for minimally invasive surgery. Alumina, PSZ and their composites are widely used. Bioglass prod-
ucts are also for sensitive tooth paste applications. In the market from bone regeneration to powders for the tooth paste production.
Silicon nitride and SiAlONs are also used experimentally. The following sections will cover these materials and their properties in
detail.
Aluminum Oxide (Al2O3)
More commonly known as alumina (Al2O3), aluminum oxide is the most widely used ceramic oxide material in both engineering
and medical applications. Using the Bayer process, alumina powder (as a raw material) is produced from the mineral bauxite in
large quantities. High-purity alumina has been developed as an alternative to surgical metal alloys for dental and orthopedic appli-
cations such as total hip replacement specifically as femoral head and acetabular components and as early tooth implants (Bioceram
and Tubingen implants), and is typically categorized as that with a purity of 99.99%. Alumina is ideal for applications as articulating
surfaces in orthopedic applications due to its low friction, excellent corrosion and wear resistance, and high hardness. Medical-grade
alumina has a narrow grain-size distribution, very small grain size, and very low concentration of sintering additives which makes
them ideal materials in clinical applications. Such a microstructure is capable of preventing slow crack growth and static fatigue
while the ceramic under load.
Many applications of ceramics are based upon unique mechanical, physical, and thermal properties. The primary important
issues for their biomedical applications are biocompatibility, wear resistance, strength, and density. Open porosity is often a crucial
measurement in bone replacement applications and it is an important density parameter. The porosity of a substance reveals the
volume of pores present in the material. It can have a strong effect on the properties as well as biological behavior of a ceramic
material. Open porosity can reduce the strength and allow permeability to gases or liquids. Therefore, it is often important to deter-
mine the nature of the porosity in addition to determining the density. The color of alumina is governed by impurities or additives.
It can also be determined by the sintering atmosphere and by the interaction with ionizing radiation. In general, alumina is white
but can sometimes be brown (96% alumina) or pink (88% alumina) or blue dependent on the additives and on the purity or
concentration of alumina. A solid solution is formed when alumina is reacted with chromium oxide (Cr2O3). The amount of chro-
mium oxide added will govern whether the color of alumina changes to pink or ruby. When medical-grade alumina is sintered in air
together with the addition of magnesia, it will appear as ivory. Alumina turns white when it is sintered in reducing atmosphere or if
it contains traces of silica (Willmann, 2003) (Figs. 4 and 5).
The high toughness of metals can generally be derived from its crack-tip plasticity. The fracture energy is absorbed in the plastic
zone, creating a tortuous pathway which makes the propagation of the crack much more difficult.
Fig. 4 Total hip replacement components. Left a ceramic on ceramic while right figure ceramic on polyethylene designs.
Fig. 5 Kyocera alumina ceramic knee prosthesis. Femoral component alumina or zirconia on polyethylene tibial insert component.
Despite the fact that a certain amount of plasticity can also occur at the tip of a crack in a ceramic material, the energy absorption
nonetheless is relatively small, resulting in a low fracture toughness value. The values of fracture toughness (KIC) for most ceramics
including alumina are roughly one-fiftieth of those of ductile materials such as titanium and its alloys. Due to their ionic covalent
bonding most ceramics have very high melting, or more appropriately, dissociation temperatures and strength. There brittleness is
based on their internal and external defects that influences their behavior. Consequently, due to these strong bonding ceramics in
general the production of ceramics can be only achieved with high-temperature sintering. The powders are typically heated to two-
thirds of their melting temperature during the sintering process.
Medical-Grade Alumina
The alumina bioceramic according to Willmann (1998) must offer a high resistance against corrosion within the human body envi-
ronment. This can be accomplished only by utilizing a high-purity oxide ceramic based on purified raw materials which are free of
impurities such as silicates and alkaline oxides. Furthermore, the bioceramic should not deform when loaded under physiological
situations. In terms of mechanical properties, the safety of ceramic components is related to its mechanical strength. Improving the
mechanical strength is the primary objective as well as all the properties which are interrelated to strength. As mentioned previously,
this can be achieved by using technologies such as hot isostatic pressing followed by the pre-pressing process. Through the use of
a sintering aids such as magnesium oxide (MgO), an excellent surface finish can be easily achieved provided that the alumina
ceramic material contains no porosity and its microstructure is homogeneous and consists of small fine grains. Yet again, process
such as hot isostatic pressing can be used to improve the microstructure and the density of the bioceramic. Corundum (Al2O3) is the
phase associated with medical-grade alumina and it is required to be a very stable phase. Although alumina was introduced un early
1970s by Boutin in France for orthopedic application for hip implants (Fig. 6) and later in 1975 in Germany for dental applications,
the first recognized requirements for alumina was first published in 1979 by the German Standards’ Institute (DIN). This standard
was later adopted by the International Standards Organization (ISO) and 2 years later by the American Society for Testing and Mate-
rials (ASTM). In 1994, the ISO standard was revised and this revision resulted in requirements for grain size, bulk density, and
biaxial flexural strength to be made tougher. A number of additional requirements were also listed in the revised standards detailing
issues such as compression strength, purity, wear resistance, fatigue strength, and allowed constituents must be satisfied. A screening
Fig. 6 The first alumina femoral component of Boutin from France in early 1970s.
test was also included in the ISO standard describing a ring-on-disk test for the wear couple alumina–alumina. The time-consuming
simulator testing is required to be carried out once a material passes this examination. The alumina ceramic has to pass this test
in vitro in order to be considered as medical-grade (Willmann, 1998).
These standard tests are essential to ensure the production of a high-quality ceramic material for biomedical applications.
The chemical, physical and mechanical requirements serve as criteria for a high-purity, consistent product that can be implanted
in the human body. These requirements provide specifications for biocompatible grades of alumina for clinical applications. The
low coefficient of friction, high hardness, and excellent corrosion resistance of alumina offer a very low rate of wear when it is used
as an articulating surface in orthopedic applications. Furthermore, alumina possesses the capacity to be polished to a high surface
finish. Other applications for alumina in orthopedic and maxillofacial applications include femoral head components, alumina
stems, alumina spacers employed specifically in revision surgery (Huckstep and Sherry, 1996) and ceramic knee prostheses. Further-
more, alumina as single crystal (Bioceram) and polycrystalline forms has been previously used in dental applications as tooth
implants (Oonishi et al., 1992, 2003; Sedel, 2000). The amount of sintering additives found in medical-grade alumina is extremely
low with a typical concentration of less than 0.5 wt%. The microstructure is composed of a narrow grain-size distribution with very
small grain size of less than 7 mm. With the current medical-grade alumina, the average grain size is 1.4 mm and surface finish is
normally controlled to a roughness of less than 0.02 mm. Such a microstructure is capable of inhibiting static fatigue and slow crack
growth while the ceramic is under load.
On the other hand, alumina suffers from a fundamental flaw when used as an implant material for dental and orthopedic appli-
cations given the fact that a non-adherent fibrous membrane may develop at the bone–implant interface similar to other “inert”
biomaterials unless its surface is modified or used directly in articulating area such as in a knee or hip prosthesis. Under certain
conditions, interfacial failure can occur resulting in loosening of the implant. This was observed in some earlier dental implant
designs. The mechanical behavior of alumina ceramics in simulated physiological environments has led to long-term survival
predictions when subjected to sub-critical stresses. The stress magnitude of medical-grade alumina was estimated to be 112 MPa
for a 50-year period with a 99.9% survival probability (Ben-Nissan, 1993).
AluminadBiolox Family
There are two general alumina compositions that are currently produced: BioloxÒ Forte is a pure alumina material and BioloxÒ
Delta is an alumina matrix composite material, both of which are manufactured under CeramTec. BioloxÒ Delta has been engi-
neered specifically with substantial improvements in mechanical properties compared to BioloxÒ Forte so that new applications
can be developed and used reliably in clinical application (Figs. 7 and 8). As far as application is concerned, BioloxÒ Delta has
a higher strength magnitudes than BioloxÒ Forte (Figs. 7 and 8). It is worth to mention nevertheless that the service life of a compo-
nent is determined by the loads applied regardless of what the strength of the material may be. The magnitude of these loads and the
resulting stresses are dependent to a large extent on variables such as the design of the entire implant system, product geometry, the
surgical technique used, and the post-operative function of the patient. Moreover, BioloxÒ Delta is the only ceramic with two
decades of successful clinical experience and with over 6 million implanted components.
BioloxÒ Forte is bioinert and is highly resistant to all chemical reactions due to its high purity and the strong chemical
bonding between aluminum and oxygen ions. This is also the primary reason components manufactured of this material provide
excellent biocompatibility with the human body. Furthermore, a Vickers hardness value of approximately 2000 can be achieved
in components produced from BioloxÒ Forte. With the exception of diamond, this constitutes the hardest material known.
During implantation, a BioloxÒ Forte component would remain undamaged if it was scratched since it is approximately three
times harder than metal. On the other hand, both a depression and a protrusion (or a groove) would form if the surface of
a metallic component was scratched. The wear resistance of BioloxÒ Forte components can be derived from factors such as
the loads encountered in vivo are not able to deform the components and the improved surface finish. It also possesses other
Fig. 7 Various alumina femoral heads and acetabulum components.

Fig. 8 New generation Biolox Ceramtec alumina femoral components.
excellent mechanical properties such as extremely high elastic modulus and high compressive strengths. This leads to BioloxÒ
Forte having excellent dimensional stability.
In addition to alumina, partially stabilized zirconia (PSZ) has also been accepted as a ceramic material for use in clinical appli-
cations. Even though both materials are effective, they have specific potential disadvantages. Alumina, for example, is a brittle mate-
rial with a risk of fracture despite the fact that it exhibit excellent wear properties and hardness. This hard but brittle property
combination means that certain design restrictions apply. On the other hand, PSZ has only half of alumina’s hardness but its frac-
ture resistance can be improved through transformation toughening. As a result, its overall bending strength and toughness are
substantially much higher than those of alumina. Consequently, an ideal material would be a ceramic which combines the best
properties of alumina and zirconia. This leads to the new generation of ceramics referred to as zirconia-toughened alumina (ZTA).
As previously mentioned, BioloxÒ Delta is an alumina matrix composite consisted of approximately 75% alumina and roughly
25% yttria-stabilized tetragonal polycrystalline zirconia (Y-TZP). BioloxÒ Delta combines the excellent material properties of
alumina ceramics in terms of hydrothermal and chemical stability with superior mechanical strength, improved fracture toughness,
and extremely low wear. While, the zirconia and other additives further contribute to enhancing its mechanical properties. The
increase in strength and toughness in ZTA is attributable to the stress-induced transformation-toughening mechanism which is
introduced with the addition of optimized amounts of fine zirconia particles dispersed throughout the alumina body. In principle
it is based on the transformation-toughening theory in that, as a crack grows through the ceramic, the crystal structure of the zirconia
particles in the region of the crack will change from the metastable tetragonal phase to the stable monoclinic phase. The change is
expected to increase the volume of the particles by about 3%–4% and produces compressive stresses in the alumina matrix. These
stresses in turn close the crack and act as an energy barrier to further crack growth. The addition of zirconia to the alumina matrix was
introduced to increase fracture toughness and strength. The very small grain size of the alumina matrix in addition to the high
density of the material were reported to be the contributing factors in the enhancements of properties related to BioloxÒ Delta.
The first toughening mechanism involves introducing nanosized Y-TZP into a stable alumina matrix. The grains of Y-TZP are
spatially separated from one another in an effort to reduce the possibility of structural transformation in addition to preventing
the initiation and propagation of cracks. A second toughening mechanism along with normal transformation-toughening is antic-
ipated to be operational through the addition of an oxide additive which forms platelet-like crystals. These platelets are reported to
dissipate energy by deflecting cracks and thereby increasing strength and toughness (Pfaff and Rack, 2000).
Zirconia-Toughened Alumina (ZTA)

A new batch-processed ZTA ceramic was developed in 2002 by Insley and co-workers (2007). The new ZTA ceramic consists of 75%
alumina and 25% zirconia with no presence of mixed oxides. In comparison to the commercially available BioloxÒ Delta, the new
ZTA has a significantly lower flexural strength in terms of mechanical properties. The primary reason for this difference as explained
by Insley et al. is the microstructure of their ceramic. As stated previously, BioloxÒ Delta has a much finer grain size that results in its
superior strength property. In addition to the grain size, the dispersion of the zirconia grains in the alumina matrix is also different.
Zirconia (ZrO2) and Partially Stabilized Zirconia (PSZ)
The utilization of zirconium dioxide (ZrO2) or more commonly known as zirconia took place after the application of alumina as
a bioceramic or a biomaterial in general. The term zirconia has been widely used in the medical field and industry. Zirconia inher-
ently is a very brittle material due to high temperature phase transformation and can only be used as fully stabilized zirconia as an
engineering material. However, the zirconia product that is used in the medical field is in fact partially stabilized zirconia (PSZ),
which is a mixture of zirconia with either yttria, calcia, or magnesia depending on the application, synthesis, and country of origin.
Magnesia stabilized zirconia is originated in Australia in the early-1970s. Zirconia and its alloys has growingly become a material of
choice in implant dentistry, not only as the dental implant abutments, but as whole dental implants (Depprich et al., 2008; Choi
et al., 2012, 2013a).
Zirconia is the fully oxidized form of zirconium and can exist in a number of phases depending on the treatment temperature. As
mentioned briefly earlier due to this transformation and related expansion and contraction issues, zirconia cannot be produced as
pure zirconia and it is typically stabilized to prevent crack formation. A small amount of additives can generate partial stabilization
which is referred to as partially stabilized zirconia (PSZ). Zirconia, or more appropriately, PSZ-based ceramic materials possesses
several advantages which make it favorable as orthopedic and dental implants and in other clinical applications compared to
alumina such as improved corrosion resistance, flexural strength, and fracture toughness which can inhibit crack growth and prevent
catastrophic failure (Giordano and Sabeosa, 2010). The crystallographic configuration of zirconia (PSZ) ceramics has a strong influ-
ence on its durability and mechanical properties. The high mechanical properties of zirconia (PSZ) is the result of a toughening
mechanism as the ceramic undergoes phase transformation.
The crystallographic structures of zirconia is comprised of three different forms. For pure zirconia, the monoclinic phase is stable
at room temperature. Once zirconia is heated to a temperature between 1170 C and 2360 C, it will undergo transformation to
a tetragonal phase. Heating zirconia to a temperature between 2360 C and its melting temperature of 2680 C causes another phase
transformation from a tetragonal phase to a cubic phase. Given the fact that both the tetragonal and cubic phase of pure zirconia
only occurs at elevated temperatures, neither of those phases will exist as bulk materials at room temperature unless they are
produced at nanoscale methods such as sol–gel. On the other hand, the addition of metallic oxides to zirconia will assist in the
stabilization of the phase changes from tetragonal to cubic at low temperature (Chevalier and Gremillard, 2008). These metallic
oxides include magnesium oxide (magnesia, MgO); calcium oxide (calcia, CaO); cerium trioxide (ceria, Ce2O3), and yttrium
trioxide (yttria, Y2O3) (Manicone et al., 2007).
Zirconia (PSZ) has been considered for biomedical implants from as early 1969 and is one of the highest-strength ceramics ideal
for biomedical applications (Clarke et al., 2003). As stated earlier, the focus of research has been centered one type of partially stabi-
lized zirconia known as tetragonal zirconia polycrystal (TZP). TZPs are obtained through the addition of metallic oxides such as
ceria or yttria to zirconia. More importantly, mechanical properties can be enhanced when yttria is used to stabilize zirconia in
comparison to other metallic oxide combinations, and it is the main type of zirconia currently used for medical purposes (Manicone
et al., 2007). The amount of yttria in TZP only equates to approximately 2–3 mol% (Clarke et al., 2003; Garvie et al., 1975). The
desirable tetragonal phase contains arrays of submicrometer-size grains. There is a net volumetric expansion in the ceramic grains
when the transformation to monoclinic phase from tetragonal phase in the zirconia occurs, which may place the surface of the mate-
rial into a compressive stress field which helps to resist the initiation and progression of cracks. The depth of this residual compres-
sive stress layer has been reported to be several micrometers (Yoshimura et al., 1987; Piconi and Maccauro, 1999). The very high
strength and fracture toughness of Y-TZP ceramic is based on this “metastability”.
The Y-TZP ceramic may also transform to the monoclinic phase in an aggressive manner with catastrophic results under more
severe environmental conditions of stress and moisture or during certain manufacturing conditions. It is obvious that such a “high
metastability” is undesirable for medical implants (Clarke et al., 2003).
Bioglass
When a person suffers from a pain, the main concern for that individual is relieving the pain and returning to a healthy and func-
tional lifestyle. Degeneration and diseases often result in the replacement of skeletal parts, such as the knees, hips, finger joints,
elbows, vertebrae and teeth, and repair of the mandible surgically.
It is anticipated that the growth in these areas will continue due to a number of factors, for instance, the need due to the aging
population, improvements in technology and lifestyle, a better understanding of body functionality, an increasing preference by
younger to middle-aged candidates for undergoing correction surgery, improved aesthetics and the need for better function (Hench
and West, 1996).
By definition, a biomaterial is a nondrug substance which is ideal to be placed in a system that can replace or enhance the roles of
bodily organs or tissues. These materials are able to be in contact with bodily fluids and tissues while showing little or if any adverse
reactions for prolonged periods of time.
Engineers and surgeons have identified, even at the initial stages, the problems related to the design and materials selection that
resulted in premature loss of implant function through mechanical failure, corrosion or inadequate tissue interactions. Depending
on the applications, bioactive glass and glass ceramics in addition to ceramic materials are ideal candidates with respect to the above
functions.
Glass and Glass Ceramics

Glass is an amorphous, hard and brittle material created from the molten product of oxides. The molten material is normally cooled
rapidly in order to prevent crystallization or devitrification.
For over thousands of years, glass has been known to mankind. A natural glass produced from silicate magna called obsidian was
known to prehistoric people long before how to make glass was discovered. The Phoenicians are thought by many to have been the
first people to make glass (Hench and West, 1996).
It is now possible from the means of how glass is manufactured to predict and control the properties. Much of this control
derives from the purity and use of appropriate raw materials. The choice of raw materials is generally based on their glass-
making properties, which will be discussed in the following sections.
Since its discovery by Hench and co-workers in 1960s of bioglasses which bond to living tissue (BioglassÒ), various types of
bioactive glasses and glass ceramics have attracted attention of many researchers because of their unique properties which can easily
be tailored by manipulating its compositions and morphology (Hench and West, 1996). Glasses that are used primarily based on
silica (SiO2) which may also contain small amounts of other crystalline phases for implantation purposes (Ben-Nissan et al., 2017).
The first-generation bioactive glass compositions fall within the Na2O–CaO–P2O5–SiO2 system. It was first developed in 1971
when “45S5 BioglassÒ” with a composition of 42% SiO2, 24.5% CaO, 24.5% Na2O, and 6% P2O5 by weight was proposed (Hench
et al., 1972). It was suggested that 45S5 BioglassÒ has greater osteoblastic activity which is attributed to a rapid exchange of alkali
ions with hydronium ions at the surface compared to hydroxyapatite (HAp) (Huckstep and Sherry, 1996; Vrouwenvelder et al.,
1993). This in turn results in the formation of a silica-rich layer over a period of time. The migration of Ca2 þ and PO43 þ is allowed
on this layer to the silica-rich surface where they combined with soluble calcium and phosphate ions from the solution and the
formation of an amorphous CaO–P2O5 layer takes place. This layer will then undergo crystallization upon the interaction with
OH, CO32 þ, and F from solution. A similar phenomenon was also observed in a number of studies in bioglass with slightly modi-
fied compositions (Ben-Nissan et al., 2017; Andersson and Kangasniemi, 1991).
The formation of an apatite layer was observed in glass ceramics with a similar composition and various degrees of crystallinity
(Li et al., 1992). It was suggested that the formation of this layer was directly influenced by the amount of glassy phase that still
remains and the formation ceases once the glassy phase constitutes less than approximately 5 wt%.
A bioactive glass with precipitated crystalline apatite and reduced alkaline oxide content can be synthesized through the use of
a specific heat treatment method. The resultant glass ceramic is referred to as Ceravitals, and it has been shown to have a higher
mechanical strength but lower bioactivity compared to BioglassÒ.
A glass ceramic known as A-W glass ceramic (Cerabone A-W) was produced by Kokubo et al. (1982) which contains wollastonite
(CaO$SiO2) and oxyfluorapatite (Ca10(PO4)6(OH,F2)) in an MgO–CaO–SiO2 glassy matrix. In the early 1990s, it was reported that
the A-W glass ceramic spontaneously bonded to living bone without the formation of fibrous tissue around the glass. In addition,
a bioactive and machinable glass ceramic called Bioverits was also developed composed of apatite and phlogophite ((Na,K)Mg3(Al-
Si3O10)(F)2) which were previously used as artificial vertebra (Kokubo et al., 1982). However, the commercial production of A-W
glass has been discontinued and its current production and application are restricted only to research.
Synthesis of Bioactive Glass

Bioactive glasses have been manufactured using conventional glass technology. The glass components of oxides or carbonates
in the form of grains are mixed, melted and then homogenized at a temperature between 1250 C and 1400 C (Li et al., 1991).
Bulk implants are manufactured by casting molten glass into graphite or steel molds. A final grind and polish is sometimes
essential in order to achieve the required tolerances. By producing different particle sizes directly, this process has a number
of advantages despite the fact that the method has been modified a number of times so that grinding and polishing can be
avoided.
Nanoparticles and nanofibres of bioactive glass have been made available several years ago, and they have been used either alone
or in conjunction with polymers in the form of a nanocomposite in the biomedical field. Nanoscale bioactive glasses have been
produced using various approaches including sol–gel synthesis method.
At the moment, one of the methods used to prepare nanoparticles are based on heating metal–organic precursor compounds at
temperatures above 1000 C via flame spray technology. Another commonly used technique is the spray drying. The basic principle
behind all gas phase synthesis methods is the formation of molecular nuclei followed by condensation and coalescence which
induce the subsequent growth of nanoparticles in high-temperature regions during the process (Boccaccini et al., 2010; Stark
et al., 2003). While flame spray technique is an energy-intensive approach, its advantage lies in the fact that additional energy is
not needed for the precursors in comparison to other gas phase techniques.
Several studies have been conducted to gain an understanding into the dynamics and key variables of flame spray process and
how they can be controlled in order to obtain nanoparticles of given size range and chemical compositions (Madler et al., 2002;
Athanassiou et al., 2010). The metal-carboxylate system was discovered to be an extremely convenient precursor as it allows for
the synthesis of oxide nanoparticles of virtually any composition (Athanassiou et al., 2010). Above all, metal–organic are fully
miscible among each other and can tolerate humidity and highly stable in air. Consequently, the fabrication of nanoparticulate
mixed oxides of different types with high chemical homogeneity is permitted using this process.
Bioactive glass nanoparticles in the range of 20–50 nm were successfully synthesized using the flame spray process and it was
reported that a pronounced increase in mineral content was observed when human dentin were treated with these nanoparticles
suggesting rapid remineralization (Vollenweider et al., 2007). In a later study, the capability of flame spray technique to synthe-
size radio-opaque bioactive glass nanoparticles was also demonstrated for potential use in root canal applications (Mohn et al.,
2010).
Laser spinning approach

The development into laser spinning technique has been extensive over the past few years with definite control of the results to
fabricate custom-made products (Quinteroa et al., 2007a,b, 2009). The advantages of laser spinning technique include that the
process is relatively fast and the nanofibres are produced within several microseconds. It is also capable of synthesizing glass nano-
fibres of compositions that would be difficult to obtain using other methods. The diameters of the fibers produced from laser spin-
ning technique range from hundreds down to tenths of microns; in addition the types of products vary from disordered mats to
continuous filaments (Quinteroa et al., 2007b). However, the major drawback of laser spinning is that high energy is required
during the production process, which consequently increases the production cost.
Laser spinning technique has been demonstrated to be an efficient approach for the production of nanofibres of bioactive glasses
and new nanostructures with potential for tissue engineering scaffolds, as fillers in bone defects and as reinforcing agents in nano-
composites. The capability of the laser spinning technique to produce nanofibres with a wide range of compositions makes evident
of its potential to create nanofibres with different rates of bioresorption to control the release of active ions that have the potential to
stimulate the gene expression and cellular response necessary for tissue regeneration (Quinteroa et al., 2009; Quintero et al., 2009).
In 2009, a technique was developed for the production of bioglass nanofibers using laser spinning. A small quantity of precursor
material is melted using a high-energy laser to produce a superfine filament which is then lengthened and cooled by a powerful gas
current (Quintero et al., 2009).
Micro-emulsion approach
In general, micro-emulsions are thermodynamically stable dispersions of oil and water stabilized by a surfactant and a cosurfactant
in many cases. The micro-emulsions can be of the droplet type, either with spherical water droplets dispersed in a continuous
medium of oil or vice versa with spherical oil droplets dispersed in a continuous medium of water. By adjusting the micro-
emulsion in addition to operation variables, researchers have discovered nanoparticle size and polydispersity can be regulated
(Arriagada and Osseo-Asare, 1999; Singh et al., 2008).
It has been well known that this method is an ideal technique which is also capable of obtaining nanometer-sized inorganic
particles with minimum agglomeration (Karagiozov and Momchilova, 2005; Sun et al., 2007). In contrast, the key drawbacks asso-
ciated with micro-emulsion techniques are the usage of a large quantity of oil and surfactant phases and the low yield in production.
Only a limited number of studies are available on the production of nanosized bioactive particles using this approach even though
micro-emulsion technique provides an alternative means for synthesizing several types of organic and inorganic nanometer-sized
particles compared to other production methods (Lim et al., 1996, 1999).
Solegel approach
The motivation for the sol–gel processing compared to traditional glass melting or ceramic powder methods is first and foremost the
potentially higher homogeneity and purity in addition to the lower processing temperatures associated with the approach (Hench
and West, 1990).
Using the sol–gel process, the production of bioglass and other ceramics has become an interesting research field for the past four
decades (Hench and West, 1990; Greenspan et al., 1997; Jie et al., 2004; Saboori et al., 2009; Chen and Thouas, 2011; Cacciotti et al.,
2012). Sol–gel process involves the synthesis of an inorganic network by mixing the metal alkoxides in solution. This is then fol-
lowed by hydrolysis, gelation and low-temperature firing to produce a dense and stable glass or ceramic powder. The network struc-
ture of the gel can be modified by controlling hydrolysis and polycondensation reactions during productions. Hence, structural
variation can be produced without compositional changes.
Bioactive glasses can be synthesized by sintering gels at temperatures ranging from 600 C to 700 C, which eliminates most of the
disadvantages of high temperature processing with much better control over purity. Additionally, a broader range and better control
of bioactivity can be achieved by either changing the composition or microstructure through processing parameters (Sakka, 1985).
It has been reported in a study by Li et al. (1991) that an increase in bioactivity can be achieved through the use of sol–gel process
to SiO2–CaO–P2O5 powders as compared to melt-derived glasses of the same composition. Also, the rates of dissolution and forma-
tions of surface layer between sol–gel and melt-derived bioglass were examined (Sepulveda et al., 2001). It was discovered that the
58S sol–gel bioglass powder displayed a higher rate than the melt-derived 45S5 BioglassÒ (Pereira and Hench, 1996; Andersson,
1992; Peitl Filho et al., 1996; Hayakawa et al., 1999; Arstila et al., 2004; Ylänen et al., 1999; Fröberg et al., 2004; Andersson
et al., 1990; Niki et al., 1991).
It has been suggested that the high bioactivity of sol–gel derived materials is interconnected to the microstructural features of the
gel. These features include grain and pore size associated with the large surface area, and higher rate of dissolution and negative
surface charge (Pereira and Hench, 1996). As a result, sol–gel derived bioactive glass has been suggested as an alternative to glasses
produced by melt and quenching methods based on their ability to exhibit excellent degradation and/or resorption properties,
improved homogeneity and purity, more rapid bone bonding, and higher rate of apatite layer formation (Li et al., 1991).
Bioactive Glasses From Various Research Groups

In 1969, the concept of a strong bonding by chemical reactions between bone and glass-based synthetic materials occurring on
a glass surface was first proposed by Hench et al. (Fujiu and Ogino, 1984; Wilson et al., 1981). The innovation concerned the chem-
ical reactivity of the surface of a silica-based material, which had the amorphous structure of silicate glass. In the early 1970s, Hench
appropriately named this BioglassÒ (Oonishi et al., 2003). In fact, bioactive glasses can be thought of as the precursors to all bioac-
tive ceramics. The amorphous structure is a major characteristic of glasses. As stated earlier the structure of silicate glass in general is
based on the SiO4 tetrahedron. The tetrahedral are only linked to the oxygen ions at the corners. In crystalline silica, the tetrahedra
are regularly arranged, which is characteristic of all crystalline materials. The tetrahedra in the structure of silica glass are present, but
they are no longer regularly arranged; however, as in crystalline quartz, each of the oxygen ions still connects two tetrahedra. Bioac-
tive glasses bear a resemblance to ordinary soda-lime-silica glass. Compared with ordinary glass, there is one main difference with
the composition of bioactive glass. The amount of the network former SiO2 in ordinary glass is 45%, and the network modifier
component consists mainly of approximately 14% Na2O and 10% CaO, while bioactive glass consists of a significantly lower
amount of network former that is replaced primarily by metal oxides or network modifiers. Usually in the bioglass compositions,
the Na2O (or K2O) content and the ratio of Ca/P are relatively high (LeGeros and LeGeros, 1993).
Clinically bioactive glasses have been used successfully as bone-filling materials in orthopedic and dental surgery (Lai et al.,
1998; Oonishi et al., 2000; Hench, 1997). This stimulates researchers to combine excellent mechanical properties of metals or poly-
mers with a bioactive phase of either particles or fibers to produce a bioactive composite with optimized properties.
For most of the bioactive glass compositions, the tensile bending strength is between 40 and 60 MPa with a low modulus of
elasticity between 30 and 35 GPa.
Hench and co-workers: Bioglass®

During the last five decades, glass and glass ceramics have attracted extensive attention of many researchers because of their unique
properties which can easily be tailored by manipulating its compositions. The pioneering work of Hench (1997) led to the first
development of a bioactive silicate glass called 45S5 BioglassÒ with a composition of 45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt%
CaO, and 6 wt% P2O5. This is also commercially available as BioglassÒ which provides an alternative interfacial bonding of an
implant with host tissues.
This group of glasses has become known as bioactive glasses based on upon the following definition: a bioactive material is one
that elicits a specific biological response at the interface of the material that results in the formation of bond between the host tissues
and the material (Hench, 1998a). Many questions regarding its interactions with both soft and hard tissues have been answered over
the years by a multidisciplinary team of materials scientists, orthopedic surgeons, dental researchers, biomechanics experts, and
biologists. Many clinical BioglassÒ devices, such as MEPÒ, ERMIÒ, HAPEXÒ, NovaBoneÒ, NovaMinÒ, and NovaTheraÒ, are being
utilized as substitute for bone augmentation and restoration, in orthopedic, dental and maxillofacial surgery as well as in the field of
tissue engineering (Vollenweider et al., 2007). They have proven to be efficient and some even outperformed other bioceramic and
metal prostheses (Lai et al., 1998; Hench, 1997).
Bioactive glasses are a class of biomaterials that bond to both soft and hard living tissues through the formation of a hydroxy-
carbonate apatite (HCA) layer on their surfaces (Oonishi et al., 2003; Strnad, 1992). Yet, it has been shown that controlled rates of
release of ionic dissolution products from bioglass surface are the key phenomenon for bioactivity or bond formation, particularly
critical concentrations of soluble silica and calcia ions (Karlsson and Ylänen, 1998; Serra et al., 2002). Reactions occurring on the
surface of the glass lead to the formation of a silica gel layer and subsequent crystallization of HCA. It was assumed many years that
formation of biologically active HCA surface reaction layer was the critical requirement for bioactive behavior (Serra et al., 2003;
Ducheyne et al., 1988; Wilson et al., 1993). Numerous investigations have shown the formation of a surface HCA layer to be useful
but not critical stage of reaction for bone regeneration (Karlsson and Ylänen, 1998; Stoor et al., 1998, 1999). As stated above, the key
phenomenon is the controlled rates of release of ionic dissolution products, especially critical concentrations of soluble silica and
calcia ions. Osteostimulation occurs only when the ions are at a particular ratio of ions and at a particular concentration range of
15–30 ppm Si and 60–90 ppm Ca.
The rate of bone bonding is dependent on the composition of the material. Compared with other glass compositions, a faster
rate of bone bonding as well as bonding to soft tissues can be achieved when the SiO2 content is less than 52% by weight (Fröberg
et al., 2004). Rapid regeneration of trabecular bone with an amount, architecture and biomechanical quality of bone that matches
that originally present in the site can be produced from compositions such as 45S5 BioglassÒ with high rates of bioactivity (Saboori
et al., 2009; Cacciotti et al., 2012; Fröberg et al., 2004; LeGeros and LeGeros, 1993; Stoor et al., 2010). The rapid regeneration of
bone is the result of a combination of processes referred to as osteostimulation and osteoconduction.
Bioactive glass from Finland: Åbo Akademi

The research at the Åbo Akademi University initiated in the early 1980s when Yli-Urpo, Ylanen and Andersson et al. (Ben-Nissan,
1993; Karagiozov and Momchilova, 2005) examined the glass transition temperature and biological behavior of glasses in or near
the bioactive compositional region in the system SiO2–Na2O–CaO–P2O5–A12O3–B2O3. Later, an alumina-free bioactive glass was
developed with a given composition of 53% SiO2, 23% Na2O, 20% CaO, and 4% P2O5, and it is available as S53P4 (Ylänen, 2000).
Contributions of the group at the early stages were on the chemistry and viscosity relationship of a range of bioglasses. An extensive
study was conducted by Brink (Lim et al., 1996; Stanley et al., 1997; Hench, 1998b) on the viscosity temperature dependence and
biological activity of several different glass compositions in the SiO2–Na2O–CaO–P2O5–A12O3–B2O3 system. She discovered that
certain glass compositions possess a wider working range compared to conventional bioactive glasses. When the SiO2 composition
is too low, bioactivity can be obtained but the glass will crystallize, and this restricts their use only in the forms of crushed particles.
On the other hand, when the SiO2 content increases to 53–56 wt%, the working range is widened, and crystallization of devitrifi-
cation can be avoided (Stanley et al., 1997). The working range of the glasses examined was also influenced by the amounts of alkali
and alkali earth oxides present. The larger working range allows the expanded medical use of the bioactive glasses due to the possi-
bility, for instance, of fiber spinning and flame spraying into microspheres (Lim et al., 1996; Stanley et al., 1997).
The development of third generation of bioactive glasses denoted 13-93 has enabled spinning of high-quality thin bioactive
glass fibers. From these fibers, a variety of bioactive glass fabrics can be manufactured. Furthermore, an impulse laser beam can
be used to melt a fine powder made from the new bioactive glass. As a result, a glass coating on a titanium implant can be produced
relatively easily. This coating consists of very small glass droplets firmly attached to the titanium. The glass shows excellent bioac-
tivity in spite of the repeated high temperature procedures involved in the process (Suominen and Kinnunen, 1996; Kinnunen et al.,
2000).
Bioactive glass from Japan: A-W glass

Various research groups in the 1980s and 1990s such as Gross et al., Bromer et al., Kitsugi et al., Nakamura and Yamamuro and
Kokubo et al. (Chen and Thouas, 2011; Heikkilä et al., 1993; Suominen et al., 1996; Aho et al., 2004; Lee et al., 2004; Lim
et al., 2010; Schepers et al., 1991; Gatti and Zaffe, 1991; Garcia and Ducheyne, 1994; Gatti et al., 1996; Heikkilä et al., 1995) intro-
duced a range of phosphate-based glass and glass ceramics. Kokubo and Nakamura and their colleagues steadily heated glass
powder in the MgO–CaO–P2O5–CaF2–SiO2 system up to 1050 C. Consequently, they produced a material consisting of crystalline
wollastonite (CaO$SiO2) and oxyfluoroapatite (Ca10(PO4)6(O,F)2 within a homogenous glassy phase (Heikkilä et al., 1993;
Suominen et al., 1996). As mentioned earlier, this bioactive glass ceramic was referred to as A-W, derived from the names of the
crystalline phases. The bioactive A-W glass ceramic (A-W-GC) is a two-phase structure and reported to resemble the composite struc-
ture of bone, which can be machined into various shapes with diamond tools. The bending strength of the A-W-GC is almost double
that of dense HAp and even higher than that of human cortical bone (Aho et al., 2004). Other groups of researchers introduced
different glass ceramics (Ceravitals, Bioverit I-IIIs, Ilmaplants, BAS-O) during the 1970s and 1980s (Lee et al., 2004; Lim et al.,
2010; Schepers et al., 1991; Gatti and Zaffe, 1991). All these glass ceramics consist of apatite crystals or apatite/wollastonite/phlog-
opite crystals within a homogenous glassy matrix. It was reported that A-W-GC elicits the highest mechanical strength and bioac-
tivity (Class A) of all the glass ceramics (Insley et al., 2007; Chen and Thouas, 2011).
The dissolution of calcium from wollastonite or the glassy phase is believed to be the reason for the bioactivity of glass ceramic.
The bonding between glass ceramic and bone is formed by the precipitation of the dissolved calcium from the material and phos-
phate originating from the body fluid (Garcia and Ducheyne, 1994; Gatti et al., 1996). It was believed that the nucleation sites for
the formation of calcium phosphate are provided by the silicate ions (Suominen et al., 1996; Garcia and Ducheyne, 1994). Different
calcium contents in the materials have an effect on the bioactivities of the glass ceramics. Rigid non-degradable bioactive glass
ceramics can be produced with various methods for different applications (Gatti et al., 1996). Their applications include orthope-
dics, odontology and in head and neck surgery as prostheses, spacers or granuled defect fillers.
Questions were raised regarding their appropriateness as a coating material with the development of next-generation bioactive
glass ceramics. Using various techniques, the bioactive glass ceramics was fixed on load-bearing implants in early experiments
(Heikkilä et al., 1995; Oonishi et al., 1999). Takatsuka et al. (Virolainen et al., 1997) modified A-W-GC in 1993 and was successfully
sintered on Ti-alloy due to their similar coefficients of thermal expansion. The bioactive coating showed the bonding strengths
comparable with A-W GC implants, which were used as the control. It has also been demonstrated that AW-GC coatings have
encouraging results in various other investigations (Hench and Greenspan, 2013; de Groot et al., 1987; Hench, 1991). It is believed
that the glassy part of the material is mainly responsible for the ion dissolution from the material as stated earlier and, subsequently,
the chemical reactions occurring on the material surface. Thus, one part of the coating substance is resorbed, and changes in the
material’s mechanical properties occur.
Investigation conducted by Kitsugi et al. (Hench, 1991) suggested that the use of A-W-GC coated metal implants should be
limited to short-term implantation only, due to the risk of fracture of the coating layer. Although successfully commercialized
and clinically applied, the production was stopped by the company and currently no commercial production of A-W glass is
available.
Calcium silicate bioactive glass

One of the important trace elements in the human body is silicon (Si), and they are found at a level of 100 ppm in the bone and
200–550 ppm bound to extracellular matrix compounds (Choi et al., 2013b). The location of Si was reported to be at active calci-
fication sites in the bones and directly involves in the mineralization process of bone growth (LeGeros, 1993).
In the past decade, stimulated by the Si function in human body and the bioactive compositions of silicate-based bioglass, a new
family of bioactive calcium silicate ceramics has been developed with a wide range composition. It was discovered that bioactive
silicate ceramics with specific compositions could significantly stimulate in vitro osteogenic differentiation for several stem cells and
in vivo osteogenesis and angiogenesis.
Bioactive Glass Composites

The purpose of bioactive glass composites is to combine and optimize the bioactive phase of either glass particles or fibers with the
excellent mechanical properties of metals or polymers to improve properties such as flexibility and capacity to stand to deformation
under loads. Numerous bioglass composites have been developed and tested for such biomedical applications as bone regeneration
matrix and scaffolds, dental implant and drug delivery systems (Choi et al., 2014; Choi and Ben-Nissan, 2007; LeGeros, 2002; Ben-
Nissan et al., 2004a,b; Hu et al., 2001).
Silicon Nitride as Implant Material
Silicon nitride is the preferred ceramic for high mechanical performance engineering applications however this ceramic can be
considered a great candidate for biomedical applications due to the same properties that make it so important for engineering appli-
cations such as: chemical stability, high wear resistance, low friction coefficient and mainly due to the mechanical behavior under
articulation which is considered better than that for alumina ceramics.
Silicon nitride, or Si3N4, is a strong, heat-resistant material that can be manufactured into many different forms providing
specific benefits for different types of surgical implants. Specifically for spinal and orthopedic procedures, silicon nitride’s excep-
tional properties present opportunities to develop devices that may ultimately deliver improved patient outcomes. Silicon nitride
technology has three key advantages compared to traditional implants typically antimicrobial properties due to Si3N4’s hydrophilic
surfaces inhibit bacteria growth, potentially reducing the risk of infection. Si3N4 is partially radiolucent with clearly visible bound-
aries. Additionally, it does not produce MRI or CT imaging artifacts. This is a major advantage for intraoperative implant placement
and post-op assessment.
In one of the current publications it was reported that two cylindrical implants were installed in each tibia of five rabbits and
were kept for 8 weeks. During the healing time, tissue tracers were administrated in the animals so as to evaluate the bone growth
around the implants. Eight weeks after the surgery, the animals were euthanized and histological analyses were performed. It was
reported that no adverse reactions were observed close to the implant. The osteogenesis process occurred during the entire period
defined by the tracers. However, this process occurred more intensely 4 weeks after the surgery. In addition, the histological analyses
showed that bone growth occurred preferentially in the cortical areas. Different kinds of tissue were identified on the implant
surface, characterized by lamellar bone tissue containing osteocytes and osteons, by a non-calcified matrix containing osteoblasts,
or by the presence of collagen III, which may change to collagen I or remain as a fibrous tissue.
In addition a global company recently has received 510(k) clearance for a second generation design of its spinal fusion devices,
and is pursuing clearance of an interbody fusion device featuring a porous ceramic core. It was reported that this material is an ideal
osteoconductive material. Current production techniques include: traditional reaction sintering and hot isostatic pressing methods.
Bioceramics for Drug Delivery
The particulate form of ceramic materials finds applications in medical and non-medical fields. Particles in the form of microspheres
have special applications in treating tumors located within organs supplied by a single afferent arterial blood supply. Microspheres
and structurally modified ceramics have traditionally been used for targeted drug delivery of chemotherapeutic and radio-
therapeutic agents.
In recent years, the preparation of surface modified hollow microspheres has attracted considerable attention due to their
unusual properties, such as large specific surface area due to the nanolayer modified surfaces, low density and encapsulation prop-
erties. Therefore, they are expected to be very useful for many novel applications such as drug and protein delivery systems. In certain
applications, the effectiveness of microparticulate materials can be highly improved if they can act simultaneously as carriers for
biologically active molecules. In this sense, porous and surface modified materials are advantageous, since they present additional
surface area, an important parameter that strongly influences the loading capacity and release rates.
In dental clinical applications such as the severe periodontitis treatment, where massive alveolar bone loss occurs, bone defect
filling and intensive systemic long-term antibiotics administration is required. Hydroxyapatite microspheres intended to be used as
injectable bone filling material or as enzyme delivery matrices can also be used as antibiotic releasing materials. Ferraz and co-
workers (2007) developed novel injectable drug delivery systems with the drug releasing capability for periodontitis treatment
and. Their aim is to use hydroxyapatite loaded with an antibiotic, to be used as a local drug delivery system. In this study, amox-
icillin with and without clavulanic acid and erythromycin, antibiotics already in use for oral and parenteral administration in the
treatment of periodontitis, were tested to. Their results indicated that the microspheres have shown to have chemical compositions,
porosities, and surface areas that provided adequate conditions for the sustained release profile of the drugs investigated. Bacteri-
cidal tests have indicated that during the period of this study the released antibiotics had inhibitory effect on bacteria. Osteoblastic
cells proliferate well on the surface of the microspheres, being cell growth enhanced by the presence of antibiotics. They have also
concluded that these microspheres may therefore be considered as potentially good alternative systems to act as carriers for antibi-
otics and to enhance bone regeneration while treating periodontitis.
Bioceramics for Radiotherapy
There are a number of methods to combat cancer but in general when an organ afflicted with cancer is surgically removed; the func-
tions of the diseased organ are often not recovered or restored totally. It is therefore desirable to develop a treatment for cancer that
can destroy only cancerous cells, so that normal tissue can regenerate after treatment and the healthy cells are not scarified. Radio-
therapy is a treatment that shows such potential. However, irradiation is performed from an external source in most cases. Conse-
quently, the cancer often receives an insufficient dose of radiation, especially if it is deep seated, and irradiation can cause severe
damage to healthy tissues. However microsphere assisted radiation therapy has been currently applied in clinical applications
showing a range of success.
An in situ microsphere assisted radiation method has been clinical applied using 17Y2O3–19Al2O3–64SiO2 (mol%) (YAS) glass
microspheres that are prepared by a conventional melt-quench method. The yttrium-89 (89Y) in this glass is a non-radioactive
isotope, but neutron bombardment activates 89Y to form the b-emitter 90Y, which has a half-life of 64.1 h. When radioactive glass
microspheres 20–30 mm in diameter are injected into a target organ (e.g., a liver tumor), they are trapped inside small blood vessels
in the tumor, blocking the nutritional supply to the tumor, and delivering a large, localized dose of short range, highly ionizing
b-rays. The b-rays have a short penetration range of only about 2.5 mm in living tissue, thus presenting little radiation damage
to neighboring healthy tissues. These microspheres show high chemical durability, and the radioactive 90Y remains essentially
within the microspheres inside a patient, and does not affect neighboring healthy tissues. The radioactivity of 90Y decays to a negli-
gible level within 21 days after neutron bombardment. The microspheres therefore become inactive soon after the cancer treatment.
They are already used clinically for the treatment of liver cancer in Canada, the United States, and China. Radioactive yttrium-
containing resin microspheres 30–35 mm in diameter have also been used clinically for treatment of liver cancer in Australia, China,
New Zealand, and Singapore (Ferraz et al., 2007).
Kawashita and co-workers (2006) has successfully prepared hollow Y2O3 microspheres 20–30 mm in diameter when an aqueous
carboxymethylcellulose sodium salt solution containing urease was atomized into an aqueous yttrium nitrate solution containing
urea using a spray gun located 2 m above the yttrium nitrate solution, and the resultant solid materials were heat treated at 1300 C.
The structure and chemical durability of the resulting microspheres were investigated. The outer surface of the microspheres was
smooth and dense, and the inner parts had a honeycombed structure. In simulated body fluids at pH 6 and 7, the hollow Y2O3
microspheres showed high chemical durability.
Cancer cells generally perish at around 43 C because their oxygen supply via the blood vessels is not sufficient, whereas normal
cells are not damaged at even higher temperatures of around 48 C. In addition, tumors are more easily heated than the surrounding
normal tissues, since the blood vessels and nervous systems are poorly developed in the tumor. Therefore, hyperthermia is expected
to be a very useful treatment of cancer with few side effects. Various techniques for heating the tumors, such as treatments with hot
water, infrared rays, ultrasound and microwaves have been attempted. However, deep-seated tumors cannot be heated effectively
and locally using these techniques. Ferrimagnetic microspheres 20–30 mm in diameter and recently nanospheres are useful as ther-
moseeds for inducing hyperthermia in cancers, especially in those tumors located deep inside the body. These spheres are entrapped
in the capillary bed of the tumors when they are implanted through blood vessels, and can heat cancers locally by their hysteresis
loss when placed under an alternating magnetic field.
Kokubo and co-workers (Kawashita et al., 2005) prepared Fe3O4 microspheres by melting powders in high-frequency induction
thermal plasma, and by precipitation from an aqueous solution. The preparation of microspheres in high-frequency induction
thermal plasma involves pure Fe3O4 powders to be completely melted in a plasma flame of argon gas which was produced by
high-frequency induction heating. The solidified products were sieved using a nylon mesh in order to obtain particles of 20–
30 mm in size. These particles were then heat treated to 600 C and allowed to cool under a reduced pressure of 5.1 103 Pa.
Fe3O4 powders were added into 600 mL of 1 wt% aqueous solution of hydrofluoric acid (HF) for the preparation of microspheres
in aqueous solution. Silica glass microspheres with a 12.4 mm average diameter were soaked in Fe–HF solution at 30 C for 24 days
under vigorous stirring. The products were then heat treated at 600 C for 1 h, and allowed to cool in a CO2–H2 gas atmosphere. The
result reveals that the microspheres prepared in high-frequency induction thermal plasma were mainly composed of Fe3O4, accom-
panied by a small amount of FeO. The surfaces of the microsphere were smooth before the heat treatment, but became slightly
rough after heat treatment. The increase in the surface roughness might be attributed to the formation of Fe3O4 and/or the crystal
growth of Fe3O4. The saturation magnetization of the microspheres was 92 emu g 1. The heat generation was as small as 10 W g 1,
under 300 Oe and 100 kHz. Microspheres, 20–30 mm in diameter, composed of small crystals of Fe3O4 50 nm in size, were
prepared by precipitation from aqueous solution and subsequent heat treatment. It can be seen that b-FeOOH was precipitated
on the surface of silica glass microspheres and the thickness of b-FeOOH increased with increasing soaking period. The deposited
b-FeOOH was transformed into Fe3O4 by heat treatment above 400 C in the CO2–H2 atmosphere. The silica glass microspheres
soaked in Fe–HF solution showed ferrimagnetism, with saturation magnetization of 53 emu g 1 and coercive force of 156 Oe
and a heat generation of 41 W g 1, under 300 Oe and 100 kHz, under the same magnetic field, which was four times as large as
for pure Fe3O4 microspheres prepared in high-frequency induction thermal plasma.
Concluding Remarks
Gaining a deeper insight into the production and properties of bioceramics currently used as implants as well as bone replacement
materials could significantly contribute to the design of new-generation prostheses and post-operative patient management strat-
egies. The benefits of advanced ceramic materials in biomedical application have in overall been widely appreciated, specifically
in terms of their strength and biocompatibility. Improvements in the manufacturing process such as the use of hot isostatic process
can result in small grain structures and higher densities which are required by the bioceramics. Through the use of appropriate
sintering aids and very fine grain structure, these production methods allow the density of a ceramic to reach its theoretical value
resulting in optimization of strength and prevent the propagation of cracks and fracture. Current alumina and partially stabilized
zirconia ceramics are used in both orthopedics and the maxillofacial surgery with a great success. Bioglasses can be utilized as body-
interactive materials to restore physiological functions by assisting the body to heal or promote regeneration of tissues. This
approach can further be explored in the development of next-generation bioglasses incorporating specific drugs and biogenic mate-
rials with an extended range of applications.
Currently, the field of tissue engineering has been focused on combining the use of living cells and three-dimensional bioglass or
bioceramic structures to engineer neotissue to a patient’s damaged site. Nonetheless, the challenge of providing a safe and effective
glass and glass ceramic with an acceptable biocompatibility level and the required properties remains.
Stem cells have been incorporated into a range of calcium phosphate-based scaffolds. Cultured bone marrow cells derived from
adult stem cells can be considered as mesenchymal precursor cell population and are similar to stem cells in that they can also differ-
entiate into different lineages, which are osteoblasts, chondrocytes, adipocytes, and myocytes. These cells can combine with miner-
alized three-dimensional scaffolds once implanted into the body to form highly vascularized bone tissue.
These nanoscale cultured cell-bioceramic composites can be used in the treatment of gaps in bone, thus providing excellent inte-
gration of the ceramic scaffold with bone and good functional recovery. The scope of the application of nanomedical coatings based
on bioceramics such as bioglass and calcium phosphate in diagnostics, drug delivery, and implant applications may cover a number
of important factors, such as design, engineering, and surgical aspects. At the moment, synthetic dental and orthopedic implants
suffer from a major disadvantage of failing to adapt to the local tissue environment. The purpose of utilizing nanocoatings and
nanocomposite coatings is to reduce corrosion and ion release and at the same time modifying the surfaces of implant materials
to help the body to heal and to promote regeneration of tissues, thus restoring physiological function. In biomedical materials
research, one of the key issues is the examination of the relationship between surface properties of materials and its biological
responses. Surface modification by thin film deposition has become an important tool for research intended to help us understand
how structural and chemical surface properties influence material-biosystem interactions.
It is anticipated that more research on the clinical applications of the bioceramics in both monolithic and porous forms will be
evolving in an unprecedented rate. Although the research in these areas has been very fast transfer from laboratory to clinic has been
relatively slow.
Clinical applications is expected to increase with s better understanding in bioceramics/tissue integrations is achieved. One can
expect that modifications for the purpose of controlling tissue response will open up new avenues for developing new and superior
implants and medical devices in a more systematic manner and at a faster rate than at present.
References
Aho, A. J., Tirri, T., Kukkonen, J., Strandberg, N., Rich, J., Seppälä, J., & Yli-Urpo, A. (2004). Injectable bioactive glass/biodegradable polymer composite for bone and cartilage
reconstruction: Concept and experimental outcome with thermoplastic composites of poly(epsilon-caprolactone-co-D,L-lactide) and bioactive glass S53P4. Journal of Materials
Science. Materials in Medicine, 15, 1165–1173.
Andersson, Ö. H. (1992). Glass transition temperature of glasses in the SiO2–Na2O–CaO–P2O5–Al2O3–B2O3 system. Journal of Materials Science. Materials in Medicine, 3,
326–328.
Andersson, O. H., & Kangasniemi, I. (1991). Calcium phosphate formation at the surface of bioactive glass in vitro. Journal of Biomedical Materials Research, 25, 1019–1030.
Andersson, Ö. H., Karlsson, K. H., & Kangasniemi, K. (1990). Calcium-phosphate formation at the surface of bioactive glass in vivo. Journal of Non-Crystalline Solids, 119,
290–296.
Arriagada, F. J., & Osseo-Asare, K. (1999). Synthesis of nanosize silica in a nonionic water-in-oil microemulsion: Effects of the water/surfactant molar ratio and ammonia
concentration. Journal of Colloid and Interface Science, 211, 210–220.
Arstila, H., Vedel, E., Hupa, L., Ylänen, H. O., & Hupa, M. (2004). Measuring the devitrification of bioactive glasses. Key Engineering Materials, 254–256, 67–70.
Athanassiou, E. K., Grass, R. N., & Stark, W. J. (2010). Chemical aerosol engineering as a novel tool for material science: From oxides to salt and metal nanoparticles. Aerosol
Science and Technology, 44, 161–172.
Ben-Nissan, B. (1993). Review: Reliability and finite element analysis in ceramic engineering design, mater. Forum, 17, 105–125.
Ben-Nissan, B., & Choi, A. H. (2006). Sol–gel production of bioactive nanocoatings for medical applications: Part I: An introduction. Nanomedicine, 1, 311–319.
Ben-Nissan, B., Milev, A., & Vago, R. (2004a). Morphology of sol–gel derived nanocoated coralline hydroxyapatite. Biomaterials, 25, 4971–4976.
Ben-Nissan, B., Milev, A., Vago, R., Conway, M., & Diwan, A. (2004b). Sol–gel derived nano-coated coralline hydroxyapatite for load bearing applications. Key Engineering Materials,
254–256, 301–304.
Ben-Nissan, B., Choi, A. H., & Macha, I. (2017). Advances in bioglass and glass ceramics for biomedical applications. In Q. Li, & Y. W. Mai (Eds.), Springer Series in Biomaterials
Science and Engineering (SSBSE)Biomaterials for implants and scaffolds (pp. 133–161). Germany: Springer.
Boccaccini, A. R., Erol, M., Stark, W. J., Mohn, D., Hong, Z., & Mano, J. F. (2010). Polymer/bioactive glass nanocomposites for biomedical applications: A review. Composites
Science and Technology, 70, 1764–1776.
Cacciotti, I., Lombardi, M., Bianco, A., Ravaglioli, A., & Montanaro, L. (2012). Sol–gel derived 45S5 bioglass: Synthesis, microstructural evolution and thermal behavior. Journal of
Materials Science. Materials in Medicine, 23, 1849–1866.
Chen, Q. Z., & Thouas, G. A. (2011). Fabrication and characterization of sol–gel derived 45S5 Bioglass®-ceramic scaffolds. Acta Biomaterialia, 7, 3616–3626.
Chevalier, J., & Gremillard, L. (2008). Zirconia ceramics. In T. Kokubo (Ed.), Bioceramics and their clinical applications (pp. 243–265). England: Woodhead Publishing.
Choi, A. H., & Ben-Nissan, B. (2007). Sol–gel production of bioactive nanocoatings for medical applications: Part II: Current research and development. Nanomedicine, 2,
51–61.
Choi, A. H., Matinlinna, J. P., & Ben-Nissan, B. (2012). Finite element stress analysis of Ti-6Al-4V and partially stabilized zirconia dental implant during clenching. Acta Odontologica
Scandinavica, 70, 353–361.
Choi, A. H., Matinlinna, J. P., & Ben-Nissan, B. (2013a). Effects of micromovement on the changes in stress distribution of partially stabilized zirconia (PS-ZrO2) dental implants and
bridge during clenching: A three-dimensional finite element analysis. Acta Odontologica Scandinavica, 71, 72–81.
Choi, A. H., Ben-Nissan, B., Matinlinna, J. P., & Conway, R. C. (2013b). Current perspectives: Calcium phosphate nanocoatings and nanocomposite coatings in dentistry. Journal of
Dental Research, 92, 853–859.
Choi, A. H., Cazalbou, S., & Ben-Nissan, B. (2014). Nano-biomaterials coatings in dentistry. In S. Deb (Ed.), Frontiers of oral biology series: vol. 17. Biomaterials for oral and
craniomaxillofacial applications (pp. 49–61). London: King’s College London Dental Institute.
Clarke, I. C., Manaka, M., Green, D. D., Williams, P., Pezzotti, G., Kim, Y. H., Ries, M., Sugano, N., Sedel, L., Delauney, C., Ben-Nissan, B., Donaldson, T., & Gustafson, G. A.
(2003). Current status of zirconia used in total hip implants. The Journal of Bone and Joint Surgery. American Volume, 85A, 73–84.
Depprich, R., Zipprich, H., Ommerborn, M., Mahn, E., Lammers, L., Handschel, J., Naujoks, C., Wiesmann, H. P., Kübler, N. R., & Meyer, U. (2008). Osseointegration of zirconia
implants: An SEM observation of the bone–implant interface. Head & Face Medicine, 4, 25.
Ducheyne, P., Brown, S., Blumenthal, N., Hench, L. L., Krajewski, A., Palavit, G., Ravaglioli, A., Steineman, S., & Windeler, S. (1988). Bioactive glasses, aluminum oxide, and
titanium. Ion transport phenomena and surface analysis. Annals of the New York Academy of Sciences, 523, 257–261.
Ferrage, L., Bertrand, G., Lenormand, P., Grossin, D., & Ben-Nissan, B. (2017). A review of the additive manufacturing (3DP) of bioceramics: Alumina, zirconia (PSZ) and
hydroxyapatite. Journal of the Australian Ceramic Society, 53, 11–20.
Ferraz, M. P., Mateus, A. Y., Sousa, J. C., & Monteiro, F. J. (2007). Journal of Biomedical Materials Research. Part A, 81, 994–1004.
Fröberg, L., Hupa, L., & Hupa, M. (2004). Porous bioactive glasses with controlled mechanical strength. Key Engineering Materials, 254–256, 973–976.
Fujiu, T., & Ogino, M. (1984). Difference of bone bonding behavior among surface active glasses and sintered apatite. Journal of Biomedical Materials Research, 18, 845–859.
Garcia, A. J., & Ducheyne, P. (1994). Numerical analysis of extracellular fluid flow and chemical species transport around and within porous bioactive glass. Journal of Biomedical
Materials Research, 28, 947–960.
Garvie, R. C., Hannink, R. H., & Pascoe, R. T. (1975). Ceramic steel? Nature, 258, 703–704.
Gatti, A. M., & Zaffe, D. (1991). Long-term behavior of active glasses in sheep mandibular bone. Biomaterials, 12, 345–350.
Gatti, A. M., Valdre, G., & Tombesi, A. (1996). Importance of micro-analysis in understanding mechanism of transformation in active glassy biomaterials. Journal of Biomedical
Giordano, R., & Sabeosa, C. E. (2010). Zirconia: Material background and clinical application. The Compendium of Continuing Education in Dentistry, 31, 710–715.
Greenspan, D. C., Zhong, J. P., Chen, X. F., & LaTorre, G. P. (1997). The evaluation of degradability of melt and sol–gel derived Bioglass® in-vitro. Bioceramics, 10, 391–394.
de Groot, K. R., Geesink, R., Klein, C. P. A. T., & Serekian, P. (1987). Plasma sprayed coatings of hydroxylapatite. Journal of Biomedical Materials Research, 21, 1375–1381.
Hayakawa, S., Tsuru, K., & Ohtsuki, C. (1999). Mechanism of apatite formation on a sodium silicate glass in a simulated body fluid. Journal of the American Ceramic Society, 82,
2155–2160.
Heikkilä, J. T., Aho, A. J., Yli-Urpo, A., Andersson, O. H., Aho, H. J., & Happonen, R. P. (1993). Bioactive glass versus hydroxylapatite in reconstruction of osteochondral defects in
the rabbit. Acta Orthopaedica Scandinavica, 64, 678–682.
Heikkilä, J. T., Aho, H. J., Yli-Urpo, A., Happonen, R. P., & Aho, A. J. (1995). Bone formation in rabbit cancellous bone defects filled with bioactive glass granules. Acta Orthopaedica
Scandinavica, 66, 463–467.
Hench, L. L. (1991). Bioceramics: From concept to clinic. Journal of the American Ceramic Society, 74, 1487–1510.
Hench, L. L. (1997). Glass and genes: A forecast to future. Glastechnische Berichte Glass Science and Technology, 70, 439–452.
Hench, L. L. (1998a). Bioceramics. Journal of the American Ceramic Society, 81, 1705–1727.
Hench, L. L. (1998b). Bioceramics, a clinical success. American Ceramic Society Bulletin, 77, 67–74.
Hench, L. L., & Greenspan, D. (2013). Interactions between bioactive glass and collagen: A review and new perspectives. Journal of the Australian Ceramic Society, 49, 1–40.
Hench, L. L., & West, J. K. (1990). The sol–gel process. Chemical Reviews, 90, 33–72.
Hench, L. L., & West, J. K. (1996). Biological applications of bioactive glasses. Life Chemistry Reports, 13, 187–241.
Hench, L. L., Splinter, R. J., Allen, W. C., & Greenlee, T. K. (1972). Bonding mechanisms at the interface of ceramic prosthetic materials. Journal of Biomedical Materials Research,
Part A, 2, 117–141.
Hu, J., Russell, J. J., Ben-Nissan, B., & Vago, R. (2001). Production and analysis of hydroxyapatite from Australian corals via hydrothermal process. Journal of Materials Science
Letters, 20, 85–87.
Huckstep, R. L., & Sherry, E. (1996). Replacement of the proximal humerus in primary bone tumours. The Australian and New Zealand Journal of Surgery, 66, 97–100.
Insley, G. M., Turner, I., Fisher, J., & Streicher, R. M. (2007). In In vitro testing and validation of zirconia toughened aluminaProceedings of the 7th Biolox® symposium, Stuttgart,
Germany (pp. 26–31).
Jie, Q., Lin, K., Zhong, J., Shi, Y., Li, Q., Chang, J., & Wang, R. (2004). Preparation of macroporous sol–gel bioglass using PVA particles as pore former. Journal of Sol-Gel Science
and Technology, 30, 49–61.
Karagiozov, C., & Momchilova, D. (2005). Synthesis of nano-sized particles from metal carbonates by the method of reversed mycelles. Chemical Engineering and Processing, 44,
115–119.
Karlsson, K. H., & Ylänen, H. O. (1998). Porous bone implants. In P. Vincenzini (Ed.), Materials in clinical applications, advances in science and technology 289th Cimtec-World
Forum on New Materials, Florence.
Kawashita, M., Tanaka, M., Kokubo, T., Inoue, Y., Yao, T., Hamada, S., & Shinjo, T. (2005). Biomaterials, 26, 2231–2238.
Kawashita, M., Takayama, Y., Kokubo, T., Takaoka, G. H., Araki, N., & Hiraoka, M. (2006). Journal of the American Ceramic Society, 89, 1347–1351.
Kinnunen, I., Aitasalo, K., Pöllönen, M., & Varpula, M. (2000). Reconstruction of orbital floor fractures using bioactive glass. Journal of Cranio-Maxillo-Facial Surgery, 28, 229–234.
Kokubo, T., Shigematsu, M., Nagashima, Y., Tashiro, M., Nakamura, T., Yamamuro, T., & Higashi, S. (1982). Apatite-wollastonite containing glass-ceramic for prosthetic application.
Bulletin of the Institute for Chemical Research, 60, 260–268.
Lai, W., Ducheyne, P., & Garino, J. (1998). Removal pathway of silicon released from bioactive glass granules in vivo. In R. Z. LeGeros, & J. P. LeGeros (Eds.), Bioceramics (pp.
383–386). New York: World Scientific.
Lee, Y. K., Song, J., Moon, H. J., Lee, S. B., Kim, K. M., Kim, K. N., Choi, S. H., & LeGeros, R. Z. (2004). In vitro and in vivo evaluation of non-crystalline calcium phosphate glass as
a bone substitute. Key Engineering Materials, 254–256, 185–188.
LeGeros, R. Z. (1993). Biodegradation and bioresorption of calcium phosphate ceramics. Clinical Materials, 14, 65–88.
LeGeros, R. Z. (2002). Properties of osteoconductive biomaterials: Calcium phosphates. Clinical Orthopaedics and Related Research, 395, 81–98.
LeGeros, R. Z., & LeGeros, J. P. (1993). Dense hydroxyapatite. In L. L. Hench, & J. Wilson (Eds.), vol. 1. An introduction to bioceramics (pp. 139–180). Singapore: World Scientific.
Li, R., Clark, A. E., & Hench, L. L. (1991). An investigation of bioactive glass powders by sol–gel processing. Journal of Applied Biomaterials, 2, 231–239.
Li, P., Yang, Q., Zhang, F., & Kokubo, T. (1992). The effect of residual glassy phase in a bioactive glass-ceramic on the formation of its surface apatite layer in vitro. Journal of
Lim, G. K., Wang, J., Ng, S. C., & Gan, L. M. (1996). Processing of fine hydroxyapatite powders via an inverse microemulsion route. Materials Letters, 28, 431–436.
Lim, G. K., Wang, J., Ng, S. C., & Gan, L. M. (1999). Formation of nanocrystalline hydroxyapatite in nonionic surfactant emulsions. Langmuir, 15, 7472–7477.
Lim, H. C., Sohn, J. Y., Park, J. C., Um, Y. J., Jung, U. W., Kim, C. S., Lee, Y. K., & Choi, S. H. (2010). Osteoconductive effects of calcium phosphate glass cement grafts in rabbit
calvarial defects. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 95, 47–52.
Madler, L., Stark, W. J., & Pratsinis, S. E. (2002). Flame-made ceria nanoparticles. Journal of Materials Research, 17, 1356–1362.
Manicone, P. F., Rossi Iommetti, P., & Raffaelli, L. (2007). An overview of zirconia ceramics: Basic properties and clinical applications. Journal of Dentistry, 35, 819–826.
Mohn, D., Zehnder, M., Imfeld, T., & Stark, W. J. (2010). Radio-opaque nanosized bioactive glass for potential root canal application: Evaluation of radiopacity, bioactivity and
alkaline capacity. International Endodontic Journal, 43, 210–217.
Niki, M., Ito, G., Matsuda, T., & Ogino, M. (1991). Comparative push-out data of bioactive and non-bioactive materials of similar rugosity. In J. E. Davies (Ed.), Bone-material
interface (pp. 350–356). Toronto: University of Toronto Press.
Oonishi, H., Takayaka, Y., Clarke, I. C., & Jung, H. (1992). Comparative wear studies of 28-mm ceramic and stainless steel total hip joints over a two to seven year period. Journal of
Long-Term Effects of Medical Implants, 2, 37–47.
Oonishi, H., Hench, L. L., Wilson, J., Sugihara, F., Tsuji, E., Kushitani, S., & Iwaki, H. (1999). Comparative bone growth behavior in granules of bioceramic materials of various sizes.
Journal of Biomedical Materials Research, 44, 31–43.
Oonishi, H., Hench, L. L., Wilson, J., Sugihara, F., Tsuji, E., Matsuura, M., Kin, S., Yamamoto, T., & Mizokawa, S. (2000). Quantitative comparison of bone growth behavior in
granules of Bioglass, A-W glass-ceramic, and hydroxyapatite. Journal of Biomedical Materials Research, 51, 37–46.
Oonishi, H., Clarke, I. C., Good, V., Amino, H., Ueno, M., Masuda, S., Oomamiuda, K., Ishimaru, H., Yamamoto, M., & Tsuji, E. (2003). Needs of bioceramics to longevity of total joint
arthroplasty. Key Engineering Materials, 240–242, 735–754.
Peitl Filho, O., LaTorre, G. P., & Hench, L. L. (1996). Effect of crystallization on apatite-layer formation of bioactive glass 45S5. Journal of Biomedical Materials Research, 30,
509–514.
Pereira, M. M., & Hench, L. L. (1996). Mechanisms of hydroxyapatite formation on porous gel-silica substrates. Journal of Sol-Gel Science and Technology, 7, 59–68.
Pfaff, H. G., & Rack, R. (2000). In A new material concept for bioceramics in orthopaedicsProceedings of the 5th Biolox® symposium, Stuttgart, Germany (pp. 136–140).
Piconi, C., & Maccauro, G. (1999). Zirconia as a ceramic biomaterial. Biomaterials, 20, 1–25.
Quintero, F., Pou, J., Comesana, R., Lusquiños, F., Riveiro, A., Mann, A. B., Hill, R. G., Wu, Z. Y., & Jones, J. R. (2009). Laser spinning of bioactive glass nanofibers. Advanced
Functional Materials, 19, 3084–3090.
Quinteroa, F., Mann, A. B., Pou, J., Lusquiños, F., & Riveiro, A. (2007a). Rapid production of ultralong amorphous ceramic nanofibers by laser spinning. Applied Physics Letters, 90,
153109.
Quinteroa, F., Pou, J., Lusquiños, F., & Riveiro, A. (2007b). Experimental analysis of the production of micro- and nanofibers by laser spinning. Applied Surface Science, 254,
1042–1047.
Quinteroa, F., Dieste, O., Pou, J., Lusquiños, F., & Riveiro, A. (2009). On the conditions to produce micro- and nanofibers by laser spinning. Journal of Physics D: Applied Physics,
42, 1–10.
Saboori, A., Rabiee, M., Moztarzadeh, F., Sheikhi, M., Tahriri, M., & Karimi, M. (2009). Synthesis, characterization and in vitro bioactivity of sol–gel-derived SiO2–CaO–P2O5–MgO
bioglass. Materials Science and Engineering C: Biomimetic and Supramolecular Systems, 29, 335–340.
Sakka, S. (1985). Glasses and glass-ceramics from gels. Journal of Non-Crystalline Solids, 73, 651.
Schepers, E., de Clercq, M., Ducheyne, P., & Kempeneers, R. (1991). Bioactive glass particulate material as a filler for bone lesions. Journal of Oral Rehabilitation, 18, 439–452.
Sedel, L. (2000). Evolution of alumina-on-alumina implants: A review. Clinical Orthopaedics and Related Research, 379, 48–54.
Sepulveda, P., Jones, J. R., & Hench, L. L. (2001). Characterization of melt-derived 45S5 and sol–gel-derived 58S bioactive glasses. Journal of Biomedical Materials Research, 58,
734–740.
Serra, J., González, P., Liste, S., Chiussi, S., León, B., Pérez-Amor, M., Ylänen, H. O., & Hupa, M. (2002). Influence of the non-bridging oxygen groups on the bioactivity of silicate
glasses. Journal of Materials Science. Materials in Medicine, 13, 1221–1225.
Serra, J., González, P., Liste, S., Serra, C., Chiussi, S., León, B., Pérez-Amor, M., Ylänen, H. O., & Hupa, M. (2003). FTIR and XPS studies of bioactive silica based glasses. Journal
of Non-Crystalline Solids, 332, 20–27.
Singh, S., Bhardwaj, P., Singh, V., Aggarwal, S., & Mandal, U. K. (2008). Synthesis of nanocrystalline calcium phosphate in microemulsiondEffect of nature of surfactants. Journal
of Colloid and Interface Science, 319, 322–329.
Stanley, H. R., Hall, M. B., Clark, A. E., King, C. J., 3rd, Hench, L. L., & Berte, J. J. (1997). Using 45S5 bioglass cones as endosseous ridge maintenance implants to prevent
alveolar ridge resorption: A 5-year evaluation. The International Journal of Oral & Maxillofacial Implants, 12, 95–105.
Stark, W. J., Mädler, L., Maciejewski, M., Pratsinis, S. E., & Baiker, A. (2003). Flame synthesis of nanocrystalline ceria-zirconia: Effect of carrier liquid. Chemical Communications,
5, 588–589.
Stoor, P., Söderling, E., & Salonen, J. I. (1998). Antibacterial effects of a bioactive glass paste on oral microorganisms. Acta Odontologica Scandinavica, 56, 161–165.
Stoor, P., Söderling, E., & Grenman, R. (1999). Interactions between the bioactive glass S53P4 and the atrophic rhinitis-associated microorganism Klebsiella ozaenae. Journal of
Biomedical Materials Research, 48, 869–874.
Stoor, P., Pulkkinen, J., & Grenman, R. (2010). Bioactive glass S53P4 in the filling of cavities in the mastoid cell area in surgary for chronic otitis media. Annals of Otology,
Rhinology and Laryngology, 119, 377–382.
Strnad, Z. (1992). Role of the glass phase in bioactive glass-ceramic. Biomaterials, 13, 317–321.
Sun, Y., Guo, G., Tao, D., & Wang, Z. H. (2007). Reverse microemulsion-directed synthesis of hydroxyapatite nanoparticles under hydrothermal conditions. Journal of Physics and
Chemistry of Solids, 68, 373–377.
Suominen, E., & Kinnunen, J. (1996). Bioactive glass granules and plates in the reconstruction of defects of the facial bones. Scandinavian Journal of Plastic and Reconstructive
Surgery and Hand Surgery, 30, 281–289.
Suominen, E., Aho, A. J., Vedel, E., Kangasniemi, I., Uusipaikka, E., & Yli-Urpo, A. (1996). Subchondral bone and cartilage repair with bioactive glasses, hydroxyapatite, and
hydroxyapatite-glass composite. Journal of Biomedical Materials Research, 32, 543–551.
Virolainen, P., Heikkilä, J., Yli-Urpo, A., Vuorio, E., & Aro, H. T. (1997). Histomorphometric and molecular biologic comparison of bioactive glass granules and autogenous bone grafts
in augmentation of bone defect healing. Journal of Biomedical Materials Research, 35, 9–17.
Vollenweider, M., Brunner, T. J., Knecht, S., Grass, R. N., Zehnder, M., Imfeld, T., & Stark, W. J. (2007). Remineralization of human dentin using ultrafine bioactive glass particles.
Acta Biomaterialia, 3, 936–943.
Vrouwenvelder, W. C. A., Groot, C. G., & de Groot, K. (1993). Histological and biochemical evaluation of osteoblasts cultured on bioactive glass, hydroxylapatite, titanium alloy, and
stainless steel. Journal of Biomedical Materials Research, 27, 465–475.
Willmann, G. (1998). Ceramics for total hip replacementdWhat a surgeon should know. Orthopedics, 21, 173–177.
Willmann, G. (2003). The color of bioceramics. Key Engineering Materials, 240–242, 785–788.
Wilson, J., Pigott, G. H., Schoen, F. J., & Hench, L. L. (1981). Toxicology and biocompatibility of bioglasses. Journal of Biomedical Materials Research, 15, 805–817.
Wilson, J., Yli-Urpo, A., & Happonen, R. P. (1993). Bioactive glasses: Clinical applications. In L. L. Hench, & J. Wilson (Eds.), An introduction to bioceramics (pp. 63–73). Singapore:
World Scientific.
Ylänen, H. O. (2000). Bone ingrowth into porous bodies made by sintering bioactive glass microspheres. Dissertation: Åbo Akademi University.
Ylänen, H. O., Helminen, T., Helminen, A., Rantakokko, J., Karlsson, K. H., & Aro, H. T. (1999). Porous bioactive glass matrix in reconstruction of articular osteochondral defects.
Annales Chirurgiae et Gynaecologiae, 88, 237–245.
Yoshimura, M., Noma, T., Kawabata, K., & Somiya, S. (1987). Role of H2O on the degradation process of Y-TZP. Journal of Materials Science Letters, 6, 465.
Biomedical Composites
Min Wang, The University of Hong Kong, Pokfulam, Hong Kong
Qilong Zhao, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Introduction 36
Rationale and Concepts 37
Composite Classification 38
Matrix, Reinforcement, and Interface 38
Matrix Materials 38
Polymers 38
Metals 39
Ceramics 39
Reinforcement 40
Particles 40
Whiskers and chopped fibers 40
Long fibers 40
Interface 41
Bonding mechanisms 41
Wetting of surface 42
Control of interfacial bond strength 42
Design and Architecture of Composites 42
Design of Composites 42
Major influencing factors for composite properties 42
Design philosophy 43
Architecture of Composites 43
Dispersed phase geometry and distribution 43
Fiber architecture 44
Strengthening in composites 45
Nonporous and Porous Composites 45
Mechanical Properties of Composites 45
Load Sharing 46
Biological Properties of Bioactive Composites 46
Medical Applications of Composite Materials 46
Biomedical Composites in Orthopedics 46
Biomedical Composites in Dentistry 47
Biomedical Composites for Tissue Engineering 48
Biomedical Composites for Drug Delivery and Controlled Release Applications 49
Biomedical Composites for Medical Imaging 50
Biomedical Composites for Cancer Theranostics 50
Acknowledgments 51
Further Reading 51
Glossary
BioglassÒ A family of bioactive glasses that contain SiO2, Na2O, CaO, and P2O5 in specific proportions. 45S5 BioglassÒ is the
most bioactive glass in this family of glasses and is the only bioactive biomaterial so far that can form chemical bonding with
soft tissue in the human body.
Hydrogel A polymer having a network of polymer chains and containing a large amount of water.
Hydroxyapatite A synthetic bioactive bioceramic belonging to the calcium phosphate family and having the chemical formula
of Ca10(PO4)6(OH)2. It is similar to the apatite in human hard tissues (bone and tooth).
Implant A man-made medical device placed in the human body for replacing or repairing missing, damaged or diseased
tissues/organs.
Orthopedic The medical field concerned with human musculoskeletal system.

Biomaterials: Science and Engineering j Biomedical Composites 35
Prosthesis An artificial device for replacing the missing body part lost through trauma, disease or congenital conditions.
Micromechanics The analysis of materials at the level of their individual constituent for predicting mechanical properties of
materials.
Abbreviations
2D Two-dimension or two-dimensional
3D Three-dimension or three-dimensional
AFM Atomic force microscope
AgNP Silver nanoparticle
AuNP Gold nanoparticle
AuNR Gold nanorod
BCP Biphasic calcium phosphate
BMP Bone morphogenetic protein
CAD Computer aided design
Ca-P Calcium phosphate
CF Carbon fiber
CNT Carbon nanotube
CT Computed tomography
DNA Deoxyribonucleic acid
ECM Extracellular matrix
EDTA Ethylene diamine tetraacetic acid
FDA Food and drug administration
FDM Fused deposition modeling
FTIR Fourier transform infrared
LSPR Localized surface plasmon resonance
HA Hydroxyapatite
HDPE High-density polyethylene
MRI Magnetic resonance imaging
MSC Mesenchymal Stem Cell
NIR Near infrared
PA Polyamides
PAA Poly(acrylic acid)
PAN Polyacrylonitrile
PC Polycarbonate
PCL Polycaprolactone
PE Polyethylene
PEEK Polyetheretherketone
PEG Poly(ethylene glycol)
PEO Poly(ethylene oxide)
PET Poly(ethylene terephthalate)
PGA Poly(glycolic acid)
PHB Polyhydroxybutyrate
PHEMA Poly(hydroxyethyl methacrylate)
PLA Poly(lactic acid)
PLGA Poly(lactic-co-glycolic acid)
PMMA Poly(methyl methacrylate)
PNIPAAm Poly(N-isopropylacrylamide)
PP Polypropylene
PS Polystyrene
PSU Polysulfone
PTFE Polytetrafluoroethylene
PU Polyurethane
36 Biomaterials: Science and Engineering j Biomedical Composites
PVA Poly(vinyl alcohol)

PVC Poly(vinyl chloride)
PVP Poly(vinyl pyrrolidone)
RGD Arginine/glycine/aspartic acid
QD Quantum dot
SBF Simulated body fluid
SEM Scanning electron microscopy
SERS Surface-enhanced Raman scattering
SF Silk fibroin
SLS Selective laser sintering
SPION Superparamagnetic iron oxide nanoparticle
SPR Surface plasmon resonance
SR Silicone rubber
TCP Tricalcium phosphate
TEM Transmission electron microscopy
UHMWPE Ultra-high molecular weight polyethylene
UV Ultraviolet
VEGF Vascular endothelial growth factor
Introduction
Engineering materials, including metals, ceramics, and polymers, have distinctive properties and hence have been extensively
investigated for various biomedical applications. Metallic biomaterials have a long history for prostheses and implants for
human body tissue repair in load-bearing areas owing to their high strength and toughness. Metallic prostheses and implants
(e.g., bone/teeth implants made of Co–Cr alloy or Ti alloy) cause little or limited toxicity to host tissues. But it is difficult for
them to achieve satisfactory interface with host tissues because they are bioinert. Some biodegradable metallic devices made of
iron, zinc, magnesium have also been investigated for biomedical applications but problems of their relatively low degrada-
tion rates and unsatisfactory biocompatibility exist. Normally, metallic prostheses and implants such fracture fixation plates
are removed or replaced after they have fulfilled their functions or they have undergone corrosion, fatigue, etc. Ceramics,
commonly seen as bioactive Ca-P (e.g., HA and TCP with osteoconductive property), glasses, metallic or nonmetallic oxide,
etc., are also frequently employed for various biomedical applications as bone substitution, drug delivery, and bioimaging.
They are corrosion-resistant and have excellent biocompatibility in the human body environment. However, ceramics are
brittle, which limits their scope for biomedical applications. Polymers are versatile in many biomedical applications as their
structures, which determine their properties and functions, can be designed. Compared to common metallic and ceramic
materials, polymeric materials exhibit excellent ductility and flexibility and tuneable protein and cell interactions (e.g.,
promoting or resisting protein/cell attachment under specific design) and have similar mechanical properties to soft tissues
in human bodies, making them promising candidates for applications in tissue engineering, drug delivery, bioimaging, etc.
Remarkable successes have been achieved by the use of metals, ceramics, and polymers in the biomedical field and it is certain
that these engineering materials will continue to contribute to the new generation of biomedical devices. However, many
biomedical devices are required to perform multiple functions and possess superior properties, which is challenging for
a single type of material. Consequently, composite materials, traditionally defined as a material composed of two or more
chemically distinct phases (e.g., metals, ceramics, and polymers), have been investigated and developed in the biomedical
field. Composites combine features and advantages of metals, ceramics, and polymers exhibiting improved properties or
new, unique properties.
Composites are not new and have been employed as engineering materials for thousands of years. One early example is
mud brick, which is formed by the mixture of mud and straw which have poor tensile, bending and compressive strengths,
respectively. However, excellent mechanical properties are obtained with mud brick as a composite even though these two
constituting components are weak themselves. Likewise, concrete as a composite material which is very important in the
construction industry has been utilized for a long time now. It combines different components together (e.g., sand, clay,
metals) to achieve excellent strength and stiffness in tension, bending, and compression. In recent decades, particularly
with the emergence of nanotechnology, composites with unique structures and functions have been attracting increasing
attention in almost all engineering fields including biomedical engineering. This article introduces basic concepts, provides
an overview of characteristics, manufacturing technologies and composite systems and presents biomedical applications of
composite materials.
Rationale and Concepts

A composite material, according to Kelly in “Concise Encyclopedia of Composite Materials” (Pergamon, Oxford, 1994), is “a
heterogeneous mixture of two or more homogeneous phases which has been bonded together,” and the composite material itself
can be regarded as “a homogeneous material.” Different types of materials (hence different phases) in a composite can be easily
distinguished by the interface between phases. The material forming the major and continuous phase (> 50% by volume, usually
having relatively low strength and stiffness) in the composite is termed “matrix material” and the material existing as a discontin-
uous, dispersed, minor phase (< 50% by volume, usually exhibiting relatively high strength and stiffness) in the composite is
termed “enhancement.” Composite materials can possess a combination of properties from each of constituting materials (i.e.,
metals, ceramics, and polymers) or create new properties and hence exhibit superior or unique properties, usually as light but strong
and stiff materials. Properties of composites can be controlled by changing the ratio of constituting materials, their features and the
interface.
For instance, modern composites such as metals or plastics reinforced with glass fibers or CFs have been widely used in auto-
mobile and aircraft industries, which possess better mechanical properties but have lighter weight than conventional metals or plas-
tics. Their strength and stiffness can be varied by changing the reinforcement/matrix ratio. Owing to their distinctive characteristics,
composite materials have advantages for many applications in the biomedical field. Human body tissues and organs are complicate
systems with complex compositions, structure, and functions. The tissues are generally categorized into hard tissues (i.e., bone and
tooth) and soft tissues (e.g., cartilage, skin, blood vessel, gastrointestinal tract), which have different mechanical characteristics
(Tables 1 and 2). Accordingly, metallic and ceramic biomaterials having relatively high stiffness are frequently employed for
hard tissue repair/substitution, while polymers are usually used for soft tissue repair. However, bone is a natural nanocomposite,
consisting of a hard but brittle material (i.e., nano-sized bone apatite) and a natural polymer, collagen. The composite nature of
bone makes it strong and resilient, capable of resisting high stresses, bearing high loads (for 5 times of body weight for hip joint
when walking and a peak load of more than 10 times body weight when jumping), and enduring numerous motion cycles (around
106 motion cycles for finger or hip joint in a year). Because of high stiffness of metals and ceramics and the brittle nature of ceramics,
the use of one single type of metallic or ceramic material is extremely difficult, if possible, to make bone substituting materials which
are mechanically compatible to natural bone. The mechanical mismatch between implanted materials and the natural bone leads to
unsatisfactory results such as loosening of implants since bone is remodeled due to the stress caused by the implantation of pros-
thesis with too high stiffness. According to “Wolff’s Law” and the load sharing principle in composites, bone remodels until the
stress and strain are retained at a specific level. Also, “ideal” bone substituting materials need to be biocompatible and bioactive
(i.e., osteoconductive) for achieving good interactions between the implant and host tissue. Composite materials composed of
bioactive ceramics and appropriate polymers, therefore, become attractive candidates for bone substitution which may simulta-
neously provide excellent properties in strength, biocompatibility and bioactivity. In many other areas such as drug delivery and
bioimaging, metallic or ceramic nanoparticles have shown their great potential, while their improvement by the incorporation
of polymers to form nanocomposite particles is necessary for attaining enhanced biocompatibility and functionality. Overall, it
is essential to use the composite approach to develop new biomaterials so as to meet particular and important biomedical
requirements.
Table 1 Mechanical properties of hard tissues
Hard tissue Modulus (GPa) Tensile strength (MPa)
Cortical bone (longitudinal direction) 17.7 133

Cortical bone (transverse direction) 12.8 52
Cancellous bone 0.4 7.4
Enamel 84.3 10
Dentine 11.0 39.3
Table 2 Mechanical properties of soft tissues
Soft tissue Modulus (MPa) Tensile strength (MPa)
Articular cartilage 10.5 27.5

Fibrocartilage 159.1 10.4
Ligament 303.0 29.5
Tendon 401.5 46.5
Skin 0.1–0.2 7.6
Arterial tissue (longitudinal direction) 0.1
Arterial tissue (transverse direction) 1.1
Fig. 1 Composite classification according to the geometry of the reinforcement (the second, minor phase).
Composite Classification
Numerous types of composite materials have been investigated for different biomedical applications. According to different the
matrix material (i.e., major phase), composites are classified into polymer matrix composites, metal matrix composites, and ceramic
matrix composites. Usually, the type of matrix material defines the typical characteristics of composites. For example,
polymer matrix generally yields ductile composites. Alternatively, composites are categorized by different geometries of the rein-
forcement (i.e., dispersed phase): particle-reinforced composites, fiber-reinforced composites and structural composites (Fig. 1).
Particle-reinforced composites encompass large-particle reinforced composites and dispersion-strengthened materials (with the
size of particulate reinforcements ranging from 10 to 100 nm). Typical examples include spheroidite steel (ductile ferrite matrix
reinforced by stiff Fe3C particles), automobile tires (compliant rubber matrix reinforced by stiff carbon particles) and concrete
(mixture of cement). For different fiber geometries (i.e., short fibers and long fibers), fiber reinforced composites are categorized
accordingly. While the dispersion of fibrous reinforcements in long fiber reinforced composites are mainly continuous and aligned,
fibers in short fiber reinforced composites are either aligned or randomly oriented. Fiber-reinforced composites generally provide
significant improvement in strength, with typical examples being a variety of glass fiber or CF reinforced metals or polymers.
Mechanical properties of composites are highly related to the composition, concentration, length, and orientation of fibers. Struc-
tural composites refer to the composites consisting of multiple homogeneous and composite layers, which are further classified as
laminates and sandwich panels. Laminates are usually composed of stacked and cemented anisotropic layers in a specific sequence.
Owing to the stacking nature, laminar composites usually possess excellent in-plane stiffness and one typical example is plywood.
Sandwich panels are usually composed of two stiff face sheets (e.g., metallic sheets and fiber-reinforced plastic panels) and one low-
density sandwiched core (e.g., foamed polymers with honeycomb structure). Owing to their specific structural features, sandwich
panels exhibit several significant advantages such as light weight and excellent bending stiffness.
Biomedical composites can also be categorized into bioinert composites, bioactive composites, and biodegradable/resorbable
composites. Bioinert composites are commonly formed by the mixture of two or more chemically different bioinert materials.
Bioactive composites contain a component (as either matrix or reinforcements) that elicit specific biological response for guiding
cell functions, for example, Ca-P, BioglassÒ, A-W glass-ceramic, and other bioactive bioceramics. As for biodegradable (or, resorb-
able) composites, biodegradable/resorbable materials such as biodegradable polymers (PLA, PLGA, PCL, etc.), and bioceramics
(e.g., TCP) which can be completely degraded/absorbed in the human body with no or limited side effects are employed as consti-
tuting materials, resulting in desired composites for the new generation of implants.
Matrix, Reinforcement, and Interface

Matrix Materials
Polymers
Polymers are widely used in the biomedical field in either dried, solid form or as hydrogels. They can be easily processed, as
compared to metals and ceramics, into different shapes (wire, rod, plate, film, etc.) and different micro-/nanostructures (micro-/
nanoparticle, micro-/nanoplate, etc.) for different purposes. One general requirement of polymers for biomedical applications is
good biocompatibility. “Biostable” synthetic polymers including PE, PP, PU, PTFE, PVC, PA, PMMA, polyacetal, PC, PET, PEEK,
PSU, SR, and UHMWPE are regarded as “biocompatible” materials and frequently used for making medical devices for different
applications, with the remarkable successes such as PTFE/PU artificial vascular grafts and UHMWPE tendon/ligament/joint substi-
tutes. For some implants, polymers with appropriate biodegradation property are preferred. Biodegradable polymers including PCL,
PLA, PGA, and their co-polymers such as PLGA and PDLLA have good biocompatibility and different biodegradation rates. Some of
them have been approved by FDA for biomedical uses. Natural polymers, such as collagen, gelatine, chitosan, hyaluronic acid, SF,
and alginate, are frequently used for different biomedical devices owing to their excellent biocompatibility. When polymers are
made into different structures/shapes at submicro-/nanoscale (nanoparticles, nanocapsules, etc.), unique properties such as high
surface-area-to-volume ratio and tuneable swelling behavior are obtained, which are suitable for applications such as drug delivery.
However, polymeric materials (both natural and synthetic) have relatively low stiffness and strength and lack bioactivity, which has
limited their scope of biomedical applications.
Recently, hydrogels are important materials in the biomedical field. They are formed by chemical or physical crosslinking of
polymer chains into 3D and hydrophilic polymer networks with the absorption of water or biological fluids thousands of times
the weight of polymer networks. Hydrogels with porous polymer network and excellent water swelling properties highly resemble
native human body tissues and hold great promise in various biomedical applications including tissue engineering and drug
delivery. Hydrogel alone has shortcomings in mechanical strength and functions but can be effectively improved by the incorpo-
ration of reinforcements such as nanoparticles and nanofibers. Synthetic polymers such as PHEMA, PVA, PEG, PAA, PMMA, ther-
moresponsive PNIPAAm, etc., and natural polymers such as alginate, collagen, gelatine, hyaluronic acid, etc. have been employed
for producing nanocomposite hydrogel with improved mechanical properties and tailored functions such as superior physical,
chemical, electrical, and biological properties.
Metals
Metallic biomaterials, owing to high strength and stiffness, have been widely employed for hard tissue substitution, particularly as
prostheses in orthopedic and dentistry. In 700 BC, gold dental prostheses were made by Etruscans. And in the years till present day,
numerous metallic prostheses have been developed for different hard tissue substitution purposes. Typical modern examples are
316L stainless steel, Co–Cr alloy, Ti-alloy for orthopedic surgery and amalgam for dental filling. They are bioinert and exhibit
good biostability in the physiological environment. The metallic medical devices can provide sufficient structural and mechanical
supports to assist hard tissue repair or to partially substitute tissue functions. These metallic biomaterials have excellent strength and
high stiffness (Table 3). Actually, according to “Wolff’s Law,” the stiffer nature of metallic materials over native hard tissue can cause
unsatisfactory outcomes. To address this issue, metallic biomaterials can be employed as either matrix materials or reinforcements
to form composites with improved mechanical properties closer to those of native hard tissues.
With the emergence of nanotechnology, metallic nanoparticles, due to their various unique physical properties, have gained
increasing attention as novel biomaterials in biomedicine. Typical examples are AgNPs, AuNPs, Au–Ag alloy nanoparticles which
have unique SPR and SERS properties and which have great potential in bioimaging and biosensing to, for example, detect tumor
biomarkers for cancer diagnosis. These functional metallic nanoparticles with specific geometry/shape can even convert tissue-
permeable NIR light to heat, producing “photothermal” effect which is promising as a new cancer therapy. With dual functions
in cancer detection and cancer therapy, these functional metallic nanoparticles have been therefore extensively investigated as novel
“theranostics” which are nanodevices for both cancer diagnosis and therapy. However, metallic theranostics face various challenges,
including biocompatibility, limited in-the-body circulation time, and poor cancer targeting ability. Upon functionalization with
specific polymers, the resultant metal-polymer nanocomposite can have enhanced properties. Also, anticancer drugs can be encap-
sulated in the polymeric phase of nanoparticles and subsequently released at tumor sites for achieving synergetic effects with
chemotherapy and photothermal therapy.
Ceramics
Ceramics are inorganic, nonmetallic, solid materials. High stiffness and mechanical strength (Table 4) and good biostability and
biocompatibility of bulk bioceramics such as alumina (Al2O3) make them good candidates for load-bearing medical devices in
orthopedics and dentistry. For example, zirconia ceramics is a popular dental material for crown. Bioactive bioceramics have
also been extensively investigated and widely employed for orthopedic and dental prostheses. Bioactive Ca-P, particularly HA,
have “osteoconductive” property which facilitates growth of bone cells and the attachment of implant and hence are widely
used for hard tissue repair. BioglassÒ, A-W glass-ceramic, and other bioactive glasses and glass-ceramics are also frequently used
bioactive bioceramics in orthopedics and dentistry. BioglassÒ is noncrystalline material which contains less silica and higher
amounts of calcium and phosphorous (with a 5:1 molar ratio of calcium to phosphorus) as compared to normal glass and exhibits
Table 3 Mechanical properties of metallic biomaterials
Metal Modulus (GPa) Tensile strength (MPa)
Stainless steel 190 586

Co–Cr alloy 210 1085
Ti-alloy 116 965
Amalgam 30 58
Table 4 Mechanical properties of ceramic biomaterials
Ceramic Modulus (GPa) Tensile strength (MPa)
Alumina 380 300

Zirconia 220 820
Bioglass® 35 42
HA 95 50
the ability to bond to body tissues, even soft tissue. It shows osteoconductive property and is able to get well integrated with living
bone after implantation. A common problem for bulk ceramic materials for biomedical applications is their brittleness, which must
the addresses for their successful medical applications. The composite approach provides a viable way for using bioceramics and
ceramic matrix composites have been studied and developed.
Good biocompatibility and electromagnetic isolation properties are also advantages of ceramics for biomedical applications.
Bioceramics such as alumina and BioglassÒ prove to be effective for the packaging of implantable electrical medical devices such
as cardiac pacemaker, artificial retinal prostheses, and artificial cochlea. Nanoceramics, that is, ceramics at nanoscale (primarily
ceramic nanoparticles) are also attractive. They are usually synthesized via sol-gel and sintering route, providing multiple unique
properties/functions (high surface-area-to-volume ratio, dielectric, ferroelectric, piezoelectric, pyroelectric, ferromagnetic, magneto-
resistive, etc.) in accordance with their compositions and structures. For example, silica nanoparticles/nanocapsules with mesopo-
rous shell are excellent drug delivery vehicles for different therapeutics. SPION is another example, which can be employed as MRI
contrast agent for bioimaging and can also generate heat in an alternating magnetic field for providing hyperthermia cancer therapy.
Reinforcement
Particles
For biomedical composites, rigid particulate reinforcements are usually dispersed in ductile matrices (usually polymers). Mechan-
ical and biological properties of particle-reinforced composites are significantly affected by the characteristics (e.g., composition,
size, shape) of reinforcing particles. Bioactive bioceramic nanoparticles, such as Ca-P, BioglassÒ, and A-W glass-ceramic particles
are used for making bone-mimicking bioactive composites for orthopedic and/or dental applications. Sizes of particulate reinforce-
ments in biomedical composites normally range from several nanometers to tens of micrometers, while their size distribution can
be mono-modal (narrow), mono-modal (wide, with a long tail end) or bi-modal. In the laboratory, Ca-P particles are usually
synthesized via a precipitation reaction at a suitable pH, resulting in amorphous spherical nanoparticles with sizes ranging from
10 to 30 nm or acicular shaped nanoparticles. For acicular shaped nanoparticles, the aspect ratio (i.e., the length to width ratio)
and particle orientation are key parameters affecting mechanical properties of resultant composites. Commercially available
Ca-P (e.g., HA and TCP) and BioglassÒ particles are usually irregularly shaped. Surfaces of irregular particulate reinforcements is
preferable for forming stable mechanical interlocking with the matrix, whereas smooth surfaced and spheroid shaped particulate
reinforcements are easily detached from the matrix when stressed in the absence of chemical bonding between reinforcement
and matrix. However, bioactive glass and glass-ceramic particles made by conventional melting and quenching methods usually
exhibit shape corners, leading to stress concentrations in composites. They can undergo an additional mechanical milling process
to reduce the sharp corners prior to composite manufacture. Commercial TCP particles have porous surfaces and tend to break up
into smaller particles during composite manufacture. In contrast, original small particulate reinforcements, owing to aggregation
through physical interactions (e.g., Van der Waals force, electrostatic interaction), tend to combine together, forming larger particle
agglomerates in composites manufacturing process, resulting in nonuniform distribution of particulate reinforcements in the matrix
(i.e., condensed state) and hence unsatisfactory mechanical stability of resultant composites. Ideally, individual particles should be
evenly distributed in particulate reinforced in composites, achieving the dispersed state.
Whiskers and chopped fibers

Composites with fibrous reinforcements are able to achieve significant improvements in mechanical properties, which are influ-
enced by the mechanical properties of the fibers themselves, aspect ratio (i.e., the fiber length to fiber diameter ratio), fiber diameter,
fiber volume fraction, etc. Fibrous reinforcements in fiber-reinforced composites are either continuous (aligned long fibers) or
discontinuous (whiskers or chopped fibers), and the distribution of whiskers or chopped fibers in matrix are either aligned or
randomly oriented. Mechanical properties of composites reinforced by aligned whiskers and chopped fibers are anisotropic, while
randomly oriented and discontinuous fiber-reinforced composite can be regarded as isotropic. Aligned whiskers and chopped fibers
as reinforcements are usually high-cost thin single crystals such as alumina, SiC, and SiN which have nearly perfect crystalline struc-
tures and hence extremely high stiffness, enabling significant mechanical property improvement for metal or ceramic matrix. Chop-
ped glassy fibers are used to reinforce polymer matrices.
Long fibers
Continuous fiber reinforced composites (i.e., long fibers as reinforcement) are also employed in the biomedical field. In general,
long fibers are either polymer or ceramic fibers and include aramid fibers, UHMWPE fiber, cellulose fibers, CFs, glass fibers, and
Ca-P fibers. CFs are made from a variety of precursor fibers (polymer, mesophase pitch, etc.) and are usually graphitized PAN fibers
for use in the medical field. PAN-derived CFs consist of crystallites of “turbostratic” graphite whose basal planes are parallel to the
fiber axis which produces high axial modulus and strength for CFs. Manufacturing of CFs from PAN fibers requires high-temperature
processing. The modulus of resultant CFs is affected by the processing temperature, with standard (230–240 GPa), intermediate
(250–300 GPa), and high (350–500 GPa) modulus values being achieved after treatments at 1200 C–1400 C, 1400 C–2200 C,
and above 2200 C, respectively. CFs are regarded as biocompatible and therefore were employed for investigations to, for example,
reinforce porous PTFE for soft tissue augmentation, reinforce UHMWPE as bearing surface in total joint prostheses, reinforce epoxy
resins as fracture fixation devices, and be braided into artificial ligament/tendon. Bioactive glass fibers are also used in studies of
long fiber reinforced biomedical composites. As amorphous materials, mechanical properties of glass fibers are normally isotropic,
which is different from CFs. As they are made of ductile materials, polymer fibers such as aramid fibers and UHMWPE fibers exhibit
different mechanical behaviors as compared to CFs and glass fibers. In contrast to the brittle manner of ceramic fibers under tensile
stress to fracture, polymer fibers usually show ductile fracture. Consequently, polymer fiber reinforced composites have high tough-
ness, providing application examples in the orthopedics and dentistry. Furthermore, by using biodegradable polymer fibers such as
PLA, PCL, and PLGA fibers as reinforcements, various composite-based fully biodegradable/resorbable medical devices have been
fabricated.
As stated earlier, mechanical properties, such as strength, of continuous fiber reinforced composites depend on those of the rein-
forcing fibers and the strength of brittle fibers can be treated statistically. For example, the strength of ceramic fibers (e.g., CF and
glass fibers) can be described by the three-parameter Weibull distribution equation:

l s s0 a
P ðsÞ ¼ 1 exp (1)
l0 b
where s is applied stress, P is probability of fiber failure, l is fiber length, l0 is reference length, a is shape parameter, b is scale
parameter, and s0 is location parameter. Once Weibull distribution parameters (a, b, s0) are known, the strength of a fiber of a given
length can be predicted. Single fiber tensile tests are the most direct experimental method to determine Weibull distribution
parameters. Alternatively, Weibull distribution parameters are obtained from the bundle tensile tests on the assumption that there is
no friction between the fibers.
Interface
Bonding mechanisms
The interface between the reinforcement and matrix strongly affects properties of resultant composites, which is either strong or
weak governed by different bonding mechanisms involved. Normally, a strong interface results in high strength of resultant
composites but leads to low toughness of composites. In contrast, a weak interface may result in composites with low strength
and sometimes with cracks. The interfacial bonding between reinforcement and matrix is either physical or chemical, as illustrated
in Fig. 2. For comparison, the interface formed by chemical bonding is several orders of magnitude stronger than that by physical
bonding. However, chemical bonding, that is, reinforcement chemically reacting with matrix at the interface, is generally avoided at
the interface. Hence most of composite interfaces are relying on physical bonding, including molecular entanglement following
interdiffusion, electrostatic attraction, cation-anionic interaction (e.g., in layer-by-layer assembly), and mechanical interlocking.
Consequently, surface features (surface morphology, surface electric charge, surface energy, etc.) of reinforcements and matrices
are determining factors for interface formation. The reinforcement and matrix with specific surface features such as rough morphol-
ogies and opposite surface electric charges are likely to form a strong interface.
Fig. 2 Schematic diagrams showing interfacial bonding between the reinforcement and matrix in composites.
Fig. 3 Contact angle measurement for a solid surface.
Fig. 4 Schematic diagrams showing mechanical tests for determining interfacial bond strength.
Wetting of surface
Surface energy is an important property of a material, which is usually assessed via contact angle measurement of the material. In the
contact angle measurement, a liquid drop is placed on the flat surface of a solid material and subsequently stabilized to maintain
a certain shape by the balance of forces (Fig. 3). Wetting of the solid surface is determined by interaction of three phases (materials),
that is, vapor, liquid, and solid. For an ideal solid surface, that is, a flat, rigid, perfectly smooth (nonporous) and chemically homo-
geneous surface having zero contact angle hysteresis (meaning the advancing and receding contact angles are the same), its wetting
behavior can be described by the Young equation:
gsv ¼ gsl þ glv ∙cos q (2)
where gsv, gsl, and glv are surface tensions (surface energy) between solid and vapor, solid and liquid, and liquid and vapor,
respectively, q is liquid contact angle on the solid surface. The surface energy of a solid material (solid to air, gsl) can be calculated by
measuring the contact angles of the liquid drop (q) in a series of experiments. This method for wettability studies can be applied to
constituting materials of a composite and also the composite itself. Frequently, this method is use to investigate the wetting of fibers
by the matrix in developing new composites.
As for a nonideal rough solid surface, its wetting behavior can be described using different models such as the Wenzel model:
cos q ¼ r∙cos q (3)
where q* is the apparent contact angle under the stable equilibrium state (i.e., minimum free energy state), r is the surface roughness
ratio defined as the ratio of true area of the solid surface to the apparent area, and q is the Young contact angle as defined for an ideal
surface.
Control of interfacial bond strength

Interfacial bond strength is a key factor influencing the overall mechanical properties of composites. In order to obtain desired
mechanical properties for the composites, the interfacial bond strength should be carefully controlled, which may be achieved using
different techniques. For example, for many polymer-matrix composites, their interfacial bond strength is effectively enhanced by
the use of silane coupling agents, forming strong chemical bonding between reinforcements and matrix through a local hydrolysis
reaction. As for many ceramic-matrix composites, metal of ceramic fibers are employed for increasing their toughness, not strength.
For a metal-matrix composite reinforced by SiC fibers, coating SiC fibers with a duplex C/TiB2 reaction layer is effective to enhance
the interfacial bond strength, resulting in a moderate interfacial bond strength.
The interfacial bond strength of different types of fibrous composites is investigated through tests and theoretical analysis, aim-
ing to study the shear stress that causes the debonding of reinforcement from the matrix and the debonding process. Single fiber
pull-out or single fiber push-out tests are frequently used for determining the critical interfacial shear stress for debonding
(Fig. 4), thereby measuring the interfacial bond strength.
Design and Architecture of Composites

Design of Composites
Major influencing factors for composite properties
Properties (physical, structural, mechanical, biological, etc.) of composites are affected by many factors. Major influencing factors
for biomedical composites include
(1) Matrix properties (average molecular weight of polymer, average grain size of metal or ceramic, etc.)
(2) Bioactivity of the reinforcement or matrix
(3) Stability and biodegradability of the reinforcement or matrix

(4) Reinforcement shape, size, and size distribution
(5) Reinforcement properties and volume percentage in composite
(6) Distribution (and orientation) of the reinforcement in the matrix
(7) Reinforcement-matrix interfacial state
Biodegradable materials, including biodegradable polymers such as PLA, PLGA, and PCL and biodegradable ceramics such as TCP
and some bioactive glasses are increasingly used for biomedical composites. The bioactivity of composites is usually achieved by the
use of bioactive bioceramics (Ca-P, BioglassÒ, etc.) in composites. Alternatively, the bioactivity is obtained by the incorporation of
bioactive molecules such as growth factors in composites. Compared to composite materials for general engineering applications,
biodegradable composite cannot undergo harsh manufacturing processes. Therefore, processing parameters, particularly tempera-
ture and pressure, used in the manufacturing of biomedical composites should be carefully selected.
Design philosophy
Comprehensive considerations should be taken when designing a composite for the particular biomedical application(s). Consid-
ering the specific body environment for the composite to be used, appropriate materials as reinforcement and matrix, as well as the
architecture of the composite suitable, need to be determined. A manufacturing technology is then decided for making the
composite. Therefore, designing biomedical composites can involve
(1) Choosing matrix material
(2) Selecting reinforcement
(3) Controlling interface
(4) Designing composite architecture
(5) Selecting manufacturing technique and processing parameters
For applications that require high toughness, polymers with ductile nature are usually selected as matrix to produce polymer matrix
composites. Conventional manufacturing technologies for polymer matrix composites include extrusion, injection molding, and
compression molding for making particulate (or short fiber) composites and filament winding, pultrusion, and vacuum bag-
autoclaving for fabricating fibrous composites. For applications that require excellent strength and load-bearing ability, ceramic
and metal matrix composites can be employed. They can be fabricated using different technologies. For example, ceramic matrix
composites are made through pressureless sintering, hot pressing, or hot isostatic pressing. For hard tissue repair (or regeneration)
in orthopedics and dentistry, particulate bioactive bioceramics are widely used to reinforce polymers, forming bioactive composites.
For fabricating these composites, bioceramic particles are either mixed with matrix polymers prior to composite manufacture or
formed in situ in the matrix phase. There are therefore two routes, that is, thermo-mechanical methods and physico-chemical
methods, to produce bioceramic-polymer composites for hard tissue substitution. The thermo-mechanical methods directly
incorporate as-synthesized bioceramic particles into the polymer matrix using conventional plastic processing technologies. The
physico-chemical methods involve initial in situ precipitation of mineral crystals in the polymer matrix in a suitable medium
and subsequent consolidation of the composite.
The conventional fabrication techniques mentioned above have achieved remarkable successes for biomedical composites. With
advances in nanotechnology and also manufacturing technologies in recent decades, novel techniques for fabricating composites
with well-defined micro-/nanostructures or customized shapes/architecture have been developed. They include solution-based tech-
niques (e.g., self-assembly, phase separation, and electrospinning) and additive manufacturing technologies. Self-assembly is
a potent way to form micro-/nanoscale composite materials with complicated hierarchical and ordered structures. Self-assembly
can be driven by different mechanisms and generally uses weak molecular interactions such as electrostatic attraction, hydrogen
bonding, p–p stacking interaction, and hydrophobic/hydrophilic interaction to produce micro-/nanoscale composites. Techniques
such as phase separation and electrospinning are frequently employed to fabricate porous composite materials. In particular, elec-
trospinning provides a simple but highly effective way to prepare fibrous and nanocomposite biomaterials, which are suitable as
tissue engineering scaffolds to assist human tissue regeneration. Additive manufacturing techniques are highly effective in the auto-
mated and precise manufacturing of polymer, ceramic, metal, and composite products from CAD models or 3D models built by
medical imaging (e.g., 3D image of a human’s skull captured by MRI). They have high potential in fabricating customized
composite devices for different biomedical applications. Various biomedical products made from composite materials, including
wearable biomedical devices, medical implants, tissue prostheses, and tissue engineering scaffolds, with enhanced performance
have been fabricated using different additive manufacturing techniques (SLS, FDM, etc.).
Architecture of Composites
Dispersed phase geometry and distribution
For a particle-reinforced composite, the geometry and distribution of particles (i.e., the dispersed phase) are important in deter-
mining composite properties such as mechanical properties and electrical properties. In the mathematical modeling of the mechan-
ical properties of a particle-reinforced composite, particles are usually assumed to have perfectly spherical shapes for simplicity.
However, the reinforcing particles in practical situations are usually irregular in shape. Spherical particles are also difficult to
Fig. 5 Schematic 3D views of different dispersion states of particulate reinforcements in the composite matrix: (A) dispersed state, (B) intermediate
state, (C) condensed state.
form mechanical interlocking with the matrix, thereby leading to unstable interface in the absence of other interactions between the
particles and matrix. For example, the geometry of HA particles formed by tightly bonded crystals are usually irregular and they exist
as the dispersed phase in the polymer matrix composite. Irregular surfaces of HA particles contribute to the interfacial bond strength
as molecules of the polymer matrix are able to penetrate into the troughs on the surface of HA particles to form strong mechanical
interlocking.
Particle distribution in the composite also plays a critical role in determining composite properties. In general, an even distri-
bution of particles in the matrix is a prerequisite for particle reinforced composites with high quality and desirable performance.
However, due to various factors during the preparation and/or manufacturing processes that cause particles to agglomerate, particles
sometimes tend to combine together, resulting in different particle aggregation states. For the particle-reinforced composites with
the intermediate or condensed state of particles (Fig. 5B,C), cracks are easily generated at particle contacting points under mechan-
ical stresses. The electrical properties of composites reinforced by conductive particles are also adversely affected by the uneven
distribution of particles in composites. Therefore, a high shear force is required during composite manufacture to overcome the
adhesive forces that cause aggregation of particles so as to produce composites with desirable even distribution of primary particles.
The energy input for generating the high shear force should be carefully controlled because too high energy may break up primary
particles into small fragments.
Fiber architecture
The fiber architecture, including fiber length, fiber orientation, fiber stacking arrangement, fiber texture and the angle and packing
sequence of fiber-reinforced sheets, is a critical factor affecting the performance of fiber reinforced composites and simple cross-ply
laminates. For short-fiber composites, fiber length and fiber orientation are key influencing factors. While the fiber length affects the
load transfer between short fiber and the matrix locally, fiber orientation affects the macroscopic properties of the composite. Fiber
orientation in short fiber composites can be random or aligned, resulting in isotropic or anisotropic materials. However, isotropic
short fiber composites (i.e., those with randomly oriented short fibers) can become “anisotropic” upon unidirectional tension as the
fiber orientation is changed by the elongational flow.
As for continuous and aligned long-fiber composites, the fiber stacking arrangement is an important factor in determining
their mechanical properties as interfiber spacing is varied by different fiber stacking arrangements. As seen from the cross-
sectional view in Fig. 6, long fibers in composites can be arranged in a hexagonal form (Fig. 6A) or square (Fig. 6B).
Compared to the composites with the hexagonal fiber array, those with the square array have denser fiber arrangement
(i.e., smaller interfiber spacing), resulting in larger fiber volume fraction. For fibrous composites, woven, braided, and knitted
fiber arrays also affect macroscopic properties of the composites since different texture patterns result in distinct fiber arrange-
ments. For laminates formed by sequential stacking of continuous and aligned long-fiber composite sheets, the angle and the
sequence during packing need to be considered. Each of the unidirectional sheets is stacked at a certain sequence with a certain
angle in order to meet the requirements for specific anisotropic mechanical properties in particular composite applications.
For example, blood vessels have multilayer structure with a bottom isotropic endothelial layer and two anisotropic smooth
muscle layers (i.e., with respective longitudinally arranged and circularly arranged smooth muscle cells). Therefore, laminated
structures with proper design and manufacture can be promising candidates as vascular grafts owing to their biomimetic
mechanical properties.
Fig. 6 Schematic diagrams showing cross-sectional views of different fiber packing arrangements in fiber reinforced composites: (A) hexagonal
form, (B) square form.
Strengthening in composites
The strength of a composite is determined by, among many factors, reinforcement, reinforcement volume fraction, matrix, archi-
tecture of the composite, and the interface. Composites reinforced by continuous and aligned fibers usually exhibit excellent but
directional mechanical properties. The strength and stiffness of a fiber reinforced composite reply on those of the fiber. Hence,
the prerequisite is to develop and use strong and stiff fibers: CFs, glass fibers and polymer fiber such as aramid fibers. Composites
having higher reinforcement volume fractions undoubtedly provide higher mechanical strength. The reinforcement volume fraction
in a particle reinforced composite also significantly affects its mechanical properties. While the Young’s modulus and tensile
strength of composites normally increase with an increase in particle volume fraction, the fracture strain of composites decreases
accordingly for composites with a ductile polymer matrix and a stiff bioceramic reinforcement.
Brittle materials such as ceramics need to be toughened, instead of strengthened, by using the composite approach. For example,
rubber toughening is used for “brittle” polymers, where rubber particles are incorporated in the polymer matrix. Brittle ceramics can
be toughened by the incorporation of fibers (continuous or chopped), whiskers or particles, for which toughening mechanisms such
as fiber bridging, fiber debonding, and fiber pull-out operate, These toughening mechanisms require additional energy for the cracks
initiate and propagate.
Nonporous and Porous Composites

The overall structure of a composite can be nonporous or porous, which is designed and the achieved by the manufacturing tech-
nique used. Nonporous composite materials are normally fabricated by conventional techniques (extrusion, injection molding,
pultrusion, etc.) and exhibit good structural integrity and excellent mechanical properties. They can be used for biomedical appli-
cations requiring high mechanical loading, such as bone substitution and dental filling. In contrast, a porous composite usually has
much lower strength than the nonporous composite of the same composition but exhibits advantages such as light weight and high
surface-area-to-volume ratio. Porous biodegradable composites are now particularly investigated as novel tissue engineering scaf-
folds, which provide microenvironments with suitable physical, chemical, and biological features for cell attachment and growth.
The fabrication techniques for porous composites include solvent casting, freeze drying, phase separation, electrospinning, 3D
printing, etc. All these techniques can form porous composites with interconnected pores, which are important for cell migration,
cell–cell connection, and tissue ingrowth. Through the chosen strategy, the sizes of interconnected pores in porous composites can
be tailored because different tissue engineering applications require different pore sizes. Porosity is another parameter that needs to
be controlled in porous composites for tissue engineering. A compromise may have to be made to achieve high porosity while
maintaining sufficient mechanical strength.
Mechanical Properties of Composites

Numerous investigations have been conducted to study the mechanical properties of composites, particularly on long fiber
reinforced composites. Mechanics of composites have been well established. The micromechanics of continuous fiber rein-
forced composites can be described by using relatively simple models with simplified assumptions. In the mechanics of mate-
rials approach, for calculating elastic properties (e.g., Young’s modulus) of unidirectional fibrous composites, it is assumed that
(a) continuous fibers are perfectly aligned, (b) constituent materials are perfectly bonded, and (c) initially, there are no voids
and internal stresses in composite. Voigt model is used for the situation that the composite is stretched along with the fiber
axis:
Ec ¼ Ef ∙Vf þ Em ∙Vm (4)
where Ec, Ef, and Em are elastic moduli of the composite, reinforcing fiber and matrix, respectively, and Vf and Vm are the volume
fractions of the fiber and matrix, respectively. When a tensile force is applied perpendicular to the fiber axis, Reuss model is used for
calculating the Young’s modulus of the composite:

1=Ec ¼ Ef Vf þ Em =Vm (5)
While neither of these models is rigorously correct, Voigt model and Reuss model do provide the upper and lower bounds
respectively for the Young’s modulus of a continuous fiber-reinforced composite. When the two moduli of the reinforcement
and matrix differ by no more than a factor of two or three, the simple linear superimposition of Voigt and Reuss values provides
a good approximation. And if the tension is exerted at an angle (B) with respect to the fiber axis, a modified Voigt model is used for
calculating the Young’s modulus of composites:
Ec ¼ Ef ∙Vf ∙cos4 B þ Em ∙Vm (6)
Particle reinforced composites with a relatively soft matrix (e.g., polymer) and an evenly distributed stiff particulate reinforce-
ment (e.g., ceramic particle) may be considered as isotropic, and their Young’s modulus (Ec) can be predicted by the simple Kerner
equation:
1 þ ACVp
Ec ¼ Em ∙ (7)
1 CVp
where,
Ep
7 5nm Em 1
A¼ and C ¼ (8)
8 10nm Ep
þA
Em
and nm is the Poisson’s ratio of the matrix, Vp is the volume fraction of the reinforcing particles, Em and Ep are the Young’s moduli of
the matrix and dispersed particles, respectively. Generally, the strength of particle reinforced composites decreases with an increase
in the volume fraction of reinforcing particles, which is confirmed by the results obtained from tensile tests.
Laminated composites can be regarded as the stacking of continuous fiber reinforced composites in specific sequences. The
micromechanics models are then built on the basis of behaviors of continuous fiber reinforced composites. Details of the theoretical
analysis can be found in books on mechanics of composites. Although results from micromechanics analysis have some differences
from experimentally obtained data, they nevertheless provide useful guidelines for designing composites with targeted mechanical
properties.
Load Sharing
For orthopedic and dental devices, mechanical properties are important factors affecting their clinical performance. It is well known
that bone remodeling proceeds in accordance with “Wolff’s law,” that is, bone is deposited and reinforced in areas of greatest stress.
The mismatch of stiffness between the implant and hard tissue surrounding the implant adversely affect tissue remodeling and tissue
healing. Therefore, once a much stiffer metallic or ceramic implant is placed adjacent to the bone in the body, the bone will much
lower stiffness will bear much reduced mechanical load in accordance with the load sharing principle of the composite theory, result-
ing in what is known as “stress shielding.” Consequently, bone is remodel to decreased bone mass according to “Wolff’s law.” Stress
shielding is a commonly observed phenomenon, particularly in orthopedics where many metallic implants are used.
Many efforts have been made to solve the stress shielding problem. Since Bonfield et al. proposed the concept of analogue
biomaterials in the 1980s, using the composite approach, numerous bioactive bioceramic-polymer composites (and subsequently
bioceramic-metal and bioceramic-ceramic composites) have been investigated. Considering bone is a natural nanocomposite, it is
reasonable and also natural to design a material that mimics the bone structure and provides matching mechanical properties,
particularly the stiffness, for bone substitution. The particulate HA reinforced high-density polyethylene succeeded clinically as
the first bone analogue material for bone tissue repair and many other composites followed. Bioceramics such as HA, BioglassÒ
and A-W glass-ceramic particles and polymers such as PSU, PEEK, and PU can be used to form biostable composites for bone substi-
tution. In recent decades, biodegradable composites made of resorbable bioceramics (e.g., TCP) and biodegradable polymers (e.g.,
PLGA) for orthopedics and dentistry gain much attention. They allow gradual increase of stress on the host hard tissue during the
healing process with the degradation of the composite implant. The fully biodegradable orthopedic/dental devices can also avoid
second surgery, providing great benefits to both patients and the health care system.
Biological Properties of Bioactive Composites

One major issue for implants for tissue repair and tissue engineering scaffolds for tissue regeneration is biological properties con-
cerning biocompatibility and bioactivity. The biocompatibility of a material refers to its ability to perform with an appropriate host
response in the particular host environment, which is determined by the material surface properties (morphological, physical,
chemical, electrical, etc.) and bulk properties (stiffness, strength, corrosion resistance, etc.). The evaluation of biocompatibility
of a composite material is a complex issue since two or more distinctive materials (of the composite) simultaneously exist at the
device-tissue interface and usually one or more of them are bioactive and biodegradable, resulting in dynamic device-tissue inter-
facial state. Many factors need to be considered for assessing the biocompatibility of a composite material. Various in vitro and
in vivo tests are required for systemically evaluating the biocompatibility of a composite material for the targeted application(s),
which may include assessments of cytocompatibility, acute toxicity, subchronic toxicity, genotoxicity, hemocompatibility, chronic
toxicity, carcinogenicity, reproductive, and developmental toxicity and immune responses. The surface features of a biomedical
composite should be carefully designed for meeting particular biocompatibility requirements.
The bioactivity of a composite is also an important factor for some clinical applications. It is generally endowed or increased by
the use of bioactive materials as a constituting material in the composite. Popular choices are bioactive bioceramics such as HA and
BioglassÒ which enable new bone formation at the implant-tissue interface and hence strong bonding between the composite
implant and bone. The inclusion of bioactive molecules in composites is an alternative way to functionalize biomedical composites
with particular bioactivity. However, since most bioactive molecules are proteins and peptides which are very susceptible to damage
by the “harsh conditions” during composite manufacture, to preserve the bioactivity of bioactive molecules at a desirable level is
highly challenging for producing biomolecule-incorporated composites.
Medical Applications of Composite Materials

Biomedical Composites in Orthopedics
Biomedical composites have been investigated for different orthopedic applications including bone fracture repair, total joint
replacement (for hip, knee, etc.) and repair of connective tissues such as tendon and ligament. Two different treatments are
clinically used for bone fracture, namely, internal fixation, and external fixation. External fixation does not require opening the
fracture site but employs holding devices such as casts, splints, braces or similar fixation systems to align the fractured bone.
Plaster of calcium sulfate is a holding material which can be reinforced by woven cotton fibers, CF or glass fibers for mechanical
enhancement. Holding devices for external fixation made of CF/epoxy composites have attracted attention due to their light-
weight and comparable mechanical properties to metallic devices, together with the ease to monitor the bone healing process
through medical imaging. In contrast, internal fixation involves the implantation of fixation devices such as plates, screws, pins,
and wires to hold the bone fragments in place. While metallic devices are still commonly used for internal fixation, developing
composites with matching properties to bone provides solutions to problems such as stress shielding. The composites are
divided into three categories: biostable composites, partially biodegradable composites, and fully degradable composites.
CF/epoxy and glass fiber/epoxy were early nonbiodegradable composites investigated for internal fixation but there were
concerns on potential toxicity of residual monomers. Bioinert CF/PEEK, which has comparable mechanical properties to
metallic devices, was also studied for internal fixation use. However, biodegradable composites have now gained attention
with the advantage of avoiding a second surgery to take out the device after bone healing. The matrix materials for biodegrad-
able internal fixation systems are usually FDA-approved biodegradable polymers (PLA, PLGA, PCL, etc.). These polymers have
insufficient strength and hence mechanical reinforcement by CF or resorbable bioceramic particles is usually used, resulting in
partially biodegradable composites or fully biodegradable composites. Composites for joint replacement must meet highly
demanding requirements, including high strength and toughness, excellent resistance to fatigue and wear, and in some situa-
tions, suitable surface properties for the lubricated surface in joints. Composites such as CF/PSU and CF/PEEK were investi-
gated for the stem of hip prosthesis. CF/UHMWPE composite was investigated for the acetabulum cup but caused concern
on the production of CF debris during wear of the cup. Composites have also been investigated for the substitution tendon
and ligament. Although some successes have been achieved by the use of tendon/ligament prostheses made of either nonde-
gradable fibrous composites (PET/PHEMA, aramid fiber/PE, CF/PTFE, etc.) or partially degradable composites (CF/PLA, CF/PU,
etc.) which have comparable mechanical properties (elastic properties) to those of native tendon and ligament, there are still
great challenges for these prostheses such as bonding to bone, which requires further multidisciplinary efforts in biology, chem-
istry, and materials sciences.
Since the concept of bone analogue biomaterial was introduced, a variety of bioactive ceramic-polymer composites have been
investigated for bone substitution. HA/HDPE was the first successful composite material for bone tissue repair in the clinic. Owing
to its good bioactivity and compatible mechanical properties, it has been used for orbital floor implants and middle ear implants.
Subsequently, BioglassÒ/HDPE composite and A-W glass-ceramic/HDPE composite were also developed for human body tissue
repair. Owing to the high bioactivity of BioglassÒ which provides the possibility for bonding to soft tissue, BioglassÒ/HDPE
composite is potentially useful for soft tissue repair. High strength polymers such as PSU and PEEK can provide stronger matrix
for the composite and hence they are used to develop HA/PSU and HA/PEEK composites for bone tissue repair. For high load-
bearing applications, composites of metal matrix instead of polymer matrix are investigated. A typical example of such composites
is HA/Ti-6Al-4V composite. Studies are also conducted on ceramic matrix composites such as HA/zirconia composite. Developing
biodegradable composites for tissue repair is a trend in the biomaterials field. Synthetic biodegradable polymers such as PLA, PLGA,
and PCL and natural biodegradable polymers such as collagen, chitosan and PHB are often used as matrix materials and bioactive
bioceramics such as HA and TCP are the dispersed phases. They form various bioactive and biodegradable composites with different
mechanical and biological properties for targeted applications.
Biomedical Composites in Dentistry

Almost every person encounters dental problems during his/her life. Different biomaterials are employed for different dental treat-
ments, ranging from cavity filling to tooth replacement. Dental restorative materials are used for filling tooth cavities or treating
dental caries, and traditional materials are Amalgam, gold, alumina, zirconia, acrylic resins, silicate cements, etc. Ideal restorative
materials need to have sufficiently low viscosity for completely filling the cavity, similar thermal expansion coefficient to the
dentine, good biocompatibility and resistance to fatigue, wear, creep, and water absorption. Since traditional restorative materials
have respective shortcomings in biocompatibility and mechanical properties, composites as dental restorative materials such as
barium glass/colloid silica/resin composite have been made and studied. In situations of severely damaged tooth with inadequate
structure to retain the filling or restorative materials, a dental post is inserted in the root canal for reinforcing the remaining dentine.
Metallic materials such as Co–Cr and Ti alloys are traditionally used for forming dental posts. But posts made by epoxy matrix rein-
forced by CF or glass fibers have been also studied. In addition, finite element analysis has indicated that ideal posts should possess
varied stiffness along its length (with relatively low stiffness at the apical end and high stiffness at the coronal end), which are
impossible to make using homogenous materials whereas composite posts with specific design can readily meet this requirement.
Dental implants, that is, artificial tooth roots permanently replacing the missing teeth, are often required to replace heavily damaged
teeth, which are implanted in the jawbone. Frequently used materials for dental implants include traditional metallic alloy (Co–Cr–
Mo alloys, stainless steel, Ti alloys, etc.) and ceramic materials (zirconia, alumina, etc.), and new composites (CF/carbon, SiC/
carbon, CF/PMMA, etc.) are investigated for the implants. Among these materials, composites exhibit superiority in bioinertness
(for low allergic risk), mechanical properties (high strength but low modulus, enhanced fatigue resistance, etc.) and cost-
effectiveness. In addition, composite dental implants coated by a bioactive ceramic layer (e.g., a bone-like apatite layer formed
by biomimetic coating) are able to promote bone cell growth, thereby enhancing implant-bone integration. Despite of the
achievements of biomedical composites in dentistry, there are still great challenges, leaving a large scope for further pursuing studies
on innovative materials and techniques.
Biomedical Composites for Tissue Engineering

Tissue engineering, as a new and developing multidisciplinary field, has been attracting great attention in the society. Tissue engi-
neering has significant advantages and has shown great potential over conventional tissue substitution/prosthesis implantation and
organ/tissue transplantation treatments. The design and application of appropriate tissue engineering scaffolds are critical in
scaffold-based tissue engineering. Unlike implants for tissue repair, biodegradable tissue engineering scaffolds are used as porous
structural frameworks to enable cell attachment, proliferation and differentiation, leading to new tissue formation. Advanced tissue
engineering scaffold can provide specific structural, mechanical, and biochemical features for appropriately directing cell behaviors
and cell functions. Most of tissue engineering scaffolds are made simply from biodegradable polymers such as PLA, PLGA, and PCL
but they have many limitations. Consequently, the composite approach is employed as an important strategy for developing high-
performance scaffolds. Numerous composite tissue engineering scaffolds have therefore been investigated for specific tissue regen-
eration applications.
The composite approach is an effective ways to modify conventional polymer tissue engineering scaffolds with additional func-
tion(s). As infection is a common problem in tissue repair/regeneration, different strategies for achieving the antibacterial effect of
scaffolds are investigated. A straightforward way is to incorporate antibiotics in the polymer struts of tissue engineering scaffolds.
However, the loaded drug tends to be rapidly released. In comparison, composite scaffolds fabricated by incorporating AgNPs
exhibit superior antiinfection performance. The AgNPs encapsulated in the structs of a porous scaffold provide long-term and sus-
tained release of antibacterial silver ions, resulting in nanocomposite tissue engineering scaffolds with additional antibacterial func-
tion. Carbon materials (e.g., CNTs and graphene) have been used as reinforcements in composite tissue engineering scaffolds,
which are effective for improving mechanical performance. Also, the scaffolds with graphene as the dispersed phase are able to
provide significantly enhanced electrical conductivity, which are potentially useful for neural tissue regeneration.
The composite approach is effective and important for making bioactive tissue engineering scaffolds, particularly osteoconduc-
tive scaffolds with the incorporation of bioactive bioceramics for bone tissue engineering. One strategy is to form bioactive
composite coating on polymer scaffolds through accelerated biomimetic deposition. In this route, biodegradable polymer scaffolds
with interconnected pores are firstly fabricated. They are then immersed in a collagen-containing high-strength SBF for sufficient
time, resulting in the formation of an apatite/collagen layer, which has similar structure and composition to bone, on the struts
of polymer scaffolds. The resultant composite scaffolds exhibit well-preserved porous structures and enhanced bioactivity. Another
strategy is to incorporate bioactive bioceramic particles (micro- or nano-sized) in biodegradable polymers to form directly bioactive
composite scaffolds which also have enhanced structural integrity and mechanical properties. A variety of bioactive ceramic-filled
scaffolds have thus been developed. For example, resorbable Ca-P nanoparticles (10–30 nm in diameter) are synthesized and then
used to form Ca-P/PHBV nanocomposite microspheres, with a homogenous distribution in the microspheres. These Ca-P/PHBV
microspheres are used in a SLS process to form nanocomposite scaffolds with customized shapes and programmed porous struc-
tures (Fig. 7). The Ca-P/PHBV nanocomposite scaffolds fabricated via SLS (a 3D printing technique) are promising for bone tissue
engineering owing to their advantages in architecture, biodegradability, bioactivity, and mechanical properties. In the fabrication of
fibrous tissue engineering scaffolds via electrospinning, Ca-P or carbonated HA nanoparticles are incorporated in biodegradable
polymer fibers, resulting in fibrous osteoconductive nanocomposite scaffolds for bone tissue engineering.
For successful functional regeneration of bone, developing multifunctional nanocomposite scaffolds becomes important.
The scaffolds should have desired osteoconductivity together with appropriate osteoinductivity and vascularization ability.
Fig. 7 Ca–P/PHBV nanocomposite structure and scaffold fabricated by SLS: (A) macroscopic view of a porous structure in the shape of a femur,
(B) SEM image of the struts of a scaffold.
Fig. 8 Multifunctional bone tissue engineering scaffolds fabricated by cryogenic 3D printing: (A) SEM image of a scaffold and nanopores in a strut
of the scaffold, (B) release profiles for Ca2þ ions from different scaffolds, (C) release profiles for BMP-2 from different scaffolds.
Consequently, osteoinductive growth factors (e.g., BMPs), and angiogenic growth factors (e.g., VEGF) are incorporated in bioactive
nanocomposite scaffolds with good retention of their bioactivity even though there are major obstacles in their incorporation as
delicate biomolecules cannot endure the harsh conditions in the manufacture of porous composites. Effective ways have to be
found to solve the problems. For example, SLS is an effective 3D printing technique for fabricating bioactive nanocomposite scaf-
folds but the high temperature during SLS causes denaturing of bioactive molecules to be incorporated. A strategy is developed
where the Ca-P/PHBV nanocomposite scaffolds fabricated via SLS are coated by a gelatin layer and then grafted by heparin mole-
cules, which enables effective binding and subsequent sustained release of an osteoinductive factor (BMP-2). In the case of fibrous
scaffolds, through dual-source dual-power electrospinning with concurrent emulsion electrospinning and blend electrospinning,
BMP- and VEGF-containing polymer fibers and Ca-P nanoparticle-encapsulated polymer fibers are simultaneously deposited on
a rotating fiber collecting drum, resulting in multifunctional tricomponent fibrous scaffolds offering osteoconductivity and osteoin-
ductivity together with vascularization potential for the regeneration of vascularized bone. Also for bone tissue engineering, a novel
cryogenic 3D printing technique is developed to fabricate multifunctional nanocomposite scaffolds from a multicomponent emul-
sion with co-incorporated BMP-2 and Ca-P nanoparticles. The 3D printed scaffolds exhibit desirable hierarchically porous architec-
ture (with tuneable interconnected micropores as well as surface nanopores on the struts) and provide dual delivery of BMP-2 and
calcium ions (Fig. 8), facilitating MSC osteogenic differentiation.
The composite approach has achieved remarkable success in tissue engineering, particularly for bone tissue regeneration. Never-
theless, successful tissue engineering strategies do not solely depend on scaffolds. Cells and other factors (biochemical, topograph-
ical, mechanical, etc.) are equally important. Since current composite manufacturing techniques still face challenges in the
simultaneous incorporation of cells and bioactive factors, innovative work is needed for building living and tissue-like constructs
with biomimetic structural, biochemical, and mechanical features.
Biomedical Composites for Drug Delivery and Controlled Release Applications

On-demand drug release is an important target for drug delivery. But it is a great challenge because of the lack of suitable materials
for making smart drug delivery systems. Composites can have tuneable physical and chemical properties with the variation of
constituting materials, which makes them promising candidates for forming stimuli-responsive drug delivery vehicles which are
capable of delivering drugs in specific spatiotemporal- and dosage-controlled fashion. Therefore, different smart drug delivery
systems have been prepared using the composite approach, which are able to release drug in response to different stimuli (temper-
ature, light, magnetic, electrical, ultrasound, pH, enzyme, etc.).
A typical group of smart composite drug delivery systems is hydrogel nanocomposites, which are formed by encapsulating func-
tional nanoparticles, including metallic nanoparticles and ceramic nanoparticles, in a hydrogel matrix. Hydrogels with networked
polymer and high water content are suitable for loading hydrophilic drug, as well as for providing an appropriate “shield” for the
encapsulated nanoparticles which improves the biocompatibility of the delivery system. The swelling of hydrogel nanocomposites
can be controlled by the entrapped functional nanoparticles, resulting in controlled release of loaded drug. For example, the drug
release from a hydrogel nanocomposite consisting of a thermal sensitive polymer, PNIPAAm, and encapsulated AuNRs can be
controlled by an NIR laser irradiation. The drug is released as a result of the significant shrinkage of PNIPAAm hydrogel once
the system temperature is increased above the lower critical solution temperature of the PNIPAAm hydrogel which is caused by
the photothermal effect of AuNRs under the NIR laser irradiation. Similarly, a hydrogel nanocomposite drug delivery system is
formed by using PNIPAAm hydrogel and magnetic nanoparticles. Pulsatile drug release is achieved under an alternating magnetic
field that generates heat, which promotes drug release, in the hydrogel nanocomposite through encapsulated magnetic nanopar-
ticles. Using the composite approach, other smart drug delivery systems are also developed by modifying conventional drug delivery
systems such as liposome, polymer particles and mesoporous silica. For example, liposome-based nanocomposite drug release
systems achieve controlled drug release when the liposome collapse is be triggered by either the heat generated by the encapsulated
AuNRs under the NIR laser irradiation or by the bubbles generated from the entrapped ammonium bicarbonate at a certain temper-
ature. In a graphene oxide/polymer nanocomposite drug delivery system, the release of loaded antiinflammatory drug is through
a voltage stimulation, exhibiting an interesting electrical-responsive release profile. Mesoporous silica nanoparticles with very high
surface-area-to-volume ratios are excellent for loading drugs. Through capping the mesoporous silica nanoparticles with different
“gatekeeper” species (e.g., responsive molecules and functional nanoparticles), smart drug delivery systems actuated by different
mechanisms (temperature, pH, magnetic field, etc.) have been developed, resulting in different desirable controlled drug release
profiles. Furthermore, through appropriate use of different functional species (PNIPAAm, AuNR, SPION, etc.) and specific design,
dual- or multistimuli responsive drug delivery systems capable of responding to different external stimuli can be made, significantly
broadening the development of smart drug delivery systems.
Biomedical Composites for Medical Imaging

Disease diagnosis now highly relies on modern medical imaging techniques, with CT and MRI being commonly used. Contrast
agents are important for the effectiveness of these techniques and must meet multiple requirements for biomedical applications,
for example, biocompatibility, stability, contrast intensity, and cellular uptake. The composite approach has been used to prepare
advanced contrast agents with enhanced performance. For example, SPION is an effective contrast agent for MRI enhancement at
tissue, cellular and even molecular level. However, SPION still faces the issues in contrast intensity and biocompatibility as it is
easily captured by the reticuloendothelial system. Using the composite approach, problems of SPION are effectively addressed.
With metal doping (e.g., Mn doping), T2 relaxation properties and contrast intensity of resultant SPION-based nanocomposite
are enhanced; and the nanocomposite combining Mn-doped SPION and a polymer micelle provides good magnetic response,
reduces the toxic side-effects and prolongs the circulation time.
Different imaging techniques have respective advantages and shortcomings. Optical imaging shows ultrasensitive and real-time
imaging capability but has limited depth in imaging owing to the poor tissue penetration of light. Other techniques such as MRI
have superiority in 3D imaging with good spatial resolution but show limitations in sensitivity and intensity. Multimodal imaging
approaches combing different techniques are therefore attractive as advanced diagnostic tools. By using specifically designed nano-
composites, advanced contrast agents for multimodal imaging are developed. For example, the nanocomposite with a SPION core,
capped AuNP coronas and a peptide coating can achieve good cellular uptake and is suitable for dual-modal imaging of MRI and CT
owing to the SPION core and AuNP coronas, respectively. And a nanocomposite for triple-modal imaging is also established, in
which upconversion nanoparticles, SPIONs and dye molecules are encapsulated by an amphiphilic block copolymer. This nano-
composite can be used in triple-modal imaging of upconversion luminescence, downconversion fluorescence and MRI, as well
as drug delivery capability, exhibiting excellent multiple functionality.
Biomedical Composites for Cancer Theranostics

Current cancer diagnostic techniques and treatments have significant limitations. With increasing knowledge and technological
advancement in cancer biology, material science, and particularly, nanomedicine, novel nano-platforms providing simultaneous
diagnostic and therapeutic functions for cancer, termed theranostic agents or simply theranostics, have emerged since a decade
ago. These novel nanodevices are able to target multiple cancer markers and kill cancer cells via synergetic ways, addressing the chal-
lenges of cancer heterogeneity and adaptive resistance. And also the cancer therapies by theranostics can be guided by diagnostic
imaging results, thereby leading to enhanced cancer treatment. Ideal theranostics should have appropriate small size and surface
chemistry for optimal cellular uptake, ultrasensitive imaging of multiple tumor-specific markers, desirable cancer targeting ability,
and in particular designs, high loading and controlled release of anticancer drugs. Apart from chemotherapy, other cancer therapies
are also required in most designs. These multifunctional requirements for theranostics depend on the advances of material science
and engineering, particularly the development of nanocomposites.
There are different types of nanocomposite theranostics, which are able to perform cancer diagnosis on the basis of photo-
luminescence, SERS, MRI, ultrasonography, photoacoustics or CT imaging (monomodal or multimodal imaging) and
simultaneously to provide cancer therapy by means of drug and/or gene delivery, photothermal therapy, photodynamic therapy,
magnetic hyperthermia, or combined therapy. The combinations of cancer diagnosis and therapy for theranostics are many and
hence it is impossible to comprehensively review them here. Only a few typical theranostics are introduced. Nanocomposite
theranostics are normally prepared on basis of specific nano-platforms with unique physical properties and functions. These plat-
forms are categorized as Au-based platforms, carbon-based platforms, SPION-based platforms, mesoporous silica-based plat-
forms, QD-based platforms, upconversion nanoparticle-based platforms, etc., with some of them overlapping with each
other. Since these nano-platforms face common challenges in biocompatibility, circulation, cancer cell targeting and cellular
uptake, surface functionalization through specific polymer coating or molecular grafting has been employed for synthesized
nanocomposite agents, which has little or limited influence on the unique physical properties of the particle core. For example,
PEG grafting is a popular method to improve the biocompatibility and circulation time in the human body of nanocomposite
theranostics with cores of AuNP, SPION, etc. The polymer coating layer of a nanocomposite theranostics can also act as a carrier
for anticancer drugs, providing controlled drug release in response to the stimuli from the functional core (e.g., heat generated by
AuNRs/CNTs under an NIR laser irradiation or heat generated by SPIONs in an alternating magnetic field). For example, multi-
functional core-shell structured nanoparticles with an AuNR core, a mesoporous silica shell and a PLGA outer layer are developed
as new theranostics. It is a robust nanocomposite theranostics enabling efficient cancer cellular uptake and light-mediated (from
LSPR of AuNRs) photothermal therapy combined with chemotherapy (from triggered release of doxorubicin loaded in meso-
pores in silica).
Concluding Remarks
The composite approach in developing new biomaterials as well as modifying existing biomaterials becomes more and more
important in the biomaterials field. Biomedical composites can provide unique properties, which cannot be obtained from metallic,
polymeric or ceramics biomaterials alone, to meet specific requirements for biomedical applications. A composite is a complex
system in the context of medicine. The successful development of a biomedical composite for a targeted clinical application rests
on the firm grasp of the fundamentals of composite theory, composite manufacturing technologies and knowledge in biology and
medicine. On such a solid foundation, new and novel biomedical composites can be designed and developed for the benefit of
patients and the society. There are already many biomedical composites for various biomedical applications. While there is still
a large scope for developing new composites for areas such as orthopedics and dentistry, new and exciting developments for
biomedical composites may come from areas of high demands and great challenges such as neural tissue repair, regenerative medi-
cine, and nanomedicine.
Acknowledgments
Min Wang thanks The University of Hong Kong, Hong Kong Research Grants Council and the National Natural Science Foundation of China and
Qilong Zhao thanks China Postdoctoral Science Foundation, Guangdong Innovative and Entrepreneurial Research Team Program and the Funda-
mental Research Program of Shenzhen for funding their research in the biomaterials and tissue engineering field.
Further Reading
Ambrosio, L. (2009). Biomedical composites (1st edn.). Cambridge: Woodhead Publishing.

Armentano, I., Dottori, M., Fortunati, E., Mattioli, S., & Kenny, J. M. (2010). Biodegradable polymer matrix nanocomposites for tissue engineering: A review. Polymer Degradation
and Stability, 95(11), 2126–2146.
Atala, A., & Lanza, R. P. (2002). Methods of tissue engineering (1st edn.). San Diego: Academic Press.
Boccaccini, A. R., & Ma, P. X. (2013). Tissue engineering using ceramics and polymers (2nd edn.). Cambridge: Woodhead Publishing.
Bonfield, W. (1988). Composites for bone-replacement. Journal of Biomedical Engineering, 10(6), 522–526.
Bonfield, W., Wang, M., & Tanner, K. E. (1998). Interfaces in analogue biomaterials. Acta Materialia, 46(7), 2509–2518.
Di Silvio, L. (2008). Cellular response to biomaterials (1st edn.). Cambridge: Woodhead Publishing.
Duan, B., & Wang, M. (2013). Selective laser sintering and its biomedical applications. In V. Schmidt, & M. R. Belegratis (Eds.), Laser technology in biomimetics: Basics and
applications (pp. 83–109). Heidelberg: Springer.
Francis, R., & Kumar, D. S. (2016). Biomedical applications of polymeric materials and composites (1st edn.). Chichester: John Wiley & Sons.
Hench, L. L. (1991). BioceramicsdFrom concept to clinic. Journal of the American Ceramic Society, 74(7), 1487–1510.
Hench, L. L. (Ed.). (2013). An introduction to bioceramics (2nd edn.,). Singapore: World Scientific.
Herakovich, C. T. (1998). Mechanics of fibrous composites (1st edn.). Chichester: John Wiley & Sons.
Kelly, A. (1994). Concise encyclopedia of composite materials (1st edn.). Oxford: Pergamon.
Lee, J. E., Lee, N., Kim, T., Kim, J., & Hyeon, T. (2011). Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications. Accounts of Chemical
Research, 44(10), 893–902.
Merino, S., Martin, C., Kostarelos, K., Prato, M., & Vazquez, E. (2015). Nanocomposite hydrogels: 3D polymer-nanoparticle synergies for on-demand drug delivery. ACS Nano, 9(5),
4686–4697.
Rezwan, K., Chen, Q. Z., Blaker, J. J., & Boccaccini, A. R. (2006). Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.
Biomaterials, 27(18), 3413–3431.
Scholz, M. S., Blanchfield, J. P., Bloom, L. D., Coburn, B. H., Elkington, M., Fuller, J. D., Gilbert, M. E., Muflahi, S. A., Pernice, M. F., Rae, S. I., Trevarthen, J. A., White, S. C.,
Weaver, P. M., & Bond, I. P. (2011). The use of composite materials in modern orthopaedic medicine and prosthetic devices: A review. Composites Science and Technology,
71(16), 1791–1803.
Sumer, B., & Gao, J. M. (2008). Theranostic nanomedicine for cancer. Nanomedicine, 3(2), 137–140.
Wang, M. (2003). Developing bioactive composite materials for tissue replacement. Biomaterials, 24(13), 2133–2151.
Wang, M. (2004). Bioactive materials and processing. In D. L. Shi (Ed.), Biomaterials and tissue engineering (pp. 1–82). Heidelberg: Springer.
Zhao, Q. L., & Wang, M. (2017). Smart multifunctional tissue engineering scaffolds. In Q. Wang (Ed.), Smart materials for tissue engineering: Applications (pp. 558–595).
Cambridge: Royal Society of Chemistry.
Bulk Properties of Biomaterials and Testing Techniques
Chong Wang, Dongguan University of Technology, Dongguan, China
Introduction 54
Basic Mechanical Properties and Testing Methods 54
Tensile Testing and Material Properties 54
Compression Testing and Material Properties 55
Shear Testing and Material Properties 56
Torsional Testing and Material Properties 57
Flexural Testing and Material Properties 57
Impact Testing and Material Properties 58
Dynamic Mechanical Analysis and Material Properties 58
Long-Term Mechanical Properties and Testing Methods 59
Stress Relaxation 59
Creep 59
Fatigue 59
Friction and Wear 60
Testing at the Micro- or Nano-scale 60
Microhardness Testing 60
Nanoindentation 60
Tests for Single Molecules 62
Bonding Strength and Testing Methods 62
Tensile Bonding Tests 62
Shear Bonding Tests 62
Peel Tests 63
Summary 63
Acknowledgements 63
References 63
Further Reading 64
Glossary
Biomaterial A biomaterial is a nonviable material used in a medical device, intended to interact with biological systems. It can
be a metal, polymer, ceramic or composite and must be biocompatible.
In vitro In vitro is Latin, meaning “in the glass.” In vitro experiments are performed with biological species (biological
molecules, cells, microorganisms, etc.) outside their normal biological environment. They are generally conducted in labware
such as test tubes, flasks and Petri dishes.
In vivo In vivo is Latin, meaning “within the living.” In vivo studies are those in which the effects are investigated inside the
whole, living organisms including animals and humans.
Nomenclature
AAMI Association for the Advancement of Medical Instrumentation
ASTM American Society for Testing and Materials
CEN European Committee for Standardization
DMA Dynamic mechanical analysis
ISO International Organization for Standardization

54 Biomaterials: Science and Engineering j Bulk Properties of Biomaterials and Testing Techniques
Introduction
Different human body tissues have significantly different mechanical properties in accordance with their roles in the human body
system. For example, load-bearing bones in the lower limb support the body weight and are subjected to compressive, bending, and
torsional forces when one stands, walks, and runs, while tendon and ligaments frequently bear tensile forces. Due to traumatic
injuries, diseases and tumor resections along with an aging population in the society as well as a young generation with more
outdoor activities and higher rates of accidents, more people suffer from tissue loss each year. The damaged tissues may be replaced,
repaired, or regenerated by implanting long-term prostheses, biomaterials, or cell-scaffold constructs. Importantly, the implanted
biomaterials should mechanically match the target tissue in the human body. Therefore, in developing new biomaterials, the
mechanical property requirement for the targeted application and how to achieve the mechanical properties for the new biomate-
rials should be considered. To this end, understanding how the mechanical properties of biomaterials are determined is very impor-
tant. Different mechanical properties of bulk biomaterials can be obtained by using different types of mechanical tests. This article
gives an overview on a variety of mechanical testing techniques for biomaterials. Standard testing methods should be used in con-
ducting mechanical tests. These standards are issued by national organizations such as American Society for Testing and Materials
(ASTM) and Association for the Advancement of Medical Instrumentation (AAMI) in the USA and by international organizations
such as International Organization for Standardization (ISO) and European Committee for Standardization (CEN).
Basic Mechanical Properties and Testing Methods

Tensile Testing and Material Properties
Biomaterials which are designed to repair body tissues frequently under tensile loads should have sufficient tensile strength and
stiffness. For instance, human body tissues including, but not limited to, skin, muscle, tendon, and long bone have relatively
high tensile strength as tensile loads are often applied to these tissues. Hence biomaterials including biocompatible metals, poly-
mers, ceramics, and composites for the replacement of these tissues should have sufficient tensile strength and fracture strain (or,
elongation-at-break) to avoid fracture and have matching Young’ modulus with the target tissue. These mechanical properties can
be measured using tensile testing. Tensile testing is a basic mechanical testing technique in material science and engineering in
which samples of the material are subjected to uniaxial tension until failure (Fig. 1A). Mechanical tests should be conducted accord-
ing to a test standard and in the case of tensile testing, a standard such as ASTM D638 should be followed. A test standard specifies
sample size and geometry, sample preparation, test speed, measurement requirements, etc. Normally, an electromechanical testing
machine is used for conducting tensile tests. The standard tensile sample has two shoulders and a gauge in between. The shoulders
are larger and hence can be readily gripped by the testing machine, whereas the gauge section has a smaller cross-section so that the
deformation and failure occur in this area. During tensile testing, the sample is gripped at the two ends and then a tensile force is
applied until the sample fractures. Properties such as ultimate tensile strength, elongation-at-break and area reduction can be
directly measured and other properties such as Young’s modulus, yield strength and Poisson’s ratio can be subsequently deter-
mined. The stress–strain curve of the tested sample is automatically plotted by the testing machine. The stress (s) and strain (ε)
are calculated as follows:
Fn
s¼ (1)
A
DL L L0
ε¼ ¼ (2)
L0 L0
where Fn is the applied tensile force, A is the original cross-sectional area of the gauge section, DL is the change in gauge length, L0
is the original gauge length, and L is the final length. Fig. 1A shows schematically the setup for tensile testing using an elec-
tromechanical testing machine. Fig. 2 exhibits a schematic tensile stress–strain curve of a metallic biomaterial with associated
mechanical properties. Biomedical polymers are normally ductile materials with low strength and stiffness. Bioceramics are
generally stiff and brittle materials. Metallic biomaterials possess sufficient stiffness, high strength, and good ductility. For
comparison, Fig. 3 provides schematically typical tensile stress–strain curves for biomedical polymers, bioceramics, and metallic
biomaterials. Panigrahi et al. investigated tensile properties of biocompatible Ti-45Nb alloy using a microtensile testing machine
equipped with a laser speckle-based strain sensor (Panigrahi et al., 2016). After treatments such as hydrostatic extrusion and high
pressure torsion, this metallic biomaterial has an elastic modulus of 66 3 to 69 2 GPa, while its ultimate tensile strength
increases from 446 14 MPa (the starting material) to 668 10 MPa and 986 7 MPa for the material having undergone
hydrostatic extrusion and high pressure torsion, respectively. Zhao et al. studied the tensile properties of the Ti-(15–18)Mo
metallic biomaterial which were subjected to solution treatment and cold rolling (Zhao et al., 2012). As the Mo content increases,
the tensile strength increases. The tensile strengths of all alloys subjected to solution treatment are larger than 670 MPa. After cold
rolling, the tensile strengths of all alloys increase remarkably but the elongation-at-break decreases. The increase in the tensile
strength has been attributed to a combined effect of the deformation-induced u phase transformation and work-hardening
during cold rolling.
Biomaterials: Science and Engineering j Bulk Properties of Biomaterials and Testing Techniques 55
(A) (B)
(C)
(D)
Dial for energy absorption

(E) (F)
Original height
Sample on the anvil

Fig. 1 Testing methods for measuring different mechanical properties of bulk biomaterials: (A) tensile testing, (B) compression testing, (C) shear
testing, (D) torsional testing, (E) three-point bending testing, and (F) impact testing.
Compression Testing and Material Properties

Biomaterials which are designed to repair body tissues under compression should have sufficient compressive strength. Similarly,
the compressive modulus and compressive fracture strain of these biomaterials should also be also measured. These mechanical
properties can be obtained using compression tests according to a test standard such as ASTM D695. As stated in the ASTM Hand-
book, compression testing is a useful technique to measure the plastic flow behavior and ductile fracture limits of a material. It is
also useful for measuring the compressive fracture properties of brittle materials or low-ductility materials. Compression testing is
used more often for brittle materials. For compression testing, an electromechanical testing machine is also used to determine the
material properties upon the application of a compressive load (Fig. 1B) and compressive stress–strain curves are recorded. Prop-
erties such as compressive modulus and compressive strength can be measured or determined. Puertolas et al. investigated the
Slope: Young’s modulus, E

σ Onset of necking
σ UTS
Fracture
σ FS point
Tensile strength
σ UTS
Fracture strength
Yield strength
σ FS
σy
0.2% strain ε
Plastic strain
at fracture, ε f
Fig. 2 A schematic tensile stress–strain curve of a metallic biomaterial with associated mechanical properties.
Bioceramic
Metallic biomaterial
Stress
Biomedical polymer
Strain
Fig. 3 Schematic tensile stress–strain curves for biomedical polymers, bioceramics and metallic biomaterials.
compression behaviour of biphasic calcium phosphate (BCP) and biphasic calcium phosphate-agarose scaffolds for bone regener-
ation (Puertolas et al., 2011). Both dense BCP (D-BCP) samples and designed porous architecture BCP (DA-BCP) samples are made
and further infiltrated with agarose (AG) to form D-BCP/AG and DA-BCP/AG scaffolds. Compression curves for D-BCP/AG samples
exhibit a similar qualitative shape, but with higher values of compressive modulus and compressive strength than the DA-BCP/AG
system. The compressive strengths of these scaffolds are in the range 10–50 MPa, which are between the compressive strength of
cortical bone (100–230 MPa) and that of cancellous bone (2–12 MPa). Martínez-Vázquez et al. fabricated sintered 3D b-tricalcium
phosphate (b-TCP) scaffolds which were further infiltrated with poly(lactic acid) (PLA) or poly(ε-caprolactone) (PCL) melts, form-
ing dense ceramic/polymer composites (Martínez-Vázquez et al., 2010). During compression testing, successive load drops are
observed in the uninfiltrated TCP scaffolds. These fracture events are also detectable in TCP/PCL and TCP/PLA composites, but
the compression curves are much smoother and the maximum stress values are substantially higher (20 MPa vs. 55 MPa vs.
110 MPa), which is attributed to strengthening upon infiltration of polymers.
Shear Testing and Material Properties

Total joint replacement has been the treatment for patients suffering from osteoarthritis. Similar to other endoprosthetic devices,
one of the key issues to ensure the successful clinical outcome is the creation and maintenance of a stable bond between the device
and host tissue (i.e., bone). The harsh biological conditions and repeated high mechanical load cause bond degradation and lead to
reduced implant stability which ultimately results in clinical complications such as periprosthetic fracture, aseptic loosening, pain,
and loss of bone, making subsequent revision surgery very challenging. Therefore, developing biomaterials with sufficient shear
strength is of great importance. In a shear testing, the forces are applied parallel to the upper and lower faces of the object
(Fig. 1C). Materials behave differently in shear than in tension or compression, exhibiting different values for shear strength and
shear modulus. Lap shear testing (ASTM D3163) can be performed to determine the shear strength of an adhesive that is applied to
two metal plates which are pulled in opposite directions until failure of the adhesive. Ayed and Ayadi made sintered b-TCP-TiO2
composite and investigated the variation of both Young’s modulus and shear modulus of b-TCP with different amounts of TiO2
(2.5–50 wt%) sintered at various temperatures (1000–1300 C) (Ayadi and Ayed, 2015). The moduli of b-TCP-TiO2 composite
generally increase with increases in sintering temperature and amount of TiO2. Young’s modulus and shear modulus reach the high-
est values at 1200 C with 40 wt% TiO2, being 33.1 and 15.7 GPa, respectively. Above 40 wt% TiO2, mechanical performance of the
composites is adversely affected by the increase in sintering temperature.
Torsional Testing and Material Properties

It is recognized that the hollow cylindrical geometry of human long bone in upper limbs and lower limbs is designed to resist
bending and torsion. Studies also indicate that bending and torsion primarily constitute the loading regimes for human tibia
during walking and running. Therefore, bulk biomaterials aiming at replacing human long bone should have sufficient
torsional resistance. Compared to tension and compression which have received much attention, the occurrence of torsional
loading on bone is often neglected, probably owing to the technical difficulties of assessing torsion using conventional tech-
niques. As torsional loading can induce large isochoric deformation, torsional testing is sometimes considered a complemen-
tary test to large strain uniaxial tensile testing for biomaterials. Torsional testing is conducted to measure how much of a twist
a biomaterial can withstand before cracking or breaking (Fig. 1D). Mechanical properties of biomaterials which are subjected
to torsion in vivo should be characterized through torsional testing. Buijs et al. investigated differences in maximum torque
between seven biodegradable and two titanium screw osteofixation systems and also differences of maximum torque between
“hand tight” and break of the biodegradable and the titanium screw systems (Buijs et al., 2007). It is shown that the mean
maximum torque of the two titanium screw systems is significantly higher than that of the seven biodegradable screw systems.
The use of 2.0 mm BioSorb FX, 2.0 mm LactoSorb, or the larger 2.5 mm Inion screws are recommended as biodegradable
screws in osteofixation.
Flexural Testing and Material Properties

Human hard tissues such as long bone must withstand large bending forces when heavy objects are held. Therefore, bioma-
terials aiming at repairing/replacing body tissues which are occasionally or often subjected to bending forces should have
sufficient flexural strength and stiffness, which can be measured using either three-point bending tests or four-point bending
tests. Three-point bending testing (Fig. 1E) can provide values for modulus of elasticity in bending (Ef), flexural stress (sf),
flexural strain (εf), and the flexural stress–strain response of biomaterials. The parameters and properties are calculated as
follows:
L3 m
Ef ¼ (3)
4bd3
3PL
sf ¼ f or a rectangular cross section (4)
2bd2
PL
sf ¼ f or a circular cross section (5)
pR3
6Dd
εf ¼ (6)
L2
where Ef is flexural modulus of elasticity, sf is the maximum stress at midpoint; εf is the strain in the outer surface (mm/mm),
P is the applied load at a given point on the load-deflection curve (N), L is the support span (mm), b is the width of test beam
(mm), d is the depth of test beam (mm), D is the maximum deflection of the center of the test beam (mm), m is the gradient
(i.e., slope ¼ P/D) of the initial straight-line portion of the load-deflection curve (N/mm). For load-bearing biomedical
applications, He et al. investigated flexural and compressive properties of biocompatible porous titanium with entangled wire
structures made under different sintering conditions (He et al., 2013). The flexural behavior of titanium with entangled wire
structures strongly depends on the porosity. When the porosity is less than 60%, the flexural stress–strain curves exhibit
a distinct initial elastic stage. After yielding, plastic deformation occurs, resulting in “deformation strengthening.” When stress
reaches the maximum value (i.e., the flexural strength of the material), the material fails, leading to a sharp stress drop in the
stress–strain curve. When the porosity is greater than 60%, the initial elastic limit becomes indistinct, and “deformation
strengthening” and the stress drop become gradual in the stress–strain curves. The flexural modulus and flexural strength
increase dramatically as the porosity decreases. For these porous titanium with entangled wire structures, the thicker titanium
wire (0.15 mm in diameter) leads to lower flexural elastic modulus (1.5 GPa) and lower flexural strength (86 MPa); and the
thinner titanium wire (0.08 mm in diameter) results in higher flexural elastic modulus (2.4 GPa) and higher flexural strength
(159 MPa).
Impact Testing and Material Properties

The human skeleton provides major structural support for the body. Apart from performing various roles of posture, muscular
contraction control, internal organs protection, etc., the skeleton also withstands different types of forces applied to the body in
various physical activities, some of which are already shown above. Among the different types of forces, a suddenly applied load
has been more critical to the skeleton as well as bones, because through the suddenly applied load, that is, impact, considerably
more stress is developed within a short time in comparison to other loading conditions. Therefore, determining the impact prop-
erties of biomaterials intended for repairing or regenerating the skeleton is also very important. Impact testing is also common to
evaluate the toughness and notch sensitivity of materials used in other engineering fields (mechanical, civil, etc.). It can test metals,
polymers, ceramics, and composites. In a Charpy impact test, a notched specimen is broken by the impact of a heavy pendulum or
hammer, falling from a predetermined height and hence velocity (Fig. 1F). The test measures the energy absorbed by the fractured
specimen. Wei and Ding developed acid-resistant, calcium silicate-based composite implants with high strength as a load-bearing
bone graft substitute (Wei and Ding, 2016). The impact strength of implants as a function of gelatin content is investigated. They
find that the gelatin-containing composite has a significantly higher impact energy density (1.9–2.6 kJ/m2) than that of the material
without gelatin (0.8 kJ/m2). For the composite with different gelatin contents, the impact strength of composite with 10% of gelatin
shows a remarkably high value (2.6 kJ/m2).
Dynamic Mechanical Analysis and Material Properties

Human body tissues such as articular cartilage exhibit distinctive viscoelastic characteristics with substantial loading rate depen-
dency. Although adequate stiffness is necessary for the role of cartilage in weight support, other aspects of tissue dynamic mechan-
ical properties are also important for its function, and these properties are dependent on loading rate. Therefore, dynamic
mechanical properties of many biomaterials should be investigated for matching with the target tissues. The dynamic mechanical
properties can be measured through dynamic mechanical analysis (DMA). In a DMA test, a sinusoidal stress (tensile or bending) is
normally applied to the specimen and the strain of the specimen is measured to determine the complex modulus. The temperature
of the sample chamber or the frequency of the applied stress can be varied during a DMA test for investigating its effect on the
complex modulus. DMA can also be used to locate the glass transition temperature (Tg) of a polymer. For a totally elastic solid,
the resulting strain is in phase with the applied stress. For a purely viscous fluid, there will be a 90 degree phase lag of strain
with respect to stress. For a viscoelastic material under DMA test, the stress (s) and strain (ε) can be written as
s ¼ s0 sin ðtu þ dÞ (7)
ε ¼ ε0 sin ðtuÞ (8)

where u is the frequency of strain oscillation, t is time, d is phase lag between stress and strain. For the viscoelastic material being
tested, the storage modulus measures the stored energy, representing the elastic portion, while the loss modulus measures the
energy dissipated as heat, representing the viscous portion. The tensile storage modulus (E0 ), loss modulus (E00 ), and loss tangent
(tan d) are defined as follows
d0
E0 ¼ cos d (9)
ε0
d0
E00 ¼ sin d (10)
ε0
E00
tan d ¼ (11)
E0
The complex modulus (E) is
E ¼ E0 þ iE00 (12)
where i2 ¼ 1.
Temperature sweep is a commonly used technique in DMA analysis, which involves measuring the complex modulus at a low
constant frequency while varying the specimen temperature. Using a polymeric specimen, a prominent peak in tan d appears at the
glass transition temperature (Tg) of the polymer. For frequency sweep, a specimen is held at a fixed temperature and tested at varying
frequency. Peaks in tan d and in E00 with respect to the test frequency can be associated with glass transition, which corresponds to
the ability of polymer chains to move past each other. DMA is often used to determine storage modulus, loss modulus, tan d and Tg.
Barbieria et al. investigated the effect of Ca–P content on the dynamic mechanical properties of Ca–P/PDLLA composite (Barbieria
et al., 2013). DMA tests are conducted in the three-point bending mode over a frequency sweep covering the normal physiological
frequencies from 1 to 10 Hz, which simulates walking to fast running, and the analysis is performed at 37 C with both dry and wet
samples. As expected, the storage modulus in the dry condition significantly increases with the increase in Ca–P content. When the
Ca–P containing samples are moistened, their mechanical properties change abruptly. The loss tangent increases, with the largest
change occurring in the composite containing more apatite, and decreases with increasing frequency.
Long-Term Mechanical Properties and Testing Methods

Stress Relaxation
Stress relaxation depicts how viscoelastic materials, especially polymeric materials, relieve stress under constant strain. Viscoelastic
materials behave in a nonlinear, non-Hookean fashion. This nonlinearity is indicated by stress relaxation and also by another
phenomenon known as creep, which depicts how strain of a viscoelastic material changes under constant stress. As stress relaxation
often leads to the failure of implanted biomaterials, studying stress relaxation over time under simulated clinical cyclic loading
conditions can provide information which is useful not only for selecting a specific biomaterial appropriate for the clinical appli-
cation but also for deciding the time point at which the material should be changed. Mirani et al. fabricated sandwich-like
composite by incorporating a thin layer of PLGA into porous layers of gelatin-chitosan and etched the composite to endow compos-
ites with nanoscale features (Mirani et al., 2009). They then studied the effects of loading time, temperature and also relaxation time
on stress relaxation of the composite. Under similar conditions of temperature and relaxation time, the magnitude of the initial
tensile stress changes significantly with the changing in loading rate. However, when the relaxation time increases, a significant
change in the stress accumulation is observed and value of the maximum stress for each loading cycle decreases with increased relax-
ation time. The temperature of the environment also has a significant effect on the maximum stress reached in each cycle. When the
relaxation time is 60 s, as compared to unetched samples, the composite displays a much lower maximum tensile stress and also
shows better relaxation behavior with time.
Creep
Creep refers to the phenomenon that a solid deforms at an elevated temperature slowly and permanently under the influence of
a static mechanical stress. Unlike brittle fracture, creep deformation does not occur suddenly upon the application of a stress.
Instead, strain accumulates as a result of prolonged application of a stress which is below the yield strength of the material. In
the area of total hip arthroplasty (THA), creep refers to compressive flow of prosthetic components under body weight (several times
of body weight during normal walking), especially, to the femoral head penetration into ultra-high molecular weight polyethylene
(UHMWPE) acetabular cup in the early postoperative period (i.e., in the bedding-in period). It is an issue in clinical assessments
through plain radiographic or radiostereometric analysis that creep penetration of UHMWPE cup is dominated in the first half
to 2 years after THA, and subsequently wear becomes dominant. The main type of creep testing machine, which is also call constant
load creep testing machine, consists of a loading platform, loading fixture, dial gauge, furnace (or, heating chamber), and founda-
tion. The load platform is where the specimen is subjected to a constant stress. The loading fixture includes grips and pull rods. Grips
are used to hold the specimen in position. Dial gauge is used to measure the creep strain and the heating chamber is what surrounds
the specimen and maintain the temperature. Takahashi et al. investigated the effects of size and thickness on creep behavior for
conventional and vitamin E-diffused highly crosslinked polyethylene (designated as ArCom and E1, respectively) for total hip
arthroplasty (Takahashi et al., 2016). It is found that there are statistically significant differences in the creep strain rate between
ArCom and E1 samples. The creep strain rates are reduced by 15.9% on average in E1 samples as compared to ArCom samples
of the same diameter and thickness, indicating improved creep resistance of E1 material. It is also found that the creep strain
rate of the 32-mm diameter ArCom samples is 20.1% less than the 28-mm diameter ArCom samples. The creep strain rates exhibit
linear correlations with the component size and thickness. The E1 material has significantly greater resistance to creep than ArCom
material owing to its slightly enhanced crystallinity and the mechanical response of highly crosslinked structure to the applied
compressive force.
Fatigue
Fatigue is a form of failure that occurs in structures subjected to dynamic and fluctuating stresses. Under these circumstances, it is
possible for the failure to occur at a stress level considerably lower than the tensile or yield strength of the material. Fatigue is
a progressive and localized structural damage. There is always a population of defects of various sizes, geometries and orientations
in a solid and if the stress at the tip of sharp, suitably oriented defects is above a threshold, microscopic cracks begin to form. These
cracks can grow (and coalesce) under cyclic loading and eventually a large crack reaches a critical size, causing sudden fracture of the
structure. The shape of the structure significantly affects the fatigue life (i.e., the number of loading cycles that the structure sustains
before fracture) in which square holes or sharp corners lead to elevated local stresses where fatigue cracks can initiate and propagate,
whereas circular holes and smooth transitions or fillets do not decrease significantly the fatigue strength (i.e., the maximum applied
stress for a specified fatigue life) of the structure. Fatigue life is influenced by a number of factors, including cyclic stress state (mode
and parameters), residual stress, material type, grain size (for metals and ceramics) or average molecular weight (for polymers),
geometry, surface quality, size and distribution of internal defects, mode/direction of loading, temperature, environment, etc.
For some biomaterials, fatigue fracture and wear have been identified as major problems associated with implant loosening, stress
shielding, and ultimate implant failure. Using four-point bending fatigue testing, Akita et al. investigated the fatigue behavior of
bulk b-type titanium alloy Ti-15Mo-5Zr-3Al annealed in high temperature nitrogen gas (Akita et al., 2015). The load waveform
is sinusoidal and fatigue tests are conducted in air at ambient temperature. It is found that the fatigue strength of annealed spec-
imens is lower than that of untreated specimens. Large facets and brittle fracture morphology are observed on the fatigue fracture
surface of specimens annealed in nitrogen gas owing to the severe grain coarsening.
Friction and Wear

Friction is the force resisting the relative motion of solid surfaces, fluid layers, and material elements sliding against each other.
There are several types of friction:
(1) Dry friction: it resists relative lateral motion of two solid surfaces in contact. Dry friction is subdivided into (i) static friction
between nonmoving surfaces, and (ii) kinetic friction between moving surfaces.
(2) Fluid friction: it refers to the friction between layers of a viscous fluid that are moving relative to each other.
(3) Lubricated friction: it is a case of fluid friction where a lubricant separates two solid surfaces.
Coefficient of friction is the result of friction force divided by normal force and is an important parameter for two surfaces in contact.
It can be significantly decreased by different types of lubrication. Natural human joints (the synovial joints) have very low coeffi-
cients of friction, whereas the coefficients of friction of artificial joints are much larger.
Wear is associated with interactions between surfaces and refers to the deformation and removal of material on a surface as
a result of mechanical action of the opposite surface. Wear of materials (metals, polymers, ceramics, and composites) occurs by
the plastic displacement of surface and near-surface material and by the detachment of particles (i.e., wear debris) of the material
from the surface. The size of wear particles generated may vary from the millimeter range down to an ion range. Wear may occur by
contact with other hard materials, flowing liquid, solid particles, or liquid droplets. Wear can be quantified by volumetric wear rate
which is the material volume loss in a unit sliding distance under the applied normal force. There are different laboratory methods
for wear testing (pin-on-disc, pin-on-plate, etc.). Industrial wear studies of total hip prostheses use multi-station hip wear simulators
which can simulate walking patterns of people. The amount of wear debris generated and the size and shape of wear particles signif-
icantly affect clinical outcomes of total hip replacement. Yang and Hutchinson studied the corrosion-wear of b-Ti alloy TMZF
(Ti-12Mo-6Zr-2Fe) under dry and wet conditions (Yang and Hutchinson, 2016). The coefficient of friction is measured in the
pin-on-disc wear tests. It is shown that the coefficient of fraction of the alloy under the wet condition is lower than that under
the dry condition. The wear of the alloy tested in a simulated body fluid increases significantly as compared to that in the dry condi-
tions for all test durations.
Testing at the Micro- or Nano-scale

Microhardness Testing
Microhardness testing is used to determine the hardness of a material surface at the microscopic level. For some metals and poly-
mers, there are empirical correlations between hardness and strength (or modulus) of the material. In microhardness testing, a dia-
mond indenter of a specific geometry (e.g., four-sided pyramid shape) is impressed into the surface of a specimen with a test load
and loading time and produces an indentation with the length of a side being tens of microns. The hardness number is based on
measurements made on the indent formed in the surface of the specimen, in which the applied force is divided by the surface area of
the indent itself, giving the hardness unit as kgf/mm2. Microhardness testing can be conducted using a Vickers or Knoop indenter. In
the Vickers hardness tests, both diagonals of the indent are measured and the average value is used to compute the Vickers hardness
number (HV) (Fig. 4A). In the Knoop hardness tests, only the longer diagonal is measured and the Knoop hardness is calculated
based on the projected area of the indent divided by the applied force. Vickers hardness testing can also produce indentation fracture
toughness (which is normally higher than the value obtained from standard fracture toughness tests) for ceramic materials. Wang
used microhardness tests to assess the hardness and indentation fracture toughness of dense bioceramics and Fig. 5 shows an inden-
tation on sintered dense hydroxyapatite by a Vickers indenter (Wang M., 1992, previously unpublished data). Sun et al. compared
the mechanical behavior of several bulk metallic glasses (BMGs, including ZrCuNiAl, ZrTiNiCuAl, and TiZrNiCuBe), pure Ti and
Ti-6Al-4V alloy for biomedical applications (Sun et al., 2014). The Vickers hardness of as-cast BMG alloy samples as well as
pure Ti and Ti-6Al-4 V are measured using a microhardness tester. The microhardness is ranked as HVZrCuAlAg > HVZrCuAlNi >
HVZrTiNiCuAl > HVTiZrNiCuBe > HVTi-6Al-4V > HVTi.
Nanoindentation
In nanoindentation, extremely smaller loads and a hard indenter tip (usually a diamond tip) with a geometry of three-sided
pyramid (the so-called “Berkovich tip”) are used (Fig. 4B). The indented areas are very small, being only be a few square microm-
eters or even nanometers. During the course of the instrumented nanoindentation process, unlike conventional hardness or micro-
hardness testing which only record the load applied, the depth of penetration of the indenter in the material being tested is
continuously recorded. The area of the indent is determined using the known geometry of the indenter tip. During nanoindentation,
the applied load and depth of penetration are measured to provide a load-displacement curve, which can be used to extract mechan-
ical properties of the material being tested. Wang et al. used nanoindentation to study the mechanical properties of bone-like apatite
formed in vitro on various biomaterials (Wang et al., 2002). They have demonstrated that nanoindentation is a powerful tool in
determining mechanical properties of a very thin layer of bioceramic on biomaterials surface. Across the width of the bone-like
apatite layer, the elastic modulus of bone-like apatite does not vary significantly. Fig. 6 displays a nanoindentation on bone-like
apatite and a typical nanoindentation load-displacement curve for bone-like apatite. Using nanoindentation, Sun and Tong
Fig. 4 Testing at the micro- or nanoscale: (A) microhardness testing using a Vickers indenter, (B) indenter tip for nanoindentation, and (C) single
molecule testing using optical tweezers.
Fig. 5 An indentation on sintered dense hydroxyapatite made by a Vickers indenter.
Fig. 6 Nanoindentation of bone-like apatite: (A) a nanoindentation made by a Berkovich indenter, and (B) a typical load-displacement curve for
bone-like apatite.
investigated fracture properties of three different natural materials: nacre, bovine hoof wall and beetle cuticle (Sun and Tong, 2007).
Micro/nanoscale cracks are generated by nanoindentation using a Berkovich tip. The nanoindentation results in pile-up around the
indent. The fracture toughness of bovine hoof wall is the highest, the second is nacre and the elytra cuticle of dung beetle is the
lowest.
Tests for Single Molecules

Mechanical characterization of single molecules or biological cells is very challenging, and cellular and molecular biomechanics
have made significant advances over the past two decades due to the invention and innovation in testing techniques and
testing equipment. Optical tweezers are now widely used to study various matters such as intramolecular and intermolecular
interactions in the folding/mis-folding of proteins and RNAs and the dynamics underlying the function of molecular motors
and complex macromolecular assemblies, in which mechanical forces can be applied to individual molecules by a force probe.
Optical tweezers are a powerful tool in biomechanical studies owing to the very high resolution it can achieve and also its high
sensitivity to rare and transient events. In a typical test setup using optical tweezers, the molecule of interest is attached to
polystyrene beads at the two ends of the molecule, with one bead being trapped by a laser beam which holds the bead in
position at this end. The coverslips that hold the bead at the other end of the molecule move with the motorized X–Y stage
and hence the molecule is stretched during testing (i.e., tensile testing) (Fig. 4C). Two main measurement techniques can be
used: first, non-equilibrium measurements, such as force-ramp or force-jump; and second, equilibrium measurements, where
the molecule is held under a constant force and/or the traps are kept at a constant position and then the extension of the
molecule is measured. Sun et al. stretched type II collagen molecules with optical tweezers and directly investigated
the mechanical properties in a physiologically relevant condition (Sun et al., 2004). The molecule is stretched by moving
the two-bead complex away from the optical trapping center at a constant rate through the piezo-stage. The displacement
of the trapped bead is monitored by the use of a build-in interferometer. The highly nonlinear force-extension curves for
type II collagen molecules are thus obtained. It is found that the force increases sharply when the molecules are stretched
around their contour length. The molecular parameters of collagen are obtained by fitting force-extension curves using the
worm-like chain elasticity model. The observations in this study indicate that collagen molecules are flexible molecules, rather
than rigid rod, in the neutral pH solution.
Bonding Strength and Testing Methods

Tensile Bonding Tests
Surface properties are highly important for all biomaterials. Coatings made of biocompatible metals, polymers, ceramics or
composites are often used for modifying the surface of current biomaterials or biomaterials under development. One of the impor-
tant properties of coatings is the bonding strengthdtensile bonding strength and/or shear bonding strengthdwith the substrate
(metal, polymer, ceramic, or composite). Because of the importance of bonding strength in nearly all branches of surface engi-
neering, standard testing methods for bonding strength, albeit they are not perfect and sometimes yield questionable results due
to inherent problems, have been long established. ASTM Standard D907 defines adhesion as “the state in which two surfaces
are held together by interfacial forces which may consist of valence forces or interlocking forces or both.” ASTM C633 standard spec-
ifies test details for determining tensile bonding strength and shear bonding strength. It basically requires the detection of the load
or stress to fail a standard joint. These tests need a strong bonding agent to attach the coating to a loading fixture. The test results
suffer from epoxy penetration into the coating (when epoxy is used as a bonding agent in the test fixture). Another problem for both
tensile and shear bonding strength tests is that the fracture mode can be ambiguous because mixed-mode failure often occurs in the
tests, which make it indistinguishable for adhesive strength and cohesive strength of the coating. Apart from biomedical coatings,
the bonding strength of biomaterials to body tissues is also important and similar test strategy can be adopted for the bonding
strength determination/comparison. A key issue for the successful application of new resin-based composites as filling materials
in dentistry is the bonding and performance of the composites. The composites need to infiltrate and impregnate the entire demin-
eralized and opened collagen network of dentin to overcome humidity and to form a high crosslinking polymer inside the collagen
network and hence the composite-dentin hybrid layer can have a high resistance to degradation. El-Malky and Abdelaziz studied the
effect of pre-curing waiting time of different bonding resins on the tensile bonding strength to dentin (El-Malky and Abdelaziz,
2015). The methacrylate-based two-steps-total-etch bonding system and two-steps-self-etch bonding system show significant higher
strength values than that of the one-step-self-etch bonding system. A 30-s pre-curing waiting time for bonding resins appears to
provide high tensile bonding strength to dentin.
Shear Bonding Tests

Biomedical coatings are formed on biomaterials (metals, polymers, ceramics or composites) for either enhancing biological prop-
erties, for example, biocompatibility and bioactivity, or improving physical, mechanical and/or other properties, for example,
corrosion-resistance and reduced wear, of the biomaterial (the substrate). As discussed above, the shear bonding strength for
biomedical coatings can also be measured using standard test methods. But there are problems in interpreting the test results
when mixed-mode failure occurs in the tests. Again, in many situations, the shear bonding strength of a biomaterial to the host
tissue is highly important. For example, dental adhesives must maintain their stability when shear masticatory forces are frequently
applied. Dental adhesives are composed of resins or low-filler-content composites, which function to bond dental materials to
teeth. The strength and durability of dental adhesives are the key factors for ensuring a successful and durable dental restoration.
Biocompatible dental adhesives with enhanced performances are required and different types of adhesives are investigated. Sun
et al. incorporated surface modified titania nanoparticles into dental adhesives (Sun et al., 2017). It is found that the addition
of nanoparticles significantly enhance shear bonding strength between teeth and the titania-filled adhesives.
Peel Tests
One important property of bioadhesives such as those used in surgical tapes is their adhesive strength. ASTM D1876 provides a stan-
dard for testing peel resistance for adhesives. The goal of a peel test is to determine the adhesive strength of a material or the strength
of the adhesive bond between two materials. The adhesive strength may be referred to as the “stickiness” of an adhesive as it is
a measure of the resistance of the adhesive to separation from the substrate after the adhesive has been applied. Peel tests for
measuring the adhesive strength include T-peel, 90 degree peel, and 180 degree peel. The T-peel test is a type of tensile test per-
formed upon two flexible substrates that have been bonded together and placed into peel test grips such that one substrate sticks
up and the other sticks down while the bonded area sticks out horizontally so that the entire setup forms a “T” shape. The 90 degree
test requires a 90 degree peel test fixture to determine the adhesive strength between a flexible (tape) and rigid substrate (plate),
where the plate lies horizontally with the gripped end of the tape sticking up perpendicularly while the rest is bonded to the plate
so that it forms an “L” shape. The 180 degree test is similar to the 90 degree peel test except that the bonded area between the tape
and plate is placed vertically between the peel test grips while the free end of the of plate is gripped by the bottom and the free end of
the tape is gripped by the top so that it forms a tight “U” shape. Lin et al. investigated drug-loaded wound dressing with thermo-
responsive, adhesive, absorptive and easy peeling properties (Lin et al., 2001). The tack property of the EudragitE films increases
with an increasing content of poly(N-isopropylacrylamide) (PNIPAAm) microgel beads, and the peel strength of Eudragit E films
initially decreases with the addition of PNIPAAm microgel beads but increases to a maximum value when PNIPAAm microgel beads
are added from 4% to 7.6%.
Summary
Short-term mechanical tests for tension, compression, shear, bending, torsion, etc. provide fundamental mechanical properties that
can be used to compare different biomaterials for targeted applications or during the development of new biomaterials, to study
(bio)materials processing-structure-property relationships and screen out materials. Long-term mechanical tests are highly impor-
tant for load-bearing implants/devices and hence creep (for biomedical polymers and polymeric composites, as metallic and
ceramic biomaterials do not creep at the human body temperature), fatigue, and wear tests need to be conducted under simulated
human body conditions. Bonding strength determination is needed for biomedical coatings and bioadhesives as well as for assess-
ing the biomaterial-host tissue bond. Mechanical tests at the micro- and nanoscale are performed not only for scientific research but
also for new biomaterials development. For all types of mechanical tests, if a testing standard has been developed and is available,
the test must be conducted according to the standard. This will ensure that test results can be compared and used by different groups
of people (researchers, manufacturer, medical device end users, etc.) across national boundaries.
Acknowledgements
Min Wang thanks Nanyang Technological University, Hong Kong Polytechnic University, The University of Hong Kong, UK’s Engineering and
Physical Sciences Research Council, Singapore’s Ministry of Education, Hong Kong Research Grants Council and the National Natural Science
Foundation of China for research funding. Chong Wang thanks the Office of Education of Guangdong Province for funding his Young Innovative
Talent Project and Dongguan University of Technology for support with the High-level Talents (Innovation Team) Research Project.
References
Akita, M., Uematsu, Y., Kakiuchi, T., Nakajima, M., & Tamada, K. (2015). Materials Science & Engineering A, 627, 351–359.
Ayadi, I., & Ayed, F. B. (2015). Journal of the Mechanical Behavior of Biomedical Materials, 49, 129–140.
Barbieria, D., de Bruijn, J. D., Luo, X. M., Fare, S., & Yuan, H. P. (2013). Journal of the Mechanical Behavior of Biomedical Materials, 20, 162–172.
Buijs, G. J., van der Houwen, E. B., Stegenga, B., Bos, R. R. M., & Verkerke, G. J. (2007). Journal of Oral and Maxillofacial Surgery, 65, 2142–2147.
El-Malky, W., & Abdelaziz, K. M. (2015). Tanta Dental Journal, 12, 99–110.
He, G., Liu, P., Tan, Q. B., & Jang, G. F. (2013). Journal of the Mechanical Behavior of Biomedical Materials, 28, 309–319.
Lin, S. Y., Chen, K. S., & Run-Chu, L. (2001). Biomaterials, 22, 2999–3004.
Martínez-Vázquez, F. J., Perera, F. H., Miranda, P., Pajares, A., & Guiberteau, F. (2010). Acta Biomaterialia, 6, 4361–4368.
Mirani, R., Pratt, J., Lyer, P., & Madihally, S. V. (2009). Biomaterials, 30, 703–710.
Panigrahi, A., Sulkowski, B., Waitz, T., Ozaltin, K., Chrominski, W., Pukenas, A., Horky, J., Lewandowska, M., Skrotzki, W., & Zehetbauer, M. (2016). Journal of the Mechanical
Behavior of Biomedical Materials, 62, 93–105.
Puertolas, J. A., Vadillo, J. L., Sanchez-Salcedo, V. S., Nieto, A., & Vallet-Regi, M. (2011). Acta Biomaterialia, 7, 841–847.
Sun, J., & Tong, J. (2007). Journal of Bionic Engineering, 4, 11–17.
Sun, Y. L., Luo, Z. P., Fertala, A., & An, K. N. (2004). Journal of Biomechanics, 37, 1665–1669.
Sun, Y., Huang, Y. J., Fan, H. B., Liu, F. Y., & Chen, J. J. (2014). Journal of Non-Crystalline Solids, 406, 144–150.
Sun, J., Petersen, E. J., Watson, S. S., Sims, C. M., Kassman, A., Frukhtbeyn, S., Skrtic, D., Ok, M. T., Jacobs, D. S., Reipa, V., Ye, Q., & Nelson, B. C. (2017). Acta Biomaterialia,
53, 585–597.
Takahashi, Y., Tateiwa, T., Shishido, T., Masaoka, T., & Yamamoto, K. (2016). Journal of the Mechanical Behavior of Biomedical Materials, 62, 399–406.
Wang, M., Chen, L. J., & Ni, J. (2002). Transactions of the Society For Biomaterials 28th Annual Meeting, Tampa, Florida, USA (p. p. 259).
Wei, C. K., & Ding, S. J. (2016). Journal of the Mechanical Behavior of Biomedical Materials, 62, 366–383.
Yang, X., & Hutchinson, C. R. (2016). Acta Biomaterialia, 42, 429–439.
Zhao, X. F., Niinomi, M., Nakai, M., & Hieda, J. (2012). Acta Biomaterialia, 8, 1990–1997.
Further Reading
Callister, W. D., Jr., & Rethwisch, D. G. (2014). Materials science and engineeringdAn introduction (9th edn.). John Wiley & Sons.
Hertzberg, R. W., Vinci, R. P., & Hertzberg, J. L. (2013). Deformation and fracture mechanics of engineering materials (5th edn.). John Wiley & Sons.
Meyers, M. A., & Chawla, K. K. (2008). Mechanical behavior of materials (2nd edn.). Cambridge University Press.
Corrosion of Orthopedic Implants
Qiong Wang, Felipe Eltit, and Rizhi Wang, The University of British Columbia, Vancouver, BC, Canada
Introduction 65
Metals in Hip Implants 66
Fundamentals of Corrosion Mechanisms 67
General Concepts 67
Types of Corrosions in Hip Implants 68
In Vivo Corrosion Environment 70
Corrosion-Induced Implant Fracture 70
Clinical Fracture of Implants 70
Corrosion in Fatigue Crack Initiation 71
Corrosion-Induced Tissue Failure 72
Ion and Particle Release Mechanisms 72
Ion release mechanisms 72
Particulates release mechanisms 73
Biological Reactions to Corrosion Products 74
Adverse local tissue reaction to metal implants 74
Histopathology of ALTRs 75
Roles of metal ions in tissues and fluids 75
Biological effect of metal particles 77
New Biomaterial Designs 79
Future Perspectives 79
References 80
Introduction
Metals and their alloys have been successfully used as biomaterials in a wide range of implantable medical devices, from coronary
stents and artificial hearts to dental implants and orthopedic implants (Ratner et al., 2004; Williams, 2014). Their essential roles as
core structural materials in implants are likely to remain the same for decades to come. This is because metals have the best combi-
nation of stiffness, strength, toughness, and formability among all engineering materials including polymers, ceramics, and their
composites. Despite their generally robust performance as biomaterials, metals suffer from low corrosion resistance (as compared
with ceramics). Under the combination of the corrosive physiological environment and the challenging biomechanical loading,
metallic implants may degrade. The consequences of implant corrosion can be serious and are twofold. First, corrosion may even-
tually lead to the destruction and mechanical failure of the implants. Second, metal ions and particles released from the implants
may cause adverse tissue reactions (ALTRs) that will ultimately require surgical revision to the implants. The purpose of this article is
thus to review the corrosion of metallic implants, from mechanisms to clinical issues. We further focus our discussion on total hip
implants in orthopedics. Orthopedic implants comprised nearly half of all medical device implants in use (Moore et al., 1991).
Among them, over 1 million total hip replacements (THR) are performed annually worldwide to relieve pain and restore mobility
of patients with osteoarthritis (Bozic et al., 2009; Singh, 2011; Learmonth et al., 2007). Replacing the mobile hips also represents the
most challenging biomechanical application of metallic biomaterials. It is our hope that the readers will find the basic mechanisms
applicable to other medical implants.
A THR system consists of a metallic femoral stem (mainly a Ti alloy), attached to a hemispherical femoral head articulating
against an acetabular cup with a liner (Fig. 1). One important design characteristic in hip replacements is the combination of mate-
rials that make up the bearing surface of the implanted hip joint, namely the material used for the articulating femoral head and the
acetabular liner. The femoral head is usually made of materials with high wear resistance such as CoCr alloys, and ceramics. The
liner is mainly made of polyethylene in addition to metals and ceramics. Usually, the material combination used at articulating
surfaces was used to abbreviate the types of implants. For example metal-on-polyethylene (MoP) implant means a type of implant
consisting of a metal femoral head articulating against a polyethylene liner. Modularity has been commonly used in THR allowing
the surgeons to select components independently based on patient’s individual anatomy, offering greater variety of solutions during
implantation, easy access in revision procedures, and reduced inventory (Marlowe et al., 2013).
Primary THR surgery has > 90% success rate within a 10-year follow-up, which is one of the greatest advances in the healthcare of
the 20th century (Learmonth et al., 2007; Older, 2002; Malchau et al., 2002; Williams et al., 2002). Historically, THRs fail due to
aseptic loosening, infection, and dislocation (Malchau et al., 2002; Ulrich et al., 2008; The NJR Editorial Board, 2016). However,
with the increasing use of modular components, complications related to corrosion emerged, leading to failure of modular systems

66 Biomaterials: Science and Engineering j Corrosion of Orthopedic Implants
Fig. 1 The components of a MoP hip replacement system.
and consequently increased revision rate. One complication is the ALTRs developed to some latest designs that require revision
surgeries. As a result, regulatory agencies including Health Canada and FDA have issued alerts related to MoM hip implants
(Healthy Canadians, 2012; U.S. Food and Drug Administration, 2013). ALTRs have also been observed recently in patients with
MoP implants, the most commonly used design today (Whitehouse et al., 2015; Cooper et al., 2012; Picardo et al., 2011; Perino
et al., 2014). Although the exact mechanisms of ALTR are unclear at present, the role of corrosion products released from the
metallic materials has been widely accepted (Meyer et al., 2012; Cooper et al., 2013; Langton et al., 2010). In addition to ALTRs,
corrosion may also induce mechanical failures such as fracture and loosening of components. Studies have linked these mechanical
failures to corrosion at modular interfaces (Ellman and Levine, 2013; Nima et al., 2013).
These clinical failures raised concerns over implant corrosion, and call for in-depth investigation of the underlying mechanisms.
In the following sections, we first introduce different types of metallic biomaterials, followed by common corrosion attacks and
their corresponding mechanisms. The role of corrosion in two clinical failuresdthe mechanical failure of the implants and the
ALTRsdis elaborated. Development of metallic materials used in hip implants to improve their clinical performances against corro-
sion is also discussed.
Metals in Hip Implants
Metals are the most used materials in hip replacement due to their mechanical properties. In Canada, > 90% of femoral heads used
in hip replacements are made of metals (Canadian Institute for Health Information, 2015). In the United Kingdom, the metallic
femoral heads are used in 65% of hip implants (The NJR Editorial Board, 2016). An ideal metallic material used in hip replacement
is expected to have great biocompatibility, excellent resistance against corrosion and wear, acceptable strength to endure the cyclic
loading, and a low Young’s modulus to minimize stress shielding to bone (Park and Lakes, 2007). Table 1 summarizes metallic
materials commonly used in hip implants and the required mechanical properties in the regulation.
Stainless steels (SS) are iron-based alloys containing a minimum of 12% Cr to prevent corrosion in the atmosphere. SS is easy to
work and has high strength and low price. Type 316L used in hip replacement is an austenitic SS and contains around 17% Cr, 12%
Ni, and 2.5% Mo with carbon below 0.03%.
CoCr alloys are commonly used as the bearing surface due to their high corrosion and wear resistance. Forged CoCr is also
widely used in the femoral stem for its excellent fatigue resistance. The CoCr alloys are composed of 27%–30%Cr, 5.0%–7.0%
Mo, and small amount of other elements (Mn, Si, Ni, Fe, and C) with balanced Co (ASTM, 2014a,b). Chromium is expected to
form a passive film spontaneously on the surface of the metal, offering the required corrosion resistance (Contu et al., 2003;
Yan et al., 2006). Molybdenum is added to increase pitting resistance and to produce finer grains with precipitates of hard carbide
phases together with chromium (Liao et al., 2012; Sugimoto and Sawada, 1977). Based on carbon contents, CoCr alloys are cate-
gorized into high carbon alloys with 0.05–0.35 wt% carbon, and low carbon alloys with carbon concentration < 0.05 wt%. Higher
carbon content usually is associated with more carbide phase precipitates in the alloy, and hence superior wear resistance (Affatato
et al., 2011). However, chemical and microstructural inhomogeneity caused by carbide precipitates compromises the alloy’s
Biomaterials: Science and Engineering j Corrosion of Orthopedic Implants 67
Table 1 Mechanical properties of metals used in hip implants
Materials ASTM# Condition E(GPa) sys min (MPa) suts min (MPa) d(%) min
Stainless steel F138 Annealed 200 190 490 40

30% worked 200 690 860 12
Co–28Cr–6Mo F75 Cast 210 450 655 8
F799 forged 210 827 1172 12
Co–20Cr–15W–10Ni F90 Annealed 210 310 860 30
Cold worked 210 760 1250 15
cp-Ti F67 Grade 4 110 483 550 15
Ti–6Al–4V F136 Wrought 110 795 860 10
Ti–6Al–7Nb F1295 Wrought 105 800 900 10
Ta F560 Cold worked 186 345 480 1
Annealed 186 140 210 8
resistance to localized corrosion (Montero-Ocampo and Rodriguez, 1995). In the application of hip replacement, thermal treatment
such as fast cooling is used to produce FCC (face-centered-cubic) structure in CoCr (Brooks, 1982; Davis, 1990). And the FCC phase
remains metastable due to very low kinetic transformation from FCC to HCP (hexagonal-close-packed) structure at room temper-
ature (López and Saldivar-Garcia, 2007).
Ti alloys are commonly used for its strong corrosion resistance and high biocompatibility. Commercially pure titanium
(cp-Ti) has two allotropic forms (Welsch et al., 1993). From room temperature up to 882 C, titanium exists as a phase of
hexagonal-close-packed (HCP) crystal structure. Above 882 C, it transfers to b, which has a body-centered-cubic crystal structure.
The mechanical properties depend on the relative amounts and distribution of a and b phase. To stabilize these two phases,
elements such as aluminum, tin, or zirconium are commonly added to stabilize a phase, while molybdenum, vanadium, or
niobium are often added to stabilize b phase. The most commonly used a þ b dual-phase Ti alloy in biomedical device is
Ti–6Al–4V (Rack and Qazi, 2006).
Tantalum is also used as for enhanced osseointegration, strong corrosion resistance, and lower elastic modulus (Davis, 2003;
Bobyn et al., 1999). Due to the mismatch of elastic modulus between bone (10–30 GPa) and metals used in femoral implant,
bone is not sufficiently loaded and becomes stress shielded, which eventually leads to bone resorption and compromise of clinical
performance. To solve this problem, low elastic modulus of porous titanium and tantalum is developed to allow an effective load
transfer and bone preservation (Ryan et al., 2006; Balla et al., 2010; Levine et al., 2006).
Fundamentals of Corrosion Mechanisms

General Concepts
Corrosion is an electrochemical reaction involving electron transfers. An electrochemical reaction consists of two half-reactions.
Anodic reaction is a half-reaction of oxidization releasing electrons, for example, a metal turns into metal ions in Reaction (1).
The other half-reaction named cathodic reaction is a reduction process gaining electrons, for example, a reduction of protons forms
hydrogen gas in Reaction (2). The two half-reactions coupled together form an overall electrochemical reaction occurring in
corrosion:
M # Mþ þ e (1)
2Hþ þ 2e # H2(g) (2)

The corrosion reaction can be examined by two basic theories. The first is the thermodynamic theory characterizing the driving
forces for corrosion reactions, revealing whether the reactions may happen. The other is the kinetic process related to the reaction
rates, revealing how fast the corrosion reaction happens.
For thermodynamics, the driving force for the reaction is the change in Gibbs free energy (D G). When D G is negative, its cor-
responding reaction will proceed spontaneously.
D G is also associated with electrode potential in this expression D G ¼ nFE, where n is the number of electrons transferred, F is
the Faraday constant, and E is the electrode potential (reduction expression) which electrons need to overcome to transfer in
between.
For an overall electrochemical reaction, similar D G expression can be written, where E is the cell potential given by E-
cell ¼ Ecathode Eanode. Based on thermodynamics theory, the more negative of its electrode potential, the more active it is as an
anode, and the more likely it is to be oxidized.
The other factor governing the corrosion is the kinetic process that determines how fast it happens. The well-known kinetic
barrier is the passive film (usually an oxide layer) formed on a metal surface. Cr and Ti, two commonly used elements in implant
alloys, are very active according to the thermodynamics (Table 2) (Haynes, 2004). However, the passive films spontaneously
Table 2 Standard electrode potentials (Haynes, 2004)
Half-reaction E (V)
2Hþ þ 2e # H2(g) 0

2Ni2 þ þ 2e # Ni(s) 0.25
2Fe2 þ þ 2e # Fe (s) 0.44
Cr3 þ þ 3e # Cr(s) 0.74
2Ti2 þ þ 2e # Ti (s) 1.63
formed on these two metals are very dense and intact, preventing the active dissolution of metal substrates. This passivation of Cr
and Ti enables alloys such as 316L SS, CoCr, and Ti alloys to possess strong corrosion resistance.
Types of Corrosions in Hip Implants

Corrosion may be general or localized. General corrosion involves the uniform dissolution of the metal surface, while localized
attack occurs on specific sites of a passive metal surface where there are high local dissolution rates. For alloys used in hip implants,
the most relevant types are localized corrosion such as galvanic corrosion, pitting, crevice corrosion, and mechanically assisted
corrosion.
Galvanic corrosion: Galvanic corrosion tends to occur when dissimilar materials are connected electrically and exposed to an elec-
trolyte. The active material with lower electrode potential is preferentially corroded to another due to the driving force of the elec-
trode potential difference between these dissimilar metals. In most cases, however, galvanic corrosion is not a major concern on
multi-alloy hip prostheses. Electrochemical studies showed low corrosion rate of 0.02 mA/cm2 when cobalt and titanium alloys
were coupled and no instances of extensive corrosion were found on cobalt–titanium interfaces of the retrieved implants (Lucas
et al., 1981). No detectable galvanic corrosion has been observed on (SS)-Ti alloy mixed interfaces in vitro (Serhan et al., 2004).
Without pitting or crevice corrosion of the SS, the coupling of SS-CoCr alloy remains in the stable passive state, and no significant
change of corrosion behavior was detected on the materials (Reclaru et al., 2002). Since passive film formed on the metal surface
remains intact in static environment serving as an efficient barrier to corrosion, the risk of galvanic corrosion is negligible for alloys
used in a biomedical device (Virtanen et al., 2008; Mears, 1975).
Crevice corrosion and pitting: Passive oxide films are remarkable in their ability to protect a wide variety of metals and alloys from
corrosion. However, passive films often break down in certain areas where localized corrosion attack happens. The most common
forms of localized attack are crevice corrosion and pitting. Crevice corrosion occurs within confined space or under shielded metal
surfaces. Pitting is the localized breakdown of passive films usually caused by halogen ions such as chloride, leading to the subse-
quent formation of pits at these sites. Crevice corrosion and pitting differ in their mechanisms of initiation but share similar mech-
anism of propagation (Fig. 2) (McCafferty, 2010).
Crevice corrosion occurs in geometrical clearances such as the modular interfaces of hip implants. At the very beginning of
crevice corrosion, oxygen reduction occurs both on the metal surface exposed to the bulk electrolyte and on the portion of the metal
surface within the crevice.
However, the external metal exposed to the bulk electrolyte has abundant supply of oxygen from the atmosphere so that
a steady-state concentration of O2 is maintained. On the contrary, within the narrow clearance of the crevice, the oxygen becomes
depleted due to the long diffusion path formed by the crevice. This difference in O2 concentration between the bulk solution and
crevice becomes the driving force which makes the metal exposed to lower concentration of oxygen to have a more negative poten-
tial for oxygen reduction. This electrode potential difference between internal and external metal will initiate corrosion of metals in
the crevice. The electrochemical reactions of the crevice reactions are as follows:
Cathode (on metal outside of crevice):
O2 þ 2H2 O þ 4 e #4 OH
Anode (on metal in the crevice):
Cr#Cr 3þ þ 3 e
Cr 3þ þ 2H2 O#Cr ðOHÞ3 þ 3Hþ
As the anodic reaction shows, the electrolyte composition within the crevice will become acidic due to the hydrolysis of metal
cations. This aggressive electrolyte in the crevice will break down the passive layer and continue to dissolve the metallic element.
Pitting is another localized corrosion attack. Pitting is caused by the presence of aggressive anions such as halogen ions, usually
chloride in the electrolyte. The chloride ion is a strong electron donor with relatively small volume and high diffusivity. And it tends
to interact with electron acceptors such as metal cations, resulting in the breakdown of the passive film.
Crevice corrosion and pitting share similar mechanisms in propagation. The electrolyte composition within the crevice and pits
will become acidic due to the hydrolysis of metal cations (anodic reaction above). And it will also contain concentrated amounts of
Fig. 2 Schematic illustration of crevice corrosion (A), (B), (C), and pitting (D). (D) is the possible pitting happening when Cl is present in the
crevice. Pictures are modified based on McCafferty, E. (2010). Crevice corrosion and pitting. In Introduction to corrosion science. New York, NY:
Springer New York; pp. 1–575.
cations discharged from the alloy. In chloride solutions, the crevice and pits will also become concentrated in chloride ions to main-
tain the charge neutral. This internal electrolyte is sufficiently aggressive to break down the passive film on the metal, which will
further develop corrosion.
SS 316L shows higher susceptibility to pitting and crevice corrosion than CoCr and Ti alloys (Gurappa, 2002; Walczak et al.,
1998). The resistance to pitting corrosion depends on Cr and Mo content. Moreover, relatively small variations in steel composition
or surface condition such as roughness may significantly influence the pitting behavior (Virtanen et al., 2008). Studies on retrieved
implants show that > 90% of the failure of 316L SS is due to the pitting and crevice corrosion attack (Sivakumar et al., 1995). Since
CoCr alloy has relatively higher content of Cr and Mo, pitting corrosion is not common. High-carbon CoCr alloy is more susceptible
to pitting corrosion due to the segregation of hard phases at the grain boundary (Panigrahi et al., 2014). Compared with CoCr alloy,
titanium alloys possess much higher resistance against corrosion, especially crevice corrosion (Gurappa, 2002; Gurrappa, 2003).
Indeed crevice attack of titanium alloys does not generally occur at temperature < 70 C regardless of solution pH or chloride
concentration (Welsch et al., 1993).
Mechanically assisted corrosion: In modular hip implants, corrosion usually occurs when the passive films are damaged by mechan-
ical wear. The commonly observed mechanically assisted corrosions at modular implants are fretting corrosion and tribocorrosion.
Fretting corrosion is a type of corrosion assisted by micromotion-induced wear (Gilbert et al., 1993). This wear disrupts the passive
film, exposing the alloy that is susceptible of corrosion until a new passive layer is formed (Hallab and Jacobs, 2003; Urban et al.,
2006; Swaminathan and Gilbert, 2012). This cyclic process of wear-exposure-repassivation happens at the modular junctions of the
implants, such as the head–neck junction and the neck-body junction, leading to the active corrosion and generation of debris.
The amplitude of displacement in fretting corrosion is governed by the applied bending moment and the rigidity of the taper in
implants (Gilbert and Mali, 2012). As Fig. 3 shows, the two points (solid triangle) within the taper regions are considered rigidly
connected to the opposite surface. The elastic strain associated with cyclic bending will give rise to a displacement (D) within the
taper interface in the range of 20–40 mm for typical tapers used today. At the same time, the confined taper interface is a crevice
geometry where crevice corrosion is favored, resulting in accelerated corrosion (Marlowe et al., 2013).
Studies have confirmed that modular designs are susceptible to fretting corrosion in vivo. In a study, 16 retrieved modular tita-
nium alloy stems from cementless hip replacement were analyzed, and evident corrosion sites were characterized with fretting scars,
pitting, and etching (Urban et al., 2006). Most of the retrieved modular tapers showed severe fretting corrosion both at head–neck
taper and neck-stem interfaces (Rodrigues et al., 2009; Wright et al., 2010; Lakstein et al., 2011; Brown et al., 1995). Fretting played
a major role in the initiation of the corrosion at the modular interfaces, and the long head–neck extension was more prone to fret-
ting corrosion due to the larger fretting amplitude (Brown et al., 1995). It has also been proposed that combining different alloys in
a modular junction (e.g., Ti alloy combined with CoCr alloy) increases the risk of fretting corrosion at the interface between them
(Gilbert et al., 1993; Collier et al., 1992; Cook et al., 1994).
Fig. 3 Schematic illustration of fretting at the modular junction: (A) under cyclic bending, two points (solid triangle) are at rigid contact and the
elastic strain gives rise to a displacement (D) within the taper, crevice geometry is formed at the junction. (B) Zoom-in on the contact region with the
modular taper, metal-oxide surfaces in asperity contact cause contact stresses, local surface deformation and oxide debris (Gilbert & Mali, 2012).
Tribocorrosion is a general term used to describe wear-assisted corrosion. Technically, fretting corrosion is a subcategory of tribo-
corrosion (Mathew et al., 2009). Here, tribocorrosion is used to describe the specific wear-assisted corrosion at articulating surfaces.
Compared to fretting corrosion, tribocorrosion is characterized with large amplitude of wear without assistance of evident crevice
corrosion. The direct consequence of tribocorrosion is the release of wear debris from the bearing materials (Wimmer et al., 2013;
Büscher et al., 2005; Büscher and Fischer, 2005; Illgen et al., 2008; Catelas et al., 2011; Doorn et al., 1996).
In Vivo Corrosion Environment

The normal body fluid is a relatively mild environment of buffered solution at around pH of 7.4 with temperature of 37 C
(Hanawa, 2004). However, there are some specific in vivo corrosive elements for metallic materials such as chloride ions, localized
pH drop, proteins, and cell-induced oxidizers. The concentration of chloride ion in synovial fluid is similar to that in serum (Per-
man, 1980). Drop in pH values was observed in revised implants due to crevice corrosion (Willert et al., 1996) and periprosthetic
tissues because of osteoclasts activities during bone remodeling (Konttinen et al., 2001). The decreased pH may accelerate the corro-
sion of alloys used in the hip implants. The presence of proteins in the physiological environment will affect the corrosion of
metallic materials via changing the mechanics and kinetics of the corrosion reactions on the surface (Valero Vidal et al., 2010; Igual
Munoz et al., 2015; Okazaki and Gotoh, 2005; Goldberg and Gilbert, 1997; Hallab et al., 2003). Additionally, active oxygen species
released by immunological cells such as macrophages when responding to a foreign body at acute inflammation are potential
oxidizers assisting corrosion process (Hanawa, 2004; Gilbert et al., 2015; Liu and Gilbert, 2017). Therefore, it is extremely chal-
lenging to simulate the exact in vivo environment in a laboratory setup.
Corrosion-Induced Implant Fracture

Clinical Fracture of Implants
The fatigue strengths of metallic biomaterials have been well documented and are listed in Fig. 4 (Niinomi, 2007). The majority of
these alloys (especially forged CoCr and Ti alloys) have fatigue strength > 500 MPa (in air) (Niinomi, 2007; Black and Hastings,
1998), which is much higher than the strength of human bones (100–200 MPa) (Teoh, 2000). Historically, fracture of femoral
stem was not common in THR. However, in the recent years, increasing cases of fracture associated with modular junctions of
hip replacements have been reported. Prior to the introduction of modularity in the stems of hip implants, the prevalence of fracture
in the femoral component was estimated to be 0.23% (Wright et al., 2010; Lakstein et al., 2011; Charnley, 1975). However, the
introduction of modularity in the femoral stems has raised the risk of mechanical failure up to 1.4% (Krishnan et al., 2013; Grupp
et al., 2010), and some models have been recalled from the market due to their elevated failure rate (U.S. Food and Drug
Administration, 2012).
Fatigue fracture of titanium alloys: Most of the fractures of titanium components occurred at the stem-taper junction in the form of
fatigue (Wright et al., 2010; Grupp et al., 2010; Dunbar, 2010). In a study of 5000 cases of patients with modular titanium femoral
Fig. 4 Fatigue strength at 107 cycles of biomedical alloys and bone (Niinomi, 2007).
neck, fatigue fracture was observed in up to 1.4% of them within the first 2 years of implantation (Grupp et al., 2010). Investigation
of fracture has revealed different contributing factors such as overweight of the patient (Ellman and Levine, 2013), inadequate bone
support (Lakstein et al., 2011), and stress concentration due to design (Huot Carlson et al., 2012). The most widely reported mech-
anism in the initiation of fatigue fracture of the Ti alloy components in modular hip systems is fretting (Ellman and Levine, 2013;
Huot Carlson et al., 2012; Atwood et al., 2010; Paliwal et al., 2010). Nevertheless, corrosion-induced fracture of Ti alloy component
has been reported (Gilbert et al., 2012).
Fatigue fracture of CoCr alloys: CoCr alloys have higher fatigue resistance compared to Ti alloys so that the risk of CoCr fatigue
occurrence is supposed to be lower. Unfortunately, fatigue fracture has also been observed in CoCr alloys. One mechanism for
fatigue fracture on CoCr is the crack nucleation at microstructural defects such as voids, inclusions, and laser etching accidently
introduced during manufacturing (Galante, 1980; Della Valle et al., 2005; Woolson et al., 1997; Lee and Kim, 2001). Another
possible reason for fatigue fracture of CoCr stem is the overload due to undersized component in some designs (Norman et al.,
2014; Ishaque et al., 2011). The most common factor triggering fatigue fracture of CoCr alloys in modular hip replacement system
is corrosion, such as fretting corrosion and intergranular corrosions (Norman et al., 2014; Wang et al., 2017; Mencière et al., 2014;
Gilbert et al., 1994).
Corrosion in Fatigue Crack Initiation

Corrosion is a key factor in the fatigue of alloys used in hip replacement. Corrosion attacks in a physiological environment create
surface defects and accelerate crack nucleation and propagation; hence, the fatigue strength of alloys can be significantly reduced
leading to increased risk of fracture (Niinomi, 2007; Antunes and de Oliveira, 2012). An important question regarding corrosion
fatigue is how surface cracks are initiated in the presence of corrosion.
Pitting corrosion: Pitting is the major corrosion factor responsible for crack initiation as surface defects (pits) are local stress
concentration sites under tensile stress. Although pitting corrosion in CoCr is seldom observed in laboratory polarization studies
in simulated body fluid, since it typically fails by transpassive dissolution (Bettini et al., 2011), pitting in retrieved CoCr alloys has
been reported (Gilbert et al., 1993, 1994). In one study, pitting induced by intergranular corrosion led to the removal of particles
from the surface (Gilbert et al., 1993). Recent laboratory tests suggested that pitting corrosion could happen at carbide boundaries
and high-energy grain boundaries in electrochemically corroded CoCr samples (Panigrahi et al., 2014; Bettini et al., 2012; Wimmer
et al., 2001), especially in the presence of initial high contact pressure (Hanawa et al., 2001) and proteins (Mathew et al., 2010). The
stress might tear the carbides off the surface (Hanawa et al., 2001) and the presence of proteins lowered the pitting potential, leading
to localized chemical attacks (Mathew et al., 2010). Pitting on Ti alloys in a physiological environment is extremely rare as Ti shows
very strong resistance against pitting (Gilbert et al., 2015). Corrosion features similar to pitting were observed in a retrieved Ti alloy
stem and were associated with possible hydrogen embrittlement in the alloy (Rodrigues et al., 2009).
Fretting corrosion: Another key factor in crack initiation is fretting corrosion. Fretting fatigue has been widely reported in the frac-
ture of Ti alloy components in modular hip systems (Ellman and Levine, 2013; Huot Carlson et al., 2012; Atwood et al., 2010;
Paliwal et al., 2010). However, fretting corrosion-induced fracture in modular CoCr stems is less commonly documented in the
literature (Mencière et al., 2014). The residual stress and plastic deformation caused by fretting may create possible sites for crack
initiation. The released debris due to fretting at the surface will accelerate the crack initiation stage (Hoeppner and Chandrasekaran,
1994). Fretting could also trigger the release of carbides from the surface, which may further enhance intergranular pitting around
the carbides. These corrosion pits served as preferential sites for microcrack formation (Wang et al., 2017). Fretting in modular hip
implants is usually accompanied by crevice corrosion. Electrochemical reactions in the crevice of the fretting interface might change
the electrolyte composition and drop the pH, resulting in an aggressive environment enhancing other types of corrosion such as
pitting to further facilitate crack initiation. After a crack is formed, the trapped fretting corrosion products would prevent the crack
from closing under fluctuating stresses, which may facilitate further corrosion attack at the crack tip (Wang et al., 2017; Fatigue of
Structures and Materials, 2009).
Stress-assisted corrosion cracking: Under normal physiological loading conditions, the stem of a THR is subject to fluctuating tensile
stress on the lateral side and compressive stress on medial side (Hampton et al., 1980; Cicero et al., 2007). Cyclic tensile stress in the
dynamic loading of the hip implants assists the growth of surface pits into cluster of microcracks and the coalescence of those cracks
(Wang et al., 2017). Such a stress-assisted crack initiation process is similar to stress corrosion cracking (SCC), except that the tensile
stress involved is dynamic instead of static (Fatigue of Structures and Materials, 2009). Historically, SCC was often observed on SS
since they are relatively more susceptible to pitting attack than CoCr and Ti alloys (Bundy et al., 1983; Sivakumar and Rajeswari,
1992; Bombara and Cavallini, 1977). Typical SCC responsible for fatigue crack nucleation on Ti alloys was rarely reported, although
Gilbert et al. (2012) proposed a mechanism similar to SCC on a Ti–6Al–4V taper, in which the cracks propagated due to the crack tip
stress arising from the oxide formation rather than externally tensile stress. Wang et al. reported the branching and the coalescence of
cracks on the external surfaces of the CoCr adaptor and their association with the tensile stress suggesting a similar crack initiation
mechanism to SCC (Wang et al., 2017). The risk of corrosion cracking under tensile stress (either static or dynamic) is that it could
facilitate subcritical crack growth for later fatigue fracture at a stress level that is significantly lower than the yield strength of the
material. Especially, when CoCr components are used together with Ti alloy under dynamic tensile stress, stress-assisted corrosion
cracking may happen in the presence of crevice pitting and fretting, which may accelerate the crack nucleation process leading to
early fracture.
Corrosion-induced fracture is usually a result of the combination of multiple factors. Fretting corrosion at the modular interface
accelerates crevice corrosion and pitting. The local aggressive electrolyte resulting from crevice corrosion and pitting will further
facilitate fretting corrosion. The surface defects due to multiple corrosion attacks are preferred locations for crack initiation, espe-
cially on the lateral side of the implants subjected to dynamic tensile stress. Some of these cracks coalesced and propagated to a crit-
ical size under dynamic tensile loading, leading to the later corrosion fatigue fracture.
Corrosion-Induced Tissue Failure
The described mechanisms of corrosion in the hip replacements systems, especially fretting corrosion and tribocorrosion that are
present at the metal interfaces of the different implant designs, release large amounts of metal species from the implant, in the form
of particulate materials and ions, to the synovial fluid, periprosthetic tissue, and blood of patients. The evolution of these corrosion
products and the adverse reactions of peri-implant tissues are reviewed in this section.
Ion and Particle Release Mechanisms

Ion release mechanisms
Cobalt release: Cobalt is preferentially released when passive film is formed on the CoCr surface. CoCr alloys passivate spontaneously
in air, resulting in the formation of a thin oxide film (around 2 nm) containing mainly Cr 3 þ, with a minor content of Co and Mo
(Hanawa et al., 2001; Li et al., 1999; Milosev and Strehblow, 2003). The composition of the oxide film depends on the applied
potential. In the simulated physiological solution (SPS), the passive layer consists predominantly of Cr2O3 and Cr(OH)3 at
potential < 0.3 V, but at higher potentials both Co and Mo oxides can be found in the passive layer (Milosev and Strehblow,
2003). After immersion in Hanks’ solution, cobalt is dissolved, leaving Cr 3þ and Mo 4 þ, 5þ, 6þ in the surface oxide (Hanawa
et al., 2001). Complexation of Co to proteins such as albumin will form a precipitate so that measuring Co ion concentration in
physiological solution may underestimate the total cobalt release (Hedberg and Odnevall Wallinder, 2014).
Chromium release: Chromium oxides are the main components of the passive film formed on the surface of CoCr alloys used in
hip replacement systems. In static immersion tests, the amount of Cr ions released from the implants is extremely low compared
with Co and Mo (Metikos-Hukovic et al., 2006). However, after long-term implantation of CoCr alloys, elevated levels of Co and Cr
ions were observed in blood and urine of patients with metal hip replacements (Hanawa, 2004; Metikos-Hukovic et al., 2006;
Lhotka et al., 2003). The release of chromium is believed to be associated with disruption of passive film during the mechanical
wear.
Molybdenum release: Pourbaix diagram shows that Mo species are more stable in passivity at low pH. A pH higher than 7.0 is
favorable for Mo dissolution thermodynamically (Martin et al., 2013; Takeno, 2005). Moreover, molybdenum oxides formed in
air on the metal surfaces disappear quickly after immersion in 0.15 M NaCl (Li et al., 1999). The interaction with proteins seems
to have an important role in the dynamics of molybdenum release. Latest studies revealed that Mo played an important role in
metal–protein interaction (Martin et al., 2013; Simoes et al., 2016). Electrochemical quartz crystal microbalance was used to study
the mass change in different physiological solutions with proteins. Protein deposition was only observed on Mo surface rather than
Co and Cr (Martin et al., 2013). Similarly, accelerated release of molybdenum ion was observed when bovine serum albumin was
present in the solution (Simoes et al., 2016).
It should be noted that active dissolution of metal element in the static passivation of CoCr is different from the in vivo corrosion
process. First, in vivo active dissolution is often associated with mechanically assisted corrosion at interfaces. Mechanical abrasion
could damage the Cr-rich passivation layer, reduce the pH, and accelerate the corrosion process (Gilbert et al., 1993; Cummings and
Nordby, 1966). Patients with loosen implants have substantially higher release of cobalt and chromium from possible fretting
corrosion at interfaces (Sunderman et al., 1989).
Second, the presence of proteins in the physiological environment can affect the active corrosion of CoCr, via changes in the
mechanics and kinetics of the corrosion reactions on the surfaces of the metal implants (Valero Vidal et al., 2010; Igual Munoz
et al., 2015; Okazaki and Gotoh, 2005; Goldberg and Gilbert, 1997; Hallab et al., 2003). However, it has not yet been clarified
whether biomolecules could accelerate or inhibit corrosion (Igual Munoz et al., 2015; Okazaki and Gotoh, 2005; Goldberg and
Gilbert, 1997; Hallab et al., 2003). Serum proteins of 140 KDa preferentially bind to the CoCr alloy surface (Hallab et al.,
2003) and this metal–protein interaction theoretically would enhance the dissolution rate (Steinemann, 1996). Additionally,
when fretting occurs, the deposition of proteins on the CoCr alloy will form a barrier layer retarding Cr passivation and accelerating
the active dissolution of metals (Goldberg and Gilbert, 1997). On the other hand, CoCr shows a thicker protective passive film
when proteins are present, suggesting a higher resistance against metal release (Valero Vidal and Igual Muñoz, 2008). However,
studies have shown that the difference in the amount of Co released from the CoCr casting alloy in immersion test was relatively
small regardless of proteins (Okazaki and Gotoh, 2005). Moreover, electrochemical measurements of CoCr in human synovial fluid
have revealed that the corrosion rates varied greatly depending on patients rather than the presence of proteins (Igual Munoz et al.,
2015).
Another possible factor affecting the active corrosion “in vivo” is the inflammation induced by the immunological reactions to
the presence of metal implants. The OCP (open circuit potential) measured correlated well with the estimated inflammation of the
synovial membrane (Igual Munoz et al., 2015). Cells such as macrophages generate reactive oxygen species as part of their mech-
anisms of response to a foreign agent, increasing the oxidative stress in the inflammatory environment (Hanawa, 2004). Recent
studies suggested that the oxygen-derived species produced by macrophages had a role in the corrosion process of metal alloys
(Gilbert et al., 2015; Liu and Gilbert, 2017).
Particulates release mechanisms

The generation of particles is the result of the combined action of wear and corrosion. Mechanical abrasion disrupts material surface
leading to the release of debris. Corrosion changes the nature of the debris. The migration and aggregation of debris accelerate the
abrasion in return. Although many laboratory simulation tests have been conducted on the corrosion behavior of modular implants
(Swaminathan and Gilbert, 2012; Blau et al., 2005; Roy et al., 2010; Sivakumar et al., 2011), the in vivo corrosion process has not
been fully understood at present. In the following sections, the nature of corrosion debris from hip implants is reviewed to help us
better understand the debris release mechanisms (Table 3).
Fretting corrosion products at modular interfaces: Although corrosion has been widely observed at the interface of the modular
components of the hip replacements (Gilbert et al., 1993; Collier et al., 1992; Cook et al., 1994), only a few material analyses
have been reported on the solid corrosion products at the head–neck interface in total hip implants. An early case study described
a large amount of black deposits at the head–neck interface, which were rich in Cr, Ca, and P and depleted in Co (Mathiesen et al.,
1991). Further studies have demonstrated that regardless of the design, chemical differences have been observed on the corrosion
Table 3 Summary of reported corrosion debris from CoCr hip implants
Locations Corrosion debris and representative reports
Head–neck interface Inside: Crystalline metal oxides containing Cr, Mo with depleted Co (Urban et al., 1994, 1997)
At the opening: Amorphous chromium(III) phosphate hydrate (Urban et al., 1994, 1997)
Bearing surfaces MoM implant: Nano-sized metallic particles (Catelas et al., 2004; Firkins et al., 2001)
MoP implant: Mainly polyethylene debris (Illgen et al., 2008; Catelas et al., 2011; Doorn et al., 1996; Bitounis et al., 2016)
Implant–cement interface Nano-sized crystalline chromium oxide (Bryant et al., 2013, 2014)
Peri-implant tissues MoM implants: Oxide particles containing Cr 3 þ (Catelas et al., 2004; Catelas et al., 2006; Goode et al., 2012);
nano-sized metallic particles (Catelas et al., 2004; Bitounis et al., 2016; Catelas et al., 2006; Goode et al., 2012; Topolovec
et al., 2013; Doorn et al., 1998); chromium(III) phosphate (Huber et al., 2009; Hart et al., 2010, 2012)
MoP implants: Oxide particles containing Cr with low Co (Topolovec et al., 2013; Shahgaldi et al., 1995; Urban et al.,
2000); sub-micron metallic particles (Topolovec et al., 2013; Shahgaldi et al., 1995; Urban et al., 2000)
products depending on the location in the head–neck junction. Inside the internal part of the head–neck interface, the corrosion
products were composed of highly crystalline metal oxides containing chromium and molybdenum with depleted cobalt (Urban
et al., 1994, 1997), while at the opening of the modular junction, the corrosion products were composed of amorphous chromiu-
m(III) phosphate hydrate (Urban et al., 1994, 1997). The formation mechanisms of these products and their chemical differences
are still unclear.
Tribocorrosion products at articulating surfaces: Tribocorrosion is usually identified as wear tracks on the bearing surfaces (Kwon
et al., 2010a). In MoM implants, nano-sized metallic particles and metal oxides have been observed after testing in hip simulator
(Catelas et al., 2004; Firkins et al., 2001). However, in MoP implants, there are few reports on the particles released from metallic
heads since the predominant wear debris are PE particles (Illgen et al., 2008; Catelas et al., 2011; Doorn et al., 1996; Bitounis et al.,
2016). Higher surface roughness was measured on the SS femoral head in MoP implants compared to CoCr femoral head in MoM,
suggesting a higher volume loss of SS due to its inferior wear resistance (Topolovec et al., 2014).
Fretting corrosion at implant–cement interface: High acidity as a result of crevice corrosion is present at the interface of bone cement
and Ti stem. The corrosion products have been characterized as mixed titanium oxide and hydroxide (Willert et al., 1996). On
a retrieved study of 16 modular titanium alloy stems from cementless hip replacements, evident corrosion was characterized
with fretting scars, pitting, and etching. Mixed titanium oxides were found in the corrosion deposits (Urban et al., 2006). A layer
of nano-sized crystalline chromium oxide was found at the CoCr stem–cement interface (Bryant et al., 2013, 2014).
Corrosion products in peri-implant tissues: In retrieved MoM implants, particles found were mainly oxide particles containing Cr
(Catelas et al., 2004, 2006; Goode et al., 2012). Specifically, STEM-EELS revealed that majority of debris in tissues from MoM
implant were nanoparticles of Cr 3 þ with trace amount of oxidized Co (Goode et al., 2012). In addition, metallic nanoparticles
were also observed (Catelas et al., 2004, 2006; Bitounis et al., 2016; Goode et al., 2012; Topolovec et al., 2013; Doorn et al.,
1998). The presence of metallic materials agrees with in vitro studies using hip simulator (Catelas et al., 2004; Firkins et al.,
2001). The relative proportion of these two types of particles may be different (Topolovec et al., 2013; Doorn et al., 1998). A third
type of corrosion particle rich in chromium and phosphate was also observed in femoral interface tissues from retrieved MoM
implants of a specific model (Huber et al., 2009). Synchrotron radiation (XRF and XAS) analyses showed that particles in the tissues
surrounding MoM implants were predominantly CrPO4 (Hart et al., 2010). Higher Co/Cr ratio was also found in tissues in this
specific type of MoM hip implants compared to other models, implying cobalt ions to be responsible for ALTRs in this type of
MoM implant (Hart et al., 2012).
In retrieved MoP implants, chromium- and phosphate-rich particles were the primary debris detected in the tissues and these
particles were associated with severe fretting corrosion between CoCr taper and Ti stem (Cooper et al., 2013; Hsu et al., 2012).
Corrosion in MoP implants may release various types of particles into the tissue. CoCr metallic particles as well as chromium-
rich particles with reduced cobalt were observed in tissue surrounding MoP implants suffering from aseptic loosening (Topolovec
et al., 2013; Shahgaldi et al., 1995; Urban et al., 2000). The formation of these various particles in these loosened MoP implants is
a result of abnormal wear at unintended metal interfaces, such as interface of loose metal components against bone, cement, or
bearing surface due to wear through the polyethylene liner. Those processes will result in an extensive distribution of submicron
metallic wear particles in periprosthetic tissues and abdominal lymph nodes (Urban et al., 2000).
Possible debris releasing mechanisms: The nano-sized CoCr metal particles generated by tribocorrosion are probably a direct
product of wear in the CoCr alloy. The presence of a layer of nanocrystals has been observed on the articulating surface of MoM
implants (Büscher and Fischer, 2005; Sun et al., 2009; Wimmer et al., 2010), presumably as a result of recrystallization/strain-
induced phase transformation due to wear. This layer seems to be the releasing source of nano-sized metallic particles.
On the contrary, the chromium oxide particles produced by fretting corrosion are not the direct products of fretting debris from
passive films, as the nature of fretting corrosion particles is different from the passive film formed on the alloy surface (size, crystal
structure). At stem–cement interface where fretting corrosion happens, a layer of nanocrystalline chromium oxide was found
(Bryant et al., 2013, 2014), which is different from metallic layer formed on bearing surfaces. Latest few reports studied cross-
sectional tribofilms at different locations inside head–neck interface of retrieved MoP implants, revealing multiple layers with
different crystal structures (Zeng et al., 2015a,b). Laboratory fretting corrosion test (ball on plate in tribometer) showed that the
layer of nanocrystalline chromium oxide formed on CoCr surface was much thinner compared with the retrieved analysis (Zeng
et al., 2015b). At this stage, the role of this chromium oxide nano-layer in the formation of fretting corrosion products is unclear.
In theory, the formation of fretting corrosion particles is assisted by the aggressive crevice corrosion environment. The low pH and
depleted oxygen will probably change the corrosion behavior at fretting interface and result in a different form of particles.
Biological Reactions to Corrosion Products

As described previously, the corrosion process on implant surfaces releases products in the form of ions or particulate materials.
These products have been associated with adverse reactions that affect the prognosis and general health of patients with metal
implants. The interaction of these products with the tissues and cells, as well as the effects of these products on patient’s health,
is discussed in this section.
Adverse local tissue reaction to metal implants

Adverse local tissue reactions (ALTRs) are aseptic lesions that can arise in the periprosthetic tissue of patients with metallic hip pros-
theses. Despite their benign character, they can be locally aggressive causing the destruction of muscle and ligaments, and pressure
effects on veins and nerves, affecting the prognosis of further clinical solutions (Daniel et al., 2012; Almousa et al., 2013). ALTRs are
normally related to pain, swelling, and discomfort, but they can also be found in asymptomatic patients (Williams, 2011). The prev-
alence of ALTRs is unknown, but it is estimated that between 20% and 40% of patients with MoM implants and up to 5% of patients
with MoP implants could develop ALTRs (Daniel et al., 2012; Williams et al., 2011; Feeley and Voreacos, 2013; Meier, 2013). ALTRs
are the main reason of failure of metal-on-metal implants, and a significant number of MoP systems, estimating a failure rate of 9.7/
1000 patient-years due to ALTRs for MoM systems, and < 1/1000 for MoP systems, with a peak of occurrence between 5 and 7 years
after implantation (The NJR Editorial Board, 2016). Due to the high prevalence of these lesions in MoM articulation, it was spec-
ulated that the wear from the bearing surfaces was the etiologic factor that triggers ALTRs, but the presence of similar lesions in MoP
systems suggests that corrosion on different implant surfaces, especially in the modular junctions, could be involved in the devel-
opment of ALTRs (Cooper et al., 2013).
Histopathology of ALTRs
Histologically, ALTRs have a layered structure consisting of three basic layers: an ulcerated synovial surface that may or may not be
covered by fibrin deposition with sub-superficial necrosis, a second layer characterized by the infiltration of macrophages in a dense
connective matrix, and externally an area with intense mononuclear infiltration that can vary from diffuse cellular infiltration to
large perivascular aggregates of lymphocytes (Campbell et al., 2010; Mahendra et al., 2009; Natu et al., 2012). These characteristics
vary with the severity of the lesions: lesions with limited necrosis usually present abundant macrophages and minimal lymphocytes.
Moderate necrosis is associated with a mixed infiltration of macrophages and T lymphocytes, which are a mix of C4 þ and CD8 þ
lymphocytes. In cases of extensive necrosis, the number of macrophages is reduced and lymphocytes are abundant forming highly
organized tertiary lymphoid organs in the periphery of the tissues with highly endothelial veins, and T and B cells that can be orga-
nized in germinate centers (Mahendra et al., 2009; Mittal et al., 2013) (Fig. 5).
The role of metals and their corrosion products in the etiology of ALTRs is supported by the facts that ALTRs have been observed
in different implant designs (MoM THRs and resurfacing, and MoP), and associated with elevated concentrations of metals in serum
and synovial fluid, and evident corrosion debris on implant surfaces and in the tissues (Kwon et al., 2014; Matharu et al., 2016).
Based on previous histological description, it is evident that the pathogenesis of ALTRs involves the development of chronic inflam-
mation (of which lymphocytes and macrophages are the main characteristics), with elevated concentration of proinflammatory
cytokines (Kolatat et al., 2015; Eltit et al., 2017), which lead to a non-resolved inflammatory episode. A type IV hypersensitivity
was proposed as the mechanism of activation of the immune system in ALTRs (Huber et al., 2009; Willert, 2005). However, this
hypothesis was not supported in other reports (Granchi et al., 2012; Kwon et al., 2010b). The extensive necrosis of the capsule
in ALTRs could be a result of the direct effect of Co and Cr ions in the tissues. “In vitro” studies have demonstrated that Co can
induce apoptosis in different cell populations at a concentration of 6 mg/L, which is similar to the concentration of Co in synovial
fluid in some patients with ALTRs, although the majority of them present much lower values (Catelas et al., 2006; Baskey et al.,
2017; Granchi et al., 1998). Another possible mechanism is the degeneration and destruction of blood vessels of the affected tissue,
generated by the prolonged inflammation (Fig. 6). This phenomenon would generate a hypoxic environment triggering the massive
cell death observed in the ALTR tissues (Perino et al., 2014).
Roles of metal ions in tissues and fluids

The free implant surfaces are exposed to joint fluid, a transudate composed of water and hydrophilic molecules such as hyaluronic
acid, that provides the adequate lubrication to the sliding surfaces of the joint. The synovial fluid is contained in the joint capsule,
a dense connective tissue envelope, which synthesizes the synovial fluid. In the inflammatory environment, the properties of this
synovial fluid are expected to change, and therefore its lubricant capacity could be altered. The observed concentration of Co and Cr
in synovial fluid of patients without implants on their joint is around 1 mg/L and 4 mg/L, respectively. The concentration of these
metals can increase to a range between 50 and 100 mg/L in patients with implants in good conditions, and could reach between 500
and 10,000 mg/L in patients with failed implants (Eltit et al., 2017; Lass et al., 2014).
The values of cobalt and chromium in blood have been widely studied in order to monitor the performance of hip implants.
Blood has the advantage of being easy to obtain with low risk and minimum equipment. Its analysis is based on the rationale
that the metals released from the metal implants will drain to the blood vessels in the surrounding tissues and will be found in
circulation. The UK Medicines and Healthcare Regulatory Agency has issued a blood cobalt and chromium guidance value of
7 mg/L to identify MOM hip implant patients who may need further surveillance on excessive implant wear. Similarly, in a statement
of the American Association of Hip and Knee Surgeons, the American Academy of Orthopedic Surgeons, and the Hip Society, these
institutions recommend the systematic evaluation of patients with dual modular neck systems, to include the analysis of serum ion
levels together with clinical and radiological evaluation to optimize the management of these patients (Kwon et al., 2014).
Although the serum content of cobalt and chromium has been proposed as a monitoring tool for the performance of metal
implants, it has been demonstrated that their analysis has only a modest sensitivity and specificity (60%) in identifying patients
with ALTRs in MoM (Matharu et al., 2016; Malek et al., 2012), and no standards have been established for other types of bearings.
Interestingly, the ratio of Co/Cr changes depending on the bearing surfaces. In patients with metal-on-metal hip resurfacings, in
which there is a bearing surface subject to tribocorrosion but not a modular taper subject to fretting corrosion, the concentration of
chromium in blood is slightly higher than cobalt (1.2–2.5-fold), in patients with metal-on-metal THRs, in which both events are
present (metal-bearing surfaces and modular joint), the concentration of cobalt is higher than chromium (1.5–2.5 fold), while in
the MoP THRs, in which there is no metal-to-metal-bearing surfaces, but there exist modular interfaces, the concentration of cobalt
Fig. 5 Histology of ALTRs: (A) ALTR with low degree of necrosis, synovial epithelium is absent in the joint surface (arrows), but sub-superficial
necrosis is localized within 100 mm. Abundant macrophages infiltrate (asterisks) and diffuse distribution of lymphocytes is seen (arrowheads). (B)
ALTR with extensive necrosis, the synovial surface is covered by fibrin (arrows), and the sub-superficial necrosis is evident. Scarce macrophages are
observed infiltrating the tissue (asterisks) and lymphocytes organize in densely packed structures (arrowheads).
is 2.5–5 times higher than chromium, revealing that the presence of cobalt in blood is mainly associated with the corrosion at the
modular taper junctions of the implants, while the higher chromium concentration in blood is associated to surface wear in the
articulations (Lavigne et al., 2011; Garbuz et al., 2010; Vendittoli et al., 2007).
The mechanisms of cell damage by hexavalent chromium (Cr 6 þ) have been largely studied due to its association with cancer. Cr
6þ penetrates cells through sulfate channels and is intracellularly reduced to Cr 3 þ that combines with DNA and proteins, gener-
ating mutations and reducing the cell functionality. In peri-implant tissues, Cr 6þ is not observed, and Cr 3þ (the observed form)
has no carcinogenic effect due to its difficulty to penetrate the cell membrane. However, in vitro studies have demonstrated that it
can induce cell death in high concentrations due to the induction of hypoxia in the cells exposed to Co (Vengellur and LaPres, 2004;
Nyga et al., 2015; Samelko et al., 2013). There is no higher risk of cancer in patients with Co/Cr alloy implants (Smith et al., 2012),
and Cr 3 þ is not considered a health hazard (Eastmond et al., 2008).
Cobalt ions have the capability of penetrating cells by using different ionic channels in the cell membrane. It is an important
cofactor of vitamin B12 (cobalamin), which has an important role in the metabolism of the nervous systems and the formation
of red blood cells. However, the excess of cobalt in cells has deleterious effects through affecting cell metabolism and DNA repli-
cation and repair machinery, due to the binding of cobalt ions to specific proteins and affecting their functionality and consequently
the cell metabolism as it has been demonstrated through affinity chromatography (Kwon et al., 2009; Scharf et al., 2014).
Fig. 6 Vascular changes in ALTRs: (A) initial changes in the blood vessels: intense lymphocyte infiltration of the vessel walls (arrows); however, the
lumen has normal appearance (asterisk). (B) The endothelium presents a cubic shape (arrows) (should be squamous), and the vessel’s lumen is
observed compressed with an amorphous content (asterisk).
Biological effect of metal particles

As was described previously, the particles observed in the tissues correspond mainly to wear particles, histologically observed as dark
spots of nano-micron size that tend to be accumulated by the process of phagocytosis carried out by the macrophages, and corrosion
particles that are histologically observed as large (tens to hundreds of microns) translucent structures under the light microscope
(Ricciardi et al., 2016) (Fig. 7).
The negative effects of particles in the tissues could be due to the presence of particles itself, or to the leaching of ions from the
particle to the surrounding tissues. It has been demonstrated that under exposure of similar amount of particulate material, cells
are more susceptible to damage under nano-sized particles than micron-size particles, presumably due to the increased surface
area for metal leaching from the smaller particles (Papageorgiou et al., 2007). The particles as a foreign structure can trigger
a foreign body response, in which cells capable of performing phagocytosis (mostly macrophages) are recruited in the zone aim-
ing to destroy the foreign agent. In the tissues, the macrophages are usually seen loaded with small metal particles contained in
vesicles in their cytoplasm (Figs. 7 and 8), and “in vitro” studies have shown that CoCr particles activate macrophages increasing
the secretion of cytokines (Posada et al., 2015). Due to their incapability of degrading these particles, they synthesize a higher
amount of enzymes and oxidative molecules (peroxides and superoxides), generating an elevated oxidative stress that led to
cell damage, and accelerating the ion leaching from the metal particles, especially of cobalt, thus increasing the local concentra-
tion of this ion at the time that a Cr-rich core remains insoluble in the cytoplasm of the macrophages (Goode et al., 2012; Xia
et al., 2011).
Large particles of metal oxides, on the other hand, are much bigger than a single macrophage. To be able to phagocyte them,
multiple macrophages fuse, generating giant multinucleated cells, where the multi-nuclei correspond to various individual nuclei
of the fused macrophages (Fig. 7).
Although above arguments can explain the final effects of the development of ALTRs, the relationship between metal particles
and ALTRs has not been proved. In vitro studies have tried to link the development of immune response to metal particles of CoCr
alloy, demonstrating by cell culture that they can induce phagocytosis in monocyte-derived cells, and different levels of cell damage
Fig. 7 Phagocytosis of corrosion products in ATLRs. (A) Phagocytosis of small metallic particles by macrophages. The metallic particles can be
seen in the cytoplasm of macrophages (arrows). (B) Large corrosion products (asterisks) induce the formation of giant multinucleated cells
(arrows). The giant multinucleated cells are the resultant of the fusion of macrophages, and perform the phagocytosis of large particles as seen in
the image.
Fig. 8 Metal particles in intracellular vesicles. TEM of macrophages with metal particles in endosomic compartments (Scharf et al., 2014).
in different cell populations (Samelko et al., 2013; Papageorgiou et al., 2007); however, histopathology analysis has failed in
demonstrating association between the amount of particles and the development and/or severity of ALTRs (Ebramzadeh et al.,
2014).
New Biomaterial Designs
Although Ti alloys and CoCr alloys discussed in this article have been successfully used in hip implants, there are still concerns over
their clinic performance against corrosion, specially wear-assisted corrosion. Hence, efforts are being made to improve their
mechanical properties and corrosion resistance.
Ti alloys: The most commonly used Ti alloy in orthopedic implants has been Ti–6Al–4V (Rack and Qazi, 2006). Ti–6Al–4V is
classified as a a þ b alloy for its microstructure, in which Al acts as an a stabilizer while V as a b phase stabilizer. With increasing
concerns on the biological response of potentially toxic vanadium (Maurer et al., 1994), niobium and iron are used to substitute
vanadium, leading to the development and introduction of Ti–6Al–7Nb and Ti–5Al–2.5Fe alloys (Semlitsch et al., 1985, 1992;
Niinomi, 1998). And the replacement doesn’t compromise the alloy’s electrochemical and corrosion behavior in SPS (Choubey
et al., 2004). Both show excellent biocompatibility and similar mechanical properties to Ti–6Al–4V. Another concern of the Ti
a þ b alloy is its high modulus (110 GPa) compared to bone (20–30 GPa). This creates stress shielding effect due to high stiffness
of Ti alloy, leading to bone resorption. Hence, metastable b titanium alloys such as Ti–15Mo (Zardiackas et al., 1996), Ti–12Mo–
6Zr–2Fe (TMZFTM), Ti–15Mo–3Nb–0.3O (21SRx) (Wang, 1996), Ti–13Nb–13Zr (Davidson et al., 1994) and Ti–Nb–Zr–Ta alloys
(TNZT) (Nag et al., 2005) have been developed to lower the elastic modulus. At the same time, these b titanium alloys exclude Al,
which eliminates the possible risks of Al-associated toxicity. The most challenging application of Ti alloys is their use at interfaces
involving wear. Ti alloys have poor wear resistance and the friction at the interface leads to a faster degradation of the alloy, which
limit their application in articulating surfaces. Attempts have been made on the surface modification of Ti alloys in order to improve
their wear resistance. Different hard coatings such as TiN, ZrN, and amorphous carbon have been applied and tested on Ti alloy
surfaces (Hendry and Pilliar, 2001). However, retrieved analyses revealed the presence of wear debris and delaminated asperities
of TiN-coated Ti–6Al–4V articulating surface, pointing to a weak coating-substrate interface (Lass et al., 2014). To improve the inter-
facial strength, new processing technologies such as PIRAC (Powder Immersion Reaction Assisted Coating) technology were used to
reduce the residual stresses at the interface (Shenhar et al., 2000). Studies have shown that TiN coating prepared with PIRAC was still
intact and the wear rate of the polyethylene cups was significantly lower after up to 4 million cycles wear test (Gutmanas and
Gotman, 2004). Another way to improve the interface strength is to make an interface with gradual change in composition. Up
to 86% CoCr alloy could be obtained on the surface of Ti–6Al–4V alloy and this microstructure increased surface hardness and
reduced the wear rated in wear test (Vamsi Krishna et al., 2008; Nychka et al., 2011).
CoCr alloys: CoCr alloys are mainly used at the articulating surfaces because of their high wear resistance. However, there are
concerns over ALTRs associated with metal release from the corrosion of CoCr alloys. To improve biocompatibility, toxic elements
such as Ni used in CoCr alloy were reduced (Sonofuchi et al., 2016). Another attempt is to apply a hard and biocompatible coating
on the surface of CoCr alloy to prevent toxic elements from releasing and to retain high wear resistance at the same time. Different
engineered CrN, TiN, DLC (diamond like carbon), and tantalum carbide coatings and their combinations on CoCr alloys have been
studied (Ludema et al., 2011; Türkan et al., 2006; Pham et al., 2011; Balagna et al., 2012; Liu et al., 2013). Coating delamination
remains to be a key issue to be solved (Ludema et al., 2011; Liu et al., 2013).
An alternative to CoCr alloy used in bearing surface is ceramics such as alumina and zirconia. Although ceramics have superior
wear resistance and no risk of toxic ion release, their brittle nature makes them vulnerable to fracture (Semlitsch and Willert, 1997;
Lerouge et al., 1997; Fritsch and Gleitz, 1996). A promising design is a composite material combining advantages of the superior
wear resistance in ceramics with high toughness in metals. One example is Oxinium (Smith & Nephew, Memphis, Tennessee),
a zirconium–niobium alloy with around 5 mm oxidized layer formed on the surface by thermal diffusion (Sonntag et al., 2012).
The metal substrate is tough, and the hard oxide surface has similar wear resistance to ceramics. Oxinium has lower wear rate
than CoCr alloy with similar fracture toughness (Good et al., 2003; Bourne et al., 2005). Another example is the zirconia reinforced
with biocompatible metals such as Nb and Ta (Bartolomé et al., 2016; Smirnov et al., 2017). This ceramic–metal composite exhibits
excellent wear resistance, toughness, and biocompatibility (Bartolomé et al., 2007, 2016; Rodriguez-Suarez et al., 2012; Gutiérrez-
González et al., 2012).
Future Perspectives
Corrosion-induced mechanical and biological failures affect the clinical performance of current orthopedic implants, but also
present opportunities for future biomaterial development. The most commonly used metallic alloys used in implants today
were developed more than half century ago initially for other engineering applications. Alloys were not specifically designed to
take biocompatibility into consideration. However, such a situation is about to change. Today’s biomedical implants are sitting
at the interface between two rapidly growing fields: biology and materials science (Ratner et al., 2004; Williams, 2014). Progress
in biology and medicine will provide a clear picture on the structure and functions of various tissues down to cellular and molecular
levels. In-depth understanding of tissue-biomaterial interaction will guide the design of new alloys and surfaces. This is further
strengthened by promising development in materials science and engineering, as represented by the Materials Genome Initiative
launched by the US Government in 2011 (National Science and Technology Council, 2014). Past breakthroughs in material
modeling, theory, and data mining make it possible to significantly accelerate the discovery and development of advanced materials
including new biomaterials.
References
Affatato, S., Traina, F., Ruggeri, O., & Toni, A. (2011). Wear of metal-on-metal hip bearings: Metallurgical considerations after hip simulator studies. The International Journal of
Artificial Organs, 34, 1155–1164.
Almousa, S. A., Greidanus, N. V., Masri, B. A., Duncan, C. P., & Garbuz, D. S. (2013). The natural history of inflammatory pseudotumors in asymptomatic patients after metal-on-
metal hip arthroplasty. Clinical Orthopaedics and Related Research, 471, 3814–3821.
Antunes, R. A., & de Oliveira, M. C. L. (2012). Corrosion fatigue of biomedical metallic alloys: Mechanisms and mitigation. Acta Biomaterialia, 8, 937–962.
ASTM. (2014a). ASTM F799 Standard Specification for Cobalt-28Chromium-6 Molybdenum Alloy Forgings for Surgical Implants.
ASTM. (2014b). ASTM F75 Standard Specification for Cobalt-28 Chromium-6 Molybdenum Alloy Castings and Casting Alloy for Surgical Implants (pp. 1–4).
Atwood, S. A., Patten, E. W., Bozic, K. J., Pruitt, L. A., & Ries, M. D. (2010). Corrosion-induced fracture of a double-modular hip prosthesis: A case report. The Journal of Bone and
Joint Surgery. American Volume, 92, 1522–1525.
Balagna, C., Faga, M. G., & Spriano, S. (2012). Tantalum-based multilayer coating on cobalt alloys in total hip and knee replacement. Materials Science and Engineering: C, 32,
887–895.
Balla, V. K., Bodhak, S., Bose, S., & Bandyopadhyay, A. (2010). Porous tantalum structures for bone implants: Fabrication, mechanical and in vitro biological properties. Acta
Biomaterialia, 6, 3349–3359.
Bartolomé, J. F., Gutierrez-Gonzalez, C. F., Pecharroman, C., & Moya, J. S. (2007). Synergistic toughening mechanism in 3Y-TZP/Nb composites. Acta Materialia, 55, 5924–5933.
Bartolomé, J. F., Moya, J. S., Couceiro, R., Gutiérrez-González, C. F., Guitián, F., & Martinez-Insua, A. (2016). In Vitro and in vivo evaluation of a new zirconia/niobium biocermet for
hard tissue replacement. Biomaterials, 76, 313–320.
Baskey, S. J., Lehoux, E. A., & Catelas, I. (2017). Effects of cobalt and chromium ions on lymphocyte migration. Journal of Orthopaedic Research, 35(4), 916–924.
Bettini, E., Eriksson, T., Boström, M., Leygraf, C., & Pan, J. (2011). Influence of metal carbides on dissolution behavior of biomedical CoCrMo alloy: SEM, TEM and AFM studies.
Electrochimica Acta, 56, 9413–9419.
Bettini, E., Leygraf, C., Lin, C., Liu, P., & Pan, J. (2012). Influence of grain boundaries on dissolution behavior of a biomedical CoCrMo alloy: In-situ electrochemical-optical, AFM and
SEM/TEM studies. Journal of the Electrochemical Society, 159, C422–C427.
Bitounis, D., Pourchez, J., Forest, V., Boudard, D., Cottier, M., & Klein, J.-P. (2016). Detection and analysis of nanoparticles in patients: A critical review of the status quo of clinical
nanotoxicology. Biomaterials, 76, 302–312.
Black, J., & Hastings, G. (Eds.). (1998). Handbook of Biomaterial Properties. Springer Science & Business Media.
Blau, P. J., Shaffer, S. J., Geringer, J., Forest, B., & Combrade, P. (2005). Fretting-corrosion of materials used as orthopaedic implants. Wear, 259, 943–951.
Bobyn, J. D., Stackpool, G. J., Hacking, S. A., Tanzer, M., & Krygier, J. J. (1999). Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial.
The Journal of Bone and Joint Surgery. British Volume, 81–B, 907–914.
Bombara, G., & Cavallini, M. (1977). Stress corrosion cracking of bone implants. Corrosion Science, 17, 77–85.
Bourne, R. B., Barrack, R., Rorabeck, C. H., Salehi, A., & Good, V. (2005). Arthroplasty options for the young patient: Oxinium on cross-linked polyethylene. Clinical Orthopaedics
and Related Research, 441, 159–167.
Bozic, K. J., Kurtz, S., Lau, E., Ong, K., Chiu, V., Vail, T. P., Rubash, H. E., & Berry, D. J. (2009). The epidemiology of bearing surface usage in total hip arthroplasty in the United
States. The Journal of Bone and Joint Surgery. American Volume, 91, 1614–1620.
Brooks, C. R. (1982). Heat treatment, structure, and properties of nonferrous alloys. Metals Park, Ohio: American Society for Metals.
Brown, S. A., Flemming, C. A., Kawalec, J. S., Placko, H. E., Vassaux, C., Merritt, K., Payer, J. H., & Kraay, M. J. (1995). Fretting corrosion accelerates crevice corrosion of modular
hip tapers. Journal of Applied Biomaterials, 6, 19–26.
Bryant, M., Ward, M., Farrar, R., Freeman, R., Brummitt, K., Nolan, J., & Neville, A. (2013). Fretting corrosion characteristics of polished collarless tapered stems in a simulated
biological environment. Tribology International, 65, 105–112.
Bryant, M., Ward, M., Farrar, R., Freeman, R., Brummitt, K., Nolan, J., & Neville, A. (2014). Characterisation of the surface topography, tomography and chemistry of fretting
corrosion product found on retrieved polished femoral stems. Journal of the Mechanical Behavior of Biomedical Materials, 32, 321–334.
Bundy, K. J., Marek, M., & Hochman, R. F. (1983). In vivo and in vitro studies of the stress-corrosion cracking behavior of surgical implant alloys. Journal of Biomedical Materials
Research, 17, 467–487.
Büscher, R., & Fischer, A. (2005). The pathways of dynamic recrystallization in all-metal hip joints. Wear, 259, 887–897.
Büscher, R., Täger, G., Dudzinski, W., Gleising, B., Wimmer, M. A., & Fischer, A. (2005). Subsurface microstructure of metal-on-metal hip joints and its relationship to wear particle
generation. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 72, 206–214.
Campbell, P., Ebramzadeh, E., Nelson, S., Takamura, K., De Smet, K., & Amstutz, H. C. (2010). Histological features of pseudotumor-like tissues from metal-on-metal hips. Clinical
Orthopaedics and Related Research, 468, 2321–2327.
Canadian Institute for Health Information Hip and Knee Replacements in Canada: Canadian Joint Replacement Registry 2015 Annual Report; 2015.
Catelas, I., Medley, J. B., Campbell, P. A., Huk, O. L., & Bobyn, J. D. (2004). Comparison of in vitro with in vivo characteristics of wear particles from metal–metal hip implants.
Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 70, 167–178.
Catelas, I., Campbell, P. A., Bobyn, J. D., Medley, J. B., & Huk, O. L. (2006). Wear particles from metal-on-metal total hip replacements: Effects of implant design and implantation
time. Proceedings of the Institution of Mechanical Engineers. Part H, 220, 195–208.
Catelas, I., Wimmer, M. A., & Utzschneider, S. (2011). Polyethylene and metal wear particles: Characteristics and biological effects. Seminars in Immunopathology, 33, 257–271.
Charnley, J. (1975). Fracture of femoral prostheses in total hip replacement. A clinical study. Clinical Orthopaedics and Related Research, 111, 105–120.
Choubey, A., Balasubramaniam, R., & Basu, B. (2004). Effect of replacement of V by Nb and Fe on the electrochemical and corrosion behavior of Ti–6Al–4V in simulated
physiological environment. Journal of Alloys and Compounds, 381, 288–294.
Cicero, S., Gutierrez-Solana, F., Alvarez, J., & Sanchez, L. (2007). Failure analysis of a hip implant by using the FITNET fitness for service procedure. Engineering Fracture
Mechanics, 74, 688–702.
Collier, J., Surprenant, V., Jensen, R., Mayor, M., & Surprenant, H. (1992). Corrosion between the components of modular femoral hip prostheses. The Journal of Bone and Joint
Surgery. British Volume, 74–B, 511–517.
Contu, F., Elsener, B., Böhni, H., & Böhni, H. (2003). Electrochemical behavior of CoCrMo alloy in the active state in acidic and alkaline buffered solutions. Journal of the
Electrochemical Society, 150, B419.
Cook, S., Barrack, R., & Clemow, A. (1994). Corrosion and wear at the modular interface of uncemented femoral stems. The Journal of Bone and Joint Surgery. British Volume, 76–
B, 68–72.
Cooper, H. J., Della Valle, C. J., Berger, R. A., Tetreault, M., Paprosky, W. G., Sporer, S. M., & Jacobs, J. J. (2012). Corrosion at the head–neck taper as a cause for adverse local
tissue reactions after total hip arthroplasty. The Journal of Bone and Joint Surgery. American Volume, 94, 1655–1661.
Cooper, H. J., Urban, R. M., Wixson, R. L., Meneghini, R. M., & Jacobs, J. J. (2013). Adverse local tissue reaction arising from corrosion at the femoral neck-body junction in a dual-
taper stem with a cobalt-chromium modular neck. The Journal of Bone and Joint Surgery. American Volume, 95, 865.
Cummings, N. A., & Nordby, G. L. (1966). Measurement of synovial fluid pH in normal and arthritic knees. Arthritis and Rheumatism, 9, 47–56.
Daniel, J., Holland, J., Quigley, L., Sprague, S., & Bhandari, M. (2012). Pseudotumors associated with total hip arthroplasty. The Journal of Bone and Joint Surgery. American
Volume, 94, 86–93.
Davidson, J. A., Mishra, A. K., Kovacs, P., & Poggie, R. A. (1994). New surface-hardened, low-modulus, corrosion-resistant Ti–13Nb–13Zr alloy for total hip arthroplasty. Bio-
medical Materials and Engineering, 4, 231–243.
Davis, J. (1990). Properties and selection: Nonferrous alloys and special-purpose materials. Materials Park, OH: ASM International.
Davis, J. R. (Ed.). (2003). Handbook of materials for medical devices. Materials Park, OH: ASM International.
Della Valle, A. G., Becksaç, B., Anderson, J., Wright, T., Nestor, B., Pellicci, P. M., & Salvati, E. A. (2005). Late fatigue fracture of a modern cemented forged cobalt chrome stem for
total hip arthroplasty. The Journal of Arthroplasty, 20, 1084–1088.
Doorn, P. F., Campbell, P. A., & Amstutz, H. C. (1996). Metal versus polyethylene wear particles in total hip replacements. A review. Clinical Orthopaedics and Related Research,
329, S206–16.
Doorn, P. F., Campbell, P. A., Worrall, J., Benya, P. D., McKellop, H. A., & Amstutz, H. C. (1998). Metal wear particle characterization from metal on metal total hip replacements:
Transmission electron microscopy study of periprosthetic tissues and isolated particles. Journal of Biomedical Materials Research, 42, 103–111.
Dunbar, M. J. (2010). The proximal modular neck in THA: A bridge too far: Affirms. Orthopedics, 33, 640.
Eastmond, D. A., Macgregor, J. T., & Slesinski, R. S. (2008). Trivalent chromium: Assessing the genotoxic risk of an essential trace element and widely used human and animal
nutritional supplement. Critical Reviews in Toxicology, 38, 173–190.
Ebramzadeh, E., Campbell, P., Tan, T. L., Nelson, S. D., & Sangiorgio, S. N. (2014). Can wear explain the histological variation around metal-on-metal total hips? Clinical
Ellman, M. B., & Levine, B. R. (2013). Fracture of the modular femoral neck component in total hip arthroplasty. The Journal of Arthroplasty, 28, 196.e1–5.
Eltit, F., Assiri, A., Garbuz, D., Duncan, C., Masri, B., Greidanus, N., Bell, R., Sharma, M., Cox, M., & Wang, R. (2017). Adverse reactions to metal on polyethylene implants: Highly
destructive lesions related to elevated concentration of cobalt and chromium in synovial fluid. Journal of Biomedical Materials Research. Part A, 105(7), 1876–1886.
Schijve, J. (Ed.). (2009). Fatigue of Structures and Materials. Dordrecht: Springer Netherlands.
Feeley, J., & Voreacos, D. (2013). J&J study predicted 37% hip-failure rate. Bloom.Bus. New York: Bloomberg News
Firkins, P. J., Tipper, J. L., Saadatzadeh, M. R., Ingham, E., Stone, M. H., Farrar, R., & Fisher, J. (2001). Quantitative analysis of wear and wear debris from metal-on-metal hip
prostheses tested in a physiological hip joint simulator. Bio-medical Materials and Engineering, 11, 143–157.
Fritsch, E. W., & Gleitz, M. (1996). Ceramic femoral head fractures in total hip arthroplasty. Clinical Orthopaedics and Related Research, 328, 129–136.
Galante, J. O. (1980). Causes of fractures of the femoral component in total hip replacement. The Journal of Bone and Joint Surgery. American Volume, 62, 670–673.
Garbuz, D. S., Tanzer, M., Greidanus, N. V., Masri, B. A., & Duncan, C. P. (2010). The John Charnley award: Metal-on-metal hip resurfacing versus large-diameter head metal-on-
metal total hip arthroplasty: A randomized clinical trial. Clinical Orthopaedics and Related Research, 468, 318–325.
Gilbert, J. L., & Mali, S. A. (2012). Medical implant corrosion: Electrochemistry at metallic biomaterial surfaces. In Degradation of implant materials (pp. 1–28). New York, NY:
Springer New York.
Gilbert, J. L., Buckley, C. A., & Jacobs, J. J. (1993). In vivo corrosion of modular hip prosthesis components in mixed and similar metal combinations. The effect of crevice, stress,
motion, and alloy coupling. Journal of Biomedical Materials Research, 27, 1533–1544.
Gilbert, J. L., Buckley, C. A., Jacobs, J. J., Bertin, K. C., & Zernich, M. R. (1994). Intergranular corrosion-fatigue failure of cobalt-alloy femoral stems. A failure analysis of two
implants. The Journal of Bone and Joint Surgery. American Volume, 76, 110–115.
Gilbert, J. L., Mali, S., Urban, R. M., Silverton, C. D., & Jacobs, J. J. (2012). In vivo oxide-induced stress corrosion cracking of Ti–6Al–4V in a neck-stem modular taper: Emergent
behavior in a new mechanism of in vivo corrosion. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 100, 584–594.
Gilbert, J. L., Sivan, S., Liu, Y., Kocagöz, S. B., Arnholt, C. M., & Kurtz, S. M. (2015). Direct in vivo inflammatory cell-induced corrosion of CoCrMo alloy orthopedic implant surfaces.
Journal of Biomedical Materials Research. Part A, 103, 211–223.
Goldberg, J. R., & Gilbert, J. L. (1997). Electrochemical response of CoCrMo to high-speed fracture of its metal oxide using an electrochemical scratch test method. Journal of
Good, V., Ries, M., Barrack, R. L., Widding, K., Hunter, G., & Heuer, D. (2003). Reduced wear with oxidized zirconium femoral heads. Journal of Bone and Joint Surgery, 85,
105–110.
Goode, A. E., Perkins, J. M., Sandison, A., Karunakaran, C., Cheng, H., Wall, D., Skinner, J. A., Hart, A. J., Porter, A. E., McComb, D. W., & Ryan, M. P. (2012). Chemical speciation
of nanoparticles surrounding metal-on-metal hips. Chemical Communications, 48, 8335–8337.
Granchi, D., Cenni, E., Ciapetti, G., Savarino, L., Stea, S., Gamberini, S., Gori, A., & Pizzoferrato, A. (1998). Cell death induced by metal ions: Necrosis or apoptosis? Journal of
Granchi, D., Cenni, E., Giunti, A., & Baldini, N. (2012). Metal hypersensitivity testing in patients undergoing joint replacement: A systematic review. Journal of Bone and Joint
Surgery. British Volume (London), 94, 1126–1134.
Grupp, T. M., Weik, T., Bloemer, W., & Knaebel, H.-P. (2010). Modular titanium alloy neck adapter failures in hip replacementdFailure mode analysis and influence of implant
material. BMC Musculoskeletal Disorders, 11, 3.
Gurappa, I. (2002). Characterization of different materials for corrosion resistance under simulated body fluid conditions. Materials Characterization, 49, 73–79.
Gurrappa, I. (2003). Characterization of titanium alloy Ti–6Al–4V for chemical, marine and industrial applications. Materials Characterization, 51, 131–139.
Gutiérrez-González, C. F., Smirnov, A., & Bartolomé, J. F. (2012). Aging effect on the tribological behavior of a novel 3Y-TZP/Nb biocomposite against ultra high molecular weight
polyethylene. Journal of the American Ceramic Society, 95, 851–854.
Gutmanas, E. Y., & Gotman, I. (2004). PIRAC Ti nitride coated Ti–6Al–4V head against UHMWPE acetabular cup–hip wear simulator study. Journal of Materials Science. Materials in
Medicine, 15, 327–330.
Hallab, N., & Jacobs, J. (2003). Orthopedic implant fretting corrosion. Corrosion Reviews, 21, 183–214.
Hallab, N. J., Skipor, A., & Jacobs, J. J. (2003). Interfacial kinetics of titanium- and cobalt-based implant alloys in human serum: Metal release and biofilm formation. Journal of
Biomedical Materials Research. Part A, 65, 311–318.
Hampton, S. J., Andriacchi, T. P., & Galante, J. O. (1980). Three dimensional stress analysis of the femoral stem of a total hip prosthesis. Journal of Biomechanics, 13, 443–448.
Hanawa, T. (2004). Metal ion release from metal implants. Materials Science and Engineering: C, 24, 745–752.
Hanawa, T., Hiromoto, S., & Asami, K. (2001). Characterization of the surface oxide film of a Co–Cr–Mo alloy after being located in quasi-biological environments using XPS. Applied
Surface Science, 183, 68–75.
Hart, A. J. A., Quinn, P. P. D., Sampson, B., Sandison, A., Atkinson, K. D., Skinner, J.a., Powell, J. J., & Mosselmans, J. F. W. (2010). The chemical form of metallic debris in
tissues surrounding metal-on-metal hips with unexplained failure. Acta Biomaterialia, 6, 4439–4446.
Hart, A. J., Quinn, P. D., Lali, F., Sampson, B., Skinner, J.a., Powell, J. J., Nolan, J., Tucker, K., Donell, S., Flanagan, A., & Mosselmans, J. F. W. (2012). Cobalt from metal-on-
metal hip replacements may be the clinically relevant active agent responsible for periprosthetic tissue reactions. Acta Biomaterialia, 8, 3865–3873.
Haynes, W. M. (Ed.). (2004). Handbook of chemistry and physics (85th edn.). Boca Raton, FL: CRC Press.
Healthy Canadians Metal-on-Metal Hip ImplantsdInformation for Orthopaedic Surgeons Regarding Patient Management Following SurgerydFor Health Professionals 2012.
Hedberg, Y., & Odnevall Wallinder, I. (2014). Metal release and speciation of released chromium from a biomedical CoCrMo alloy into simulated physiologically relevant solutions.
Hendry, J. A., & Pilliar, R. M. (2001). The fretting corrosion resistance of PVD surface-modified orthopedic implant alloys. Journal of Biomedical Materials Research, 58, 156–166.
Hoeppner, D. W., & Chandrasekaran, V. (1994). Fretting in orthopaedic implants: A review. Wear, 173, 189–197.
Hsu, A. R., Gross, C. E., & Levine, B. R. (2012). Pseudotumor from modular neck corrosion after ceramic-on-polyethylene total hip arthroplasty. American Journal of Orthopedics
(Belle Mead, N.J.), 41, 422–426.
Huber, M., Reinisch, G., Trettenhahn, G., Zweymüller, K., & Lintner, F. (2009). Presence of corrosion products and hypersensitivity-associated reactions in periprosthetic tissue after
aseptic loosening of total hip replacements with metal bearing surfaces. Acta Biomaterialia, 5, 172–180.
Huot Carlson, J. C.; Van Citters, D. W.; Currier, J. H.; Bryant, A. M.; Mayor, M. B.; Collier, J. P. Femoral stem fracture and in vivo corrosion of retrieved modular femoral hips. The
Journal of Arthroplasty 2012, 27, 1389.e1–1396.e1.
Igual Munoz, A., Schwiesau, J., Jolles, B. M., & Mischler, S. (2015). In vivo electrochemical corrosion study of a CoCrMo biomedical alloy in human synovial fluids. Acta Bio-
materialia, 21, 228–236.
Illgen, R. L., Forsythe, T. M., Pike, J. W., Laurent, M. P., & Blanchard, C. R. (2008). Highly crosslinked vs conventional polyethylene particles-an in vitro comparison of biologic
activities. The Journal of Arthroplasty, 23, 721–731.
Ishaque, B. A., Stürz, H., & Basad, E. (2011). Fatigue fracture of a short stem hip replacement: A failure analysis with electron microscopy and review of the literature. The Journal of
Arthroplasty, 26, 665.e17–20.
Kolatat, K., Perino, G., Wilner, G., Kaplowitz, E., Ricciardi, B. F., Boettner, F., Westrich, G. H., Jerabek, S. A., Goldring, S. R., & Purdue, P. E. (2015). Adverse local tissue reaction
(ALTR) associated with corrosion products in metal-on-metal and dual modular neck total hip replacements is associated with upregulation of interferon gamma-mediated
chemokine signaling. Journal of Orthopaedic Research, 33, 1487–1497.
Konttinen, Y. T., Takagi, M., Mandelin, J., Lassus, J., Salo, J., Ainola, M., Li, T. F., Virtanen, I., Liljestrom, M., Sakai, H., Kobayashi, Y., Sorsa, T., Lappalainen, R., Demulder, A., &
Santavirta, S. (2001). Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis. Journal of Bone and Mineral Research, 16, 1780–1786.
Krishnan, H., Krishnan, S. P., Blunn, G., Skinner, J. A., & Hart, A. J. (2013). Modular neck femoral stems. Bone and Joint Journal, 95–B, 1011–1021.
Kwon, Y.-M., Xia, Z., Glyn-Jones, S., Beard, D., Gill, H. S., & Murray, D. W. (2009). Dose-dependent cytotoxicity of clinically relevant cobalt nanoparticles and ions on macrophages
in vitro. Biomedical Materials, 4, 25018.
Kwon, Y.-M., Glyn-Jones, S., Simpson, D. J., Kamali, A., McLardy-Smith, P., Gill, H. S., & Murray, D. W. (2010a). Analysis of wear of retrieved metal-on-metal hip resurfacing
implants revised due to pseudotumours. The Journal of Bone and Joint Surgery. British Volume, 92, 356–361.
Kwon, Y.-M., Thomas, P., Summer, B., Pandit, H., Taylor, A., Beard, D., Murray, D. W., & Gill, H. S. (2010b). Lymphocyte proliferation responses in patients with pseudotumors
following metal-on-metal hip resurfacing arthroplasty. Journal of Orthopaedic Research, 28(4), 444–450.
Kwon, Y.-M., Lombardi, A. V., Jacobs, J. J., Fehring, T. K., Lewis, C. G., & Cabanela, M. E. (2014). Risk stratification algorithm for management of patients with metal-on-metal hip
arthroplasty. Journal of Bone & Joint SurgerydAmerican Volume, 96, e4.
Lakstein, D., Eliaz, N., Levi, O., Backstein, D., Kosashvili, Y., Safir, O., & Gross, A. E. (2011). Fracture of cementless femoral stems at the mid-stem junction in modular revision hip
arthroplasty systems. The Journal of Bone and Joint Surgery. American Volume, 93, 57–65.
Langton, D. J., Jameson, S. S., Joyce, T. J., Hallab, N. J., Natu, S., & Nargol, A. V. F. (2010). Early failure of metal-on-metal bearings in hip resurfacing and large-diameter total hip
replacement: A consequence of excess wear. Journal of Bone and Joint Surgery. British Volume (London), 92, 38–46.
Lass, R., Grübl, A., Kolb, A., Stelzeneder, D., Pilger, A., Kubista, B., Giurea, A., & Windhager, R. (2014). Comparison of synovial fluid, urine, and serum ion levels in metal-on-metal
total hip arthroplasty at a minimum follow-up of 18 years. Journal of Orthopaedic Research, 32, 1234–1240.
Lavigne, M., Belzile, E. L., Roy, A., Morin, F., Amzica, T., & Vendittoli, P.-A. (2011). Comparison of whole-blood metal ion levels in four types of metal-on-metal large-diameter
femoral head total hip arthroplasty: The potential influence of the adapter sleeve. The Journal of Bone and Joint Surgery. American Volume, 93(Suppl 2), 128–136.
Learmonth, I. D., Young, C., & Rorabeck, C. (2007). The operation of the century: Total hip replacement. Lancet, 370, 1508–1519.
Lee, E. W., & Kim, H. T. (2001). Early fatigue failures of cemented, forged, cobalt-chromium femoral stems at the neck-shoulder junction. The Journal of Arthroplasty, 16, 236–238.
Lerouge, S., Huk, O., Yahia, L., Witvoet, J., & Sedel, L. (1997). Ceramic-ceramic and metal-polyethylene total hip replacements: Comparison of pseudomembranes after loosening.
Journal of Bone and Joint Surgery. British Volume (London), 79, 135–139.
Levine, B. R., Sporer, S., Poggie, R. A., Della Valle, C. J., & Jacobs, J. J. (2006). Experimental and clinical performance of porous tantalum in orthopedic surgery. Biomaterials, 27,
4671–4681.
Lhotka, C., Szekeres, T., Steffan, I., Zhuber, K., & Zweymüller, K. (2003). Four-year study of cobalt and chromium blood levels in patients managed with two different metal-on-
metal total hip replacements. Journal of Orthopaedic Research, 21, 189–195.
Li, Y.-S., Wang, K., He, P., Huang, B. X., & Kovacs, P. (1999). Surface-enhanced Raman spectroelectrochemical studies of corrosion films on implant Co–Cr–Mo alloy in bio-
simulating solutions. Journal of Raman Specroscopy, 30, 97–103.
Liao, Y., Pourzal, R., Stemmer, P., Wimmer, M. A., Jacobs, J. J., Fischer, A., & Marks, L. D. (2012). New insights into hard phases of CoCrMo metal-on-metal hip replacements.
Journal of the Mechanical Behavior of Biomedical Materials, 12, 39–49.
Liu, Y., & Gilbert, J. L. (2017). The effect of simulated inflammatory conditions and pH on fretting corrosion of CoCrMo alloy surfaces. Wear, 390–391, 302–311.
Liu, J., Wang, X., Wu, B. J., Zhang, T. F., Leng, Y. X., & Huang, N. (2013). Tribocorrosion behavior of DLC-coated CoCrMo alloy in simulated biological environment. Vacuum, 92,
39–43.
López, H. F., & Saldivar-Garcia, A. J. (2007). Martensitic transformation in a cast Co–Cr–Mo–C alloy. Metallurgical and Materials Transactions A: Physical Metallurgy and Materials
Science, 39, 8–18.
Lucas, L. C., Buchanan, R. A., & Lemons, J. E. (1981). Investigations on the galvanic corrosion of multialloy total hip prostheses. Journal of Biomedical Materials Research, 15,
731–747.
Ludema, K. C., Shaffer, S. J., Ortega-Saenz, J. A., Hernandez-Rodriguez, M. A. L., Ventura-Sobrevilla, V., Michalczewski, R., Smolik, J., & Szczerek, M. (2011). Tribological and
corrosion testing of surface engineered surgical grade CoCrMo alloy. Wear, 271, 2125–2131.
Mahendra, G., Pandit, H., Kliskey, K., Murray, D., Gill, H. S., & Athanasou, N. (2009). Necrotic and inflammatory changes in metal-on-metal resurfacing hip arthroplasties. Acta
Orthopaedica, 80, 653–659.
Malchau, H., Herberts, P., Eisler, T., Garellick, G., & Söderman, P. (2002). The Swedish total hip replacement register. The Journal of Bone and Joint Surgery. American Volume,
84–A(Suppl), 2–20.
Malek, I. A., King, A., Sharma, H., Malek, S., Lyons, K., Jones, S., & John, A. (2012). The sensitivity, specificity and predictive values of raised plasma metal ion levels in the
diagnosis of adverse reaction to metal debris in symptomatic patients with a metal-on-metal arthroplasty of the hip. Journal of Bone and Joint Surgery. British Volume (London),
94, 1045–1050.
Marlowe, D. E., Parr, J. E., Michael, B., American Society for Testing and Materials, Marlowe, D. E., Parr, J. E., & Michael, B. (2013). Modularity of orthopedic implants. West
Conshohocken, PA: ASTM International.
Martin, E. J., Pourzal, R., Mathew, M. T., & Shull, K. R. (2013). Dominant role of molybdenum in the electrochemical deposition of biological macromolecules on metallic surfaces.
Langmuir, 29, 4813–4822.
Matharu, G. S., Berryman, F., Brash, L., Pynsent, P. B., Treacy, R. B. C., & Dunlop, D. J. (2016). The effectiveness of blood metal ions in identifying patients with unilateral
Birmingham hip resurfacing and Corail-pinnacle metal-on-metal hip implants at risk of adverse reactions to metal debris. Journal of Bone and Joint Surgery, 98, 617–626.
Mathew, M. T. M., Srinivasa Pai, P., Pourzal, R., Fischer, A., Wimmer, M. A., & Pai, P. S. (2009). Significance of tribocorrosion in biomedical applications: Overview and current
status. Advances in Tribology, 2009, 250986.
Mathew, M., Pourzal, R., Fischer, A., & Hallab, N. (2010). Electrochemical behavior of CoCrMo alloy: Effect of protein on corrosion kinetics. In 56th Annual Meeting of the
Orthopaedic Research Society (p. 2269).
Mathiesen, E. B., Lindgren, J. U., Blomgren, G. G., & Reinholt, F. P. (1991). Corrosion of modular hip prostheses. The Journal of Bone and Joint Surgery. British volume, 73,
569–575.
Maurer, A. M., Merritt, K., & Brown, S. A. (1994). Cellular uptake of titanium and vanadium from addition of salts or fretting corrosionin vitro. Journal of Biomedical Materials
Research, 28, 241–246.
McCafferty, E. (2010). Crevice corrosion and pitting. In Introduction to corrosion science (pp. 1–575). New York, NY: Springer New York.
Mears, D. C. (1975). The use of dissimilar metals in surgery. Journal of Biomedical Materials Research, 9, 133–148.
Meier, B. (2013). Maker aware of 40% failure in hip implant. New York City, NY: Arthur Ochs Sulzberger, Jr. New York City, NY: New York Times.
Mencière, M.-L., Amouyel, T., Taviaux, J., Bayle, M., Laterza, C., & Mertl, P. (2014). Fracture of the cobalt-chromium modular femoral neck component in total hip arthroplasty.
Orthopaedics & Traumatology, Surgery & Research, 100, 565–568.
Metikos-Hukovic, M., Pilic, Z., Babic, R., Omanovic, D., Metikos-Hukovic, M., Pilic, Z., Babic, R., & Omanovic, D. (2006). Influence of alloying elements on the corrosion stability of
CoCrMo implant alloy in Hank’s solution. Acta Biomaterialia, 2, 693–700.
Meyer, H., Mueller, T., Goldau, G., Chamaon, K., Ruetschi, M., & Lohmann, C. H. (2012). Corrosion at the cone/taper interface leads to failure of large-diameter metal-on-metal total
hip arthroplasties. Clinical Orthopaedics and Related Research, 470, 3101–3108.
Milosev, I., & Strehblow, H.-H. (2003). The composition of the surface passive film formed on CoCrMo alloy in simulated physiological solution. Electrochimica Acta, 48, 2767–2774.
Mittal, S., Revell, M., Barone, F., Hardie, D. L., Matharu, G. S., Davenport, A. J., Martin, R.a., Grant, M., Mosselmans, F., Pynsent, P., Sumathi, V. P., Addison, O., Revell, P.a., &
Buckley, C. D. (2013). Lymphoid aggregates that resemble tertiary lymphoid organs define a specific pathological subset in metal-on-metal hip replacements. PLoS One, 8,
e63470.
Montero-Ocampo, C., & Rodriguez, A. S. (1995). Effect of carbon content on the resistance to localized corrosion of as-cast cobalt-based alloys in an aqueous chloride solution.
Journal of Biomedical Materials Research, 29, 441–453.
Moore, R. M., Hamburger, S., Jeng, L. L., & Hamilton, P. M. (1991). Orthopedic implant devices: prevalence and sociodemographic findings from the 1988 National Health Interview
Survey. Journal of Applied Biomaterials, 2, 127–131.
Nag, S., Banerjee, R., & Fraser, H. L. (2005). Microstructural evolution and strengthening mechanisms in Ti–Nb–Zr–Ta, Ti–Mo–Zr–Fe and Ti–15Mo biocompatible alloys. Materials
Science and Engineering: C, 25, 357–362.
National Science and Technology Council. Materials Genome Initiative Strategic Plan, National Science and Technology Council, Committee on Technology, Subcommittee on the
Materials Genome, USA. https://www.nist.gov/document-3206.
Natu, S., Sidaginamale, R. P., Gandhi, J., Langton, D. J., & Nargol, A. V. F. (2012). Adverse reactions to metal debris: Histopathological features of periprosthetic soft tissue
reactions seen in association with failed metal on metal hip arthroplasties. Journal of Clinical Pathology, 65, 409–418.
Niinomi, M. (1998). Mechanical properties of biomedical titanium alloys. Materials Science and Engineering A, 243, 231–236.
Niinomi, M. (2007). Fatigue characteristics of metallic biomaterials. International Journal of Fatigue, 29, 992–1000.
Nima, M., Trevor, N., & Michael, L. (2013). Failure of a modular hip implant at the stem-sleeve interface. Orthopedics, 36, e978–e981.
Norman, P., Iyengar, S., Svensson, I., & Flivik, G. (2014). Fatigue fracture in dual modular revision total hip arthroplasty stems. Failure analysis and computed tomography
diagnostics in two cases. The Journal of Arthroplasty, 29, 850–855.
Nychka, J. A., Kruzic, J. J., Bandyopadhyay, A., Sarikaya, M., Dittrick, S., Balla, V. K., Davies, N. M., & Bose, S. (2011). In vitro wear rate and co ion release of compositionally and
structurally graded CoCrMo-Ti6Al4V structures. Materials Science and Engineering: C, 31, 809–814.
Nyga, A., Hart, A., & Tetley, T. D. (2015). Importance of the HIF pathway in cobalt nanoparticle-induced cytotoxicity and inflammation in human macrophages. Nanotoxicology, 9,
905–917.
Okazaki, Y., & Gotoh, E. (2005). Comparison of metal release from various metallic biomaterials in vitro. Biomaterials, 26, 11–21.
Older, J. (2002). Charnley low-friction arthroplasty: A worldwide retrospective review at 15–20 years. The Journal of Arthroplasty, 17, 675–680.
Paliwal, M., Gordon Allan, D., & Filip, P. (2010). Failure analysis of three uncemented titanium-alloy modular total hip stems. Engineering Failure Analysis, 17, 1230–1238.
Panigrahi, P., Liao, Y., Mathew, M. T., Fischer, A., Wimmer, M. A., Jacobs, J. J., & Marks, L. D. (2014). Intergranular pitting corrosion of CoCrMo biomedical implant alloy. Journal
of Biomedical Materials Research. Part B, Applied Biomaterials, 102, 850–859.
Papageorgiou, I., Brown, C., Schins, R., Singh, S., Newson, R., Davis, S., Fisher, J., Ingham, E., & Case, C. P. (2007). The effect of nano- and micron-sized particles of cobalt-
chromium alloy on human fibroblasts in vitro. Biomaterials, 28, 2946–2958.
Park, J., & Lakes, R. S. (2007). Biomaterials: An introduction. New York: Springer.
Perino, G., Ricciardi, B. F., Jerabek, S. A., Martignoni, G., Wilner, G., Maass, D., Goldring, S. R., & Purdue, P. E. (2014). Implant based differences in adverse local tissue reaction in
failed total hip arthroplasties: A morphological and immunohistochemical study. BMC Clinical Pathology, 14, 39.
Perman, V. (1980). Synovial fluid. In J. Kaneko (Ed.), Clinical biochemistry of domestic animals (pp. 68–93). New York: Academic Press.
Pham, V.-H., Yook, S.-W., Lee, E.-J., Li, Y., Jeon, G., Lee, J.-J., Kim, H.-E., & Koh, Y.-H. (2011). Deposition of TiN films on co-Cr for improving mechanical properties and
biocompatibility using reactive DC sputtering. Journal of Materials Science. Materials in Medicine, 22, 2231–2237.
Picardo, N. E., Al-Khateeb, H., & Pollock, R. C. (2011). Atypical pseudotumour after metal-on-polyethylene total hip arthroplasty causing deep venous thrombosis. Hip International,
21, 762–765.
Posada, O. M., Tate, R. J., & Grant, M. H. (2015). Effects of CoCr metal wear debris generated from metal-on-metal hip implants and co ions on human monocyte-like U937 cells.
Toxicology In Vitro, 29, 271–280.
Rack, H. J., & Qazi, J. I. (2006). Titanium alloys for biomedical applications. Materials Science and Engineering: C, 26, 1269–1277.
Ratner, B. D., Hoffman, A. S., Schoen, J. F., & Lemons, J. E. (2004). Biomaterials science, an introduction to materials in medicine (2nd edn.). Cambridge, MA: Elsevier Academic
Press.
Reclaru, L., Lerf, R., Eschler, P. Y., Blatter, A., & Meyer, J. M. (2002). Pitting, crevice and galvanic corrosion of REX stainless-steel/CoCr orthopedic implant material. Biomaterials,
23, 3479–3485.
Ricciardi, B. F., Nocon, A. A., Jerabek, S. A., Wilner, G., Kaplowitz, E., Goldring, S. R., Purdue, P. E., & Perino, G. (2016). Histopathological characterization of corrosion product
associated adverse local tissue reaction in hip implants: A study of 285 cases. BMC Clinical Pathology, 16, 1–17.
Rodrigues, D. C., Urban, R. M., Jacobs, J. J., & Gilbert, J. L. (2009). In vivo severe corrosion and hydrogen embrittlement of retrieved modular body titanium alloy hip-implants.
Rodriguez-Suarez, T., Bartolomé, J. F., & Moya, J. S. (2012). Mechanical and tribological properties of ceramic/metal composites: A review of phenomena spanning from the
nanometer to the micrometer length scale. Journal of the European Ceramic Society, 32, 3887–3898.
Roy, M., Pauschitz, A., Stack, M., Kumar, S., Sankara Narayanan, T. S. N., Ganesh Sundara Raman, S., & Seshadri, S. K. (2010). Evaluation of fretting corrosion behaviour of CP-Ti
for orthopaedic implant applications. Tribology International, 43, 1245–1252.
Ryan, G., Pandit, A., & Apatsidis, D. P. (2006). Fabrication methods of porous metals for use in orthopaedic applications. Biomaterials, 27, 2651–2670.
Samelko, L., Caicedo, M. S., Lim, S.-J., Della-Valle, C., Jacobs, J., & Hallab, N. J. (2013). Cobalt-alloy implant debris induce HIF-1a hypoxia associated responses: A mechanism for
metal-specific orthopedic implant failure. PLoS One, 8, e67127.
Scharf, B., Clement, C. C., Zolla, V., Perino, G., Yan, B., Elci, S. G., Purdue, E., Goldring, S., Macaluso, F., Cobelli, N., Vachet, R. W., & Santambrogio, L. (2014). Molecular analysis
of chromium and cobalt-related toxicity. Scientific Reports, 4, 5729.
Semlitsch, M., & Willert, H. G. (1997). Clinical wear behaviour of ultra-high molecular weight polyethylene cups paired with metal and ceramic ball heads in comparison to metal-on-
metal pairings of hip joint replacements. Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine, 211, 73–88.
Semlitsch, M., Staub, F., & Weber, H. (1985). Titanium-aluminium-niobium alloy, development for biocompatible, high strength surgical implants. Biomedizinische Technik.
Semlitsch, M. F., Weber, H., Streicher, R. M., & Schön, R. (1992). Joint replacement components made of hot-forged and surface-treated Ti–6Al–7Nb alloy. Biomaterials, 13,
781–788.
Serhan, H., Slivka, M., Albert, T., & Kwak, S. D. (2004). Is galvanic corrosion between titanium alloy and stainless steel spinal implants a clinical concern? The Spine Journal, 4,
379–387.
Shahgaldi, B. F., Heatley, F. W., Dewar, A., & Corrin, B. (1995). In vivo corrosion of cobalt-chromium and titanium wear particles. Journal of Bone and Joint Surgery. British Volume
(London), 77, 962–966.
Shenhar, A., Gotman, I., Radin, S., Ducheyne, P., & Gutmanas, E. (2000). Titanium nitride coatings on surgical titanium alloys produced by a powder immersion reaction assisted
coating method: Residual stresses and fretting behavior. Surface and Coating Technology, 126, 210–218.
Simoes, T. A., Bryant, M. G., Brown, A. P., Milne, S. J., Ryan, M., Neville, A., & Brydson, R. (2016). Evidence for the dissolution of molybdenum during tribocorrosion of CoCrMo hip
implants in the presence of serum protein. Acta Biomaterialia, 45, 410–418.
Singh, J. (2011). Epidemiology of knee and hip arthroplasty: A systematic review. The Open Orthopaedics Journal, 5, 80–85.
Sivakumar, M., & Rajeswari, S. (1992). Investigation of failures in stainless steel orthopaedic implant devices: Pit-induced stress corrosion cracking. Journal of Materials Science
Letters, 11, 1039–1042.
Sivakumar, M., Kumar Dhanadurai, K. S., Rajeswari, S., & Thulasiraman, V. (1995). Failures in stainless steel orthopaedic implant devices: A survey. Journal of Materials Science
Letters, 14, 351–354.
Sivakumar, B.; Kumar, S.; Sankara Narayanan, T. S. N. Fretting corrosion behaviour of Ti–6Al–4V alloy in artificial saliva containing varying concentrations of fluoride ions; Wear,
2011; 270, 317-324.
Smirnov, A., Beltrán, J. I., Rodriguez-Suarez, T., Pecharromán, C., Muñoz, M. C., Moya, J. S., & Bartolomé, J. F. (2017). Unprecedented simultaneous enhancement in damage
tolerance and fatigue resistance of zirconia/Ta composites. Scientific Reports, 7, 44922.
Smith, A. J., Dieppe, P., Porter, M., & Blom, A. W. (2012). National Joint Registry of England and Wales risk of cancer in first seven years after metal-on-metal hip replacement
compared with other bearings and general population: Linkage study between the National Joint Registry of England and Wales and hospital episode statistics. BMJ, 344, e2383.
Sonntag, R., Reinders, J., & Kretzer, J. P. (2012). What’s next? Alternative materials for articulation in total joint replacement. Acta Biomaterialia, 8, 2434–2441.
Sonofuchi, K., Hagiwara, Y., Koizumi, Y., Chiba, A., Kawano, M., Nakayama, M., Ogasawara, K., Yabe, Y., & Itoi, E. (2016). Quantitative in vivo biocompatibility of new ultralow-nickel
cobalt-chromium-molybdenum alloys. Journal of Orthopaedic Research, 34, 1505–1513.
Steinemann, S. G. (1996). Metal implants and surface reactions. Injury, 27(Suppl 3), SC16–SC22.
Sugimoto, K., & Sawada, Y. (1977). The role of molybdenum additions to austenitic stainless steels in the inhibition of pitting in acid chloride solutions. Corrosion Science, 17,
425–445.
Sun, D., Wharton, J. A., Wood, R. J. K., Ma, L., & Rainforth, W. M. (2009). Microabrasion–corrosion of cast CoCrMo alloy in simulated body fluids. Tribology International, 42,
99–110.
Sunderman, F. W., Hopfer, S. M., Swift, T., Rezuke, W. N., Ziebka, L., Highman, P., Edwards, B., Folcik, M., & Gossling, H. R. (1989). Cobalt, chromium, and nickel concentrations
in body fluids of patients with porous-coated knee or hip prostheses. Journal of Orthopaedic Research, 7, 307–315.
Swaminathan, V., & Gilbert, J. L. (2012). Fretting corrosion of CoCrMo and Ti6Al4V interfaces. Biomaterials, 33, 5487–5503.
Takeno, N. (2005). Atlas of Eh-pH diagrams. In Geological Survey of Japan Open File Report No. 419. Natl. Inst. Adv. Ind. Sci.
Teoh, S. (2000). Fatigue of biomaterials: A review. International Journal of Fatigue, 22, 825–837.
The NJR Editorial Board 13th Annual Report-National Joint Registry for England, Wales, Northern Ireland and the Isle of Man; 2016.
Topolovec, M., Milosev, I., Cör, A., & Bloebaum, R. D. (2013). Wear debris from hip prostheses characterized by electron imaging. Central European Journal of Medicine, 8,
476–484.
Topolovec, M., Cör, A., & Milosev, I. (2014). Metal-on-metal vs. metal-on-polyethylene total hip arthroplasty tribological evaluation of retrieved components and periprosthetic tissue.
Journal of the Mechanical Behavior of Biomedical Materials, 34, 243–252.
Türkan, U., Öztürk, O., & Eroglu, A. E. (2006). Metal ion release from TiN coated CoCrMo orthopedic implant material. Surface and Coating Technology, 200, 5020–5027.
U.S. Food and Drug Administration, Stryker Initiates Voluntary Product Recall of Modular-Neck Stems Action Specific to Rejuvenate and ABG II Modular-Neck Stems, 2012.
U.S. Food and Drug Administration Metal-on-Metal Hip Implants: FDA Safety Communication 2013.
Ulrich, S. D., Seyler, T. M., Bennett, D., Delanois, R. E., Saleh, K. J., Thongtrangan, I., Kuskowski, M., Cheng, E. Y., Sharkey, P. F., Parvizi, J., Stiehl, J. B., & Mont, M. A. (2008).
Total hip arthroplasties: What are the reasons for revision? International Orthopaedics, 32, 597–604.
Urban, R. M., Jacobs, J. J., Gilbert, J. L., & Galante, J. O. (1994). Migration of corrosion products from modular hip prostheses. Particle microanalysis and histopathological findings.
The Journal of Bone and Joint Surgery. American volume, 76, 1345–1359.
Urban R, Jacobs J, Gilbert J, Rice S, Jasty M, Bragdon C, Galante J (1997) Characterization of solid products of corrosion generated by modular-head femoral stems of different
designs and materials. In Modularity of orthopedic implants; ASTM International: 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428–2959, Vol. 1301, pp.
33–44.
Urban, R. M., Jacobs, J. J., Tomlinson, M. J., Gavrilovic, J., Black, J., & Peoc’h, M. (2000). Dissemination of wear particles to the liver, spleen, and abdominal lymph nodes of
patients with hip or knee replacement. The Journal of Bone and Joint Surgery. American Volume, 82, 457.
Urban, R., Gilbert, J., & Jacobs, J. (2006). Corrosion of Modular Titanium Alloy Stems in Cementless Hip Replacement. In L. Zardiackas, H. Freese, & M. Kraay (Eds.), STP37558S
titanium, niobium, zirconium, and tantalum for medical and surgical applications, STP37558S (pp. 215–224). West Conshohocken, PA: ASTM International.
Valero Vidal, C., & Igual Muñoz, A. (2008). Electrochemical characterisation of biomedical alloys for surgical implants in simulated body fluids. Corrosion Science, 50,
1954–1961.
Valero Vidal, C., Olmo Juan, A., & Igual Muñoz, A. (2010). Adsorption of bovine serum albumin on CoCrMo surface: Effect of temperature and protein concentration. Colloids and
Surfaces, B: Biointerfaces, 80, 1–11.
Vamsi Krishna, B., Xue, W., Bose, S., & Bandyopadhyay, A. (2008). Functionally graded co–Cr–Mo coating on Ti–6Al–4V alloy structures. Acta Biomaterialia, 4, 697–706.
Vendittoli, P.-A., Mottard, S., Roy, A. G., Dupont, C., & Lavigne, M. (2007). Chromium and cobalt ion release following the Durom high carbon content, forged metal-on-metal
surface replacement of the hip. Journal of Bone and Joint Surgery. British Volume (London), 89, 441–448.
Vengellur, A., & LaPres, J. J. (2004). The role of hypoxia inducible factor 1 alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts. Toxicological Sciences, 82,
638–646.
Virtanen, S., Milosev, I., Gomez-Barrena, E., Trebse, R., Salo, J., & Konttinen, Y. T. (2008). Special modes of corrosion under physiological and simulated physiological conditions.
Acta Biomaterialia, 4, 468–476.
Walczak, J., Shahgaldi, F., & Heatley, F. (1998). In vivo corrosion of 316L stainless-steel hip implants: Morphology and elemental compositions of corrosion products. Biomaterials,
19, 229–237.
Wang, K. (1996). The use of titanium for medical applications in the USA. Materials Science and Engineering A, 213, 134–137.
Wang, Q., Parry, M., Masri, B. A., Duncan, C., & Wang, R. (2017). Failure mechanisms in CoCrMo modular femoral stems for revision total hip arthroplasty. Journal of Biomedical
Materials Research. Part B, Applied Biomaterials, 105(6), 1525–1535.
Welsch, G., Boyer, R., & Collings, E. W. (1993). Materials properties handbook: Titanium alloys. Materials Park, OH: ASM International.
Whitehouse, M. R., Endo, M., Zachara, S., Nielsen, T. O., Greidanus, N. V., Masri, B. A., Garbuz, D. S., & Duncan, C. P. (2015). Adverse local tissue reactions in metal-on-
polyethylene total hip arthroplasty due to trunnion corrosion: The risk of misdiagnosis. The Bone & Joint Journal, 97–B, 1024–1030.
Willert, H.-G. (2005). Metal-on-metal bearings and hypersensitivity in patients with artificial hip Joints_A clinical and histomorphological study. Journal of Bone and Joint Surgery,
87, 28.
Willert, H. G., Brobäck, L. G., Buchhorn, G. H., Jensen, P. H., Köster, G., Lang, I., Ochsner, P., & Schenk, R. (1996). Crevice corrosion of cemented titanium alloy stems in total hip
replacements. Clinical Orthopaedics and Related Research, 333, 51–75.
Williams, D. H. (2011). Prevalence of Pseudotumor in asymptomatic patients after metal-on-metal hip arthroplasty. Journal of Bone and Joint Surgery, 93, 2164.
Williams, D. (2014). Essential biomaterials science. Cambridge, England: Cambridge University Press.
Williams, H. D. W., Browne, G., Gie, G. A., Ling, R. S. M., Timperley, A. J., & Wendover, N. A. (2002). The Exeter universal cemented femoral component at 8–12 years. A study of
the first 325 hips. The Journal of Bone and Joint Surgery. British Volume, 84, 324–334.
Williams, D. H. D., Greidanus, N. N. V., Masri, B. A., Duncan, C. P., & Garbuz, D. S. (2011). Prevalence of pseudotumor in asymptomatic patients after metal-on-metal hip
arthroplasty. The Journal of Bone and Joint Surgery. American Volume, 93, 2164–2171.
Wimmer, M., Loos, J., Nassutt, R., Heitkemper, M., & Fischer, A. (2001). The acting wear mechanisms on metal-on-metal hip joint bearings: In vitro results. Wear, 250, 129–139.
Wimmer, M. A., Fischer, A., Büscher, R., Pourzal, R., Sprecher, C., Hauert, R., & Jacobs, J. J. (2010). Wear mechanisms in metal-on-metal bearings: The importance of tri-
bochemical reaction layers. Journal of Orthopaedic Research, 28, 436–443.
Wimmer, M. A., Mathew, M. T., Laurent, M. P., Nagelli, C., Liao, Y., Marks, L. D., Pourzal, R., Fischer, A., & Jacobs, J. J. (2013). Tribochemical reactions in metal-on-metal hip
joints influence wear and corrosion. In Metal-on-metal total hip replacement devices (pp. 292–309). West Conshohocken, PA: ASTM International.
Woolson, S. T., Milbauer, J. P., Bobyn, J. D., Yue, S., & Maloney, W. J. (1997). Fatigue fracture of a forged cobalt-chromium-molybdenum femoral component inserted with cement.
A report of ten cases. The Journal of Bone and Joint Surgery. American Volume, 79, 1842–1848.
Wright, G., Sporer, S., Urban, R., & Jacobs, J. (2010). Fracture of a modular femoral neck after total hip arthroplasty: A case report. The Journal of Bone and Joint Surgery.
American Volume, 92, 1518–1521.
Xia, Z., Kwon, Y.-M., Mehmood, S., Downing, C., Jurkschat, K., & Murray, D. W. (2011). Characterization of metal-wear nanoparticles in pseudotumor following metal-on-metal hip
resurfacing. Nanomedicine, 7, 674–681.
Yan, Y., Neville, A., Dowson, D., & Williams, S. (2006). Tribocorrosion in implantsdassessing high carbon and low carbon Co–Cr–Mo alloys by in situ electrochemical
measurements. Tribology International, 39, 1509–1517.
Zardiackas, L., Mitchell, D., & Disegi, J. (1996). Characterization of Ti–15Mo Beta titanium alloy for Orthopaedic implant applications. In S. A. Brown, & J. E. Lemons (Eds.), Medical
applications of titanium and its alloys: The material and biological issues (pp. 60–75). West Conshohocken, PA: ASTM International, 16.
Zeng, P., Rainforth, W. M., & Cook, R. B. (2015a). Characterisation of the oxide film on the taper interface from retrieved large diameter metal on polymer modular total hip
replacements. Tribology International, 89, 86–96.
Zeng, P., Sharp, J., Rana, A., Thompson, R., Rainforth, W. M., Cook, R. B., et al. (2015b). Sub-surface characterisation of tribological contact zone of metal hip prostheses. Journal
of Physics Conference Series, 644, 12029.
Decellularized Extracellular Matrix
Paul Frank Gratzer, School of Biomedical Engineering, Dalhousie University, Halifax, NS, Canada
Introduction 86
Decellularization Techniques 87
Physical 87
Chemical 87
Inhibition of Enzymes 89
Example Decellularization Processes 90
Assessing Decellularization 91
Sterilization Issues 93
Example Decellularization Applications 93
The Future of Decellularization 96
Further Reading 96
Glossary
Allograft Donor tissue or organ transplanted from one human to another.
Critical micelle concentration The concentration at which detergents form a spherical cluster.
Decellularization Techniques or processes whereby tissues or organs are treated to remove cellular materials while preserving
the non-living, structural scaffold.
Monoclonal antibody An antibody produced by a single clone of cells or cell line and consisting of identical antibody
molecules.
Xenograft Donor tissue or organ transplanted from an animal to a human.
Abbreviations
CMC Critical micelle concentration
DAPI 40,6-Diamidino-2-phenylindole
DSC Differential scanning calorimetry
EO Ethylene oxide
FITC Fluorescein
GAG Glycosaminoglycan
GI Gastrointestinal
H&E Hematoxylin and eosin
HIT Hydrothermal isometric temperature testing
HLA Human leukocyte antigens
IPSC Induced pluripotent stem cells
MHC Major histocompatibility complexes
PMSF Phenylmethanesulfonylfluoride
SDS Sodium dodecyl sulphate
SIS Small intestinal submucosa
TnBP Tri-butyl phosphate
UBM Urinary bladder matrix
Introduction
The term “decellularization” is used to describe techniques or processes whereby tissues or organs obtained from human (allograft)
or animal (xenograft) donors are treated to remove cellular materials while preserving the non-living, structural scaffolddreferred

Biomaterials: Science and Engineering j Decellularized Extracellular Matrix 87
to as the extracellular matrix (ECM). By doing so, the main source of rejection of the donor materialdthe cellsdare removed.
Further, by maintaining the ECM of the tissue or organ, the remaining non-living donor material can be used as a scaffold to regen-
erate living, functional replacements that can be used to treat patients with diseased, dysfunctional, or damaged parts of their body
without being rejected. The idea of decellularization has been around since 1975, however, it has seen increased attention and
research activity within the last 20 years as the areas of Tissue Engineering and Regenerative Medicine look to develop living replace-
ments for parts of the human body.
Decellularization Techniques
The main objectives of decellularization techniques are to remove as much donor cellular materials (genetic, cell membrane, cyto-
skeletal proteins) as possible while at the same time keeping the native structure and composition of the ECM as intact as possible.
These objectives, in theory, seem relatively simple and straight forward, however the implementation of techniques to achieve the
goals of decellularization are far more difficult. The goal of decellularization is akin to removing the white and yolk (i.e., cellular
materials) from an egg without damaging or disrupting the egg shell (i.e., the ECM).
Decellularization techniques, as described below and summarized in Table 1, are combined in various configurations to yield
decellularization processes that are applied to donated tissues and organs. Almost every decellularization process that has been
developed by researchers to date involves at least two basic techniques: (i) a lysis step to burst and break up cells and (ii) washing
step(s) to remove disrupted and exposed cellular materials from within the ECM.
Physical
At the start of most decellularization procedures, cells are lysed physically using osmotic gradients, mechanical compression/
massage, or freeze-thaw cycles. Hypertonic (high salt concentration) and hypotonic (low salt concentration) treatments put hydro-
static pressure on cell membranes in an aqueous environment causing them to burst and release their cell contents. The contents of
the cell are then more accessible to later treatments with detergents or isotonic (balanced salt) washout procedures. Mechanical
compression or massage is used to encourage membrane degradation and the gradual exposure of more cell membranes to extrac-
tion solutions. Freeze-thaw cycles are used to kill cells and then fracture their cell membranes so that subsequent washout proce-
dures can access internal cell contents and fragmented membranes. Vacuum techniques, use of ultrasound (sonication), and
increased pressure, for example use of supercritical liquid CO2, can also be used to lyse cells or aid in the removal of cellular compo-
nents. Care must be taken with cell lysis methods, however, in order to avoid damaging the ECM. This is especially true for mechan-
ical and freeze/thaw techniques.
Chemical
Critical to the success of many decellularization strategies are the use of detergents or surfactants. Detergents are a special group of
phospho-lipid compounds that are soluble in aqueous environments. Detergents are amphiphiles in that they have both a polar
and a non-polar domain. They are useful for decellularization when they are added in a sufficient concentration to form micelles.
A micelle is a cluster of detergent monomers, often spherical, that is oriented so that the non-polar domains of the detergent mole-
cules are interacting internally, and the polar domains are interacting with water molecules externally. The concentration at which
detergents form micelles is called the critical micelle concentration (CMC). The CMC varies with decellularization conditions,
including ionic strength, pH, temperature and the presence of protein and lipids (including other detergent molecules).
Micelles occupy space in the aqueous environment, and as such, must disrupt hydrogen bonds between water molecules. The
interaction of polar head groups of the detergent monomers in the micelle is usually more than sufficient to satisfy the energy
requirements for keeping water molecules apart, thus maintaining the micelle as an intact stable structure in the fluid. The “pro-
tected” non-polar tails of the detergent molecules in the center of the micelle essentially provide a second phase where non-
polar lipid and non-polar protein domains can “dissolve.” During decellularization, the extraction of cell components, including
lipids and proteins from tissue, is accomplished when these micelles enter the tissue, dissolve non-polar components, and are
washed out with solution changes. Detergents can be classified by one of three designations: ionic, nonionic, and zwitterionic. Ionic
detergents are either anionic or cationic, although cationic detergents are not used in decellularization processes due to their strong
protein denaturing tendencies. Anionic detergents contain a negatively charged (polar) head group. Anionic detergents, such as
sodium dodecyl sulphate (SDS), are highly efficient at solubilizing proteins and are generally denaturing to some extent. A
subgroup of ionic detergents are the bile acid salts, found in the intestine where they solubilize fats. Bile acid salts, such as sodium
deoxycholate, have a rigid steroidal backbone and lack a defined polar head group. Instead, they have their polar residues arranged
all on one side of the rigid steroid backbone and form kidney-shaped micelles, unlike the spherical micelles formed by detergents
with linear non-polar backbones. Bile acids are milder than other anionic detergents such as SDS. Nonionic detergents, such as
Triton X-100, have neutral polar head groups and are nondenaturing to proteins. Nonionic detergents break lipid–lipid and
lipid–protein interactions. Finally, zwitterionic detergents, such as CHAPS, have properties of both ionic and nonionic detergents.
Zwitterionic detergents are generally milder than ionic detergents and more denaturing to proteins than nonionic detergents.
88 Biomaterials: Science and Engineering j Decellularized Extracellular Matrix
Table 1 Decellularization techniques and reagents
Mode of action Potential drawbacks
Physical methods
Osmotic gradient Bursts or contracts cells, disrupts cell Lyses cells, releasing proteases that can attack
membranes ECM
Freeze/thaw Disrupts cell membranes Ice crystals can destroy ECM (flash freezing)
Mechanical delamination Separates tissue layers along natural planes of Excessive forces can damage ECM
dissection
Agitation/compression/vacuum Increases exposure of cell membranes to ECM can be damaged by sonication or excessive
extraction solutions forces
Chemical methods
Anionic detergents
Bile acid salt
Sodium deoxycholate Solubilizes phospholipids Precipitates at pH 6.9 to form bile acid
Disrupts protein–lipid interactions Potential growth factor removal
Generally non-denaturing, mild
Synthetic
SDS (sodium dodecyl sulfate) Disrupts protein–protein interactions May denature collagen and elastin in ECM
Denatures and solubilizes proteins Removes GAGs, growth factors
Binds virus particles (incl. HIV) May contribute to swelling
Reagent is cytotoxic
Triton X-200 (alkylaryl polyether sulfonate) Solubilizes cellular components May need to use concurrently with zwitterionic
detergents for highest effectiveness, potential
growth factor removal
N-lauroyl-sarcosinate Ruptures cells May leave cell remnants, potential growth factor
Solubilizes cell membrane proteins removal
Nonionic detergents
Triton X-100 (PEG tert-octylphenyl ether) Non-denaturing protein solubilization Low removal efficiency in some tissues
May disrupt ECM organization, potential growth
factor removal
Tween 20 or 80 (PEG-sorbitan monolaurate/ Solubilizes peripheral membrane proteins Low efficiency in isolation
oleate) Tween 20 less effective than Tween 80
Potential growth factor removal
Igepal CA630 (formerly sold as Nonidet P40) Non-denaturing protein solubilization Less hydrophilic than Triton X-100
(Octylphenyl-PEG) May sediment during storage
N-octyl-b-D glucopyranoside Solubilization of membrane proteins Requires cold storage
Zwitterionic detergents
Sulfobetain-10 and -16 Help decrease micelle size with anionic Hygroscopic
detergents Potential growth factor removal
Retain zwitterionic nature over wide pH range
CHAPS Disrupts protein–protein interactions High critical micelle concentration (6–10 mM,
Non-denaturing, forms small micelles depending on temperature)
Alcohols
Glycerol Destroys bacteria
Solubilizes cell components
Isopropanol Disrupts cell membranes, dissolves lipids Volatile, ECM mechanical property changes
Destroys bacteria and viruses
Ethanol Destroys bacteria Volatile, causes matrix shrinkage, change in
mechanical properties
1-Butanol Extracts lipids Severely contracts ECM, change in mechanical
properties
Acids
Peracetic acid Solubilizes cytoplasm components Can denature ECM components and growth
Disrupts DNA factors (especially above 0.1% v/v)
Bases
NaOH Disrupt DNA/RNA, cell membranes Loosens, frays, and fragments elastin fibers
Destroy viruses, inactivates prions
NH4OH Weakens lipid–protein bonds High vapor pressure
Chelators
Table 1 Decellularization techniques and reagentsdcont'd
Mode of action Potential drawbacks
EDTA Bind to metallic cofactors, inhibiting enzymes Can inhibit enzymes used in decellularization
EGTA Bind ions cells need to attach to substrates Processes
Enzymatic methods
Membrane/attachment enzymes
Trypsin Disrupts desmosomes, focal adhesions Does not remove all active MMPs
Cleaves peptide bonds on Arg and Lys Removes fibronectin, laminin, elastin, GAGs,
growth factors
Can damage ECM after prolonged exposure
DISPASE Attacks type IV collagen, helps lift cells Can completely disrupt basement membranes
DISPASE II
Phospholipase Digests phospholipids in cell membranes Requires special conditions (pH, temperature)
Antigen-targeted enzymes
Thermolysin Attacks antigen in the hemidesmosome of the
basal layer of keratinocytes
a-galactosidase Digests a-1,3-galactose antigen Requires special conditions (pH, temperature)
Exonucleases
DNAse I Facilitates hydrolysis of terminal DNA strands Sensitive to mechanical denaturation
Presence of EDTA inhibits activity
RNAse A Facilitates hydrolysis of terminal RNA strands May be degraded by trypsin or thermolysin
Endonucleases
Benzonase Facilitates degradation of internal bonds in DNA Presence of EDTA inhibits activity
(single and double stranded) and RNA
Solvents, such as tributyl phosphate (TnBP), can be useful in decellularization as they are capable of disrupting protein and lipid
interactions by destabilizing hydrophobic interactions. An added benefit to the use of TnBP in a decellularization protocol is its
proven anti-viral action. While not widely used in current decellularization protocols, other solvents have also shown good decel-
lularization performance with very little damage to the ECM.
Alkaline bases have been used to denature DNA. Commonly used alkaline bases include ammonium hydroxide, sodium sulfide,
sodium hydroxide, and calcium hydroxide. Such compounds have been used in the decellularization of dense tissues such as
dermis, but alkalines can degrade structural component of the ECM including collagen. Alkaline treatment results in a dramatic
reduction in GAG content and altered mechanical properties of tissues. Similarly, acids are used to dissociate DNA from the
ECM by solubilizing cytoplasmic components and disrupting nucleic acids. Acids can also denature ECM proteins including
GAGs, collagen, and growth factors. It is important to optimize the dose and exposure time when acids are used for decellulariza-
tion. Peracetic acid, applied at 0.1% (v/v) in a single wash for 2 h, and combined with appropriate mechanical methods and rinsing,
can thoroughly decellularize thin tissues such as small intestinal submucosa (SIS) and urinary bladder matrix (UBM). Acids
commonly used for decellularization include deoxycholic acid and acetic acid. However, acetic acid has been shown to cause
damage to and removal of collagens from the ECM.
Alcohols can be effective in decellularization processes if cell membranes are permeabilized. If the polar hydroxyl groups of alco-
hols can diffuse into the cell, the alcohols replace intracellular water and lyse the cell by dehydration. Ethanol or methanol may also
be used as a final wash to remove residual nucleic acids from tissue. The non-polar carbon chain of alcohols can also help to dissolve
non-polar substances such as lipids. Care must be taken, however, at the use of alcohols has been shown to significantly alter the
mechanical properties of the ECM.
Inhibition of Enzymes
During decellularization, the lysis of cells releases the contents of lysosomes into the extracellular space. Many of the enzymes
released, especially proteases, are highly effective at degrading the ECM. The goal of decellularization is to preserve the intact
ECM while removing cellular components, so the action of degradative enzymes is counterproductive. Most decellularization proce-
dures make use of protease inhibitors at least during the initial cell lysis phase of the procedure while some continue the use of
protease inhibition throughout. Protease inhibition is often achieved through a combination of methods. Many procedures inhibit
lysosomal proteases with elevated pH ( pH 8). Chelating agents, such as EDTA or 1,10-phenanthroline, that bind metallic enzyme
cofactors such as magnesium, iron, or zinc are also used. Chemical inhibition of proteases can be achieved by the addition of any
one of a number of common protease inhibitors. Chemical inhibitors include phenylmethanesulfonylfluoride (PMSF), aprotinin,
or leupeptin. PMSF (irreversible binding) and aprotinin (reversible binding) inhibit serine proteases such as trypsin and chymo-
trypsin, whereas leupeptin (low stability in aqueous environments) inhibits both serine and cysteine proteases. The inhibition
of bacteria is also essential, as most tissues processed with decellularization are not procured under aseptic conditions. Common
antibiotics used for decellularization treatments include gram positive bacteria cell wall synthesis inhibitors (penicillin or vanco-
mycin), bacteria and mycoplasma protein synthesis inhibitors (streptomycin or gentamicin) and agents that induce ribosomal mis-
coding in gram positive/negative bacteria and mycoplasma (kanamycin). Sodium azide (NaN3) is also often added to
decellularization solutions to inhibit microbial growth.
Example Decellularization Processes

At first glance, there appears to be a bewildering array of different decellularization processes presented in the scientific literature.
Fortunately, there are only a few truly unique decellularization processes with others being variations of these basic methods. Most
decellularization processes can be classified as a (i) physically-based process, (ii) chemically-based process, or (iii) biochemically-
based process. There are many protocols that borrow from these three basic processes and include combinations of various decel-
luarlization techniques outlined above.
An example of a biochemically-based decellularization protocol involves the use of enzyme(s) such as Trypsin. Trypsin is
commonly used in laboratories throughout the world to release cells from substrates during routine cell culture. It is also used
in enzymatic assays studying the “nativity” or how intact collagen is within tissues, as it will preferentially degrade only denatured
collagen. It has been used in decellularization protocols as it digests desmosome complexes holding cells together and disrupts cell
adhesions that link cells to the ECM. A representative protocol using trypsin for decellularization of tissues is shown in Table 2A. In
general, studies using trypsin for cell extraction have shown poor extraction efficiency and significant ECM damage when compared
with detergent processes.
Mechanical delamination can be used to accomplish decellularization. Tissues from the gastrointestinal (GI) tract and the
urinary system have been treated by this process. Tissues from the GI tract and the bladder have the same basic organization
from deep to superficial of mucosa, submucosa, muscularis, and serosa. In most strategies using GI tissues, the tubular tissue is
opened longitudinally and the mucosa delaminated. The remaining serosa and muscularis are then also delaminated, leaving
Table 2 Example decellularizaation protocols
Treatment Conditions Duration
(A) Representative trypsin-based decellularization protocol

1 0.05%–0.5% trypsin digest (optional repeat) pH 7.4, 0.02%–0.2% EDTA 24–96 h
2 DNAse/RNAse digest (optional) HBSS, pH 7.4, 37 C 2h
3 PBS washout and storage 4 C, antibiotics Discretionary
Treatment Conditions
(B) Representative mechanical delamination decellularization protocol

1 Bladder: osmotic gradient removes urothelial cells 1 N NaCl
2 Delamination:
Bladder: delaminate serosa, muscularis, and submucosa
(three superficial layers)
GI tract: split longitudinally, delaminate mucosa, turn over
GI tract: delaminate muscularis and serosa (two superficial
layers)
3 Cell lysis/disinfection of 0.1% peracetic acid 4% ethanol
Bladder: urinary bladder matrix, UBM (mucosa and lamina Sterile water
propria remain)
GI tract: small intestinal submucosa, SIS (submucosa and
lamina propria remain)
4 Phosphate buffered saline rinse pH 7.4, 2 15 min
5 Distilled water rinse 2 15 min
Treatment Conditions Duration
(C) Representative Triton/SDS detergent decellularization protocol

1 Hypotonic tris buffer pH 8, 4 C-room temp., 5 mM EDTA, PMSF, antibiotics, 24–36 h
agitation
2 Hypertonic tris buffer þ nonionic detergent: pH 8, 4 C-room temp., 1.5 M KCl, PMSF, antibiotics, 24–48 h
1% (v/v) Triton X-100 agitation
3 DNAse/RNAse digest Hank’s Balance Salt Solution (HBSS), pH 7.4, 37 C 1–5 h
4 Anionic or nonionic detergent: pH 8, antibiotics, agitation 24–48 h
1% (v/v) SDS or Triton X-100 in tris buffer
5 Washout: ethanol or HBSS Antibiotics, agitation 48 h
6 PBS washout (optional) and storage 4 C, antibiotics Discretionary
the submucosal layer and the basement membrane of the mucosa. In the case of the bladder, the submucosa, muscularis, and serosa
are often microdissected away from the mucosa with the help of mechanical delamination. The few cells that remain in the submu-
cosa/lamina propria (small intestinal submucosa, SIS) or the mucosa/lamina propria (urinary bladder matrix, UBM) are lysed and
removed in a washout procedure and peracetic acid treatments. A representative protocol useful for processing intestine or bladder
tissue is presented in Table 2B. Such methods, however, lead invariably to the damage of the ultrastructure and the integrity of the
ECM scaffold. Due to the thin sheet-like nature of SIS materials, specialized load-bearing tissues such as heart valves and ligaments
may not be the ideal application for this class of material. It has recently been reported, however, that the mechanical strength of SIS
can be improved by layering of the material. SIS and UBM are ideally suited to patching applications such an in the urinary bladder
and general tissue augmentation.
The most widely used decellularization protocols are based on synthetic detergents that solubilize proteins. The anionic deter-
gent SDS and the non-ionic detergent Triton X-100 are the most widely used synthetic detergents for decellularization. A represen-
tative protocol is given in Table 2C. This decellularization concept has been widely used and is one of the most studied to date in
terms of its effects on the properties of the ECM. The effectiveness of cellular extraction with Triton-SDS procedures is generally
excellent, although this treatment is not as mild as that obtained with sodium deoxycholate. There are also numerous other exam-
ples of the combination or replacement of Triton X-100 and/or SDS with other detergents such as CHAPS, Tween 20/80, sodium
deoxycholate, trypsin and the organic solvent tri-n-butyl phosphate (TnBP). Many of these protocols are reported to achieve high
levels of cellular extraction with varying levels of associated changes in the properties and composition of the ECM.
No one decelllarization process can be used as a universal treatment for all tissues or organs. Generally, each tissue or organ is
unique in terms of the conditions and techniques used to successfully decellularize them. While successful decellularization treat-
ment processes have been found for specific applications, these have largely been determined by empirical knowledge and
experience.
Assessing Decellularization
The most important step in assessing the effectiveness of a new decellularization process or the application of a decellularization
process to a new tissue or organ source requires the use of multiple characterization assays and techniques. There have not been any
regulations or standards developed or accepted for describing what constitutes minimal or even optimal requirements to ensure that
any tissue or organ processed with decellularization will be safe and effective as a replacement for a part of the human body. That
being said, over the last 20–25 years, research has shown that there are areas of measurement to assess the effects of decellularization
on tissues and organs that directly affect performance when implanted into a recipient.
These measurement criteria for decellularized tissues and organs should include:
1. the lack of visible nuclear material in a histological section stained with hematoxylin and eosin (H&E) or 40,6-diamidino-2-
phenylindole (DAPI)
2. < 50 ng ds DNA per mg of dry weight of ECM
3. < 200 bp DNA fragment length
4. the lack of intracellular components (e.g., cytoskeletal materials, cell organelles)
5. the lack of cell membrane elements, specifically known immunogenic ones such as major histocompatibility complexes (MHC)
or human leukocyte antigens (HLA)
6. the maintenance of ECM elements in their native state and quantities (collagen, glycosaminoglycans (GAGs), elastin, etc.)
7. the resulting ECM scaffold does not contain nor release cytotoxic elements
The removal of cellular nuclear elements in decellularized materials is important as DNA has been shown to elicit immune
responses and promote calcification in certain applications. At minimum, routine histologic staining can be used as a rough detec-
tion of DNA/RNA. Histologic stains, such as H&E or trichrome, provide a relatively insensitive measure for identifying DNA within
the ECM (Fig. 1). A second more accurate and sensitive quantifiable method of DNA detection can be achieved using commercially
available dsDNA intercalators, such as PicoGreen, propidium iodide, or bisbenzimide, as well as by gel electrophoresis. As shown in
Fig. 2, DNA content of tissues treated with different decellularization processes can vary dramatically.
Not only is the removal of cellular genetic materials important, but the removal of intracellular and cytoplasmic debris, such as
cytoskeletal proteins a- and b-actins and vimentin, are important as they may be involved in adverse host reactions such as increased
inflammation. Further, cytoskeletal protein removal has been shown as a good indicator of a high level of decellularization having
been achieved. Most essential to the reduction of immunogenicity in allo- and xenograft materials via decellularization, however, is
the complete removal of cell membrane elements (i.e., MHC or HLA). Both MHC and HLA cytoplasmic proteins have been shown
to be directly responsible for adverse immune and inflammatory reactions in transplanted donor materials. Methods best suited to
detect immunogenic cytoplasmic proteins in decellularized materials involve the use of antibodies that recognize them and chro-
mogenic or immunofluorescent markers to detect the antibodies such as peroxidase or fluorescein (FITC) based staining (Fig. 3).
Equally as important as removing immunogenic donor cellular materials, is the maintenance of the composition and structure
of the ECM of decellularized tissues and organs. Untreated tissues and organs are used as the comparative control to determine if
decellularization has affected the ECM. As stated previously, certain decellularization techniques can have particular detrimental
effects on ECM elements such as collagen, GAGs, and elastin that could remove or damaged them during processing. Maintaining
Fig. 1 Pig bone-anterior cruciate ligament (ACL)-bone graft before (left) and after (right) decellularization processing. Note loss of red/pink colora-
tion (above right) as cells and blood products are removed during decellularization resulting in a whitish color (above left). The removal of cells is
also indicated by the loss of blue/purple stained cell nuclei in histological sections of untreated tissue (below left) when compared to decellularize
tissue (below right) using the stain hematoxylin and eosin.
Fig. 2 DNA content for commercially available and laboratory produced ECM scaffold materials as determined with the PicoGreen assay. (Color
version of figure is available online.) From Journal of Surgical Research 152, 135–139 (2009), “Quantification of DNA in Biologic Scaffold Materials”,
Thomas W. Gilbert, Ph.D., John M. Freund, B.S., and Stephen F. Badylak, D.V.M., Ph.D., M.D.
the native composition and structure of the original donated tissue or organ is key to the decellularized ECM interacting with and
guiding cells, either in vitro or in vivo, to repopulate the ECM matrix and reform living functional tissues. The role of the ECM
includes providing a repository for growth factors via binding to matrix molecules, cell instructive cues via cell integrins, and
mechanical and physical properties of tissues The assessment of the composition and degree of intact structure of tissues and organs
should include biochemical, immunohistochemical, structural, and mechanical property measurements where appropriate.
Biochemical assays may include hydroxy-proline content for collagen quantitation, nin-hydrin elastin detection, or di-methylene
blue assay for GAGs. Monoclonal antibodies may also be used to assess the quantity and location of specified ECM elements in
the processed ECM matrix. For the assessment of possible changes to the native structure of ECM components, methods that detect
macro- and micro-molecular changes can be used. For example, conducting mechanical tests, transmission electron microscopy
(TEM), scanning electron microscopy (SEM), or histological microscopy using stains such as Masson’s trichrome or Van Geisen
elastin stain can provide information on the macromolecular structure of ECM components.
Fresh DermGEN™ GraftJacket™
HLA-DR
HLA-A,B,C
Fig. 3 Immunohistochemical staining (brown peroxidase) for the presence of two main cellular immunogenic proteins responsible for allograft skin
rejection and increased inflammation: HLA-DR and HLA-A,B,C. Note significant staining for both fresh (unprocessed) human skin and Graftjacket™
(Acelity) Decellularized Human Dermal Matrix. In contrast, DermGEN™ (DeCell Technologies Inc.) decellularized human dermal matrix shows
complete removal of all immunogenic cell components. 400 magnification.
For micromolecular structural information, techniques such as thermal denaturation assays for collagen stability (e.g., hydro-
thermal isometric testing (HIT) or differential scanning calorimetry (DSC)) can be used. Further, more sensitive measures of
collagen structural changes can be detected using enzyme-based assays (e.g., trypsin or chymotrypsin) where the enzyme only inter-
acts with and solubilizes collagen with a non-native structure.
A final criterion for ensuring a positive outcome when using decellularized materials is the verification of a non-toxic material.
Cytotoxic effects are mainly attributed to residual chemicals or reagents used in the decellularization process. For example, sodium
dodecyl sulphate (SDS) has been shown to be cytotoxic and it binds very tightly with ECM components. Therefore, its removal from
the decellularized ECM is both critical and difficult to achieve without disruption of protein structures in the ECM. Assays conducted
to asses possible cytotoxicity of decellularized tissues and organs can be done with samples of intact material or with homogenized
extract samples. Multiple standard cell lines, such as mouse derived immortalized 3T3 cells, can be cultured in the presence of decel-
lularized materials and commercial cell viability assays (e.g., MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)) can be used to detect possible cytotoxicity.
Sterilization Issues
As with any material implanted into the human body, sterility is of the utmost concern to ensure patient safety. Unfortunately, most
standard methods used to sterilize medical instruments and devices, such as surgical instruments, cannot be used with biological mate-
rials. Use of exposure to high energy sources, such as gamma irradiation, has been shown to have detrimental effects on the structure
and chemical composition of ECM components. For example, collagen is shown to lose thermal stability indicative of it denaturing
whereby it goes from a very organized helical form to a random globular (gelatin) structure (Fig. 4). This loss of collagen’s native struc-
ture in irradiated tissue is also indicated by its increased susceptibility to degradation by the enzyme trypsin (Fig. 4). Other non-
energetic standard sterilization techniques, such as treatment with the chemical ethylene oxide (EO) in gaseous form, have been shown
to bind and react chemically to proteins in the ECM thereby altering their structure and chemical composition. These changes have
been linked to immune responses, increased inflammation, and accelerated degradation of EO treated tissues. Further, both irradiation
and EO sterilization have been shown to significantly alter the mechanical properties of tissue-based materials.
One method of sterilization recently developed for decellularized materials that has been shown to be both effective and non-
detrimental to its properties is the chemical reagent peracetic acid. Peracetic acid has been and is still used to sterilize donated
human bone. The concentrations and conditions that are used to treat bone however, destroy soft tissues and organs. Therefore,
a combination of unique treatment conditions and lower peracetic acid concentrations have been developed to successfully sterilize
decellularized materials without altering their structure or properties.
Example Decellularization Applications
Initially, decellularization processes as far back as the 1970s were aimed at soft-tissues such as skin, heart valves, arteries and
veins, ligaments, and tendons. Organ decellularization, however, has only developed in the last decade. Even so, to date, almost
Fig. 4 Rabbit trachea before (A) and after (B) decellularization. Trachea begins with red coloration indicating presence of cells and blood compo-
nents (A) which are removed during decellularization resulting in a whitish appearance (B). Histological sections (Masson’s trichrome staining) of
untreated (C) and decellularized trachea (D). Note removal of blue/black nuclei of cells after decellularization and the maintenance of the pink/purple
stained extracellular matrix.
every soft-tissue and organ in the mammalian body has been exposed to a decellularization process. For example, ligament
(Fig. 1), skin (Fig. 3), trachea (Fig. 4), ear cartilage (Fig. 5), Heart (Fig. 6) and Liver (Fig. 6). Clinical application and commer-
cialization of decellularized materials has so far remained in the area of soft-tissue with applications mainly in the areas of
wound care and reconstructive surgeries. Commercial wound care products for the treatment of chronic wounds such as diabetic
ulcers, venous stasis ulcers, and pressure ulcers include GraftjacketÔ (Acelity), DermamatrixÔ (Synthes), DermacellÔ (Lifenet),
DermapureÔ (Tissue Regenix), and DermGENÔ (DeCell Technologies Inc.) (Fig. 7). Other commercial products are being used
for breast reconstruction (e.g., AllodermÔ (Aceltity)), Orthopedic surgery (e.g., OrthopureÔ (Tissue Regenix), ArthroflexÔ
(Arthrex)), and dental surgery (AllodermÔ (Acelity), OracellÔ (Lifenet)). To date, the use of decellularized organs has not passed
animal model studies.
Fig. 5 Human ear cartilage histological sections (hemotoxylin and eosin staining) before (A) and after (B) decellularization. Note removal of blue
nuclei of cells after decellularization and the maintenance of the pink stained extracellular matrix.
Fig. 6 (Above) Decellularization of an adult porcine heart over 48 h. A 6-month-old porcine heart undergoing perfusion decellularization over
a period of 48 h. The native structure and vasculature are preserved after decellularization. After 48 h, the heart is completely decellularized. (Below)
Medical decellularization of an adult porcine liver over 24 h. A 6-month-old porcine liver undergoing perfusion decellularization over a period of 24 h.
The native structure and vasculature remain preserved after decellularization. This figure is taken an Elsevier publication: Eliminating the organ trans-
plant waiting list: The future with perfusion decellularized organs. Dominique Seetapun, PhD, Jeffrey J. Ross, PhD. Surgery Volume 161, Issue 6,
Pages 1474–1478 (June 2017).
Fig. 7 Application of decellularized human dermal graft (DermGEN™) on chronic diabetic foot ulcers. Patient 1 (upper) presented with a large non-
healing ulcer on the heel. After DermGEN™ was applied, the ulcer was completely healed in 8 weeks. Patient 2 presented with a small, deep ulcer on
the top of the big toe. After DermGEN™ was applied, the ulcer was close after 2 weeks and remained closed at 7 weeks with continued epithelial
renewal.
The Future of Decellularization
The future for the application of decellularization technology is promising. Already, successful applications of decellularization
technology to solve clinical problems exist. As a better understanding develops as to what is required for the removal of immuno-
genic donor cellular materials and the preservation of the remaining ECM, more effective decellularization processes will be devel-
oped. Further, as new cell sourcesdsuch as adult stem cells and induced pluripotent stem cells (IPSC)dare used for the
repopulation of decellularized tissues and organs, more successful applications of decellularization technology to treat human
disease and dysfunction.
Further Reading
Badylak, S. F., & Gilbert, T. W. (2008). Immune response to biologic scaffold materials. Seminars in Immunology, 20, 109–116.
Dyck, C. R., & Gratzer, P. F. (2007). Decellularized tissues in tissue engineering. In D. R. Bloomington (Ed.), Chapter 11, New research on biomaterials (pp. 281–320). Hauppauge,
NY: Nova Science Publishers Inc. Chapter 11.
Fu, R.-H., Wang, Y.-C., Liu, S.-P., et al. (2014). Decellularization and recellularization technologies in tissue engineering. Cell Transplantation, 23, 621–630.
Gilpin, A., & Yang, Y. (2017). Decellularization strategies for regenerative medicine: From processing techniques to applications. BioMed Research International, 2017, 1–13.
Gratzer, P.F. (2017) U.S. Pat. 9 566 369 B2 (Methods for tissue decellularization) issued February 14, 2017.
Kawecki, M., Labu, W., Klama-Baryla, A., et al. (2018). A review of decellurization methods caused by an urgent need for quality control of cell-free extracellular matrix scaffolds
and their role in regenerative medicine. Journal of Biomedial Materials Research Part B Applied Biomaterials, 106B, 909–923.
Keane, T. J., Swinehart, I. T., & Badylak, S. F. (2015). Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods, 84, 25–34.
Morris, A. H., Chang, J., & Kyriakides, T. R. (2016). Inadequate processing of decellularized dermal matrix reduces cell viability in vitro and increases apoptosis and acute
inflammation in vivo. BioResearch Open Access, 5(1), 177–187.
Seetapun, D., & Ross, J. J. (2017). Eliminating the organ transplant waiting list: The future with perfusion decellularized organs. Surgery, 161, 1474–1478.
Diamond, Carbon Nanotubes and Graphene for Biomedical Applications
Aaqil Rifai, Elena Pirogova, and Kate Fox, RMIT University, Melbourne, VIC, Australia
Diamond 98
Common Methods of Diamond Production 98
Diamond in Medicine 99
CVD diamond 99
Nanodiamonds 100
Diamond-Like Carbon 101
Carbon Nanotubes 101
Graphene 103
Specific Applications 104
Antibacterial Applications 104
Hard Tissue Implants 105
Drug Delivery 106
Bio-Imaging and Bio-Sensing 106
Conclusion 106
Further Reading 107
Abbreviations
CVD Chemical vapor deposition
CNT Carbon nanotube
DLC Diamond like carbon
MSC Mesenchymal stem cells
MWCNT Multiwalled carbon nanotube
PCD Polycrystalline diamond
PDMS Polydimethylsiloxane
PVD Physical vapor deposition
SWCNT Single walled carbon nanotube
UNCD Ultrananocrystalline diamond
Emerging trends show that carbon-based materials are rapidly taking place in medical applications. Carbon materials in current
biomedical use are diamond, diamond-like carbon, graphene, and carbon nanotubes. These allotropes of carbon have distinctive
chemical and physical characteristics owing to their spatial arrangement of the elements. Materials like diamond, carbon nanotubes
and graphene are used as a material for optics, medical electronics, tissue engineering, medical implants, medical devices, sensors
and other biomedical applications. In this reference module, we focus on broad applications of diamond, carbon nanotubes and
graphene. Diamond is used in advanced biomedical applications due to its hardness, wear resistance and biocompatibility prop-
erties. Diamond like carbon (DLC) is used for coating hip joints and other articulating surfaces, with an improvement to the overall
longevity of the implants. Silicon carbide (SiC) has been utilized in heart stents as well pyrolitic carbon used within artificial hearts.
Carbon nanotubes are more commonly used for drug delivery and sensing capabilities. Graphene is used in biomedicine with focus
on drug delivery, cancer therapy and biological imaging as well as in optics as an electrically insulating material.
In addition, carbon is popular due to its capacity to be formed across both the nano and micro scales. Carbon nanomaterials are
revolutionizing nanomedicine due to their controllable chemistries and properties. Zero-dimensional (0-D) carbon allotrope mate-
rials have all the dimensions in the nanoscale such as particulate diamonds, fullerenes and carbon blacks. Alternatively, one-
dimensional materials (1-D) and two-dimensional materials (2-D) have two and one nanoscale dimension, respectively, and these
are usually restricted to the thickness or diameter of the carbon material. An example of each may be carbon nanotubes (CNT),
diamond nanorods or carbon nanofibers for 1-D and graphene or diamond nanoplatelets for 2-D. A large number of the biomedical
carbons are however three-dimensional materials, for example, nanocrystalline diamond (NCD) films, nanodiamond, nanostruc-
tured diamond-like carbon (DLC) films and fullerene. Amongst these materials mentioned, diamond, graphene, and CNT have
gained the most attention in medicine. A number of deposition techniques have been explored to produce various allotropes of
carbon depending on the bonding nature (sp3 or sp2) and whether it is crystalline or amorphous. The carbon bonds can be
Encyclopedia of Biomedical Engineering, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.99874-X 97

98 Biomaterials: Science and Engineering j Diamond, Carbon Nanotubes and Graphene for Biomedical Applications
Table 1 Comparison of the advantages and disadvantages of some typical carbon materials used in biomedical
applications
Material Advantages Disadvantages
Amorphous carbon Moderate hardness Hardness is lower for

(Graphite-like carbon GLC, Wear resistance GLC than for DLC
diamond-like carbon DLC) Lower friction than diamond
High load bearing capacity
Good Adhesion
Low internal stress
Diamond Hard Brittle
Low friction Difficult to upscale
Corrosion resistant
Chemical inertness
High electrical resistance/resistivity
Optical transparency
Biocompatible
CNTs Biocompatible Safety concerns
High surface area Mostly impure
High sensitivity Lack of selectivity
Soluble High sensitivity to humidity
Can be functionalized
Graphene Flexible Cost
Optical transparency Difficult to upscale
Conductive
Hard
classified in terms of amorphous carbon (DLC), nanocrystalline diamond (NCD), and ultra nanocrystalline diamond (UNCD).
Referring to Table 1, it is clear that each carbon material offers different properties and benefits towards biomedical implantation.
Biomedical materials or biomaterials can be used to assist, treat, repair, or replace any function in the tissue, organ or body.
Carbon-based biomaterials are known to be biocompatible and have become increasingly common within the past decade. Poly-
mers comprising carbon are also of interest in biomedical applications. For instance, Poly-L-lactide (PLLA) is a polymer commonly
used for biodegradable coronary stents and bone plates. Contact lenses made from poly(methyl methacrylate, PMMA) and poly-
mers such as polycaprolactone (PCL) are used for resorbable applications. As nanomedicine has gained traction, these polymers are
readily being used as housing for nanoparticulate and drug delivery. However, for the purpose of this reference work we have not
focused on polymers rather the forms of carbon graphene, diamond and carbon nanotubes.
This article emphasizes on the importance of these materials and presents the major research undertaken in this area.
Diamond
Diamond is an exciting material for biomedical applications with broad and diverse medical applications from orthopedics to
medical bionics. Diamond is a material composed of carbon in its sp3 form. Diamond has a reported biocompatibility and bioac-
tivity making it a very suitable material for biointerfacing applications in areas such as orthopedics, dental and cardiovascular engi-
neering. The first instance where diamond provided the clear advantage to more common metallic biomaterials was in wear
resistant coatings for hip implants. Diamond is used in medicine in a number of different materials, predominantly nanocrystalline
diamond (NCD), polycrystalline (PCD), particulate nanodiamonds (PNDs), and nanodiamonds. PNDs are commonly fabricated
using detonation synthesis, however other forms of nanodiamonds can be synthesized to a high purity in to obtain specifically
tuned materials. The unique characteristics of PND are hardness, high thermal conductivity, chemical stability, fluorescence and
optical properties. More importantly, PNDs are highly biocompatible and non-toxic compared to other materials. Nanodiamonds
are also highly versatile types of diamond as they can be functionalized and optimized according to the type of bioapplication
required.
Common Methods of Diamond Production

Diamond is predominantly applied as a biomedical coating. These coatings are applied using the chemical vapor deposition (CVD)
technique in which materials to be coated are exposed to a plasma containing hydrogen, and carbon to deposit a secondary dia-
mond coating. In order to do so, nanodiamond powder is required to seed or provide a thin film in order to nucleate to form
the thicker CVD diamond coating. Where a freestanding diamond sheet is required sacrificial templates can be used (usually silicon)
and removed using acid dissolution to leave the remaining diamond. Nanodiamonds are generated by either the bottom up
Biomaterials: Science and Engineering j Diamond, Carbon Nanotubes and Graphene for Biomedical Applications 99
Table 2 Types of diamond
Crystal size Appearance Biomedical use
Single crystals mm–cm Clear–colored • No current biomedical application but possibilities in

sensing
Microcrystalline diamond (MCD) films 0.5–50 mm Black–clear • Implantable electrodes
• Implant encapsulation
• Heat dissipation
• Wear resistant coating
Nano crystalline diamond (NCD) 10–500 nm Black • Wear resistance
• Anti-corrosion barrier films
• Barrier film (biomedical)
Ultra-nano crystalline diamond (UNCD) 5–10 nm Black • Implantable electrodes
• Wear resistance
• Barrier film (biomedical)
Nanodiamond (ND) 5 nmþ Black • Antibacterial coatings
• Sensing
• Drug delivery
Modified from Garrett, D. J., Tong, W., Simpson, D. A. and Meffin, H. (2016). Diamond for neural interfacing: A review. Carbon 102, 437–454. doi: https://doi.org/10.1016/
j.carbon.2016.02.059 with permission.
approach of TNT detonation in a pressurized container or by the top down approach of milling a larger diamond. CVD presents
a form of diamond growth in micro and nano-crystalline form, in which the crystalline form can define the diamond surface
morphology. Outside of the requirement for homogeneous diamond coatings, nanodiamonds themselves can provide an excellent
biomaterial. Nanodiamonds are however restricted to cellular uptake and tracking applications as far. As an example, fluorescent
nanodiamonds have been used successfully in biomarking and cellular tracking applications. Diamond coatings using CVD are
limited to line of sight as well as chamber size. Interestingly, however, the CVD technique of diamond growth has provided superior
applications for medical bionics applications. By changing the elemental content within the gas plasma to include elements such as
nitrogen and boron, conductive channels can be produced in the atomic diamond lattice to change what is normally an insulating
material into a conductive diamond material. However, as with any secondary coating, the thermal mismatch between diamond
and its underlying substrate material provides an opportunity for coating delamination, particularly when coated using a high
temperature, high pressure environment such as the CVD chamber. A table summarizing the main types of diamond and its
common medical applications are shown in Table 2.
Diamond in Medicine
CVD diamond
Examples of CVD diamond coatings in current medical implant applications are seen in tissue contacting areas mandibular plates,
heart valves, neural stimulators and joint replacements where biological interaction is necessary. The use of diamond is however
somewhat restricted by its inherent properties. In particular, its extreme hardness provides limitations in its interfacing with
hard tissue in load bearing applications such as that of hip implant stems or fracture fixation where stress-sheilding would be
a problem. As a result, its hardness restricts it to contact surfaces where mechanical wear may occur or where the implant requires
its material to provide an avenue to guard against corrosion. Furthermore, despite its hardness, diamond is a brittle material. As
a result, it has found a role in tissue engineering (see e.g., heart valves) where biocompatibility and biointerfacing is paramount
due to diamond’s reported bioactivity, which provides an improved interaction between diamond-containing implants and the
neighboring soft tissue. Both nanostructured diamond coatings such as ultra-smooth nanodiamond and PCD have been used to
reduce the wear debris in orthopedic and dental implants providing high hardness, low surface roughness and excellent fracture
toughness and adhesion. Interestingly, these characteristics are especially favored towards titanium alloys compared to other
common biomedical metals such as cobalt chromium. The reason behind this is unknown, however, it is possible that the diamond
has difficulty forming a carbide layer with some metals. Typically, in total hip replacement surgeries, cobalt chromium alloys have
been used, due to their superior qualities mechanically, resistance to corrosion and biodegradation. Cobalt chromium alloys
however are susceptible to ion release under prolonged wear. Diamond has been shown to limit the release of toxic ions and there-
fore increase the longevity of the implant. Although diamond offers many opportunities for biomedical use, the progress in dia-
mond and diamond-coated implantables remains at a steady pace, with future avenues for diamond-breakthroughs being in
bionics and in hermetic capsules where micro/nano devices are currently at high need.
The advantages of diamond in medical bionics were highlighted by its selection for the electrode material in Australia’s
contribution to the race towards the bionic eye. Diamond was selected as the electrode material due to the fact that it could
be made to be both conducting and insulating within the same substrate (Fig. 1). The diamond electrode array, which inter-
faced directly with the retinal ganglion cells, was the key implanted material enable when placed to epiretinally interface with
Fig. 1 Diamond based bionic eye device using ultrananocrystalline diamond for the electrodes and polycrystalline diamond for the implantable
capsule: (A) shows the diamond electrode; (B) the packaging system; and (C) the implanted epi-retinal component. Reproduced with permission
from Ahnood, A., Meffin, H., Garrett, D. et al. (2017). Diamond devices for high acuity prosthetic vision. Advanced Biosystems 1(1), 1600003.
both the neural cells but also with head mounted camera offering the best performance and safety for patients. The stimulating
array is entirely fabricated using diamond to maximize the longevity and to increase biocompatibility due to the increased elec-
trochemical surface area offered by the diamond itself when compared to traditional materials such as platinum. In order to
fabricate the device, insulating polycrystalline diamond was used for the housing and nitrogen-incorporate ultrananocrystalline
diamond was grown via CVD into laser drilled feedthrough channels. By using the CVD technique and with both conducting
and insulating components made from diamond, hermetic sealing of the electrodes was achieved. As diamond can be difficult
to fabricate, the limitations of diamond-based device in retinal prosthesis are apparent, however, this technique presents a suit-
able method to produce a large number of conductive diamond feedthroughs in monolithic polycrystalline films (256
electrodes).
Biosensing is vastly expanding in the medical field, especially, with increased pressure for biomedical solutions for detection
and biomarking. Previously, CVD diamond has been commercialized outside of the biomedical field for applications in heat
sinks, X-ray windows, particle detectors, solar-blind UV detectors and electrodes. However, translation of these devices towards
medical use has been limited. Not unlike the retinal electrodes, the potential of diamond has not been explored fully due to the
difficulty in producing wafer-size devices. Fabrication of wafer-size crystals may potentially enhance the properties of diamond
for its further biomedical applications. Researchers showed that nanocrystalline diamond can exhibit improved properties in
micromechanical machines (MEMS and NEMS devices), surface acoustic wave devices, biological cell cultures and for DNA
detection.
Nanodiamonds
As with diamond films, nanodiamonds exhibit favorable characteristics in the biomedical applications due to its enhanced cellular
adhesion. Nanodiamonds (ND) are emerging as a novel class of nanomaterials due to their inexpensive cost, fluorescent capability,
non-cytotoxicity and biocompatibility. In fact, of all the nanocarbons, nanodiamond has the reported highest biocompatibility
compared to the alternative carbon nanomaterials such as carbon black, single walled nanotubes and multiwalled nanotubes.
NDs can be largely scaled as well as tunable with functionalized surfaces. Common biomedical avenues of NDs include drug
delivery, cell interaction, cancer therapy, protein and gene delivery, biomarking and antibacterial applications. These NDs are highly
versatile and are able to deliver antigens, water insoluble drugs, antibodies, nucleic acids and imaging agents into target cells, where
the therapeutic or imaging/diagnostic molecules would be released, and efficiently utilized. Nanodiamonds have found a specific
role in biomedical applications for their capacity to provide bio-sensing within individual cells. Intersecting between quantum
physics and biology, the capability to track the vacancies within the diamond lattice using a laser provides revolutionary capacities
for nanodiamonds in vivo. This will provide an excellent bio-sensing platform as individual nanodiamonds can specifically interact
with specific cells providing information as to cellular behavior and as a drug-delivery vehicle. These biomedical systems can be
scaled up for industrial production and easily altered to create designated functionality and terminations. Studies comparing nano-
diamond powder to nanodiamond films suggest that both provide a solid substrate for cellular interactions. In a study by Lechleit-
ner, published in the esteemed journal biomaterials (see Further Reading) it was shown that for a borosilicate glass sample coated
with either nanoparticulate diamond powder or with nanocrystalline diamond film, both diamond coating techniques provided an
improved bioscaffold for epithelial cells, with neither producing any changes to epithelial phenotype (Fig. 2). As discussed however,
previously in this article, the termination of the nanodiamond powder and films dictated the epithelial response. Both hydrogen
terminated and oxygen terminated powder were assessed finding that hydrogen termination provides a restrictive and inhibiting cell
attachment surface compared to the more biosuitable oxygen terminated surface. As a result, the functional polar groups of nano-
diamonds provide a method of controlling cell adhesion. This is important in biomedical environment where both hydrophilic and
hydrophobic surfaces have a role.
Fig. 2 Fluorescence images of the cell–surface interface of epithelial (HK-2) cells grown on glass, nanoparticulate diamond powder (DP) and
oxygen terminated nanocrystalline diamond (NCD-O). Images on the left side: Alexafluor 594 labeled activated Focal adhesion kinase stain. Images on
the right side: Rhodamin/Phalloidin labeled actin filaments (taken 24 h after seeding). Bars indicate 10 mm. Adapted with permission from Lechleitner,
T., Klauser, F., Seppi, T., et al. (2008). The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as
cell growth support surfaces. Biomaterials 29(32), 4275–4284.
Diamond-Like Carbon
Amorphous carbon with a level varying in sp3/sp2 content is called diamond-like carbon (DLC). As the sp3/sp2 ratio increases in
the favor of sp3, the amorphous carbon material shows diamond-like properties. Various methods exist to form DLC, but like dia-
mond, CVD and physical vapor deposition (PVD) are favored. DLC shows promise for biomedical applications with reported excel-
lent wear resistant properties. However the strength of DLC remains a concern. DLC exists in numerous forms of carbon, such as
amorphous carbon with hydrogen (a-C:H), amorphous carbon without hydrogen (a-C) and amorphous carbon pure sp3-
hybridized (ta-C). The hardness between the carbons forms also differs. The biggest downfall of DLC coating has been the load
bearing, residual stress and the level of adhesion with substrate materials with coating delamination prevalent. DLC has been shown
to have excellent bio- and haemocompatibility. It is thought that this may be due to the carbon bonds of DLC react with oxygen
removing the harmful super-oxide radicals often linked to tissue damage, strokes and cancer.
Carbon Nanotubes
Carbon-based nanotechnological advances made use of such a platform for a variety of biomedical applications. Carbon nano-
tubes (CNTs) are composed of carbon atoms and arranged in a benzene ring forming sheets to display a seamless cylinder. CNT
have superior structural, mechanical and electronic properties by diameter, length and chirality or twist. Fundamentally, CNTs
are a series of cylindrical carbon tubes with a high aspect ratio (Fig. 3). CNTs can be composed of a single tube, commonly called
Fig. 3 Scanning electron micrograph of multi-walled carbon nanotubes showing that the tubular structure is a nanometer in diameter and a microm-
eter in length. Adapted with permission from Harrison, B. S. and Atala, A. (2007). Carbon nanotube applications for tissue engineering. Biomaterials
28(2), 344–353. https://doi.org/10.1016/j.biomaterials.2006.07.044.
the single-walled carbon nanotube (SWNT) or the multiple cylinders, commonly known as multiwalled carbon nanotubes
(MWNTs). These two types of CNT differ in term of the sheet orientation. Some of the common applications of CNTs include
DNA and protein biosensors, ion channel blockers, bioreceptors and biocatalysts. As a result of the current trend in miniaturi-
zation and nanomedicine, CNTs are becoming more prominent in both neuroscience and tissue engineering research. CNT are
aligned in the top tier of carbon-based materials for both research and industrial applications. CNT are ordered, hollow nano-
structures composing of carbon atoms bonded around each other through sp2 bonds. There is a high demand for the use of CNTs
in biomedical applications for drug delivery, as antioxidants, for biosensors and for mechanical reinforcements. CNTs are typi-
cally produced using the CVD technique. CVD has benefits in CNT production, however limitations exist in the technique when
repeatability is sought.
CNTs appear promising as a scaffold for neuronal and ligamentous tissue growth for the CNS and orthopedic loci. Further, CNTs
have also been used as new structures capable of detecting antibodies (e.g., in autoimmune disease) or in combination with DNA or
peptide nucleic acid used for ultrasensitive complementary DNA strand detection. CNTs appear to be well suited towards bioma-
terials and may become more interesting as a tool for tissue engineering. The CNTs have the capacity to be combined in cellular
imaging, biological and chemical sensing, bioactive agent delivery, and matrix engineering. While there is still potential and devel-
opment of CNT for biomedical applications, concerns arise from their cytotoxicity. The lacking aspect of CNT is the non-
biodegradability, however excretion from the body is not a problem as it was shown in vivo. In addition, the capability of creating
nanosized sensors may provide key information regarding tissue microenvironments, thus yielding new insight into the cell–matrix
interface. CNT have been shown as promising material for drug delivery, electrical stimulus and cellular feedback. In summary, CNT
serve as a carbon-based material for novel applications on the rise including tissue engineering. CNTs for tissue engineering repre-
sent a challenging but potentially satisfying opportunity to develop the next generation of engineered biomaterials.
Improved tissue engineered mechanisms are new and versatile in tunability. The possibilities of nanosensors monitoring tissues
would open up pathways for tracking the performance, tissue and cellular responses. For example, the ability to monitor diseases,
and changes in critical biochemical processes such as apoptosis and angiogenesis leading to progressive disorders with high spatial
resolution would be beneficial for tissue engineering and sensing. An approach to monitoring engineered tissues might be to take
advantage of implantable sensors capable of relaying information outside the body. Real-time data is ideal for relating to the phys-
iological relevant variables such pH, PO2 (partial pressure of oxygen) and glucose levels. Sensing particles in the nanoscale can be
a significant advantage due to a number of life threatening diseases. The sensor also reduces a need for revision surgeries and the cost
of this procedure. Several features make CNTs ideal mechanisms for nanosensors including their electrical properties, large surface
area, and the capacity to immobilize DNA or other proteins.
It should be noted, however, that there remains doubt over the safety of CNTs within the body. CNTs are generally insoluble in
all types of solvents and it remains unknown how this translates to its solubility within the body itself. Introducing foreign bioma-
terials into the body can be challenging with risks of cytotoxicity involved. At any particular time, understanding the organism’s
response to the foreign substance is crucial. Currently, there are a number of studies that debate whether fullerene nanomaterials
such as buckyballs and CNTs are cytotoxic. Up to date research is contradictory and patchy, with some research studies reporting that
CNTs are toxic while others stating CNTs demonstrate excellent properties for cellular growth. Several in vitro studies, however, have
indicated that CNTs may be cytotoxic. For instance, Harrison and Atala reported in Biomaterials their 2007 study which showed that
when SWNTs and MWNTs were incubated with the macrophages of alveolar, a significant increase in cytotoxicity was shown after
6 h of growth. The impairment of phagocytosis of alveolar macrophage was shown to be significant with low doses of SWNT and
with high doses of MWNT, necrosis and degeneration were observed.
In contrast, however, CNTs have been shown to also improve both neural signal transfer and support dendrite elongation.
Further subdermal implantation showed minimal inflammation or noted irritation. As a result, it remains clouded as to the real
toxicity of CNTs. Nanotoxicity is of great interest at the moment. Consequently, many new reports are forming on the in vivo
toxicity. It is currently considered that the CNTs themselves are non-toxic but that toxicity is generated by accumulation and agglom-
eration of CNTs (as well as other non’carbon nanoparticles used today in nanomedicine). The toxicity due to CNT agglomeration, in
airways and vessels is not unlike the buildup of cholesterol within the body. Whilst there are concerns of the toxic effects of CNT,
newly developed fabrication methods are likely able of mitigating some of the risk. By functionalizing CNTs with glycopolymers,
the CNTs have been shown to be indistinguishable. This development gives rise to the use of CNTs for advanced tissue engineering
applications.
Graphene
Unlike diamond, graphene is a 2D atomic crystal that only comprises of a single layer of carbon atoms and typically has a honey-
comb structure. Graphene is the individual two-dimensional crystal structures made from a layer of atomic layers of graphite
(Fig. 4). Since the discovery of graphene sheet crystals, the exploration into its properties has been exponentially increasing. There
have been many recent advances in biomedical graphene as the material has the attributes of supreme mechanical stiffness, elas-
ticity, strength, thermal and electrical conductivity, all features sought after in medicine. Further, graphene has been used for
advanced applications in optics, electronics and thermal therapy. Another notable recent development is graphene oxide. Graphene
oxide is derived from sheets with oxygen functional groups. The most common method of graphene oxide production is known by
Hummer’s Method that involves the oxidation of graphite in potassium permanganate in sulfuric acid. Graphene is composed of
entirely sp2 hybridized carbon. The van der Waals forces keep the graphene sheets tightly packed together in graphite. Some of the
most popular biomedical applications of graphene oxide are schematically represented in Fig. 5. Biologically, graphene is recog-
nized for the high surface area and delocalized electrons serving as a site for drug delivery for cancer. Due to the oxidation of gra-
phene, there are several groups in graphene that provide suitability. Like other carbon biomaterials, currently graphene is
synthesized using the CVD technique.
Graphene however exerts some non-desirable features when implanted in the biomedical environment. The van der Waal forces
in graphene results in aggression under aqueous conditions. Therefore, caution must be taken when using graphene as opposed to
other carbon-based materials for biomedical applications. Alternatively, the oxidative functional groups are hydrophilic and the
water molecules can communicate with each other. The interaction of graphene oxide and water is advantageous when using as
a bio-lubricant.
Fig. 4 The different structures of graphene from the 2D sheet to 3D graphite (right) or 1D CNTs (center). Figure reproduced with permission from
Geim, A K. and Novoselov, K S. (2007). The rise of graphene. Nature Materials 6(3), 183–191.
Fig. 5 Most common biomedical applications of graphene related materials including sensing applications, drug delivery, and photothermal therapy.
Reproduced with permission from Bitounis, D., Ali-Boucetta, H., Hong, B. H., Min, D. H. and Kostarelos, K. (2013). Prospects and challenges of gra-
phene in biomedical applications. Advanced Materials 25(16), 2258–2268.
In the field of nanotechnology, new materials are continuously being sought for advanced biosensing purposes where accurate,
sensitive and selective detection of biomarkers are becoming a necessity. Recently, graphene-based sensors have shown great poten-
tial. Graphene-based sensors, used to detect thrombin and the protein, caspase-3, are effective tools for diagnosis and monitoring of
chronic and acute conditions. Also, they are effective in screening for genetic disorders such as Alzheimer’s disease. Here using gra-
phene, abnormalities can be traced for early identification and determination of the level of severity of a genetic disorder. Graphene-
based nanomaterial solutions have two alternate technologies. One that involves the use of a probe molecule on the graphene sheet
and other uses a label-free approach with measurements for electrical properties and interactions with an analyte. In addition to
biosensing applications, graphene-based materials were also shown a great promise for different applications in tissue engineering.
Graphene oxide, as an example, has been reported to show good biocompatibility (cellular growth and proliferation) with osteo-
blast cells as well as increased bone bioactivity and hydroxyapatite formation in vitro.
Graphene materials have also provided some advantage to both stem cell growth and differentiation. Comparative studies have
shown that the proliferation and mineralization of mesenchymal stem cells (MSC) on graphene and graphene oxide substrates was
higher when compared to the standard PDMS substrates. Hence, it is likely that graphene provides an improvement in osteogenic
properties than more traditional substrates. For a more detailed analysis of graphene’s interactions with other cell types (e.g., human
mesenchymal stem cells, human neural stem cells, mouse hippocampal neurons) refer to the studies listed in the references and
further readings at the end of this article. Interestingly, graphene appears to provide an advanced material in neural sensing with
graphene reportedly promoting adherence and the inducement of neural stem cells towards neurons instead of ganglion cells as
well as increasing neuronal outgrowth in the early stages of development. Fig. 6 shows the enhancement of neural stem cells on
fluorinated graphene substrates showing the preferential attachment of mesenchymal stem cells on graphene. As a result,
graphene-based substrates can be designed to be an advanced biomedical material for the next generation of neural chips and
implantable substrates for neurodegenerative diseases. Overall, the mechanical, electrical, cellular properties of graphene can be
significant for developing biomedical systems, scaffolds, and other applications.
Specific Applications
Antibacterial Applications
Antibacterial materials or even materials with some antibacterial advantage are critical for biomedical applications. Antibacterial
coatings can be classified into two groups, passive where only upon contact will the coating effect bacterial adhesion or active where
antibacterial agents are delivered locally. Reducing the risk of bacterial adhesion is highly recommended in the modern day
Fig. 6 Graphene nanomaterials in tissue engineering. (A) Enhanced neuronal differentiation of neural stem cells (NSCs) on fluorinated graphene in
2D; (i) schematic showing mesenchymal stem cell (MSC) patterning by printing PDMS barriers on graphene films directly; (ii and iii) MSCs preferen-
tially attached on the fluorinated graphene with aligned F-actin (red) and expression of neural specific markers-Tuj1 and MAP2 (green) (scale bar
50 mm). (B) (i) SEM of 3D graphene foam; (ii) fluorescence images of NSCs proliferated on 3D-GFs for 5 days stained for nectin (green), nuclei (blue)
and (iii) fluorescence images of differentiated NSCs under differentiation conditions, Tuj-1 for neuron (green), GFAP for astrocyte (red), O4 for oligo-
dendrocyte (green) and DAPI for nuclei (blue). Reproduced with permission from Goenka, S., Sant, V. and Sant, S. (2014). Graphene-based nano-
materials for drug delivery and tissue engineering. Journal of Controlled Release 173, 75–88.
orthopedic implantation. As carbon materials have become more prevalent in biomedical implantation, carbon nanomaterials have
shown toxicity in some environments to both eukaryotic (cells with a nucleus) and prokaryotic (cells without a nucleus like
bacteria) cells. It is unknown whether this is due to the hydrophilicity of these materials or due to their nanosize which both enable
the nanoparticles to cross the cell membrane. As a result, these moderately toxic carbon nanomaterials such as fullerene CNTs and
graphene oxide have gained momentum for antibacterial applications. The antibacterial effect appears to be related to surface treat-
ment, reactivity and mechanical interaction which is more likely to affect bacterial adhesion than the carbon material itself. In any
case, antibacterial effects have been reported for most carbon biomaterials, particularly graphene and diamond. Graphene oxide
“nanowalls” showed some toxicity to bacteria whilst graphene, graphite, graphene oxide, reduced graphene have a reported effect
on Escherichia coli bacterial growth. Diamond, on the other hand, appears to provide resistance to bacterial colonization when
compared to more orthodox biomaterials (e.g., stainless steel and titanium).
Hard Tissue Implants

Bone comprises highly porous hard tissues with variable mechanical properties. Where hard tissue replacement is required, tissues
such as bone and teeth can be replicated using carbon-biomaterials. For hard tissue implants, the biggest concern is trauma, infec-
tion, tumor, and general wear. Carbon biomaterials, such as diamond are strong applicants for an implant due to its hardness,
strength, biocompatibility and chemical stability to provide the longevity for implantation, particularly as a coating material. As
a coating material, diamond shows osteogenic differentiation, biocompatibility and osseointegration. Graphene has also showed
similar osteogenic capabilities with reported accelerated bone growth upon these substrates. When combined with polymers like
polycaprolactone, functionalized MWCNTs suggest similar osteogenic properties with reported increases in osteoblast proliferation.
These carbon materials may provide both the implant and the fixture to support bone formation. Similarly, nanodiamond can also
improve the mechanical properties of the scaffold. Functional termination changes the properties of the material. Well dispersed
oxygen terminated nanodiamond can increase the hardness of the underlying substrate for implantation. This modification can
significantly increase the cellular attachment as well as the bone formation. The osteogenic, cytotoxic and mechanical properties
are key criteria’s that must be evaluated against to promote a scaffold. Good osteogenic factors will improve the bonding strength
of the implant and the bone tissue. With a sound fixation, the bone tissue repair as well as the wear will be long lasting. Without
a good interface between implant and bone, the implant can be a subject to aseptic loosening and subsequent implant failure.
Further, both diamond and CNT coatings onto more traditional metal implants can provide a protective anticorrosive layer. The
anticorrosive nature of the diamond and CNTs serves as a critical element to reduce metallic ions that may be lost during the
Fig. 7 Demonstration of the use of the NV center in the diamond lattice for sensing applications. Reproduced with permission from Suter, D. and
Jelezko, F. (2017). Single-spin magnetic resonance in the nitrogen-vacancy center of diamond. Progress in Nuclear Magnetic Resonance Spectroscopy
98–99, 50–62.
wear of implants. Some carbon-based nanomaterials play a crucial role for treatment of those who suffer from arthritis. The water
soluble nature of carbon molecules can act to prevent the loss of fluids in joints. Ultimately, carbon-based materials have shown
benefits that supersede other materials in hard tissue implants.
Drug Delivery
For drug-delivery, nanocarbon biomaterials are widely used due to their nanoscale size and unique physiochemical properties
allowing the material to be taken up by individual cells. As a result, many reports exist of carbon-based materials being used for
the treatment of cancer. The most significant carbon materials used for drug delivery appear to be nanodiamond, CNT and gra-
phene. Given the strong advantages carbon appears to offer in hard tissue applications, it is logical that nanocarbons have a role
in tracking bone morphogenetic protein-2 (BMP-2). Using graphene oxide coatings on titanium, appears to influence BMP-2
drug delivery. As a result, in vivo bone formation can be enhanced through graphene use. Similarly, particulate nanodiamonds
are also capable of delivering BMP-2 which can induce the differentiation and enhancement of bone formation. The protein release
may be regulated by the pH or delayed responses, which give surgeons more control during an operation. The steady release of the
protein makes it desirable for areas that require longer bone healing. For the repair of tissue, carbon nanotubes (SWCNT) can be
used. In the case of Alzheimer’s disease, nanocarbon materials have been used to deliver particular drugs such as acetylcholine in
mice brains. Likewise, functionalized MWCNT can deliver growth factors through the use of non-covalent grafting and by dispersing
the growth factors provide a reduction in toxicity.
Bio-Imaging and Bio-Sensing

Carbon-based materials are typically presented in form of bio-imaging agents or biosensors for diagnostic purposes. CNTs have
amazing electrochemical properties, making them a popular form of biosensors. Unlike conventional glassy carbon, CNTs have
the ability to undergo fast electron transfer. Hence, they are able to negate most disadvantages faced by the other carbon or metal
electrodes for amperometric or voltammetric analyte detection. CNT have also used optical biosensors to design new infrared fluo-
rescence and Raman scattering of the sensors. The other material with good electrochemical and conductive properties is graphene.
Graphene uses two types of systems for bio-sensing. The first uses a probe molecule which communicates with the analyte, whilst
the second measures the change in the electrical properties and how it interacts with the analyte. The major advantage, when using
graphene-based electrochemical sensors, is that the measurements are only affected by the electrodes. Further, carbon nanomaterials
in bio-sensing platforms can be utilized for the detection of cancer biomarkers. Due to the biocompatibility of carbon based mate-
rials, it is easier to disperse the agents and markers in the body. They have lower chances of toxicity with other cells. The most
exciting breakthrough however is the recent intersection of quantum physics and medicine. Diamond has long being of interest
in quantum computing due to the vacancies in the diamond lattice which exhibit a detectable color center. At the present, nano-
diamonds have been used to track individual cells that have taken up the single nanodiamond through phagocytosis. As a result of
tracking the color center within that diamond, individual cells can be probed for activity and movement (Fig. 7). In summary, as the
research evolves diamond- and graphene-based bio-sensors will be more prevalent.
Conclusion
Carbon-based materials are becoming extensively used in biomedical applications. As reported throughout this article, various dia-
mond, carbon nanotubes and graphene materials can be fabricated and manipulated to generate unique properties and provide
a unique biomedical material. The unique properties of carbon material have the potential to improve the current status-quo in
biomaterial design, tissue engineering, drug delivery, cytotoxicity, biosensing, bioelectronics and many more biomedical applica-
tions. The use of carbons in biosensing, bioelectronics and implants have risen. Advances such as the utilization of diamond for
stimulating retinal electrodes demonstrate the optimization of carbon for biomedical performance. Such research complements
other advances that have followed breakthroughs in the biomedical field. Carbon nanotubes provide a novel material for applica-
tions in drug delivery, whilst functionalized CNTs produce highly soluble biomaterials. Although there are some safety concerns for
the nanocarbons, there remains benefits for exploring carbon in nanomedicine pathways. Similarly, graphene has thrived in
biomedical industries with advantages over other non’carbon materials in implantable biosensors. Overall, numerous biomedical
applications can be envisaged as the growth of carbon-based materials continues to thrive.
Further Reading
Ahnood, A., Meffin, H., Garrett, D., et al. (2017). Diamond devices for high acuity prosthetic vision. Advanced Biosystems, 1(1), 1600003.
Bitounis, D., Ali-Boucetta, H., Hong, B. H., Min, D. H., & Kostarelos, K. (2013). Prospects and challenges of graphene in biomedical applications. Advanced Materials, 25(16),
2258–2268.
Cha, C., Shin, S. R., Annabi, N., Dokmeci, M. R., & Khademhosseini, A. (2013). Carbon-based nanomaterials: Multifunctional materials for biomedical engineering. ACS Nano, 7(4),
2891–2897.
Chen, H., Müller, M. B., Gilmore, K. J., Wallace, G. G., & Li, D. (2008). Mechanically strong, electrically conductive, and biocompatible graphene paper. Advanced Materials, 20(18),
3557–3561.
Correa-Duarte, M. A., Wagner, N., Rojas-Chapana, J., et al. (2004). Fabrication and biocompatibility of carbon nanotube-based 3D networks as scaffolds for cell seeding and
growth. Nano Letters, 4(11), 2233–2236.
Garrett, D. J., Ganesan, K., Stacey, A., et al. (2011). Ultra-nanocrystalline diamond electrodes: Optimization towards neural stimulation applications. Journal of Neural Engineering,
9(1), 016002.
Garrett, D. J., Tong, W., Simpson, D. A., & Meffin, H. (2016). Diamond for neural interfacing: A review. Carbon, 102, 437–454. https://doi.org/10.1016/j.carbon.2016.02.059.
Geim, A. K., & Novoselov, K. S. (2007). The rise of graphene. Nature Materials, 6(3), 183–191.
Goenka, S., Sant, V., & Sant, S. (2014). Graphene-based nanomaterials for drug delivery and tissue engineering. Journal of Controlled Release, 173, 75–88.
Hadjinicolaou, A. E., Leung, R. T., Garrett, D. J., et al. (2012). Electrical stimulation of retinal ganglion cells with diamond and the development of an all diamond retinal prosthesis.
Biomaterials, 33(24), 5812–5820.
Harrison, B. S., & Atala, A. (2007). Carbon nanotube applications for tissue engineering. Biomaterials, 28(2), 344–353. https://doi.org/10.1016/j.biomaterials.2006.07.044.
Hauert, R. (2004). An overview on the tribological behavior of diamond-like carbon in technical and medical applications. Tribology International, 37(11), 991–1003.
Jaatinen, J. J., Korhonen, R. K., Pelttari, A., et al. (2011). Early bone growth on the surface of titanium implants in rat femur is enhanced by an amorphous diamond coating. Acta
Orthopaedica, 82(4), 499–503.
Kim, S., Ku, S. H., Lim, S. Y., Kim, J. H., & Park, C. B. (2011). Graphene–biomineral hybrid materials. Advanced Materials, 23(17), 2009–2014.
Lechleitner, T., Klauser, F., Seppi, T., et al. (2008). The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as cell growth
support surfaces. Biomaterials, 29(32), 4275–4284.
Lee, W. C., Lim, C. H. Y., Shi, H., et al. (2011). Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide. ACS Nano, 5(9), 7334–7341.
Li, N., Zhang, X., Song, Q., et al. (2011). The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates. Biomaterials, 32(35),
9374–9382.
Lin, X., Clasky, A., Lai, K., & Yang, L. (2016). Carbon-based nano biomaterials: Design, fabrication and application. In , 49. In: Biomedical nanomaterials: From design to
implementation. London: Institution of Engineering and Technology.
McGuinness, L. P., Yan, Y., Stacey, A., Simpson, D., Hall, L., et al. (2011). Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells. Nature
Nanotechnology, 6(6), 358–363.
Park, S. Y., Park, J., Sim, S. H., et al. (2011). Enhanced differentiation of human neural stem cells into neurons on graphene. Advanced Materials, 23(36), H263–H267.
Passeri, D., Rinaldi, F., Ingallina, C., et al. (2015). Biomedical applications of nanodiamonds: An overview. Journal of Nanoscience and Nanotechnology, 15(2), 972–988.
Siew, P. S., Loh, K. P., Poh, W. C., & Zhang, H. (2005). Biosensing properties of nanocrystalline diamond film grown on polycrystalline diamond electrodes. Diamond and Related
Materials, 14(3), 426–431. https://doi.org/10.1016/j.diamond.2004.11.016.
Suter, D., & Jelezko, F. (2017). Single-spin magnetic resonance in the nitrogen-vacancy center of diamond. Progress in Nuclear Magnetic Resonance Spectroscopy, 98-99, 50–62.
Tang, L., Tsai, C., Gerberich, W., Kruckeberg, L., & Kania, D. (1995). Biocompatibility of chemical-vapor-deposited diamond. Biomaterials, 16(6), 483–488.
Gold Nanoparticles for Colorimetric Detection of Pathogens
Paul Z Chen and Frank X Gu, University of Waterloo, Waterloo, ON, Canada
Introduction 109
Classical Methods for Pathogen Detection 109
Principles of Biosensing 110
Gold Nanoparticles as Colorimetric Biosensing Agents 110
Nucleic Acid-Based Pathogen Detection 111
Complementary Base Pairing 111
Aptamers 112
Antibody-Based Pathogen Detection 112
Nonfunctionalized Pathogen Detection 113
Outlook 114
Acknowledgements 114
Further Reading 114
Glossary
Absorbance Energy loss of photons passing through a material due to dissipation through inelastic processes.
Extinction The combined effects of absorbance and scattering. For nanoparticles, especially those greater than 40 nm,
a spectrophotometer generally measures extinction rather than absorbance due to nonnegligible scattering processes.
Multiplex Synonymous with “many”. Used as an adjective to describe the simultaneous detection of multiple pathogens,
usually through the use of a corresponding number of specific biorecognition elements.
Scattering An incident photon energy causes a photon to be emitted at the same frequency or a shifted frequency.
Sensitivity A commonly used homonym: In analytical contexts, it is the smallest concentration change that can be detected,
which is sometimes, but not always, the same as the limit of detection. In diagnostic contexts, it is the true positive rate.
Surface plasmon resonance The resonant, or maximal, oscillation of conduction electrons at a negative permittivity/positive
permittivity interface under illumination. Dielectric/metal interfaces satisfy the permittivity requirement, which leads to
a significant enhancement in the absorption and scattering properties of metals.
UV/Vis spectrophotometer A key instrument used to characterize colorimetric nanoparticles. It is used for the measurement, at
a single wavelength or spectral range, of the attenuation of a beam of light when passing through or reflecting off of a sample.
Abbreviations
Au NP Gold nanoparticle
Au NR Gold nanorod
bp Base pair
CTAB Cetyltrimethylammonium bromide
LSPR Localized surface plasmon resonance
mL Milliliter, 10 3 L
nm Nanometer, 10 9 m
PCR Polymerase chain reaction
PNA Peptide nucleic acid
POC Point-of-care
RNA Ribonucleic acid
SPR Surface plasmon resonance
ssDNA Single-stranded deoxyribonucleic acid
ssRNA Single-stranded ribonucleic acid

Biomaterials: Science and engineering j Gold Nanoparticles for Colorimetric Detection of Pathogens 109
Introduction
Over the last 20 years, nanomaterial-based pathogen detection has witnessed an explosion in interest. Publications in the area
increase yearly significantly each year; intense research has fuelled academic and clinical significance explorations. Seminal studies
from the late 1990’s and early 2000’s have laid the foundation for a number of novel methods for pathogen detection, of which
some are undergoing clinical validation or are even available commercially.
Such progression is due to two primary reasons: First, the study of nanomaterials has itself exploded to uncover desirable prop-
erties that are unavailable to bulk-scale materials. As one example, at the bulk scale gold is, simply, the color gold. As nanoparticles,
however, their color can be tuned throughout the visible spectrum (red, orange, green, purple, blue, etc.) and beyond. Second, infec-
tious diseases continue to be one of the world’s most pressing issues. Once thought to be a solved problem, mortality and morbidity
mount. Infectious diseases still account for 20% of global deaths and 30% of disability-adjusted life years, a measure of overall
disease burden. Moreover, the crisis of drug resistance threatens the efficacy of treatment. Effective diagnosisdusually through
the detection of pathogens, the causative agents of infectious diseasedis classically insensitive, slow, unreliable and difficult but
remains as the key step in improving outcomes and reducing drug resistance.
Classical Methods for Pathogen Detection
Detecting pathogens has been a formidable challenge that has thus far limited disease outcomes. Classically, pathogen detection is
preformed using one of three methods: culturing and inspection, immunological assays or polymerase chain reaction (PCR)-based
assays (Fig. 1).
Culturing and inspection isolates and grows pathogens from a clinical specimen. For detection, cells are then grown in selective
media and/or undergo an indefinite series of biochemical tests (Gram staining, catalase test, hemolysis, etc.). Thus, turnaround
times are slow (generally days, sometimes 2 weeks for certain pathogens), and the method is prone to contamination and inaccurate
results. Moreover, some pathogens cannot be cultured in the lab.
The second classical method, immunological assay, is primarily preformed in the clinic for biomolecule detection but is occa-
sionally used for pathogens. The enzyme-linked immunosorbent assay (ELISA) is a well-known example. ELISA uses antibodies
bound on a substrate, usually a microwell plate, to capture and isolate the target analyte. In sandwich ELIZA, the most popular
format, secondary antibodiesdusually a polyclonal antibodydthat target a surface epitope of the analyte are then added. These
secondary antibodies are conjugated to enzyme that catalyzes the conversion of chromogenic substrate for a colorimetric
response. The intensity of colorimetric response is proportional to the concentration of target analyte. Immunological assays
are, thus, highly specific but are costly and still require many operational steps by trained personnel and have a long sample-
to-answer time.
PCR-based assays amplify and then detect a target nucleic acid sequence through the design of primer and probe nucleic
acid sequences. If the target sequence is specific to a species or even strain of pathogen, then detection can be conferred. A
number of variations of PCR have been developed. Reverse transcription-PCR (RT-PCR) can be used to detect RNA viruses.
It also takes advantage of the rapid degrading nature of RNA to distinguish between live and dead cells. Quantitative real-
time PCR (qPCR) uses fluorescent molecular probes to monitor amplification in real-time and thus can confer detection
during amplification. PCR-based methods can decrease run time but require extensive sample preparation by highly trained
personnel and are susceptible to inaccuracies due to contamination. Interestingly, culturing and inspection remains the
“gold standard” for pathogen detection. Some hospitals have begun to adopt qPCR but a clear need exists for improved
methods.
Culturing & Immunological

Inspection assays
PCR-based assays
Fig. 1 Schematic representations of the three classical methods for pathogen detection.
110 Biomaterials: Science and engineering j Gold Nanoparticles for Colorimetric Detection of Pathogens
Principles of Biosensing
A biosensor is comprised of a biorecognition element coupled to a transducer. The former enables binding of the nanomaterial to
target pathogen; the latter converts a binding event to some measurable readout (e.g., color). Generally, nanomaterials are function-
alized with a biorecognition element and employed as transducer or a component of the transduction process.
The performance of a biosensor depends on six key characteristics: sensitivity, dynamic range, accuracy, sample-to-answer time,
robustness and amenability. Sensitivity is the smallest concentration change that can be detected. Sometimes, but not always, it is
equivalent to the limit of detection, the lowest concentration that can be detected. Dynamic range is the working, linear scoped
from lowest concentration to highest concentrationdwhich the biosensor operates. Accuracy is a key factor for validity and
considers false results. Sample-to-answer time is the integral metric for rapidity, as it considers the entire method (sample prepa-
ration, amplification, detection time, etc.), which is of greatest diagnostic importance. A robust biosensor is not easily perturbed
by potential changes in conditions and can dictate overall performance. Amenability depends on a host of factors such as ease
of use, requirements for trained personnel, number of operational steps and amenability for use at the point-of-care (POC).
The introduction of nanomaterials has led to novel, improved biosensors. These biosensors harness unique properties of nano-
materials for improved pathogen detection. Reports have demonstrated them to be more sensitive, accurate, rapid, robust and
utilizable.
Gold Nanoparticles as Colorimetric Biosensing Agents
For biosensing, gold nanoparticles (Au NPs) are the most prevalent nanomaterial used. In general, Au NPs are the most stable type
of metal nanoparticle. They have also garnered much attention due to facilely for synthesis and modification. Importantly, Au NPs
can be prepared in a high quality, high yield and size-controllable manner with notable colloidal stability. In addition, the forma-
tion of spontaneous, covalent thiol’gold bonds is often used for modification. For example, antibodies or nucleic acids with exposed
thiol groups can chemisorb onto and functionalize Au NPs in a facile manner.
Interesting optical properties of Au NPs further increase their attractiveness for pathogen detection. Many of these optical prop-
erties rely on surface plasmon resonance (SPR). In SPR, coherent electron oscillations (called plasmons) are excited by the electric
field of incident light. To transfer momentum from a photon to a plasmon, a negative permittivity/positive permittivity interface
must be present. Metal-dielectric interfaces satisfy this requirement (i.e., the phenomenon occurs at a surface), and these excitations
are strongest at a resonant frequency, hence SPR. For nanoparticles, plasmons are confined or localized to the significantly smaller
nanoparticle surface, hence LSPR, which leads to an enhanced, local electromagnetic field. Nanoparticles composed of a noble
metal, such as gold, show strong LSPR bands in the visible range thereby enabling colorimetric utility. In addition to composition,
shape, size, local environment and interparticle distance can strongly influence LSPR (Fig. 2). Changes in the latter two factors,
through binding or aggregation upon recognition of analyte, are exploited in the design of colorimetric biosensors.
Colorimetric biosensing enables a number of modalities. For example, color change in response to a pathogen can be observed
by eye, increasing amenability. Moreover, it can be assessed quantitatively via camera or spectrophotometer for more robust
measurements. Furthermore, small changes in local environment and interparticle distance are observed with real-time changes
in visual color to potentially increase rapidity. For colorimetric pathogen detection, and general biosensing, Au NPs are
(A) (B)
Increasing size
Increasing size
More spherical More spherical

Fig. 2 (A) Transmission electron micrographs of gold nanoparticles with varying sizes and shapes. Scale bars are 50 nm each. (B) Corresponding
image of visual colors of those gold nanoparticles. Reproduced from Verma, M. S., Chen, P. Z., Jones, L. and Gu, F. X. (2014). Branching and size
of CTAB-coated gold nanostars control the colorimetric detection of bacteria. RSC Advances 4, 10660, with permission from the Royal Society of
Chemistry.
functionalized with nucleic acids or antibodies or are nonfunctionalized. The following sections will elucidate these three methods
and while highlighting seminal, impactful and promising reports of Au NPs.
Nucleic Acid-Based Pathogen Detection

Complementary Base Pairing
Biosensors can utilize the highly conserved, specific binding between nucleic acid base pairs (bps) for pathogen detection. Through
thiol’gold chemistry, Au NPs can be functionalized with probes of DNA, RNA or peptide nucleic acid (PNA), noncharged oligomers
with a peptide bond-linked backbone that present various purine or pyrimidine bases. A target pathogen can be detected through
binding of a species- or strain-specific complementary sequence. Small changes in refractive index local to the surface of the Au NPs
can lead to a colorimetric response. Such an approach, however, is insensitive. That is, innumerably large concentrations of target
nucleic acid must be present for a sufficient colorimetric response. In addition, nonspecific binding or simple changes in the compo-
sition of solution can lead to false positive readings. For greater sensitivity, specificity and robustness, an aggregation-based
approach utilizing the dependence of LSPR on interparticle distance can be employed.
In a seminal report, Mirkin et al. showed three key results: Au NPs can be stably functionalized with ssDNA probes; recog-
nition of target sequence can lead to a visually discernible colorimetric response; and this response is highly specific, distin-
guishing target from single-bp mismatches. Spontaneous aggregation is achieved through the use of two ssDNA probes that
are complementary to each half of the target sequence (Fig. 3). Detection is conferred only upon binding to both halves and
formation of dsDNA between Au NPs. This enables the facile colocalization of Au NPs. A significantly stronger and more
sensitive colorimetric response can be observed. Since small, spherical Au NPs were used, the characteristic red-to-blue color
change occurs.
Many reported biosensors have employed this foundational strategy, two probe-functionalized Au NPs for target recognition.
Some modifications have used a portable spectrophotometer for quantitative POC analysis. Other use camera phones and apps
for image-based colorimetric analysis. For clinically relevant pathogen detection, however, additional factors must be considered.
First, the target sequence must be accessible. DNA or RNA of the pathogen (bacteria, virus, fungi, etc.) must be extracted. Second,
amplification must be preformed. Although, a relatively sensitive method, target sequences are usually too dilute at clinically rele-
vant concentrations. Targeting sequences with many copies per pathogen (e.g., 16S rRNA) can help but even these nucleic acids are
too dilute. In addition, reducing sample volumes (e.g., in very small microfluidic devices) can improve sensitivity, but this comes
with a trade-off in the observable colorimetric response and a smaller dynamic range. Amplification before detection is usually pre-
formed via PCR or a related technique, such as rolling circle amplification (RCA). Amplification after binding can be preformed by
enriching the signal of Au NPs, for example, by catalytically growing the AuNPs. Third is a constraint on all colorimetric approaches:
changes in color must be observable. Many clinical samples (e.g., blood) have their own color and varying degrees of opacity. Thus,
for these, pathogens must be first be isolated.
(A)
Target DNA
(B)
i iv
ii v
iii vi
25 38 42 53 60 25 38 42 53 60
Temperature (°C)
Fig. 3 (A) Schematic representation of nucleic acid-functionalized gold nanoparticles used for the colorimetric detection of complementary
sequence. (B) Visual image of the colorimetric response after aggregation in response to (i) target nucleic acid, (ii) no target, (iii) complementary to
one probe, (iv) a 6-bp deletion, (v) a 1-bp mismatch, and (vi) a 2-bp mismatch. Reproduced from Elghanian, R., Storhoff, J. J., Mucic, R. C., Let-
singer, R. L. and Mirkin, C. A. (1997). Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold
nanoparticles. Science 277, 1078–1081, with permission from the American Association for the Advancement of Science.
Interestingly, the use of PNA probes generally increases sensitivity, accuracy and robustness. Their neutral backbone enables
PNA-DNA binding without the electrostatic repulsion that accompanies DNA–DNA binding. In addition, PNAs are more resistant
to nucleases and proteases. But, some PNAs may be slightly more hydrophobic than their DNA or RNA counterpart, although this
does not typically affect Au NP stability.
Approaches alternative to the two half-complementary functionalized probes exist for pathogen detection. In one approach,
after amplification, free ssDNA probe can be added to a sample before the addition of Au NPs and salt. ssDNAs, generally adsorb
more significantly onto the surface of Au NPs than do dsDNAs. Au NPs coated with ssDNA have improved stability over bare Au
NPs. Thus, in the presence of salt, which is applied at a concentration surpassing colloidal stability, bare Au NPs aggregate while
ssDNA-coated ones do not. In other words, a solution changing from red to blue indicates the detection of target pathogen. A similar
approach uses short thiol-modified probes. Rather than absorb onto the surface of Au NPs, the thiol allows for covalently binding.
Target sequence-binding leads to a much longer dsDNA. Thus, in the presence of salt, dsDNA-coated Au NPs remain red whereas
aggregation and blue color indicate a lack of target. A third approach uses DNAzymes, nucleic acids that can catalyze the cleavage of
other nucleic acids. DNAzyme-functionalized Au NPs can cleave DNA or RNA. The cleaved nucleic acids can then induce aggrega-
tion in the presence of salt and heat.
Aptamers
Aptamers are synthetic oligomers of ssDNA, ssRNA or peptides that bind to some target with high specificity and affinity. Analogous
to antibodies, aptamer binding leads to structurally conform to their target, with the bound state as thermodynamically favorable.
They are often used for the detection of small molecules or metal ions, which are difficult to detect using antibodies (too large
compared to small molecules or metal ions) or nonfunctionalized approaches (no specificity for targets at that size scale). And,
indeed, aptamers can be used to detect pathogen byproducts enabling indirectly detection of pathogens, such as for the Shiga
toxin-producing Escherichia coli. However, their specificity and high stabilitydrelative to antibodiesdalso allow them to be used
for direct pathogen detection.
In one approach, aptamers first adsorb onto the surface of Au NPs. In the presence of target pathogen, aptamers dissociate from
the surface of gold nanoparticles. Thus, their specificity and higher affinity for target enables a red-to-blue color change that signals
the presence of pathogen. In another approach, Au NPs can be modified with two ssDNA probes. Each probe is complementary to
part of the DNA aptamer to yield an initial blue color. Importantly, some aptamer bases remain unpaired. In the presence of target
pathogen, the aptamer binds and changes conformation thereby leading to dissociation and therefore de-aggregation of the Au NPs.
A color change to red signals pathogen detection.
Antibody-Based Pathogen Detection
Antibodies can be stably functionalized, without loss of structure and while presenting paratope, onto Au NPs. Antibodies can be
modified to present thiol groups or Au NPs can be modified to present carboxylate or amine groups. The biotin–avidin or biotin–
streptavidin method can also be used to functionalize modified Au NP. Thus, while anchored by a Au NP, binding antigen leads to
a change in the local refractive index and associated colorimetric response. Increased packing of Au NPs on the surface of pathogens
can lead to interparticle coupling for a corresponding increase in sensitivity; dynamic range may increase as well.
Similar factors must be considered for antibody-based pathogen detection. A colorimetric response must be observable in
sample of interest. Additionally, like DNA and RNA, antibodies are prone to denaturation from heat. Thus, antibody-based
approaches must considered elevated temperatures. Due to the amplification step present in DNA-based approaches, when
compared antibody-based ones are generally less sensitive. Colorimetrically, antibody-functionalized Au NPs have been used for
the detection of foodborne infections. Food matrices are usually amenable for nanoparticles to retain stability, and after slight prep-
aration, samples are void enough of conflicting color for the direct detection of pathogens. Thus, the steps required for nucleic acid-
based detection (extraction, isolation, amplification) are not necessary for such applications if the Au NPs are sensitive enough.
Antibody-functionalized Au NPs are more often used for direct detection without a large number of preliminary steps.
As previously mentioned, the structure of Au NPs is highly tunable. For example, rather than a spherical shape, rods can be
synthesized. These are called gold nanorods (Au NRs). Since LSPR strongly depends on it, different structures have different optical
properties. Thus, two distinctly different structures can be used for multiplex detection (Fig. 4). That is, Au NPs, or more specifically
Au NRs in this case, that contrast largely can detect one or more pathogens from the same sample. As one example, the aspect ratio of
Au NRs (length vs. width) can be increased to change extinction spectra. With nonoverlapping signals and appropriately function-
alized each Au NR with antibody, multiplex pathogen detection can be achieved based on aggregation around the surface of the
pathogen(s) (where the epitope[s] should be presented). Visual detection for this method is possible; but the presence of multiple
Au NRs in the same solution usually necessitates the use of an instrument for accurate utility.
An interesting format using antibody-functionalized Au NPs is the lateral flow immunochromatographic assay, a POC assay that
is colloquially known from the home pregnancy test. Indeed, the colored lines (usually red) that appear on some home pregnancy
tests are due to the collection of Au NPs. In these products, urine is applied to the sample zone. Once soaked, fluid travels through
capillary action. As it travels up the pad a collection of antibody-functionalized Au NPs is rehydrated and bind to target protein. If
present, target protein is then captured on the test zone in a sandwich format by pad-bound antibodies. Thus, the development of
Pathogen 1
Fig. 4 (Top) Schematic representation of two antibody-functionalized gold nanorods used for the multiplex detection of whole-cell pathogens. Gold
nanorods vary in aspect ratio to enable. Spectrophotometric responses (A) before and (B) after recognition of a target pathogen. Transmission elec-
tron micrographs showing the aggregation of gold nanorods with (C, D) differing surface coverage around target bacteria. Reproduced from Wang,
C. and Irudayaraj, J. (2008). Gold nanorod probes for the detection of multiple pathogens. Small 4, 2204–2208, with permission from Wiley-VCH.
red color on the test zone signals the presence of target protein and thus pregnancy. Remaining Au NPs are captured on a control
zone to ensure robustness. This approach has also been demonstrated for the detection of pathogens. Antibodies are replaced with
ones suitable for target pathogen. The sample of interest is applied similarly to the sample zone, and rehydrated Au NPs develop
color to detect pathogen.
Nonfunctionalized Pathogen Detection
The last main method uses nonfunctionalized Au NPs. Without nucleic acids or antibodies presented, the surface of Au NPs are
usually stabilize with a capping agent, such as citrate, surfactant, such as CTAB, or thiol-ligand, such as an alkanethiol. These agents
are usually present during, to help direct, synthesis and self-assemble onto the Au NP surface. If implemented properly, these agents
can also aid in the detect pathogens.
Nonfunctionalized Au NPs can be used for the general detection of pathogens. For example, using a positively charged thiol-
ligand can impart a positive surface charge onto Au NPs. b-Galactosidase, an anionic enzyme near neutral pH, can then self-
assemble onto the surface. While on the surface of the Au NP, the enzyme is inhibited. Thus, even in the presence of chromogenic
substrate, no color change occurs. In the presence of bacteria, which present many negative surface charges (teichoic acids, lipopoly-
saccharides, phospholipids, etc.), Au NPs bind, and b-galactosidase is displaced. Thus, activity is restored; substrate is quickly con-
verted and color develops. Moreover, similar to ELISA, multiple turnovers, a characteristic feature for an enzyme, provide
amplification for color development; and an impressive limit of detection can be reached (100 cells mL 1). Interestingly, these
Au NPs can be printed onto paper strips for a cost-effective POC test.
The previous report highlights major advantages and disadvantages of nonfunctionalized Au NPs. Interestingly modifica-
tions can enable novel, effective biosensing strategies. However, nonfunctionalized Au NPs are generally limited from utility
in complex environments due to nonspecific interactions. For example, positively charged Au NPs can change color due to the
presence of any sufficient negative charge (e.g., protein, salt, erythrocytes), leading to significant false positives. Relevance in
some clinical samples is currently limited in contrast to nucleic acid- or antibody-based approaches. Simple environments,
which are expected to be sterile and void of sufficient negative charge, such as drinking water, are best suited for these non-
functionalized Au NPs. In addition, species- or strain-level identification, rather than general detection of pathogens, can be
challenging.
One interesting report employs a “chemical nose” strategy. Here, Au NPs are coated with CTAB to present a positive charge. They
interact with the negative surface charge of pathogens. Since the amount and density of negative surface charge is unique for each
Fig. 5 (A) Schematic representation of nonfunctionalized gold nanoparticles used as a “chemical nose” for the detection of whole-cell pathogens.
(B) Colorimetric responses of “chemical nose” biosensor to various bacterial pathogens. Reproduced from Verma, M. S., Chen, P. Z., Jones, L. and
Gu, F. X. (2014). “Chemical nose” for the visual identification of emerging ocular pathogens using gold nanostars. Biosensors & Bioelectronics 61,
386–390, with permission from Elsevier.
bacterial species, the Au NPs produce a unique degree of color change when testing ocular bacterial pathogens. Thus, colorimetric
identification at the species-level can be achieved in an accurate manner using a single set of Au NPs (Fig. 5).
Outlook
Au NPs enable a variety of biosensing approaches for the colorimetric detection of pathogens. Importantly, when compared with
classical methods, these Au NP-based ones are more sensitive, accurate, rapid and robust. The use of colorimetry also maximizes
amenability; pathogen detection can be conferred visually. And, POC formats can be developed naturally. With varying advantages
and disadvantages, Au NPs can be employed in three ways: they can be functionalized with nucleic acids or antibodies or can avoid
functionalization. As these products pass clinical trials and are introduced to real-world application, they will provide exceptional
diagnostic capabilities and therefore improve disease outcomes while curbing the development of drug resistance.
Acknowledgements
This work was financially supported by the Natural Science and Engineering Research Council of Canada (NSERC). P.Z.C. is supported by the NSERC
Vanier Canada Graduate Scholarship and WIN Nanofellowship.
Further Reading
Ahmed, A., Rushworth, J. V., Hirst, N. A., & Millner, P. A. (2014). Biosensors for whole-cell bacterial detection. Clinical Microbiology Reviews, 27, 631–646.
Bell, S. E., & Sirimuthu, N. M. (2006). Surface-enhanced Raman spectroscopy (SERS) for sub-micromolar detection of DNA/RNA mononucleotides. Journal of the American
Chemical Society, 128, 15580–15581.
Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L., & Mirkin, C. A. (1997). Selective colorimetric detection of polynucleotides based on the distance-dependent optical
properties of gold nanoparticles. Science, 277, 1078–1081.
Giljohann, D. A., & Mirkin, C. A. (2009). Drivers of biodiagnostic development. Nature, 462, 461–464.
Hamula, C. L., Hughes, K., Fisher, B. T., et al. (2016). T2Candida provides rapid and accurate species identification in pediatric cases of candidemia. American Journal of Clinical
Pathology, 145, 858–861.
Huang, S. H. (2006). Gold nanoparticle-based immunochromatographic test for identification of Staphylococcus aureus from clinical specimens. Clinica Chimica Acta, 373,
139–143.
Kelley, S. O., Mirkin, C. A., Walt, D. R., et al. (2014). Advancing the speed, sensitivity and accuracy of biomolecular detection using multi-length-scale engineering. Nature
Nanotechnology, 9, 969–980.
Kelly, K. L., Coronado, E., Zhao, L. L., & Schatz, G. C. (2003). The optical properties of metal nanoparticles: The influence of size, shape, and dielectric environment. Journal of
Physical Chemistry B, 107, 668–677.
Li, X., Kong, H., Mout, R., et al. (2014). Rapid identification of bacterial biofilms and biofilm wound models using a multichannel nanosensor. ACS Nano, 8, 12014–12019.
Liu, J., & Lu, Y. (2006). Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nature Protocols, 1, 246–252.
Miranda, O. R., Li, X., Garcia-Gonzalez, L., et al. (2011). Colorimetric bacteria sensing using a supramolecular enzyme-nanoparticle biosensor. Journal of the American Chemical
Society, 133, 9650–9653.
Murray, C. J., Vos, T., Lozano, R., et al. (2012). Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: A systematic analysis for the global
burden of disease study 2010. Lancet, 380, 2197–2223.
Neu, H. C. (1992). The crisis in antibiotic-resistance. Science, 257, 1064–1073.
Verma, M. S., Chen, P. Z., Jones, L., & Gu, F. X. (2014a). Branching and size of CTAB-coated gold nanostars control the colorimetric detection of bacteria. RSC Advances, 4,
10660.
Verma, M. S., Chen, P. Z., Jones, L., & Gu, F. X. (2014b). “Chemical nose” for the visual identification of emerging ocular pathogens using gold nanostars. Biosensors &
Bioelectronics, 61, 386–390.
Verma, M. S., Chen, P. Z., Jones, L., & Gu, F. X. (2015). Controlling “chemical nose” biosensor characteristics by modulating gold nanoparticle shape and concentration. Sensing
and Bio-Sensing Research, 5, 13–18.
Verma, M. S., Wei, S. C., Rogowski, J. L., et al. (2016). Interactions between bacterial surface and nanoparticles govern the performance of “chemical nose” biosensors.
Biosensors & Bioelectronics, 83, 115–125.
Verma, M. S., Tsuji, J. M., Hall, B., et al. (2016). Towards point-of-care detection of polymicrobial infections: Rapid colorimetric response using a portable spectrophotometer.
Sensing and Bio-Sensing Research, 10, 10–19.
Wang, C., & Irudayaraj, J. (2008). Gold nanorod probes for the detection of multiple pathogens. Small, 4, 2204–2208.
Manufacture of Biomaterials
Min Wang, Lin Guo, and Haoran Sun, The University of Hong Kong, Pokfulam, Hong Kong
Introduction 117
Special Requirements for Making Biomaterials 117
Good Manufacturing Practice 117
Manufacture of Metals for Biomedical Applications 117
Chemical Metallurgy 117
Metal Fabrication Techniques: Forming Operations 117
Metal Fabrication Techniques: Casting 118
Other Metal Fabrication Techniques 119
Physical Metallurgy 119
Machining of Metallic Biomaterials 119
Manufacture of Porous Metals 119
Manufacture of Biomedical Polymers 120
Polymer Classification and Architecture 120
Polymer Synthesis 121
Step-growth polymerization 121
Addition polymerization 121
Copolymerization 121
Amorphous Polymers and Semi-Crystalline Polymers 122
Polymer Additives 122
Polymer Blends and Interpenetrating Networks 122
Polymer Forming Techniques 123
Compression molding and transfer molding 123
Injection molding 123
Extrusion 124
Blow molding 124
Casting 124
Fabrication of Hydrogels 124
Manufacture of Porous Polymers 125
Manufacture of Bioceramics 125
Fabrication of Dense Bioceramics 125
Manufacture of Porous Bioceramics 126
Fabrication of Pyrolytic Carbon 127
Fabrication of Bioactive Glasses 127
Fabrication of Bioactive Glass-Ceramics 128
Manufacture of Biomedical Composites 128
Composite and Composite Classification 128
Fabrication of Particulate Biomedical Composites 129
Polymer matrix composites 129
Metal matrix composites 129
Ceramic matrix composites 129
Fabrication of Fibrous Biomedical Composites 129
Short-fiber composites 129
Long-fiber composites 129
Fabrication of Laminated Structures 130
Fabrication of Porous Composite Scaffolds for Tissue Engineering 130
Manufacture of Nano-Biomaterials 130
Fabrication of Nano-Structured Biomaterials 130
Fabrication of Nano-Sized Biomaterials 131
Surface Modification Techniques for Biomaterials 131
Surface Modification Techniques for Biomedical Polymers 132
Surface Modification Techniques for Metallic Biomaterials 133
Biomimetic Deposition 133
Summary 134
Acknowledgments 134
Further Reading 134
116 Encyclopedia of Biomedical Engineering, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.11027-X

Biomaterials: Science and Engineering j Manufacture of Biomaterials 117
Introduction
Special Requirements for Making Biomaterials
Materials (metals, polymers, ceramics, and composites) are made and used in nearly all engineering fields. Many technologies for
making these materials and their products are also suitable for the manufacture of biomaterials (metallic biomaterials, biopolymers,
bioceramics and biomedical composites). Biomaterials can be considered as a sub-set of engineering materials and the paramount
criterion for turning engineering materials into biomaterials is the biocompatibility of materials. Many materials meet the biocom-
patibility requirement and also possess required properties (mechanical, electrical, biological, etc.) and hence they are used as
biomaterials. However, any or slight deviation in composition or microstructure for the material may instantly disqualify the mate-
rial from the biomedical application as such changes can result in the loss of the required property or in significantly lowered perfor-
mance in the biological environment. Therefore, the manufacturing technologies should be carefully selected and used for
biomaterials, avoiding change in composition and/or microstructure for the finished products. Another point that must be borne
in mind in biomaterials manufacture is the purity of biomaterials. Any synthesis or fabrication process should not introduce impu-
rity into the biomaterial. Accidental inclusion of impurity/impurities in biomaterials during their fabrication can alter their biolog-
ical performance to be below an acceptable level. Therefore, commercial production of biomaterials and manufacture of implants
and medical devices most likely take place in the clean room, avoiding any contamination. The final point is about sterilization.
Implant and medical devices are used for patients in the sterilized state, which is a major difference for biomaterials and engineering
materials. The product sterilization rendered by currently used sterilization methods can cause deterioration of mechanical prop-
erties and shortened shelf-life of implants, especially polymeric devices. Manufacture of biomaterials and their sterilization may
need to be considered together.
Good Manufacturing Practice

Different from the production of engineering materials and their products, the manufacture of biomaterials and their products must
follow good manufacturing practices (GMPs). GMPs are practices issued by government agencies that control the authorization and
licensing of the manufacture and sale of medical devices. They provide minimum requirements that a manufacturer must meet to
assure that their products are consistently high in quality, from batch to batch, for their intended use. They provide guidance for
manufacturing, testing, and quality assurance in order to ensure that the product is safe for human use. One important aspect in
following GMP is that the manufacture is well documented. In the United States, GMPs are issued and enforced by the U.S.
Food and Drug Administration. GMPs, along with good laboratory practices and good clinical practices, are overseen by regulatory
bodies in many countries.
Manufacture of Metals for Biomedical Applications

Chemical Metallurgy
Metallic biomaterials include noble metals (Au, Pt, etc.), 316L stainless steel, Co–Cr alloys, Ti and Ti alloys, and other biocom-
patible metals. They are essential for implants and devices in many medical fields (orthopedics, dentistry, cardiovascular, etc.).
Metals exist in combination with other elements in the Earth as minerals (compounds) and need to be extracted from ores, which
are dug out in mines in different geographical regions of the world. Metallurgical principles in extractive metallurgy are followed
to remove a metal from an ore and refine the extracted raw metal into a purer form. Metal alloys are made by adding other
elements into pure metals, resulting in materials with improved or desired properties. For example, to make 316L stainless steel
with excellent corrosion resistance for orthopedic implants, iron is alloyed with specific amounts of Cr, Ni, Mo and other
elements while keeping the carbon content below a limit. These processes (extraction, alloying, etc.) involve chemical reactions
and this branch of science is termed chemical metallurgy. (The other branch is physical metallurgy, which deals mainly with
mechanical, thermal, electrical, magnetic and other properties of metals in the solid state.) Both physical and chemical processes
including mining, extraction, melting, re-melting, alloying and controlled solidification are used to make metal-containing ore to
raw metal products in the bulk form such as ingots, from which stock metal shapes (powders, wires, plates, rods, tubes, etc.) are
subsequently produced. The stock materials are purchased and used by medical device manufacturers to fabricate various types of
products for biomedical applications. Metallurgy is distinguished from metalworking although metalworking relies on metal-
lurgy. Metalworking processes make stock materials into useable shapes and sizes, and they include casting, forging, rolling, sin-
tering, 3D printing, etc.
Metal Fabrication Techniques: Forming Operations

Metals and alloys are made into products of different shapes (plates, rods, tubes, etc.) with desired properties by different metal
fabrication techniques. These techniques include metalworking operations (e.g., forging), powder metallurgy, welding, etc. Practi-
cally, the reason for choosing a particular fabrication technique depends on several major factors such as properties of the metal, the
size and shape of the finished products, and the cost. Major metalworking operations, that is, forging, rolling, extrusion and
118 Biomaterials: Science and Engineering j Manufacture of Biomaterials
(C)
(A)
Force Billet
Die Die
Ram
Pre-heated
metal blank Extruded product
Container
Force
(B) (D)
Roll
Die
Tensile force
Roll
Fig. 1 Schematic diagrams showing four metal forming operations: (A) forging, (B) rolling, (C) extrusion, and (D) drawing.
drawing, are generally used to form metal pieces whose shapes can be formed by large plastic deformation. The four operations are
depicted in Fig. 1.
Forging utilizes large plastic deformation of metals to form objects and is normally conducted at an elevated temperature. A high
force is applied to the hot metal which deforms and conforms to the internal geometry of the die. Rolling can be performed at either
room temperature (“cold rolling”) or an elevated temperature (“hot rolling”). Hot rolling can have the advantage of obtaining fine-
grain structures for the products. Metal sheets and thin films are commonly produced by rolling. In extrusion, a metal bar or rod is
pushed through a die orifice by a high force acting on the ram. Metal rods and tubes with complicated cross-sectional geometries can
be produced by extrusion. Drawing uses tensile force to pull a material through a die orifice. Thin metal wires can be made through
drawing.
Metal Fabrication Techniques: Casting

Casting is the manufacturing process of pouring fully molten metal into a mold cavity, with the liquid metal undergoing solidifi-
cation into the desired shape. There are a number of casting techniques, including sand casting (e.g., for magnesium alloys), invest-
ment casting (e.g., for stainless steel alloys), die casting (e.g., for zinc alloys), and continuous casting (e.g., for aluminium alloys).
Casting is extensively used for fabricating metal products with complex shapes which are difficult or uneconomical to make by other
methods.
Sand casting, which is the simplest and most common type of casting, allows for fabricating metal products with low tooling and
equipment costs. The sand, either moist or dry, is mixed and bonded together with chemical binders and/or polymerized oils to
form a sand mold with internal cavity in the shape and dimensions of the finished metal product. The metal, such as Ti alloys,
is melted first and then poured into the cavity of the sand mold. Subsequently, the sand mold is separated and the solidified casting
is removed. As the sand mold needs to be destroyed in order to obtain the metal product, this fabrication technique has a low
production rate. Sand casting is extensively used for the fabrication of metals with complex structures (e.g., engine blocks of auto-
mobiles) or some small metal parts such as gears and pulleys.
Another important technique is investment casting (also known as “lost-wax casting”). In this process, a wax pattern with full
design specifications of the product is firstly made using a metal mold. The wax patterns are then assembled and coated with a refrac-
tory ceramic material (e.g., Al2O3). De-waxing is conducted to remove wax from the assembly, and the molten metal is poured into
the cavity of the assembly and undergoes solidification. The ceramic shell is then knocked down and individual castings are thus
produced. The whole process of investment casting is complicated as compared to simple sand casting and hence it is relatively
expensive. This technique is suitable for the fabrication of products with intricate shapes for almost all alloys, for example, dental
fixtures.
Die casting is a fabrication process that requires pressure to force molten metal into the cavity of a mold. In this process, the
steel mold is not destroyed. Die casting can be classified into different groups and die casting with high pressure is most widely
used. The die casting technique is simple but the equipment, dies and other related components for die casting are expensive. It is
suitable for producing very large quantities of castings with small or medium sizes for non-ferrous metals (zinc alloys, tin alloys,
etc.).
In continuous casting, a molten metal is poured into a mold and the semi-finished product continuously solidifies while new
molten metal is constantly poured into the mold. Thus a continuous length of metals is produced as the casting keeps going
downward. Continuous casting is time-saving as it can be fully automated. It is extensively used for the production of steel, copper
or aluminium in industries.
Other Metal Fabrication Techniques

Beside the metal fabrication techniques described above, several other methods such as power metallurgy and welding are also
well developed. Powder metallurgy produces metal products from metal powders in basically three steps: powder blending, die
compaction at room temperature, and sintering. It can be used to fabricate metal products that cannot be made through melting
or formed by other methods. For example, porous Ti alloys that can be used for orthopedic implants can be made via powder
metallurgy.
Welding is a fabrication technique that joins metal parts by causing fusion through heat alone or together with pressure. In
conventional welding, an electric arc between the metal and an electrode is created and the metals are melted in the welding
area. After solidification, metal parts are joined. Shielded metal arc welding and gas metal arc welding are commonly welding tech-
niques. Welding can be used to make joints between dissimilar metals (e.g., titanium-stainless steel dissimilar material pair) in
implantable medical devices such as stents and hip replacement implants.
Physical Metallurgy
Physical metallurgy is based on the fundamentals and applications of the theory of phase transformations in metals and employs
heat treatment to alter microstructures of metals for achieving desired mechanical, electrical, magnetic, thermal and other proper-
ties. The words “heat treatment” are general and indicate that a cycle of heating and cooling is performed on a metal to change its
property (which is caused by the microstructural change(s) in the metal).
Commonly employed heat treatment techniques are annealing, quenching, tempering, normalizing, case hardening, precip-
itation strengthening, etc. In annealing, a metal is heated up at a pre-determined heating rate to a pre-set high temperature,
dwells at this temperature for a required duration, and is finally cooled down (or quenched) at a desired cooling rate. Metals
after heavy “cold work”, for example, forging at room temperature and cold rolling, have high strength but low ductility. They
require heat treatment for stress relief and recovery of ductility and hence partial annealing or full annealing is performed to
soften the metal. During annealing, microstructural changes (producing “recovery,” and “recrystallization”) occur in cold-
worked metal, resulting in mechanical property changes. The yield strength of metals depends on the average grain size (the
“Hall-Petch equation”). A heat treatment above a certain temperature can increase the grain sizes of a metal (“grain growth
law”) and hence change the mechanical properties of the metal. A heat treatment can also change phase(s) in metals. For
example, two phases, a (ferrite, BCC crystal structure) and g (austenite, FCC crystal structure), can exist in steels. a is a soft
phase and g phase has high strength. 316L stainless steel with g phase is used for making orthopedic implants. For steels,
subcritical annealing can be performed at 540–650 C where there is no change of crystal structure. Intermediate annealing
can take place at 650–760 C and some transformation to austenite occurs. Full annealing for complete austenitization may
be conducted at 810–930 C.
Machining of Metallic Biomaterials

Machining is a process in which a piece of metal (sometimes, “hard” polymer; very rarely, bioceramic) is made into desired shape
and dimensions through controlled material removal by using a machine. Machining including turning, milling, drilling, etc., and
cutting is the most common method. The machines must have cutting tools made of very hard materials for performing machining
operations. Metallic biomaterials are often machined into final products. However, care must be taken in machining certain bioma-
terials, for example, Ti-based alloys, due to characteristics of these materials. Different machining conditions based on the types of
cooling process and metal cutting parameters are used for metallic biomaterials, and the machining techniques are generally dry
machining, wet machining, minimum quantity lubricant (MQL) machining, cryogenic machining, high speed machining
(HSM), and air cooling machining. Among all these, wet machining is the most widely used material removal process for metals
with the advantages of high efficiency and low production time and cost. The surface properties of metals, cutting tools and cutting
fluids play important roles in the machining process of metallic biomaterials.
Manufacture of Porous Metals

Porous metals were firstly attempted in 1943 when pores were intentionally introduced into aluminium by adding mercury into the
molten aluminium. The concept of using porous metals in biomedical applications was investigated much later, and the earliest
work was conducted in 1972 when employing porous metals for osseointegration was studied. There have been continuous efforts
for using porous Ti alloys in orthopedics. In recent years, developing porous metallic scaffolds for regenerating human hard tissues
is making good progress, which involves materials such as Ti alloys and Mg alloys. At present, a number of techniques are used to
fabricate porous metals, which include powder metallurgy, space holder method, plasma spraying, and additive manufacturing
technologies.
Manufacture of Biomedical Polymers
A polymer is a long chain macromolecule composed of repeating subunits termed “monomers.” The monomers are con-
nected by covalent bonds in polymers. The term “polymer” is derived from ancient Greek words poly and meres, which
mean “many” and “parts,” respectively. Polymers play an important role in daily life owing to their broad range of prop-
erties, availability and low cost. Polymers have been widely used for many types of biomedical devices (e.g., dental, ortho-
pedic, and cardiovascular implants). Knowledge in polymer synthesis and product manufacture is essential in the biomedical
field.
Polymer Classification and Architecture

Polymers can be classified in a number of ways. They can be classified according to the origin of materials: natural or
synthetic. Natural polymers such as RNA, DNA, proteins and polysaccharides have existed since life began, and they play crit-
ical roles in all forms of life. In the history of mankind, since the earliest times, people have been exploiting natural polymers
for clothing, writing materials, weapons, etc. Natural polymers can possess either relatively simple structures, such as natural
rubber, cellulose and starch, or very complex structures, including enzymes, proteins and nucleic acids. Synthetic polymers
appeared much later as compared with natural polymers but have been playing crucial roles in the modern world. In
1909, the first synthetic polymer phenol-formaldehyde (“Bakelite”) was invented, which was soon followed by many other
synthetic polymers.
Polymers can also be classified into two groups, thermoplastic polymers and thermosetting polymers, based on their
thermal response properties. Thermoplastic polymers can be thermally softened or plasticized repeatedly. Polymers such as
polyolefins, nylons and linear polyesters belong to this group. For thermosetting polymers, in the product manufacture
process, chemical changes occur upon heating of this type of polymers and convert them into an infusible mass. The curing
or setting process leads to growth and cross-linking of chain molecules, producing giant molecules. After product manufac-
ture, thermosetting polymers cannot be re-melted. Polymers including resins, urea, diene rubbers and phenolic are thermo-
setting polymers.
On the basis of polymer chain structure, polymers can be classified as linear, branched, cross-linked, and network. Fig. 2 depicts
the structures of these polymers. In linear polymers, the monomers are joined together in a linear manner, while in branched poly-
mers, some monomers are joined as branches on the polymer backbone. If the monomer units are joined in multiple chains and
form interconnections between chains, cross-linked polymers are made. When a cross-linked polymer includes plentiful intercon-
nections between chains in 3D, a network polymer is formed.
The interlinking ability of a monomer, or the number of available sites for bonding with other monomers during poly-
merization, is termed the functionality of a monomer. A molecule can therefore be monofunctional (one site), bifunctional
(two sites), or polyfunctional (more than two sites). Monomers with bifunctional structural units can only have two link-
ages with other monomers. Thus the array of linkages between bifunctional monomers results in a linear polymer. Polyfunc-
tional monomers can be linked together to form nonlinear structures. If the side growth during polymerization is
terminated before the polymer chain can link up with another polymer chain, a branched polymer is formed. If the growing
polymer chains of polyfunctional monomers can be linked with each other, cross-linked polymers or network polymers are
produced.
For biomedical applications, natural polymers such as collagen, gelatin, chitosan, hyaluronic acid and alginate are used.
Synthetic polymers such as poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE) and ultra-high molecular weight
polyethylene (UHMWPE) are widely used for various implants and medical devices. Biodegradable polymers such as poly(lactic
acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL) are extensively used for tissue engineering
applications.
(A) (B)
Linear Branched
(C) (D)
Cross-linked Networked
Fig. 2 Structures of polymer chains.
Polymer Synthesis
Two main types of polymerization are used in polymer synthesis: step-growth polymerization and addition polymerization. Ring-
opening polymerization is also used for some polymers. If polymerization involves multiple types of monomers, copolymerization
takes place, forming copolymers.
Step-growth polymerization
Step-growth polymerization, or condensation polymerization, is commonly employed to produce polyesters and nylons. The poly-
merization reaction occurs between the functional groups of molecules. Small molecules such as water are eliminated by the chem-
ical reaction in step-growth polymerization. Below is an example for making nylons:
R NH2 þ R 0 COOH /R 0 CONHR þ H2 O (1)
In step-growth polymerization, one or more types of monomers can be involved, and each monomer should have at least two
sites for bonding. For polymerization with more than one type of monomers, for example, involving A and B monomers, A–B step-
growth polymerization or A–A/B–B step-growth polymerization can occur.
Addition polymerization
Addition polymerization, or chain reaction polymerization, requires the monomers to have at least one double bond. In addition
polymerization, no molecule is eliminated and no by-product is generated. The molecular weight of the formed polymer is exactly
the same as the sum of all monomers included in the polymerization. A chain reaction links monomers together by rearranging the
bonds with each monomer. For example, the reaction to synthesize ethylene is
nfCH2 ¼ CH2 g/–CH2 –ðCH2 –CH2 Þ–CH2 – (2)
The initiation of addition polymerization (in above case, the breaking of a double bond) requires catalysts, pressure, heat or UV
light. The growing polymer chain is a free radical in the polymerization. Terminal radicals are required for ending the reaction.
There are three steps in this type of polymerization: initiation, propagation, and termination. During initiation, the monomer
acquires an active site to become a free radical. The addition of initiators or other approaches such as absorption of heat, light or
irradiation can trigger the initiation process. In propagation, the initiated monomers add other monomers in rapid succession. This
step continues until the active site, which is continuously relocated at the end of the growing chain during propagation, is deacti-
vated by chain termination or chain transfer. The termination step involves the reaction of a polymer chain radical with another free
radical. These three steps constitute the normal step-growth polymerization process. However, in many cases, a fourth step, namely,
chain transfer, is also included. In chain transfer, the growing activity of a polymer chain is transferred to previously inert species.
Copolymerization
Copolymerization is the polymerization using two or more types of monomers and produces copolymers. The composition of
copolymers can vary widely, resulting in vastly different properties. Possible arrangements of monomers in different copolymers
are shown in Fig. 3.
In copolymerization, the number of reactions increases geometrically with the number of monomer types, that is, four reactions
for two types of monomers. The composition and structure of copolymers are determined by the relative rates of different chain
propagation reactions.
(A) Alternating
(B) Random
(C) Block
(D) Graft
(E) Random tripolymer
Monomer A Monomer B Monomer C
Fig. 3 Arrangements of monomers in copolymers.

Amorphous Polymers and Semi-Crystalline Polymers

The structure of polymers in the solid state can be either amorphous or semi-crystalline. (Totally crystalline polymers, e.g., polymer
crystals, are only made for scientific research.) When polymers are forming the solid structure (either cooling from the molten state
or concentrated from a solution), polymer molecules tend to aggregate together. For some polymers, some individual polymer
chains are folded and packed in an ordered arrangement during this process while other polymer chains are not, resulting in
semi-crystalline structures. Polymers with semi-crystalline structures are sometimes referred to as crystalline polymers. Since poly-
mer chains are normally long, there are always amorphous regions in polymers. If individual chains of polymers cannot be folded
and packed in an orderly fashion during the solid forming process, the chains are arranged randomly and even entangled, forming
amorphous polymers. Typical examples of amorphous polymers include polycarbonate and poly(methyl methacrylate). Compared
with amorphous polymers, semi-crystalline polymers tend to possess enhanced mechanical properties. Polyethylene and polyacry-
lonitrile are typical examples of semi-crystalline polymers.
Polymer Additives
Additives are widely used to modify polymer behaviors. Only a few polymers are used in the chemically pure form in real appli-
cations. In most cases, additives are incorporated in polymers to adjust their physical or chemical properties.
A major category of additives is stabilizer, which can enhance the stability of polymers against the environmentally degradative
effects. Some polymers undergo rapid deterioration in mechanical properties under normal environmental conditions. The deteri-
orative effects are often caused by oxidation, thermal exposure or UV radiation. To prevent such deteriorative processes, antioxidants
and thermal/light stabilizers are frequently incorporated in commercial polymer products. A major concern for most polymers is
that they are flammable in the chemically pure form. To reduce the flammability of polymers, another type of additives, flame retar-
dants, is used in polymer products. Flame retardants either initiate chemical reactions to cool the combustion region or interfere the
combustion process to cease burning. Fillers and plasticizers are additives that are widely used to modify physical properties of poly-
mers. Various fillers can be incorporated in polymeric materials to improve thermal stability, abrasion resistance, tensile or
compressive strength, toughness, etc. Plasticizers are used to enhance the ductility, toughness, and flexibility of polymers while
reducing their hardness and stiffness. Some polymers are difficult to process due to their high melt viscosity or adhesion with
machine surfaces. Processing additives such as lubricants are therefore added to improve the manufacture of the products of these
polymers.
There are also many other types of additives, such as colorants (imparting specific colors to polymers), blowing agents (forming
rigid or flexible foams), antistatic agents (reducing or eliminating static electricity), and biocide (control or destroy harmful
organism).
Polymer Blends and Interpenetrating Networks

Polymer blends are materials that are obtained by mixing two or more polymers. Such physical mixtures are made when the poly-
mers are in the molten state or as polymer solutions. Polymer blends are produced to achieve desired properties, which can be
tailored to meet specific application requirements. Another purpose of using polymer blends is to reduce the cost of expensive poly-
meric materials by introducing low-cost polymers in the blends.
Polymer blends can be classified into two groups: compatible (miscible) polymer blends and incompatible (immiscible)
polymer blends. Compatible polymer blend is a homogeneous (single-phase) system with a single value for a property. For
example, the glass transition temperature (Tg) of a compatible polymer blend is generally the weighted average of the values
of individual polymer components. Some compatible polymer blends possess superior properties than those of the individual
components alone. This is a result of the high level of thermodynamic compatibility caused by strong intermolecular attraction.
Incompatible polymer blends are phase-separated systems consisting of heterogeneous mixtures of polymer components. Such
system has multiple values for a property. For example, multiple glass transition temperatures related with each individual
components of the blend exist for these polymer blends. Factors that decide whether a particular polymer blend is a single phase
or phase-separated system include thermodynamic of the system, kinetics of the mixing process, processing temperature, solvent,
and incorporation of additives. The primary factor to determine miscibility of two polymers is thermodynamics which is gov-
erned by Gibbs free energy.
Another approach for developing new polymeric materials with desirable properties from multiple polymer components is to
create interpenetrating networks (IPNs), which are the combinations of two or more polymers in the network form. At least one
polymer in an IPN is crosslinked and/or is synthesized in the presence of the other. Simply mixing two or multiple polymer compo-
nents does not create IPNs. A schematic diagram for IPN structures is shown in Fig. 4. IPNs have advantages of both network poly-
mers and polymer blends. Polymers include polystyrene, poly(ethyl acrylate), polyurethanes, and poly(methyl methacrylate) has
been used to form IPNs.
IPNs can be classified as several types: sequential IPN, simultaneous IPN (SIN), latex IPN, gradient IPN, thermoplastic IPN, and
semi-IPN. Sequential IPN is the case that polymer networks are formed one by one. One polymer network is fabricated first, and
then the monomers and their crosslinker and activator of the other polymer network are swollen into the first polymer network for
in situ polymerization, forming the final IPN. In contrast, SIN is produced by simultaneous polymerization of different monomers
Fig. 4 A schematic diagram showing the structure of polymer interpenetrating networks.
and/or prepolymers that are mixed together before reactions. Latex IPN has a structure similar with latexes, with each particle consti-
tuting a micro-IPN. Gradient IPN is the case that the crosslinking density or overall composition varies geometrically on the macro-
scopic level. A general process to prepare gradient IPN is to partially swell the initially formed polymer network by the other type of
monomers, followed by rapid polymerization before the larger-scale diffusion occurs. Thermoplastic IPNs are the polymer networks
that are produced mainly by physical crosslinks. The physical crosslinks make thermoplastic IPNs flowable at high temperatures.
Semi-IPN is the case that one or more polymers are crosslinked in the IPN, while the rest of polymers in the IPN is not crosslinked.
IPNs have been investigated for many industrial applications including food packaging and fuel cells. They have high potential for
applications in tissue engineering and controlled drug delivery.
Polymer Forming Techniques

Various techniques have been employed in polymer forming and new methods are continuously developed. The selection of a pro-
cessing method for a polymer depends on the following factors. The first factor is whether the material is thermoplastic or thermo-
setting. For thermoplastic polymers, the use of a processing method also depends on the temperature at which the polymer softens,
the atmospheric stability of the polymer, and the geometry and size of the finished product. For thermosetting polymers, the first
step is the preparation of a linear polymer, which is normally in liquid state due to its low molecular weight. The linear polymer is
then cured in a mold, forming polymer product.
Compression molding and transfer molding

Compression molding and transfer molding are widely used for processing thermosetting polymers. A schematic diagram of
compression molding is shown as Fig. 5A. Compression molding can be used to fabricate both thermoplastics and thermosets
and requires heating up of raw materials (in the form of powders or pellets) in the mold. For thermoplastics, the temperature should
be higher than Tg during the forming process. A pressure is applied in compression molding and should be maintained during cool-
ing. For compression molding, an appropriate amount of the polymer and necessary additives are thoroughly mixed and loaded
between upper and lower parts of the mold. Both parts are heated up and only one is movable. After the mold is closed, heat
and pressure are applied. The polymer in the mold is melted and flows into all space of the mold cavity. After a pre-set time,
the mold is cooled and opened up to eject the product with desired shape. A major advantage of compression molding is the
low capital cost due to the simplicity of the process.
Transfer molding is a variation of compression molding for processing thermosetting polymers. In this forming process, the solid
ingredients are initially melted in a heating chamber. The melted viscous polymer is then injected into the mold cavity and a pressure
is applied. Transfer molding can process thermosets with complex parts.
Injection molding
Injection molding of polymers is similar to die casting for metals. It converts polymers from the power or pellet form to various
polymer products, such as radio cabinets, computer frames and many other commodities. It is usually used for processing thermo-
plastic polymers. A schematic diagram of this technique is shown as Fig. 5B. Generally, polymer pellets or powders are fed into
a heating chamber and heated up until they melt. The melted viscous liquid is then injected into an enclosed mold under pressure
and cooled for solidification. After the polymer becomes solid, the mold is opened and the product is ejected. Injection molding is
a continuous process and can be automated. The production rate is high and hence it is used for the mass production of polymer
products.
(A) Compression moulding
Mould: Mould plunger

heated &
cooled
Guide pins
Moulding
Mould cavity Mould Close
compounds
Mould Open
(B) Injection moulding

Hopper
Heating zone
Nozzle
Rotating
screw
Mould
Fig. 5 Schematic diagrams showing compression molding and injection molding processes for polymers.
Thermosetting polymers can also be processed through injection molding, employing what is called reaction injection molding.
Curing reaction of thermosets takes place while the material is under pressure in the heated mold. Longer cycle time as compared
with injection molding for thermoplastic polymers is required in reaction injection molding.
Extrusion
Extrusion is for converting granules or pellets of thermoplastic materials into solids by simply injection molding through an open-
ended die. Polymeric materials with continuous lengths and constant cross-sectional geometries can be produced through extru-
sion. The schematic illustration of polymer extrusion is similar to that of extrusion for metal alloys (Fig. 1C). A polymer in the
form of granules or pellets is fed into an extruder and melted. The molten polymer is then forced through a die with an orifice
of designed shape, which enters a sizer and cooler to develop the correct size and shape. The solid polymer is pulled by motor-
driven rolls and transferred to a cutter to make the final product (pellets, wires, rods, etc.).
Blow molding
Blow molding is frequently used to fabricate plastic containers with thin walls. The process is similar to blowing glass bottles. Blow
molding involves two essential stages: forming polymer parison, and blowing the parison into the desired hollow shape. A ther-
moplastic is melted first and forms the parison, which in most cases is hollow tubes, and a compressed air is blown into the
soft molten parison to expand it until it reaches the contours of the mold. The product is then cooled and removed from the mold.
Casting
Casting is a technique which pours a liquid material into a mold and then solidifies the material to form a rigid product that repro-
duces the detailed shape and dimensions of the mold cavity. In casting, the mold is not heated. The liquid for casting can be molten
polymers or polymer solutions. Solidification of the material in the mold is through a physical (cooling) or chemical (polymeri-
zation) process. This technique can be used for both thermoplastic and thermosetting polymers.
Solvent-casting is often used in the laboratory for developing new materials and products. In this process, polymer is dissolved
by a solvent to form a polymer solution. The polymer solution is then poured into a mold. The solvent evaporates from the solution
in the mold in a controlled environment and a solid polymer product is obtained.
Fabrication of Hydrogels
Hydrogels, also known as gels, are crosslinked three-dimensional macromolecular networks obtained from hydrophilic polymers
which can absorb and retain a considerable amount of water. Because of its favorable biocompatibility and other advantages, hydro-
gels have been intensively investigated in the biomedical field and find applications such as soft contact lenses and drug delivery
vehicles. Several fabrication technologies have been developed to produce hydrogels, including physical crosslinking, chemical
crosslinking, radiation crosslinking, and grafting polymerization. Only two fundamental methodsdphysical crosslinking and
chemical crosslinkingdare briefly covered here.
In physical crosslinking, no cross-linking agents are included in the reaction. This feature gives advantages to the physical cross-
linking approach since using cross-linking agents can affect the integrity of substances to be entrapped. In certain applications, the
cross-linking agents even need to be removed before application. The formation of physically crosslinked hydrogels is largely depen-
dent on hydrocolloid type, concentration, pH, salt type, temperature, and other thermodynamic parameters. With careful selection
of these parameters, a broad range of hydrogel textures and properties can be obtained. Many methods have been developed for
physical crosslinking, such as heating/cooling a polymer solution, H-bonding, complex coacervation, ionic interaction, freezing-
thawing, etc.
In chemical crosslinking, hydrogels are formed through covalent bonding. Hydrogels fabricated through chemical crosslinking
cannot be dissolved in solvents unless the covalent bonds formed in the crosslinking process are cleaved. Chemical crosslinking
includes three major approaches: (1) crosslinking of water-soluble polymers with irradiation, (2) crosslinking of water-soluble
polymers with cross-linker, (3) copolymerization of a monomer with cross-linker. For these three approaches, many crosslinking
methods have been explored, such as chemical grafting, radiation grafting, aqueous state radiation, solid state radiation, radiation in
paste, etc.
Manufacture of Porous Polymers

With the emergence of tissue engineering, making porous polymer structures becomes highly important. Porous polymers are also
very useful in other biomedical applications, such as filtration and drug controlled release. Many methods are available for fabri-
cating porous polymers, including gas foaming, solvent casting and porogen leaching, phase separation, templating via self-
assembled structures, electrospinning, and 3D printing. With the increasing demand for porous polymers with specifically required
characteristics/properties, new manufacture techniques are continuously investigated and developed.
Manufacture of Bioceramics
Fabrication of Dense Bioceramics
Bioceramics are biocompatible ceramic or glassy materials that are designed to repair or reconstruct the damaged parts of human
bodies. Bioceramics can be fabricated into a variety of forms (e.g., powder, coating, and bulk) to provide different functions in
human tissue repair or replacement. They include ceramics, glasses, glass-ceramics, and composites. Bioceramics can be mainly
divided into three groups, as illustrated by Fig. 6. Quite a few members in the calcium phosphate (Ca–P) family are attractive
ceramic biomaterials, such as hydroxyapatite (HA, Ca10(PO4)6(OH)2) and b-tricalcium phosphate (b-TCP, Ca3(PO4)2). Both HA
and TCP are weak bioceramics and cannot be used directly for load-bearing applications.
For many applications, bulk, non-porous bioceramic products are required. Bioceramic products start with the synthesis of bio-
ceramic powders. For example, for HA, several methods can be used for making its powders, with wet synthesis being the most
common technique utilized. For producing bars, rods or other types of products, bioceramic powders or powder mixtures of desired
composition together with (or without) chemical additives are pressed into desired shape and size in dies using a hydraulic press to
form powder compacts, known as the greenbody, which are not physically strong. The greenbody is then sintered at a high
Bioceramics
Bioactive ceramics,
Bioresorbable
Bioinert ceramics glasses and glass-
ceramics
ceramics
Tricalcium
Carbon Hydroxyapatite
phosphate
Calcium
Alumina Bioglass®
sulphate
Zirconia A-W glass-

Others
ceramics ceramic
Others Others
Fig. 6 Classification of bioceramics according to their bioactivity and biodegradability.

(A)
MO MO MO
SiO 2 SiO2 SiO2

SiO2 SiO2 SiO2
MO
MO
MO
MO MO MO
SiO2 SiO2 SiO2
2
2
2
O
O
O
Si
Si
Si
Initial stage Intermediate stage Final stage
(B)
Al2O3 Al2O3 Al2O3
Al 2O 3 Al 2O 3 Al 2O 3
Al2O3 Al 2O 3 Al2O3
Al 2O 3 Al2O3 Al 2O 3 Al 2O 3
Al2O3 Al 2O 3
Al2O3 Al2O3
3
Al2O3
3
2O
2O
2O
Al
Al
Al
Initial stage Intermediate stage Final stage
Fig. 7 Common ceramic processing technologies for making dense bioceramics: (A) liquid-phase sintering, and (B) solid-state sintering.
temperature to bond particles to form dense bioceramic products. The purpose of sintering is to use thermal energy to form chem-
ical bonds between bioceramic particles, resulting in strong, bulk bioceramic products. There are two important conventional
methods for producing bulk bioceramics, liquid-phase sintering and solid-state sintering, as illustrated in Fig. 7. Liquid-phase sin-
tering uses a small amount of an additive (e.g., a glass, which must be biocompatible) which melts below the bioceramic melting or
decomposition point. The molten additive can flow and enter the space between bioceramic particles, making dense, almost non-
porous bioceramic products. Liquid-phase sintering has the advantage of sintering ceramics at relatively low temperatures, thus
avoiding bioceramic decomposition, and of producing highly dense (non-porous) bioceramics and hence has been used to
make bioceramics such as glass-toughened HA, which is a composite. Solid-state sintering can be conducted for both single-
component system and multi-component system bioceramics without using a liquid phase in sintering. It is used for making
alumina or zirconia ceramic products. Solid-state sintering normally requires relatively high temperatures for sintering and may
not be able to totally eliminate pores in the sintered products. Both liquid-phase sintering and solid-state sintering can be performed
without the application of pressure.
Hot pressing is a manufacturing method that combines the forming and sintering steps to produce bulk bioceramics with rela-
tively simple shapes. The ceramic powder is put into a die and subjected to sintering with the simultaneous application of uniaxial
pressure. The sintering temperature can be lowered due to the application of pressure, which helps to avoid decomposition of bio-
ceramics at elevated temperatures. Hot isostatic pressing (HIPing) uses uniform pressure from all directions, instead of uniaxial
pressure, to sinter bioceramics into both simple and complex shapes. The bioceramic powder in a container is put into a high pres-
sure vessel and is subjected to both high temperature and isostatic gas pressure for sintering. The most widely used pressurizing gas
is argon, an inert gas that does not cause chemical reaction. When the chamber is heated, the pressure inside the vessel increases.
HIPing can use still lower temperature for sintering the bioceramic and the product can be almost non-porous. However, the HIPing
equipment is expensive.
Manufacture of Porous Bioceramics

Porous bioceramics can be used for non-load bearing implants in orthopedic and craniofacial applications. The porous structure
can allow the tissue to infiltrate, which enhances the attachment of implant and the tissue. To introduce pores into the bioceramic
bodies, a variety of methods are developed, such as incorporating pore-creating additives (e.g., amino-acid derivatives), gel-casting,
freeze casting, direct foaming, 3D printing, etc.
Porous calcium phosphate bioceramics are extensively studied as bone scaffolds for bone tissue engineering due to their excel-
lent biocompatibility and bioactivity and suitable degradation behavior. When calcium phosphate bioceramics have open porous
structures with three-dimensional interconnected pores, cells and biomolecules such as growth factors can penetrate into them,
resulting in better bone regeneration. However, one of the shortcomings that needs to be considered is the severe reduction in
mechanical properties when pores are introduced into bulk bioceramics, especially at the high porosity levels. Thus exploring ways
to achieve suitable mechanical properties of porous bioceramics is highly important. Compared with conventional techniques for
producing porous bioceramics with a random porous structure, 3D printing of porous bioceramics, which has distinctive advan-
tages in several aspects, now gains increasing attention.
Fabrication of Pyrolytic Carbon

Pyrolytic carbon is a synthetic biomaterial and the term was first introduced in 1960s. Generally, pyrolytic carbon is produced
through thermal deposition of hydrocarbon compounds at its decomposition temperature without the presence of oxygen. Pyro-
lytic carbon can have different microstructures (isotropic or lamellar) depending on the fabrication condition (temperature,
concentration of carbon precursor, gas flow rate, etc.). In the majority of biomedical applications, pyrolytic carbon is used as
a versatile coating due to its good blood and tissue biocompatibility. It can be deposited onto synthetic blood vessels to form
a thin layer that has excellent blood compatibility without affecting the flexibility of the grafts. It is also used in mechanical heart
valves.
The most important forms of pyrolytic carbon for biomedical applications are low-temperature isotropic (LTI) pyrolytic
carbon and ultra low-temperature isotropic (ULTI) pyrolytic carbon. Dense and high strength LTI pyrolytic carbon is generally
deposited onto implants via chemical vapor deposition (CVD) from a hydrocarbon gas at a high temperature in a fluidized
bed. The ULTI pyrolytic carbon is usually deposited from a carbon precursor via a hybrid vacuum process in the presence of
a catalyst.
Fabrication of Bioactive Glasses

The excellent biocompatibility and bioactivity of bioactive glasses make them excellent materials for implants for repairing or recon-
structing damaged bones. Bioactive glasses are also promising materials for porous scaffolds for bone tissue regeneration owing to
their high osteogenic potential and in recent years, the controlled biodegradation of new bioactive glasses.
Bioactive glasses were first discovered by Hench and his associates in Florida, United States, in 1969. Comparing with
other bioactive materials, one of the most distinctive characteristics of bioactive glasses is the formation of an HA-like layer
between the glass implant and host tissue, resulting in a firm and stable bonding which has evolved from a series of time-
dependent surface reactions after implantation. Currently, the most common manufacturing techniques of bioactive glasses
are conventional melt quenching and the sol–gel method. The flowcharts in Fig. 8 describe the processes of these two
methods. There are various bioactive glasses developed for biomedical applications. BioglassÒ was invented by Hench and
is a family of bioactive glasses that contain SiO2, Na2O, CaO, and P2O5 in specific proportions. Different from traditional
soda-lime-silica glasses, BioglassÒ has low SiO2 content (less than 60 mol%), high Na2O and CaO contents and a high
CaO/P2O5 ratio. Hence BioglassÒ is a weak bioceramic and cannot be used for load-bearing applications. BioglassÒ can be
fabricated using the conventional glass-making technique via melt quenching. Certain amounts of weighted raw materials
including SiO2, Na2CO3, CaCO3, and Ca2P2O7 are thoroughly mixed first, followed by melting at 1300–1450 C and anneal-
ing at 450–550 C. Impurities such as Al2O3 must be avoided. Bulk BioglassÒ can be formed by casting or molding using
graphite or steel molds.
(A) Melt quenching (B) Sol-gel

Raw materials Raw materials
method method
Mixing Preparation of sol
Melting Gelation
Casting or injection Aging

moulding
Drying
Annealing
Stabilization
Bioactive
glass Sintering
Bioactive glass
nanoparticles
Fig. 8 Production processes for bioactive glasses: (A) melt quenching technique, and (B) sol–gel method.
Fabrication of Bioactive Glass-Ceramics

Bioactive glass-ceramics are crystal-glass nanocomposites which have one or more crystalline phases with controlled size and
morphology in a glass matrix and are made through controlled crystallization of glasses. Consequently, they possess the advantages
of glasses as well as specific properties of sintered crystalline ceramics. They can be stronger and tougher than the parent glass or glass
matrix owing to the reinforcement and toughening by the small crystals. Various types of glass-bioceramics have been developed
after the invention of BioglassÒ, and CeraboneÒ A-W glass-ceramic which contains apatite and b-wollastonite (CaO$SiO) crystals
was invented Kokubo and his co-workers in Japan in the early 1980s. A-W glass-ceramic is considered the most outstanding bioac-
tive glass-ceramics for hard tissue repair.
A-W glass-ceramic (with the commercial brand name of CeraboneÒ) is manufactured from a parent glass in a pseudoternary
system 3CaO $ P2O5 CaO $ SiO2 MgO $ CaO $ 2SiO2 and has the contents of 38 wt% apatite, 34 wt% wollastonite and
28 wt% residual glass. The parent glass is prepared using the conventional melt-quenching method, and nano-sized oxyfluoroapa-
tite crystals and b-wollastonite crystals are precipitated during controlled crystallization of the parent glass at 870 C and 900 C and
are homogenously distributed in the glass matrix to form the homogeneous bioactive glass-ceramic. The material is slowly cooled
from high temperature to avoid cracking. A-W glass-ceramic possesses excellent mechanical properties owing to the reinforcing
effect of wollastonite as well as good bioactivity rendered by apatite. These characteristics make A-W glass-ceramic implants very
successful for bone tissue repair.
Manufacture of Biomedical Composites

Composite and Composite Classification
Composites are heterogeneous materials consisting of two or more homogeneous phases which are separated by interface(s). The
distinctive constituent phases are bonded together by chemical or physical bonds at the interface. Unlike traditional materials, the
physical, chemical, biological and other properties of composites can be tailored according to the requirements in specific applica-
tions. This is a distinctive and highly important advantage for this group of materials and is greatly enhanced by carefully modu-
lating the composition, interfacial bonding and physical arrangement of constituent phases in composites. This advantage should
be utilized to the fullest extent when developing new materials for targeted applications. A composite is designed to combine the
best properties of each constituent in the composite or to create new materials with properties that current materials cannot provide.
In most cases, a composite consist of two distinct phases: the matrix phase and the reinforcing phase (or, “second phase” or
“dispersed phase”). The matrix phase is a continuous phase that surrounds the other phase and provides the overall form. The rein-
forcing phase is a dispersed phase and is generally stronger and stiffer than the matrix phase.
Many human body tissues are natural composites such as bone. Artificial composites have been investigated such as composites
for dental filling and reinforced bone cement. In biomedical applications, each component in a composite must be biocompatible.
If the composite degrades in the body, the degradation products must also be biocompatible.
There are different ways to classify composite materials. One common way is to categorize composites according to the geometry
of the reinforcing phase. Hence there are particulate composites, fibrous composites and laminated structures (Fig. 9). In each cate-
gory, composites are further divided into different sub-groups. For example, fibrous composites can belong to either of the two
groups: continuous fibers and short/chopped fibers, on the basis of the ratio of fiber length over its diameter (the so-called “aspect
Composite
Particle-reinforced Fiber-reinforced Structural
Large Continuous long

Laminates
particles fibers
Dispersion Short fibers / Sandwich

strengthened Chopped fibers panels
Others Others Others
Fig. 9 Classification of composite materials according to the geometry of the reinforcing phase.
ratio”). Continuous fibers generally have a ratio greater than 1 105, while aspect ratios of short/chopped fibers are between 5 and
200.
Composites can also be categorized by the matrix material used. Therefore, there are metal matrix composites, ceramic matrix
composites, and organic matrix composites. Among these three groups, polymer matrix composites are more commonly developed
for biomedical applications. The trend now is to develop biodegradable biomedical composites for human tissue repair or
regeneration.
Fabrication of Particulate Biomedical Composites

Polymer matrix composites
Particulate polymer-matrix composites are the largest group of biomedical composites. Using standard equipment in the plastics
industry, they can be fabricated using one of the three methods: extrusion, injection molding, and compression molding. These
three techniques have already been introduced in the previous section on the manufacture of biomedical polymers. Extrusion
and injection molding are limited to the manufacture of composites with particulates or short/chopped fibers only. Injection
molding is suitable for the mass production of particulate or short fiber composites. Compression molding can be used for partic-
ulate composites with either thermoplastic or thermoset polymer matrix.
Metal matrix composites

Typically, metal-matrix composites for biomedical applications consist of a metal alloy matrix and a particulate ceramic second
phase. Such composites are investigated primarily for having the strength and stiffness provided by the metal matrix which are
required for load-bearing implants. The particulate second phase is normally a bioactive bioceramic. The techniques for fabricating
particulate metal-matrix composites can be classified on the basis of the temperature used in the manufacturing process: (1) liquid
phase process, (2) solid state process, and (3) solid–liquid process. Liquid phase processes involve the incorporation of dispersing
the particulate second phase in a molten matrix metal, followed by the solidification of the composite. Stir casting can be applied in
the liquid phase process. In solid state processes, both phases in the composites are kept at the solid state but the high temperature
and pressure exerted provide the energy needed to form bulk composites. Powder metallurgy can be used in the solid state process.
Solid–liquid processes involve the mixing of the particulate second phase and metal matrix in a region of the phase diagram where
both liquid and solid phases exist in the matrix. If the temperature in all these processes is too high, chemical reaction(s) between
the particulate second phase and metal matrix can occur, which may be detrimental to composite performance.
Ceramic matrix composites

Ceramic-matrix composites for biomedical applications generally consist of a bioceramic matrix and particulate, bioceramic second
phase. These composites can be made using processing technologies for bioceramics, which have been presented in the previous
section. Pressureless sintering, hot pressing, and HIPing. The important issue in the manufacture of ceramic matrix composites is
the densification of composites during manufacture. Efforts are required to make fully or highly dense (zero porosity or near-
zero porosity) ceramic products, which is difficult to achieve.
Fabrication of Fibrous Biomedical Composites

Short-fiber composites
Two commonly used fabrication processes for particulate polymer matrix composites, namely, extrusion and injection molding, can
also be used for the manufacture of short-fiber composites with polymer matrices. However, due to high shear forces generated in
these manufacturing processes, fiber breakage is often encountered. The short fibers are also oriented in the extrusion or injection
direction in the final products, which results in anisotropy of the material. Compression molding does not encounter these two
problems but its production rate is low.
Long-fiber composites
Producing long-fiber composites requires specialized equipment. Filament winding is a common process for fabricating long/
continuous-fiber composites. A schematic illustration of this process is shown as Fig. 10A. In this process, the long fibers are pulled
through a low viscosity resin bath for polymer impregnation. Then the fibers are precisely wound onto a rotating mandrel. Succes-
sive layers are laid on the mandrel until product specification is reached. Subsequently, the fibrous composite on the mandrel is
cured, and the product is removed from the mandrel. Filament winding is a good and efficient technique for manufacturing parts
with rotational symmetry such as cylinders and tubes. This process can control fiber orientation and fiber content. The highly
aligned fibers provide the final products with high tensile strength in the fiber direction. Filament winding has been investigated
to make medical products such as prosthetic hip stems, arterial grafts, intervertebral disks, intramedullary rods for fracture fixation
and ligament prostheses.
Pultrusion (Fig. 10B) is another fabrication technique for many long-fiber composites. During this process, the long fibers are
also pulled through a bath of liquid resin for impregnation. The material then goes through a heated die, which has a cavity of
constant cross-sectional area along most of its length, for curing. After being cured in the heated die, the solid, hot composite is
cooled and cut into required size. This process can be used for both thermoset and thermoplastic polymer matrix composites. In
(A) (B)
Reinforcing fibers
Heated die Cutter
Mandrel Reinforcing fibers
Resin bath Pulling system
Resin bath
Fig. 10 Schematic diagrams showing two common technologies for the production of long-fiber composites: (A) filament winding, and
(B) pultrusion.
the case of thermoset composites, the feedstock is long fibers and the bath is filled with liquid thermosetting resin. For thermoplastic
composites, the feedstock consists of both long fibers and thermoplastic polymer. The feedstock is preheated to a temperature near
or above the softening point of the polymer. It then enters a heated die for melting and forming the composite with designed cross-
sectional shape and size. Pultrusion is widely used for the mass production of parts with a constant cross-section such as rods, pipes,
beams, sheets, and tubes.
A new technique that can be used for making long-fiber composite is electrospinning. Over the past 20 years, electrospinning is
intensively investigated for fabricating porous, nanofibrous scaffolds for tissue engineering applications. With the proper modification
of the basic electrospinning technique, composite scaffolds can be produced via electrospinning. Electrospun nanofibers themselves
can actually be used as reinforcing fibers in non-porous long-fiber composites. There are reports on research in this direction.
Fabrication of Laminated Structures

Laminated structures are composites composed of 2D sheets or panels which are stacked and bonded together with the orientation
of the high-strength direction (the fiber direction) varying with each successive layer. The general fabrication process is to use auto-
claves/ovens to apply heat and pressure for curing stacked 2D sheets or panels into final or near-final products. Various methods
have been developed to manufacture laminated structures, such as vacuum bag-autoclave and resin transfer molding.
Vacuum bag-autoclave is a process for fabricating high-performance laminates such as fiber-reinforced epoxy. In this process,
a prepreg is first produced with a fundamental structure of a thin sheet. This sheet consists of a resin matrix embedded with uni-
axially oriented long fibers. For some matrix material, such as epoxy, the matrix in the thin sheet of is partially cured. Pieces of
the thin sheets are cut out and placed on top of each other according to the composite design. The stacked prepreg sheets are moved
to a shaped tool to form a laminate. Those sheets can be placed in different directions to achieve various strength, stiffness and other
properties. Afterwards, the laminate is applied with a vacuum through a vacuum pump for vacuum-bagging and the entrapped air in
the laminate is removed. The laminate enclosed in the vacuum bag is placed in an autoclave for curing. When the curing process is
completed, the product is removed from the autoclave and taken out from the vacuum bag for other operations.
Fabrication of Porous Composite Scaffolds for Tissue Engineering

Porous polymer-, metal- or ceramic-matrix composites (the so-called “scaffolds”) are made and studied for potential tissue engineering
applications. Polymer matrix composite scaffolds are far more common that composite scaffolds based on metals or ceramics. The
techniques for producing polymer scaffolds, such as solvent casting and porogen leaching, phase separation, electrospinning and
3D printing, can be suitably modified for fabricating polymer matrix composite scaffolds that contain bioactive bioceramic particles.
However, with the addition of bioceramic particles (micro- or nano-size) in the polymer solution for scaffold fabrication, technical
difficulties may be encountered, such as homogeneous distribution of bioceramic particles in composite scaffolds and sufficient
amounts of bioceramic particles in the scaffolds. Nevertheless, composite scaffolds hold great promise for the regeneration of certain
types of human body tissues and new and better techniques should be developed for making novel nanocomposite scaffolds.
Manufacture of Nano-Biomaterials
Fabrication of Nano-Structured Biomaterials
Advances in nanoscience and nanotechnology have now made a huge impact in the biomedical field. Nano-biomaterials, either
nano-structured biomaterials or nano-sized biomaterials, are increasingly investigated owing to their distinctive advantages, and
sometime unmatched advantages, for biomedical applications. Nano-biomaterials can be broadly defined as biomaterials that
have typical structural features or physical sizes of at least one dimension within the range of 1–100 nm.
Crystalline metals and ceramics made via conventional manufacturing technologies have grain sizes normally in the micrometer
range. Various technologies are available to change the grain size for achieving targeted mechanical properties but the grain size
cannot be reduced to the nanometer range. With new nanotechnology, bulk materials with nano-sized grains can now be made,
which provide outstanding mechanical properties. For example, nano-structured HA has been produced for bone tissue repair.
For producing nano-structured HA, HA nanoparticles, instead of HA microparticles, are firstly synthesized. The HA nanoparticles
are compacted to form the greenbody which are subsequently sintered in a carefully controlled process. In this HA sintering, grain
growth is avoided and hence sintered dense HA has only nano-sized grains. This nano-structured HA possess much high strength
than dense HA made through the conventional route. Furthermore, nano-structured HA exhibits much improved biological perfor-
mance than conventional dense HA. For some Ti alloys and Mg alloys, there are reports that new technology can help to decrease
their grain size to the sub-micron range, which significantly improves their mechanical properties.
Another important area for developing nano-structured materials is nanocomposites. R&D in nanocomposites has progressed
very fast over the past decade. Nanomaterials such as HA nanoparticles, metal nanoparticles, carbon nanotubes (CNT), and gra-
phene have been incorporated into polymers for various applications. These nanocomposites can be either non-porous or porous.
The fabrication techniques for nanocomposites are mostly based on manufacturing technologies which have been described in
previous sections. Obviously, there are particular problems in dealing with nanomaterials for making nanocomposites. Extensive
investigations are required to overcome obstacles in nanocomposite manufacture.
Fabrication of Nano-Sized Biomaterials

Nano-sized biomaterials (nanoparticles, nanofibers, etc.) have shown great potential in various areas including drug delivery, cancer
diagnosis and treatment, and tissue regeneration. These nano-sized biomaterials can have different shapes and geometries (nano-
spheres, nanorods, nanotubes, etc.) and can be inorganic (e.g., HA), metallic (e.g., Au), polymeric (e.g., PLGA) or composite/hybrid.
There are already a great variety of techniques for synthesizing or fabricating nano-sized materials or biomaterials. It is expected that
there will be more new technologies for producing nano-sized materials.
For controlled drug delivery, polymer nanoparticles (polymeric micelles, nanocapsules and nanospheres) as drug nanocarrier
are fabricated either from an already-synthesized polymer or by polymerization of monomer units. Drugs can be loaded into/
onto polymer nanoparticles by either physical entrapment or chemical conjugation and later released in vivo for providing the ther-
apeutic function. Some biomolecules such as growth factors can also be incorporated in polymer nanoparticles for other functions.
For example, using the double emulsion technique, spherical polymer particles can be produced and a sustained release of incor-
porated biomolecules can be obtained.
Nano-sized bioceramic particles, particularly HA and TCP nanoparticles, with high surface areas can be used for different appli-
cations. In general, HA nanoparticles can be made using a number of techniques. Conventional wet chemical synthesis with subse-
quent oven-drying or freeze-drying can produce HA nanoparticles but with many technical problems. HA nanoparticles with very
small sizes and a narrow size range can be successfully synthesized using the “water-in-oil” emulsion technique.
Nano-sized metals have shown high potential as diagnostic or therapeutic tools. Magnetic iron oxide nanoparticles can be used
for magnetic resonance imaging (MRI imaging), drug-delivery, and hyperthermia. They can be synthesized with controlled size, size
distribution and shape by using several different methods, including co-precipitation from of iron salts in aqueous media, micro-
emulsion, and thermal decomposition and high temperature reaction. Gold nanoparticles have also received great attention owing
to their unique properties such as localized surface plasmon resonance, which leads to their multiple applications such as labeling,
sensing, imaging and photothermal therapy. There are a number of ways to synthesize gold nanoparticles and the simplest ways is
the reduction of metal salt precursors with a reducing agent (e.g., chitosan) under controlled conditions in either organic solvents or
water. However, more sophisticated ways need to be used to produce gold nanoparticles with desired shape and structure (highly
branched nanoparticle, nanorod, nano-cage, etc.).
CNTs have gained popularity in research and they can be potentially used in the biomedical field. CNTs can be composed of
a single tube or concentric cylinders of carbon, termed single-walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes
(MWNTs), respectively. CNTs are generally prepared using three different methods: arc-discharge, laser ablation, and chemical
vapor deposition. Due to their high strength and unique electrical and physical properties, CNTs may find applications in cell
labeling and tracking, chemical and biological sensing, and bioactive agent delivery.
Another important area for developing nano-size biomaterials is the fabrication of nanofibers. Nanofibers can be used to
construct tissue engineering scaffolds which mimic the nanofibrous extracellular matrix (ECM) of human body tissues. Nanofibrous
scaffolds can serve as temporary ECM for cells and influence cell behaviour. Various methods have been developed for producing
nanofibers of polymers (natural or synthetic) and typical techniques include self-assembly, phase separation, electrospinning and
newly developed 3D printing technologies, as illustrated in Fig. 11. Electrospinning is described in detail in another article of this
encyclopedia.
Surface Modification Techniques for Biomaterials
Surface properties are crucial for biomaterials since they are closely associated with the reaction of the host to implanted biomate-
rials. For many biomedical applications, surface modification of biomaterials is frequently required. Surface modification is
(A) Self-assembly (B) Phase separation
(1) Single peptide (2) Formation of a micelle

in the initial phase
Temperature
Bioactive head change
Hydrophilic domain
Hydrophobic tail
(3) A cylindrical micelle
(C) Electrospinning (D) 3D printing
Pressure
Fig. 11 Technologies for producing nanofibrous biomaterials.
a process that changes the composition and structure of the surface of bulk materials to obtain improved surface properties while
maintaining the mechanical properties of the bulk material. Surface modification can be applied to biomaterials to improve cell
adhesion, cell proliferation, ECM-secretion function of cells, tissue compatibility, blood compatibility, corrosion resistance, and
many other characteristics/behavior/properties for the material. Therefore, many surface modification techniques are developed
for all types of biomaterials.
Surface Modification Techniques for Biomedical Polymers

When biomedical polymers interact with the human body environment, a process similar to the interactions between cells and their
environment takes place on the polymer surface. Such a process is initiated by protein adsorption on the polymer surface. Interac-
tions between cells and the material then occur on this newly formed protein layer. With the understanding of this process, two
general surface modification strategies are often adopted:
1. modulating the material surface so that the adsorbed proteins can preserve their normal bioactivity, and
2. immobilizing desired biomolecules directly on the polymer surface to incur specific cellular response.
Another strategy, micro- or nano-patterning is also often used in surface engineering of biomedical polymers.
In the first strategy, many surface properties can be adjusted to improve biomedical polymers. These properties include surface
roughness or surface micro- or nano-features after patterning, surface stiffness, surface charge, chemical composition, hydrophi-
licity/hydrophobicity, and other properties. Among them, hydrophilicity/hydrophobicity has been widely studied to improve
adsorption or desorption of proteins. Generally, surface with moderate wettability, instead of extreme hydrophilicity/hydropho-
bicity, can adsorb a proper amount of proteins and maintain the natural bioactivity of proteins.
Since most biomedical polymers are hydrophobic, several technologies have been investigated to increase the hydrophilicity
of these polymers. Plasma treatment of polymer surface is one of the widely used methods. It uses various reaction gases, such
as air, NH3, CO2, SO2 or other gases, to introduce polarized groups such as amino, hydroxyl, carboxyl, and sulfate groups on
polymer surfaces. Grafting copolymerization of hydrophilic polymers onto biomaterial surface is another popular method. The
initiation of copolymerization (introduction of initiators) can be achieved using different approaches, such as laser treatment,
ozone oxidation, electron beam or g-ray irradiation, and oxidation by cerium (IV). Among these initiation methods, Ce(IV)
induced grafting polymerization do not require irradiation source, plasma or ozone generator, making it a convenient method.
Photo-oxidization is another method that can introduce peroxide groups onto polymer surface without specialized equipment.
Simply immersing materials in hydrogen peroxide solutions under UV irradiation also achieves biomaterial surface
modification.
In the protein immobilization strategy, two types of proteins can be employed:

1. adhesive proteins derived from ECM, which include fibronectin, collagen, laminin, vitronectin and others. Cell adhesion and
attachment can be improved through immobilization of this type of proteins.
2. growth factors, such as epidermal growth factor (EGF) and bone morphogenetic protein (BMP). Cell proliferation, differenti-
ation, and other cell behaviors can be modulated by these growth factors.
Proteins can be either covalently or physically immobilized on biomedical polymers. For covalent immobilization, reactive groups
such as amino, carboxyl and hydroxyl groups should be initially introduced as coupling sites. Grafting poly(acrylic acid) or poly(-
methacrylic acid) is a widely used method to generate carboxyl groups. Water soluble carbodiimide is then applied to activate the
carboxyl groups. Finally, the activated carboxyl groups react with the amino groups of the target proteins, resulting in protein immo-
bilization. Hydrolysis and aminolysis can be applied to yield reactive groups for polyesters. An example of the aminolysis method is
the treating of ester-containing polymers in diamino compounds solution, with subsequent use of glutaraldehyde as coupling
reagent to graft proteins. One potential drawback of covalent immobilization is the natural conformation of the grafted proteins
may undergo certain changes. Therefore, physical protein immobilization techniques are developed. Layer-by-layer (LBL), grafting
and coating method (covalent grafting a protein layer, followed by physical coating of proteins), and entrapment of bio-
macromolecules through surface swelling with subsequent solvent transfer are examples of physical immobilization technologies.
Surface Modification Techniques for Metallic Biomaterials

Metallic biomaterials are usually covered by a metal oxide film, which plays a vital role in both corrosion resistance and tissue
compatibility. When a metallic biomaterial is in contact with living tissues, proteins are immediately adsorbed on the material
surface. The adsorption of proteins can affect material corrosion, subsequent cells adhesion, and other properties/events. Protein
adsorption, cells adhesion, corrosion resistance, and other properties determine the tissue compatibility of implanted metallic
biomaterials. To make metallic biomaterials suitable for the targeted applications, surface modification has been widely used to
improve wear resistance, corrosion resistance, antibacterial property, and tissue compatibility of materials. Many surface modifica-
tion techniques have been developed for metallic biomaterials. These technologies can be classified into two major categories: dry
processes and hydro-processes. Dry processes usually require sophisticated equipment to conduct modifications of the material
surface, while hydro-processes share a basic process of immersion or electrolysis in aqueous solutions without the need to use
sophisticated equipment or incur high costs.
Ion beam technology is a representative for dry processes. The surface modification can be categorized into two types: thin film
formation, and surface-modified layer formation. Thin film formation has the advantage of controlling the film composition but
lacks strong adhesion to the substrate. This technology is usually used for forming apatite or other type of films to improve wear
resistance, corrosion resistance, and osteoconductivity. The other route, surface-modified layer formation, cannot control the
composition of the surface layer as easily as thin film formation technologies. However, it has the advantage of the formation of
a fracture-resistant interface between the surface layer and substrate. Surface-modified layers with nitrogen and calcium ions
implanted can improve wear resistance and osteoconductivity of metallic biomaterials. Another example of dry processes is the
industrialized process of plasma spraying of HA coating on orthopedic or dental implants.
Hydro-processes are performed in aqueous solutions. By controlling certain parameters in hydro-processes, such as composition
and pH of the aqueous solution, current density of electrolysis and potential gain due to electrolysis, specific properties of resultant
surface layers can be achieved. Examples of hydro-processes include apatite formation on titanium through electrochemical treat-
ment, formation of surface-modified layer for bone implants produced by immersion in various solutions (such as alkaline solu-
tion, hydrogen peroxide solution, and calcium-containing solution; some of them need thermal treatment at same time), titanium
oxide layer formation, modification with biomolecules or polymers, etc.
Biomimetic Deposition
In general, biomimetic deposition is a solution-based method conducted in an environment that mimics the human body condi-
tion. In most cases, such body-like environment is provided by a simulated body fluid (SBF) at 37 C. The temperature, pH, and
other parameters of the conditions for biomimetic deposition are carefully controlled to simulate the body environment. Tradi-
tional surface modification techniques, such as plasma spraying, ion beaming technologies, and hydrothermal-electrochemical
treatment are usually conducted under non-physiological and extreme conditions. Bioactive agents are entrapped on the surface
merely by the formed coatings through these techniques, resulting in their rapid release upon exposure to a physiological environ-
ment. To overcome this disadvantage, surface modification of implants is conducted under the biomimetic condition, such as
immersing implants in a simulated body fluid at approximately 37 C and a physiological pH. Apatite (or other compound) can
then be formed on the metal surface. With biomimetic deposition, metals exhibit enhanced biocompatibility and bioactivity,
and proteins can be not only deposited on the surface but also incorporated into the coating. The proteins are thus released grad-
ually instead of in a single burst.
Biomimetic deposition generally consists of two steps: initial nucleation, and formation and growth of the coating. Various
deposition methods have been developed for metals, as well as for bioactive glasses, glass-ceramics and polymers. A typical case
of biomimetic deposition for metals is the coating of a calcium phosphate (Ca–P) layer on the surface of titanium alloys as
bone implants. In this case, a titanium alloy implant is immersed in an SBF for Ca–P nucleation on the metal surface. After the
nuclei have reached the critical size, crystal growth starts, gradually forming the final coating. The apatite coating achieved by biomi-
metic deposition can make implanted materials osteoconductive, leading to improved bone tissue repair by metal implants. Poly-
meric biomaterials have also been well investigated for biomimetic deposition, and a few coating techniques are developed, such as
alternate soaking process. These techniques can be applied to produce osteoconductive polymeric scaffolds for bone tissue
engineering.
Summary
Many manufacturing technologies for metals, polymers, ceramics and composites employed in other industries can still be used in
the biomedical industry for fabricating metallic biomaterials, biopolymers, bioceramics and biomedical composites, as well as their
products. However, care needs to be taken when using these technologies due to requirements and restrictions for biomedical prod-
ucts. Additionally, many other manufacturing technologies are investigated and developed specifically for biomaterials manufac-
ture. In using any of these fabrication techniques, general principles in materials science and engineering and in materials and
product manufacture must be followed. Good manufacturing practice must also be adopted. With the increasing demand for newer
and better biomaterials for medical applications, it is expected that many novel manufacturing technologies will appear. Existing
manufacturing technologies can also be modified and/or combined for producing new biomaterials to meet clinical requirements.
Acknowledgments
Min Wang thanks Nanyang Technological University, Hong Kong Polytechnic University, The University of Hong Kong, UK’s Engineering and
Physical Sciences Research Council, Singapore’s Ministry of Education, Hong Kong Research Grants Council and the National Natural Science
Foundation of China for research funding.
Further Reading
Ducheyne, P., Healy, K., Hutmacher, D. W., & Grainger, D. W. (Eds.). (2011). Comprehensive biomaterials. Amsterdam: Elsevier.
Lanza, R. P., et al. (Eds.). (2013). Principles of tissue engineering (4th edn.). Burlington, MA: Academic Press.
Ratner, B. D., et al. (Eds.). (2013). Biomaterials science: An introduction to materials in medicine (3rd edn.). Amsterdam: Elsevier.
Wang, M. (2004). Bioactive materials and processing. In D. L. Shi (Ed.), Biomaterials and tissue engineering (pp. 1–82). Berlin: Springer.
Materials and Their Biomedical Applications
Bin Duan, University of Nebraska Medical Center, Omaha, NE, United States
Introduction 136
Materials Classification 136
Primary Bonds and Secondary Bonds 136
Structure of Materials 136
Crystalline Solids 136
Common crystal structures 137
Crystalline defects 137
Polycrystalline structures 138
Anisotropy of materials 138
Amorphous Materials 139
Glasses 139
Metallic glasses 139
Polymers 139
Monomer and chain structures 139
Amorphous polymers 139
Semi-crystalline polymers 140
Homopolymers and copolymers 140
Hydrogels 140
Composite Materials 140
Membranes and Thin Films 140
Natural Materials 140
Processes for Materials 141
Solidification of Metals and Phase Transformation 141
Recovery, Recrystallization and Grain Growth of Metals 141
Polymer Crystallization 142
Glass Transition of Amorphous Polymers 142
Diffusion in Solids 143
Processing–Structure–Property Relationships for Materials 143
An Introduction to Biomedical Materials 144
History of Biomaterials 144
Interdisciplinary Nature of Biomaterials Development 144
Human Body Environment 144
Biocompatibility of Materials 145
In Vitro Assessment of Biomaterials 145
In Vivo Evaluation of Biomaterials 146
Ex Vivo Evaluation of Biomaterials 146
Standards for Biomaterials 146
Government Regulations 147
Biomaterials in Action 147
Materials in Orthopedics 147
Materials in Wound Dressings 148
Materials in Dentistry 148
Materials in Ophthalmology 148
Materials for Cardiovascular Devices 149
Materials in Drug Delivery and Controlled Release 149
Materials in Reconstructive Plastic Surgery and Cosmetic Surgery 149
Materials in Bioelectrodes and Biosensors 150
Looking Into the Future 150
Cancer Theranostics 150
Bioprinting 150
Organs-on-Chips 151
Acknowledgments 151
Further Reading 151

136 Biomaterials: Science and Engineering j Materials and Their Biomedical Applications
Glossary
Biomaterial A biomaterial is a non-viable material used in a medical device, intended to interact with biological systems.
Biocompatibility Biocompatibility is the ability of a material to perform with an appropriate host response in a specific
application.
Ex vivo Ex vivo is Latin, meaning “out of the living.” Ex vivo experiments refer to investigations or measurements conducted in
or on tissue from an organism in an external environment with minimal alteration of natural conditions.
In vitro In vitro is Latin, meaning “in the glass.” In vitro experiments are performed with biological species (biological
molecules, cells, microorganisms, etc.) outside their normal biological environment. They are generally conducted in labware
such as test tubes, flasks and Petri dishes.
In vivo In vivo is Latin, meaning “within the living.” In vivo studies are those in which the effects are investigated inside the
whole, living organisms including animals and humans.
Introduction
The history of mankind is characterized by the materials used and the naming of ages of civilizations effectively emphasizes the
importance of materialsdthe stone age, bronze age, iron age, cement age, steel age, silicon age and now new materials age. Looking
at our society as it is now and as it will be in the future, every field in the engineering world requires new materials, with the biomed-
ical field being no exception. Novel biomaterials and structures are needed for improving the performance of existing medical
devices and for developing new devices to tackle currently insurmountable medical problems. Modern biomaterials science and
engineering rests on two pillars: materials science and engineering, and biological and clinical sciences. A good understanding of
fundamentals of materials science and engineering paves the way for the successful development of new biomaterials.
Materials Classification
For whichever the engineering field, materials are classified into four categories: metals, polymers, ceramics, and composite mate-
rials. Metals are strong and ductile; polymers are light, easily processable and flexible; ceramics are hard and brittle. When the prop-
erty of metals, polymers or ceramics are not good enough for the targeted application or new property is needed for the application,
a composite material is designed and developed. Needlessly to say that Nature, the best designer and maker of materials, makes
natural composites such as wood and nacre. Metals, polymers, ceramics and composites can be made into different forms (particles,
fibers, films and membranes, non-porous bodies, porous bodies, etc.) for different functions. The structures made of these materials
can be as small as having nanometer dimensions and as large as structures covering kilometers.
Primary Bonds and Secondary Bonds

Properties (mechanical, electrical, thermal, acoustical, optical, etc.) of metals, polymers and ceramics differ greatly. What makes
them different lies in the interatomic forces that bind the atoms together in these materials. Atoms form bonds because the
compounds formed by them are more stable than the alternative arrangements of isolated atoms. For all materials, there are three
types of primary interatomic bonds: ionic bonding, covalent bonding, and metallic bonding. Molecules are formed when atoms are
bonded together by strong primary bonds. Polymer long chains have covalent bonds, metals possess metallic bonds and in
ceramics, the bonds can be either ionic, covalent or a combination of both. While the primary bonds in materials are strong bonds
with high bonding energies, there are secondary bonds which are weak physical bonds such as hydrogen bonding and exist between
virtually all atoms or molecules. Hydrogen bonding forms between some molecules that have hydrogen atoms as one of the constit-
uents. Hydrogen bonding is important in the biological systems and hence in the biomedical field.
Structure of Materials
Crystalline Solids
The physical structure of solid materials highly depends on the arrangements of atoms, ions or molecules that form the material.
Crystalline solids are materials in which atoms are arranged in a repeating 3D pattern over large atomic distances (i.e., long-range
order). Examples of crystalline solids include metals and ceramics. Crystalline solids are in contrast to amorphous/noncrystalline
materials, whose atoms or ions are not arranged in long-range repeating 3D patterns. Liquid water is a representative material of
this group.
The repeated 3D arrangement of atoms in crystalline solids is called space-lattice, which means a 3D array of points coinciding
with atom positions. The geometrical configuration of a space lattice can be described through a unit cell. A unit cell is normally the
Biomaterials: Science and Engineering j Materials and Their Biomedical Applications 137
smallest group of atoms that form a repetitive pattern of the crystalline solid and maintains the characteristics of the overall crystal.
In a perfect crystal, all the atom positions can be generated by translations of the unit cell at integral distances along each of its edges.
Three lattice vectors a, b, and c is used to describe the geometrical features of the unit cell. The lattice constants of the unit cell
include the axial lengths (a, b, and c) and the interaxial angles (a, b, and g). Fig. 1 exhibits a unit cell with the lattice vectors
and constants.
Common crystal structures

There are 14 different unit cells for all crystalline materials that are used today, representing 14 primary crystal structures. Among them,
body-centered cubic (BCC), face-centered cubic (FCC), and hexagonal close-packed (HCP) are the most common structures in metals.
Fig. 2 shows schematic drawings (unit cells) of these three crystal structures. In FCC and BCC which are both cubic, there is only one
axial length a, In HCP, there are two axial lengths: a and c. FCC and HCP are the densest unit cells. They have the highest “atomic
packing factor (APF).” APF is used to calculate the percentage of space occupied by atoms in a unit cell, assuming the atoms are spher-
ical. In the BCC structure, a central atom is surrounded by eight atoms which are located at all eight corners. The center and corner
atoms touch one another along the cube diagonals. Each BCC unit cell includes two atoms: the central atom is a complete one, and
each corner atom is shared by eight unit cells. The APF of BCC crystals is 0.68. FCC is also cubic, with atoms existing at the corners and
centers of all the cube faces. An FCC unit cell has the equivalent of four atoms, with each corner atom accounting for a 1/8 atom
and each face center atom accounting for a 1/2 atom. The APF of FCC crystals is 0.74. Therefore, FCC crystals are more closely packed
than BCC crystals. HCP structure is also important but not often encountered. Each HCP unit cell has six atoms. The APF of HCP is the
same as that of FCC, that is, 0.74. A value of 0.74 for APF signifies the closest packing possible of “spherical atoms.”
Crystalline defects
In reality, perfect crystalline solids do not exist. Various types of defects are present in crystals and considerably affect their physical
and mechanical properties. The classification of crystalline defects is made according to their geometry and shape. The four types,
χ β α
b
y
γ α
a
x
Fig. 1 A unit cell with x, y, z axes, showing lattice vectors and constants.
Fig. 2 Three common crystal structures (from left to right: BCC, FCC, and HCP).
from zero dimension to three dimensions, of crystalline imperfections are: (1) point defects, (2) linear defects, (3) interfacial
defects, and (4) bulk or volume defects.
Vacancies, the simplest point defects, are the absence of atoms at the regular lattice points and can be generated during solid-
ification as a result of local disturbances. Another type of point defects is interstitials, which means a small atom can occupy an
interstitial site between surrounding regularly-sited atoms in a crystal. Both vacancies and interstitials are important for diffusion
in crystalline solids.
Linear defects are also known as dislocations. Dislocations can be generated during material solidification. Other processes
(vacancy condensation, plastic deformation, etc.) can also create dislocations. There are two types of dislocations: edge dislocation
and screw dislocation (Fig. 3). The combination of these two types is called mixed dislocation. An edge dislocation can be consid-
ered as the insertion of an additional half-plane of atoms in an otherwise perfect crystal. A screw dislocation can be created by
applying shear stresses with opposite directions to perfect crystalline structure.
Interfacial defects, or “planar defects,” include external surfaces, grain boundaries, and stacking faults. Grain boundaries and
stacking faults are two major types of interfacial defects. Grain boundaries exist in polycrystalline materials (“Grains” are individual
crystals in polycrystalline materials). They are surface defects that separate grains with different crystallographic orientations. This
region is about two to five atomic diameters in width and is a region of atomic mismatch between adjacent grains. The atomic
packing in grain boundaries is lower than within the grains. Grain boundaries also have some atoms in strained positions that raise
the energy of the grain boundary region. Grain boundaries can be generated during solidification of metals when different nuclei
grow simultaneously to form different grains and meet each other. Stacking faults mainly occur in HCP and FCC structures. HCP
and FCC crystals are formed by stacking of atomic planes (e.g., A, B, and C planes): ABABAB. for HCP structures and
ABCABCABC. for FCC structures. During the growth of these structures, one or more stacking planes may be missing, resulting
in stacking faults.
Bulk or volume defects include pores, cracks, and foreign inclusions in crystalline materials. Pores can occur when a cluster of
point defects combine together to form a three-dimensional imperfection. This type of defects has a tremendous effect on the perfor-
mance of materials.
Polycrystalline structures
Polycrystalline materials are solids that consist of many small crystals (the “grains”). The grains are separated by grain boundaries
and normally have random crystallographic orientations. The size of the grains may vary from nanometers to millimeters. During
the solidification of polycrystalline materials, small nuclei initially form at different positions of the liquid with random crystallo-
graphic orientations. These nuclei grow into larger crystals by absorption of atoms in surrounding liquid. Eventually, the crystals
impinge on one another forming a granular or polycrystalline structure. In such a structure, a region grown out from the nucleus
with the same crystal orientation is called a grain. Grains are separated by grain boundaries, the interfaces across which the crystal
orientation suddenly changes. The atoms are packed loosely in the region of grain boundaries, making them mechanically and
chemically unstable. Thus cracks and corrosion occur more frequently at grain boundaries.
Anisotropy of materials
Anisotropy is the directionality of properties, which implies different values of the same property in different directions. On the
contrary, isotropy is the situation that properties are independent of directions. For single crystals, physical properties, such as elastic
modulus and electrical conductivity, of many substances are anisotropic. These properties vary by the crystallographic direction in
which the measurements are taken. In many polycrystalline materials, the orientations of individual grains are entirely random.
Therefore, even though each grain is anisotropic, most polycrystalline materials are isotropic.
Fig. 3 Edge dislocation (left) and screw dislocation (right). (For the edge dislocation, the red dotted line refers to the linear defect. In the right
figure, the red arrows indicate the forces that cause the screw dislocation, which occurs on the blue plane.)
Amorphous Materials
In contrast to crystalline solids, the atomic structures of amorphous materials lack long-range order because of factors that inhibit
the formation of a periodic arrangement of atoms. Atoms in amorphous materials are sited on random positions instead of distinct
spatial positions like the atoms in crystals. Fig. 4 provides schematic drawings for crystalline materials and amorphous materials.
Most polymers, glasses and some metals (“metallic glasses”) are amorphous materials.
Glasses
Glasses are amorphous solids that are usually transparent. They are mainly used for technological and decorative applications. The
most common type of glasses is silicate glass for windows, which generally consists of the chemical compound silica and has the
oldest history for glass applications. Other types of glasses include fiber glasses, network glasses, organic glasses, etc. In the viscous
liquid state of glasses, the molecules have limited mobility. The formation of the crystalline structure is thus inhibited and the amor-
phous structure dominates after solidification.
Metallic glasses
Metallic glasses are a type of metals that are formed as non-crystalline solids under specific conditions. Compared to normal glasses,
metals have high mobility in the molten state and are very difficult to form amorphous structures. Metal alloys with a high
percentage of semi-metals, such as Si and B, can form metallic glasses through hyper-quenching. The cooling rate is usually between
105 and 109 degrees celsius per second, which is high enough so that the atoms do not have enough time to form crystalline struc-
tures. Metallic glasses are ductile with exceptional mechanical strength. They also possess excellent anticorrosion properties since
grain boundaries do not exist in the amorphous structures.
Polymers
A polymer is a macromolecule that contains many chemically bonded subunits. Natural polymers, such as rubber and silk, have
been used by humans for centuries. Various natural polymers including DNA, proteins, enzymes and cellulose play vital roles in
biological processes of living creatures. From last century, many synthetic polymers have been invented and widely utilized in
many industries and daily life due to their broad range of properties.
Monomer and chain structures

A polymer can be synthesized through chemical combinations of many small molecules, which is termed as monomers. For
a monomer, the number of its available sites for bonding with other monomers under specific polymerization conditions is termed
as functionality. A monomer must have at least two active sites so that it can bond with two other monomers to form polymer
chains. Bifunctional monomers can only enter into two linkages with other monomers since each of them only has two sites avail-
able. Polyfunctional monomers can be linked together as nonlinear structures.
According to the chain structure, polymers can be categorized into four groupsdlinear, branched, cross-linked, and network
polymers. If the monomers are linked together in a linear manner, the resulting structure is called a linear polymer. For branched
polymers, the monomers are joined together in a branched manner. Cross-linked polymers are formed when the monomer units are
linked in multiple chains and have interconnections between chains. Network polymers are the cases that cross-linked polymers
possess sufficient interconnections between chains. The chain structures of polymers are related to the functionality of monomers.
The combination of bifunctional monomers produces a linear polymer. Polyfunctional monomers can be linked together to form
nonlinear polymers, include branched, cross-linked, and network polymers.
Amorphous polymers
The structure of polymers can also be categorized as amorphous and semi-crystalline. Most polymers have non-crystalline (“amor-
phous”) structures. Their chains are randomly entangled like noodles in a large scale. The physical entanglement of the chains with
Fig. 4 Schematic drawings for crystalline materials (left, crystalline silica) and amorphous materials (right, amorphous polymers).
the dipole secondary bonds between chains enhances the strength of amorphous polymers. For branched polymers, their side
branches can create loose packing of the chains, leading to amorphous structures.
Semi-crystalline polymers
In the crystalline parts of polymers, individual polymer chains are folded and packed in ordered arrangement. However, an entirely
crystalline structure cannot be achieved for polymers since there are always amorphous region in the long polymer chains. A semi-
crystalline polymer consists of many small crystalline regions interspersed with amorphous regions. Each crystalline region
possesses a particular alignment. Semi-crystalline polymers generally have enhanced mechanical properties, increased fatigue
strength and distinctive thermal behaviors as compared to amorphous polymers.
Homopolymers and copolymers

Polymers that are formed by single repeating units are termed homopolymers, while polymers made up of two or more different
repeating units are called copolymers. The sequence of a homopolymer molecular chain is like AAAAA. (“A” represents the single
repeating unit). For copolymers, four types of arrangement exist: random, alternating, block, and graft. For a random copolymer,
different monomers are randomly arranged in the molecular chains, such as ABBAAABABBB. (“A” and “B” represent different
types of repeating units). In an alternating copolymer, the monomers are arranged alternatively, like ABABABAB.. Block copoly-
mers are the molecules that different monomers are arranged in long blocks of the same type, like AAAAABBBBAAABBBBB.. Graft
copolymers are the cases that chains consisting of one type of monomer are grafted on the long chain of another type of monomer.
Hydrogels
Hydrogels, as a group of polymeric materials, are semi-solid, cross-linked macromolecular networks made from hydrophilic poly-
mers. Because of the hydrophilic functional groups attached to the polymer backbone, the three-dimensional networks of hydrogels
can absorb and retain significant amounts of water in the cross-linked structures. The resistance of hydrogels to dissolution arises
from the cross-links between network chains. The crosslinks in hydrogels can be formed by physical cohesion forces, ionic bonds or
covalent bonds. Hydrogels can be classified as homogeneous or heterogeneous according to their network structure. Homogeneous
hydrogels possess an isotropic (random distribution) network structure, with relatively mobile chains and pores in the network.
Heterogeneous hydrogels exhibit an anisotropic network structure caused by the strong interpolymer interaction.
Composite Materials
Composite materials are solids that consist of two or more chemically distinct phases which are separated by interface(s). In most
cases, composites consist of two phasesdthe matrix phase (metal, polymer or ceramic), which is the continuous phase that provides
the overall structure, and the reinforcing phase in the form of particulates or fibers. Composites can be categorized by the form of the
reinforcing phase. There are three major forms of reinforcing phases: particle-reinforced, fiber-reinforced, and structural-reinforced.
In particle-reinforced composites, the particles (reinforcing phase) are dispersed in the matrix. For fiber-reinforced composites, the
reinforcing phase has the geometry of a fiber. Structural-reinforced composites include structures such as laminates. With the matrix
and reinforcing phase, composites can combine the desirable properties from both constituents to meet specific application
requirements.
Membranes and Thin Films

Membranes are often used for purification and separation applications. They are often developed to possess high permeability and
sufficient selectivity while matching the process conditions such as temperature and pressure. Therefore, membranes often have
certain amounts of pores with specific pore sizes. Common membrane materials include polymers, inorganics (e.g., nanoporous
silica), and metal-organic framework (MOF). Each type membrane has its own structural features. For example, silica membranes
can be mesoporous with high uniformity. In biomedical field, membranes have great potential for biosensing, biosorting, immu-
noisolation, and drug delivery.
Thin films are layers of materials (metals, polymers, and ceramics) with a thickness ranging from nanometers to micrometers. In
many applications including biomedical applications, coating a thin film on the substrate is highly important. A stack of thin films
is termed a multilayer and multilayered structures can provide distinctive properties. Thin films are vital for many industries since
they can provide excellent properties of the materials to fulfill application requirements.
Natural Materials
Numerous materials from the Nature have been used by human beings since the stone age and many of them (stone, wood, cotton,
etc.) still play important roles nowadays. Structures of natural materials vary tremendously, from crystalline structures like the dia-
mond to composites like wool. Among the natural materials, some are attractive for biomedical applications. A good example is
silks, which possess high mechanical strength and good biocompatibility. Silk materials are produced by different species with
unique properties. For silk made by silkworms, two main proteins, sericin and fibroin, form the main body of the silk. Fibroin
is the structural center of the silk, which is surrounded by sericin. The polymer chains in silk materials are bonded by both cross-
links and mechanical adhesion. Another natural material which is as biomaterials is collagen, a group of structural proteins that
widely exists in human and animals. Collagens are used in medical treatments for humans and have been intensively studied
for tissue engineering applications.
Processes for Materials

Solidification of Metals and Phase Transformation
In industrial applications, metals are usually melted and then cast into a mold to form products of designed shapes. The solidification
of metals is thus crucial for metal processing. Two major steps are included in the solidification process: (1) nucleation, which is the
formation of stable nuclei in the molten metals, and (2) growth of nuclei into crystals to form the granular structure. In the nucleation
process, the formation of stable nuclei in liquid metals can be either homogeneous or heterogeneous. In homogeneous nucleation,
metal itself provides the atoms for nuclei formation. When the temperature of a pure molten metal is below a solidification point,
many slow-moving atoms bond together to form numerous homogeneous nuclei. Heterogeneous nucleation can occur on the nucle-
ating agent, such as insoluble impurities, surfaces of the container of liquid metal and other materials in a liquid. The nucleating agent
must be wetted by the molten metal. Compared with heterogeneous nucleation, homogeneous nucleation requires a large amount of
undercooling, which may not be achievable in the industrial casting process. After the formation of stable nuclei in liquid metal,
nuclei begin to grow into crystals with different orientations. During the solidification process, the crystals grow and join together
to form grains and grain boundaries in the final solidified metal. With more nuclei in the liquid metal, the average grain size will
be smaller. Most engineering metal products have small grain sizes, which is beneficial for mechanical strength and uniformity. In
general, two major types of grain structure can be produced without using grain refiners: equiaxed grains and columnar grains. Equi-
axed grains form when crystals in the liquid metal grow equally in all directions during solidification, while columnar grains are long,
thin, coarse grains that form in the presence of a steep temperature gradient during slow solidification.
Phase is a homogeneous portion with uniform physical and chemical properties in a system such as a metal alloy. If two or more
phases exist in a given system, there will be a boundary separating the phases, and each phase has its specific characteristics. For
example, steam, water, and ice in a container are considered to be three phases since they are physically different. Phase diagram
is the chart that exhibits conditions, such as pressure and temperature, under which distinct phases coexist at equilibrium. A general
phase diagram for this three-phase system is shown in Fig. 5. Phase transformations happen when phase boundaries (the red curves)
are crossed due to the changes of conditions. The arrow in Fig. 5 is an example of phase transformationdice melts into water.
Phase transformation occurs during processing of materials and affects greatly their properties. In metal processing, control of
phase transformation can produce distinct materials to achieve desired properties. The driven force of phase transformation is the
difference of free enthalpy between two phases. Free enthalpy, or Gibbs free energy, is a function of the enthalpy (internal energy of
the system) and entropy (randomness of the atoms/molecules). The temperature for a phase transformation to occur is the ther-
modynamic equilibrium temperature shown in the phase diagram. Phase transformations frequently start by the nucleation
process. The fluctuations of large amplitude of the structure/composition can lead to nucleus formation. With the growth of
nucleus, atoms/molecules will be absorbed to the nucleus at the interfaces between transforming phases, just like crystal growth
during the solidification of metals.
Recovery, Recrystallization and Grain Growth of Metals

Cold working, such as rolling, forging, extrusion and other metal forming processes that are conducted under the “cold” condition,
strengthens metals through plastic deformation. The cold-worked metal has many dislocations and other defects. Metals that have
Liquid (Water)
Pressure
Solid (Ice)
Gas(Steam)
Temperature
Fig. 5 Schematic phase diagram for the ice-water-steam phase system.
undergone cold working possess superior mechanical strength but are much less ductile. To enhance the ductility of cold-worked
metals, a process called annealing can be conducted for the metals. During annealing, the cold-worked metal is heated to a suffi-
ciently high temperature with adequate time, and three steps of changes occur: recovery, recrystallization, and grain growth.
A cold-worked metal has higher dislocation density and internal energy than the original one. When the cold-worked metal is
heated in the recovery temperature range, sufficient thermal energy is supplied to the dislocations to increase atomic diffusion. As
a result, some dislocations are annihilated and some other dislocations move and rearrange themselves into states with lower
energy. The internal stress of the metal is thus partially relieved. The recovery temperature range is just below the recrystallization
temperature range. After recovery, the mechanical strength of the metal is reduced slightly, while the ductility is often greatly
increased.
Recrystallization is the process that new strain-free grains form and grow in the metal after the recovery process. The newly
formed grains have low dislocation densities and are characteristic of conditions before cold working. The driving force of the nucle-
ation of strain-free grains is the difference of internal energy between the strained and unstrained material. The progress of recrys-
tallization depends on both temperature and time. The mechanical strength of metals decreases notably during recrystallization.
Grain growth starts after recrystallization. If the temperature is high enough, the newly formed strain-free grain continue to grow.
With a sufficiently long time and proper temperature, the new grains grow and completely replace the previous ones, resulting in an
entire reduction in the internal energy. The internal energy is associated with the area of grain boundaries, which decrease when the
grains increase in size. The reduction of internal energy is the driving force for grain growth. Large grains grow at the expense of small
ones which shrink and eventually disappear. The Hall–Petch equation is applied to correlate the average grain size with yield
strength of polycrystalline materials.
Polymer Crystallization
Crystallization in polymers is different from that in metals and ceramics. Polymer crystallization is a process which transforms an
amorphous, crystallizable polymer into a semi-crystalline material that its molecular chains are partially aligned. The folded and
aligned polymer chains constitute an area called lamellae. Fig. 6 shows schematically structures of amorphous and semi-
crystalline polymers. Polymer crystallization can be conducted through different processes, such as solidification from the melt
and solvent evaporation.
The solidification of molten polymers starts by nucleation in which some chains in the polymer become parallel. Like the solid-
ification of metals, nucleation for semi-crystalline polymers also has two mechanisms: homogeneous, and heterogeneous. Due to
heat motion, some polymer chains occur parallel and result in homogeneous nucleation. Heterogeneous nucleation is initiated by
impurities or additives. After nucleation, crystal growth occurs at the temperature between the crystalline melting temperature Tm
and the glass transition temperature Tg. At this step, more folded chain segments are added on the nuclei. When the temperature
gradient is high enough, the direction of crystal growth correlates with the steepest gradient. If the polymer has an isotropic and
static temperature distribution, the crystal (lamellae) grows radially and forms a large aggregated called spherulite.
Solvent evaporation can also crystallize polymers. Generally, the polymer is dissolved in a solvent to form a dilute solution,
where the polymer chains are separated from each other, and the polymer chains can fold to form single-chain crystals. When
the solvent evaporate, the concentration of the solution increases and interactions between polymer chains occur to form semi-
crystalline polymers. The solvent evaporation process may generate polymers with the highest degree of crystallinity.
Glass Transition of Amorphous Polymers

Glass transition, or glass-liquid transition, is the transition of a hard and brittle glassy state into a softer and rubbery state for amor-
phous and semi-crystalline polymers. Glass transition occurs at a narrow temperature range termed as glass transition temperature
lamellae
Fig. 6 Schematic drawings for structures of amorphous polymers (left) and semi-crystalline polymers (right).
Specific volume
Temperature Tg Tm
Fig. 7 Specific volume-temperature curves of amorphous and semicrystalline polymers. (The blue zone is the region of the glassy state. Yellow
zone represent the range of rubbery state. Red zone is the liquid region.)
Tg, which is usually lower than the crystalline melting temperature Tm. For semi-crystalline polymers, the glass transition only occurs
at the amorphous regions, while this process happens in the entire structure of amorphous polymers.
The glass transition can be illustrated by a typical specific volume-temperature curve as shown in Fig. 7. The green line refers to
the curve of an amorphous polymer, while the black line represents a semi-crystalline polymer. In the blue zone, the amorphous
polymer is heated at a low temperature. Here, the polymer expands at a constant rate with an increase in temperature. When reach-
ing Tg, the volume expansion rate increase instantly to a higher level, meaning the hard and brittle glassy state changes to the soft
and rubbery state in the yellow zone. Upon further heating, the amorphous polymer gradually transforms to the liquid state in the
red zone. For the semi-crystalline polymer upon heating, the change at Tg is less intense since glass transition does not happen in the
crystalline regions. In the yellow zone, the semi-crystalline polymer is in a state that crystals dispersed in a rubbery amorphous
matrix. At Tm, the volume of the semi-crystalline polymer expands drastically due to melting of the crystalline regions in the
polymer.
Diffusion in Solids
The phenomenon in which matter is transported through matter is defined as diffusion. Diffusion is achieved by atomic motion.
Atomic movements in gases and liquids are relatively easy and rapid. In solids, such motion is restrained because of bonding of
atoms to the equilibrium position. Thermal vibrations in solids can enhance atomic mobility and thus allow some atoms to
move from high concentration areas to low concentration areas. Solid-state reactions are usually related to diffusion. The recrystal-
lization of cold-worked metals is an example of diffusion.
The diffusion in a crystalline solid is the stepwise migration of atoms from lattice sites to lattice sites. Vacancy diffusion and
interstitial diffusion are the two major mechanisms of diffusion in a crystalline lattice. Vacancy diffusion, also termed as substitu-
tional diffusion, is a mechanism by which atoms with sufficient thermal energy move from original sites to vacancies or other crystal
defects in the lattice. Interstitial diffusion is the mechanism by which atoms migrate from interstitial positions to neighboring empty
ones. In this mechanism, the moving atoms must have sufficiently small sizes as compared to the matrix atoms. Atoms such as
hydrogen, carbon, oxygen, and nitrogen can diffuse in metals via this mechanism.
Steady-state and non-steady-state diffusions are often involved in the study of diffusion. Steady-state diffusion is the situation
that the diffusion flux and concentration gradient do not vary with time. On the contrary, in non-steady-state diffusion, the diffu-
sion flux and concentration gradient change with time. Steady-state diffusion is only suitable for specific situations that a high-
pressure non-reacting gas diffuses through a metal foil to a region where the pressure of this gas is low. Non-steady-state diffusion
works for most practical diffusion situations. Steady-state diffusion and non-steady-state diffusion are dealt with by Fick’s first law
and Fick’s second law of diffusion, respectively.
Processing–Structure–Property Relationships for Materials

Processing, structure, and property are three essential components in materials science and engineering. The relationships among
these components play a vital role in the design and application of materials. Basically, how a material is processed result in its
specific structure, while the structure of a material leads to distinct properties. A linear progression can be made from processing
to structure, then to the properties, resulting finally in the specific performance and applications of a material. If a specific property
is needed for the application, the structure of the material must be suitably tuned, and hence the appropriate processing route must
be selected and processing parameters must be controlled. This article has mainly discussed structures of materials. Other article in
this encyclopedia will discuss manufacture and properties of materials in the context of biomedical applications.
An Introduction to Biomedical Materials
Many materials, including metals, polymers, ceramics and composites, are now used for biomedical applications and hence they are
called “biomedical materials.” Some of these materials were not intended as biomedical materials originally. But their use in the
biomedical applications has been successful and therefore they extended their service into the biomedical field. In the middle of
the last century, modern biomaterials science and engineering began. Since then, many materials are designed and have been devel-
oped specifically for biomedical applications.
History of Biomaterials
The concept of biomaterials is a relatively new idea and term, although biomaterials have actually been used for a very long time. A
biomaterial is a material that is capable of being introduced into the body or living tissue without inducing a harmful biological
response. Today, they are extremely widely used in the healthcare field for many different purposes, ranging from sutures to perma-
nent implants. For many years, people were using biomaterials to help solve medical problems without realizing what a biomaterial
was and why they worked or did not work. For a long time, there were no medical device companies producing biomaterials,
besides some externally used materials, and materials were not being made for the purpose of using them as biomaterials. This
means that there were no regulations or standards to follow when using materials for use in the human body, and there were mixed
results in the safety and efficacy of the materials.
The earliest used biomaterials were made from basic materials found in nature that were easily usable. There were sutures made
of linen or catgut, as well as evidence of using seashells as dental implants. Some groups of people also used the jaws of large ants to
hold wounds closed in lieu of sutures. As technology and the processing of materials advanced, so too did the biomaterials used in
medical procedures. The use of metals as biomaterials allowed for stronger devices, such as metal sutures and a variety of implants,
including dental implants. The longer these metals were used as biomaterials, people realized that there were problems with some
of them, and the concept of biocompatibility was born. Many metals were tested and some were kept for use as biomaterials and
some were discarded due to their adverse effects on the body. Eventually, plastics were developed, which allowed for a wider variety
of properties for biomaterials from which to make medical devices. This leads to an advancement in the types of devices that could
be created from the biomaterials. The polymeric biomaterials are tested for their biocompatibility, now that it is standard for testing.
There are constantly new biomaterials, like hydrogel for example, being created and tested for different applications. Hydrogels are
polymers that can swell but not dissolve in the water, which brings more new ideas to the world of biomaterials. The 1980s and
1990s were the golden era of ceramic biomaterials with many inventions and innovations on bioceramics. Biodegradable bio-
ceramics are found to be useful for hard tissue regeneration in this century when tissue engineering and regenerative medicine
have attracted great attention. Composite biomaterials play no lesser role in the biomedical field. They have found many biomed-
ical applications, ranging from biosensors, bioseparation, drug delivery devices, to human tissue repair and regeneration.
Interdisciplinary Nature of Biomaterials Development

Developing new biomaterials is a complicated process that requires a variety of skills and knowledge. To create and use new bioma-
terials, knowledge is needed in chemistry, biology, engineering, medicine, and other related areas. Due to this fact, it unlikely that
one person has obtained all of the knowledge and skills necessary to develop a new biomaterial. This leads to people working
together to develop, test, and use biomaterials.
Chemistry and materials science and engineering knowledge is used in the development of new types of biomaterials and in
optimizing existing materials. It helps with knowing how materials react in the presence of other materials and bodily substances.
Biological and medical knowledge is key in understanding the reaction of the body to the biomaterials. It also helps in being able to
determine what biological processes can be avoided and which to use to the materials advantage. Engineering skills are used to
utilize biomaterials into devices or processes that can be used to solve a problem. These skills can be used to determine the prop-
erties of the biomaterials and how those properties are suited for certain biomedical applications. Experience in medicine enable
people to know what types of biomaterials and devices are needed to make their jobs easier and to make clinical outcomes better.
They can lead to looking at new ways to do things. The people with medical skills also have the ability to test the new biomaterials
and devices made from them and are in charge of using them most of the time.
The fact that so many different pieces are put into making a biomaterial, and a device from the biomaterial, makes a need for
developing them to be interdisciplinary. Different sets of knowledge contribute to the development at different stages, and some
contribute throughout the process. If people with all of these sets of knowledge can work together seamlessly, then the development
of a biomaterial and its associated devices can run smoothly and quickly and could turn into a quality product.
Human Body Environment

The human body is a complex system and environment. It has many different types of tissues, cells, proteins, growth factors, and
other biological components. To add to the complexity, each tissue has a unique extracellular matrix (ECM) that affects the local
environment of cells and other biological components. There are many different types of proteins in the body, all of which can
adsorb, desorb, denature, and increase or decrease in activity in the presence of different biological materials and chemicals. Blood
contains proteins that are reactive and can adhere to foreign bodies and injury sites and cause clotting and inflammatory responses.
Blood also contains cells that have the job of fighting and removing foreign bodies. Along with the biological components of the
body, the body parts feel mechanical forces. These mostly come from body motion and blood flow, and can affect the function and
wear of parts of the body. The goal of a working biomaterial is to not induce any unwanted reaction from any components of the
body, whether it is toxicity or unwanted wear and tear.
Biocompatibility of Materials
For a biomaterial to be safe and effective, it must be biocompatible. If a material is biocompatible, it means that a material does not
cause any harmful effects when it is in contact with the body. It also means that the material should perform its specific function
without any unplanned effects. In general, the harmful effects that should be avoided are producing or supporting toxic substances,
irritation due to movement, rubbing, or improper mechanical forces, and improper host reactions to the biomaterial. There are
biomaterials that are said to be bioactive. Not only do these biomaterials not cause an adverse reaction, but they actually cause
wanted reactions that may be used to enhance the positive healing responses to the material. Bioabsorbable materials degrade
over time into safe substances in the body, allowing healing to occur naturally in the implantation site. A biomaterial that is bioac-
tive, bioabsorbable, or both can have enhanced biocompatibility. A biomaterial must be viewed as a piece of a device. A biomaterial
by itself may cause a different reaction than it does in the context of a full device.
The goal of a biocompatible material is to have the minimized amount of inflammation possible. The initial reaction from the
body when an implant, or any foreign body, is introduced is to undergo a foreign body reaction, which is a form of non-specific
inflammation. If the foreign body is recognized as a foreign substance, then inflammation occurs as macrophages attempt to break
down and remove the substance. Even if it does not occur instantly, if particles break off of the biomaterial, this can occur. In some
cases cells that respond in inflammation can kill tissue around the biomaterial. Reducing inflammatory reactions reduces the risk of
damage.
Another important factor in biocompatibility is for the material to be non-toxic. This means that the material itself, or its prod-
ucts, should not harm or kill cells or tissues. There are multiple types of toxicity that all must be avoided. These include genotoxicity,
carcinogenicity, reproductive toxicity, and cytotoxicity. Genotoxicity is the tendency of a chemical to mutate the genes of a cell, car-
cinogenicity is the tendency to cause a cell to become cancerous, reproductive toxicity is the tendency to cause death of reproductive
cells, and finally, cytotoxicity is the tendency to kill living cells. The chemicals released by materials can cause any of these forms of
toxicity. Some materials are susceptible to bacterial growth and may cause an infection. A material cannot be considered biocom-
patible if it has any form of toxicity or infection.
When a material comes in contact with blood, it can cause thrombosis, embolization, and the consumption of platelets and
coagulation factors. The biomaterials developed so far are never as resistant to thrombosis as natural endothelium in the body.
The materials that are exposed to blood and the vascular system for an extended period of time are especially susceptible to throm-
bosis. Thrombosis can cause major problems in the function of the biomaterials and for the health of the person who received the
implant. For a material to be considered biocompatible, it must not cause any harm to the cells or tissue while performing as
intended in the body.
In Vitro Assessment of Biomaterials

The goal of in vitro assessment of biomaterials is to mimic how the biomaterials and cells react to each other, as they would in the
body, as closely as possible so that the biocompatibility of the material can be determined and also cell behaviors be assessed. The
focus of the tests is to identify the chemical components of the materials that might be release in vivo, and determine if those chem-
icals are toxic to cells. Assays are used to determine the genotoxicity, carcinogenicity, reproductive toxicity, and cytotoxicity. All of
these can occur in multiple different ways, so they must be assessed thoroughly.
There are many standardized methods for the in vitro testing of biomaterials. Tests are designed to assess the characteristics of
chemicals that may be released by the material under development during its time in the body. This includes how the material reacts
to cells and fluids, as well as how the cells and fluids react to the material. A toxicology risk assessment is performed on the material
to identify hazardous chemicals coming from it, predict the potential exposure to the chemicals, determine the dose-response rela-
tionship of the chemicals, and then use all of the information to characterize the potential risk to patient from the material. The test
is done with a worst case scenario in mind for the amount of chemicals a patient could be exposed to, in order to be sure that the
material is safe for less than the worst case. The tests for genotoxicity test for gene mutations in bacteria, as well as chromosomal
damage in mammalian cells. If either one is positive for damage, then an in vivo test most likely is needed. Since all genotoxic chem-
icals are carcinogenic, a test for carcinogens should be performed before any testing on live animals is done. The carcinogenicity tests
check for change in cell morphology, anchorage-independent replication, and discorded colony growth, which are indicators of
malignant cells that have been formed. Reproductive toxicity testing does not need to be performed unless the biomaterial is
able to come in contact with an area of the body or cells that are associated with reproduction. In vitro cytotoxicity tests determine
whether or not a chemical from a material cause death to cells or disruption of essential process. Cytotoxic chemicals can do this by
changing the environment around the cells so that they do not work or by altering a specific part of the cell that disrupts the proper
function. Cytotoxicity can also lead to chronic inflammation. There is no single test that can fully characterize the toxicities for all
chemicals and hence multiple must be performed to assess the full extent of the material toxicity.
Due to the components and overarching importance of blood in the body, it is important to test the interaction between the
biomaterial and blood in vitro. The material should be tested for whether or not it causes thrombosis, how it interacts with platelets,
whether it damage red blood cells, or if it stimulates an immune response. The assessment models are designed to mimic the condi-
tions inside the body. This means that the constituents of the test fluid should have the coagulation factors, correct temperature,
flow, and other components that make the blood function in the body. Getting an assay to mimic clinical conditions as closely
as possible is important in the assessment of a material for any type of test.
There are needs to develop more in vitro toxicity tests to ensure that biomaterials are safe and effective without having to spend
a lot of time and money on testing. The tests need to be developed to mimic as closely as possible the in vivo environment, so that,
hopefully, an in vivo test would not be needed. It might not be completely realistic due to complexity differences between being in
a body and outside of one, but being able to test materials in vitro with accuracy and completely free of any in vivo tests would be
the ultimate goal.
In Vivo Evaluation of Biomaterials

In vivo evaluation of biomaterials is centered on testing the biocompatibility of the biomaterial in a complex biological environ-
ment and in many cases, determining the efficacy of the medical device made of the material. The evaluation of biomaterials in vivo
gives a better idea of how a biomaterial will react with the human body than in vitro tests. This is due to the complex nature of the
body environment. The in vivo tests help to determine whether or not a medical device and its associated biomaterials perform as
intended without any safety risks. The government regulatory bodies that have control over the use and testing of medical devices
issue protocols, guidelines, and standards that should be used for the in vivo assessment of medical devices. The in vivo tests are
different depending on what the intended use of the medical device is. They can also be used to test the individual biomaterials and
their reactions or the device as a whole.
The tests are based on the intended use of the device, and devices are grouped into different categories. These include the area of
tissue contact, which includes surface contact, external communicating devices, and implant devices, as well as the duration that the
device is in contact with the tissue, which includes limited (less than 24 h), prolonged (24 h–30 days), and permanent (more than
30 days). Biocompatibility tests in vivo include tests for sensitization reactions, irritation, intracutaneous reactivity, toxicity in all
forms, blood compatibility, carcinogenicity, degradation, and other immune responses.
Choosing the animal for tests is important when determining the safety and efficacy of a biomaterial or device. Some animals are
more closely related to humans in certain areas than in others, which means that using that animal for one test might work well, but
for another test it could mean nothing. For example, using a mouse model for tests with bone could work well to replicate humans
to an extent, but using a mouse for vascular tests would not translate well at all. Selecting the right animal model is crucial in deter-
mining the safety and efficacy of the biomaterials. Certain knowledge of animal biology and the similarities and differences between
humans and animals is needed for determining which model(s) should be used for which purpose(s). The important part of testing
biomaterials in vivo is to match the human tissue as closely as possible in order to accurately and correctly model the interaction of
the biomaterial in the human body.
Ex Vivo Evaluation of Biomaterials

Ex vivo evaluation of biomaterials allows the testing to be done without having to have a large number of animals. It is done by
taking tissue from the animal to test against the biomaterial outside of the body. An ex vivo test can be done using a shunt to test the
blood interaction of the biomaterial. In ex vivo tests, the physical, chemical, mechanical and other properties of the tissue and
biomaterials that have interacted with them after being implanted for a time or during the interaction are analyzed. Ex vivo eval-
uations of biomaterials are another way for ensuring the safety and efficacy of biomaterials.
Standards for Biomaterials

Standards for biomaterials are needed so that there is consistency in biomaterials R&D and in quality of biomaterials is maintained
around the world. Without the standards, there would be major problems with the manufacturing, quality control and use of the
biomaterials. The standards developed for a material are used by manufacturers, users, researchers, etc. to consistently define and
use a material every time. They specify what the chemical, mechanical, physical, and electrical properties of a material should be.
There are standards developed for material specifications, material uses, material testing, biocompatibility, and other important
standards. Material standards allow multiple manufacturers to make the same product, as well as letting the user know exactly
what they are getting every time.
Most standards are consensus standards, meaning they are developed by using popular opinion from a committee/community.
A test method standard is developed to specify the conditions of the test, type of and how many test subjects, and what data is avail-
able to be analyzed from the test. These test method standards can be used to test a material by anyone after the standard has been
developed and approved. Getting a standard approved can be a long process. It starts with a need for a standard and a group of
people is formed and tasked to decide how a test should be done. They then send out materials to be tested to multiple laboratories
and write the draft standard. They review information from multiple sources and finally test and produce the document. From
beginning to end, it can take about 3–5 years to produce a standard for a material or test.
The main organization in the United States that makes standards for biomaterials is the American Society for Testing and Mate-
rials (ASTM). Some other standardization bodies are the Association for the Advancement of Medical Instrumentation (AAMI),
which deals with medical electronics, sterilization techniques, vascular and cardiac valve materials, the American National Stan-
dards Institute (ANSI), which deals with the reviewing and acceptance of standards documents, and the American Dental Associ-
ation (ADA), which deals with dental materials standards. The ANSI also stays in contact with the other standards organizations, as
well as the International Standards Organization (ISO) in order to help produce standards for international use. Other countries
have standards organizations similar to the ones in the United States that also work with ISO. ISO gathers the standards from
all of the standards organizations to create international standards. The international standards make it easier for companies to
manufacture materials and devices that can be sold and used internationally. The standards organizations make their approved stan-
dards public so that they are available for use by everyone who needs them.
Government Regulations
Government regulations on medical devices, including biomaterials, are important in that they provide a way to ensure the safety
and efficacy of devices by providing a legally regulated path of approval. In the United States, the Food and Drug Administration
(FDA) was given the regulatory powder of medical devices in 1976. Along with the regulatory authorities in other countries, the FDA
regulates biomaterials on the basis of the risk associated with the intended use of the biomaterial. It is tasked to make sure that the
device or biomaterial is safe and functions as intended in the United States. This means that the biomaterial could be regulated one
way for one use and a different way for another use.
Since the regulations are based on the risk associated with use of the biomaterial, they are grouped into three main classes in the
United States. Class I medical devices and biomaterials are determined to be low risk. These devices and materials are mostly
external devices and are not intended to support life and failure of the device would not cause any real harm. The Class I medical
devices and biomaterials require the lowest regulatory control, as the risk of harm from them is very low. This allows them to be
quickly and easily passed through the regulatory process. Examples of Class I devices are bandage and dental floss. Class II medical
devices are found to pose moderate risk. These devices and materials are generally in contact with the body for a short period of time
or detect internal components of the body. They are devices and materials that are safe for the function they are designed for and will
not cause any major injury if failure occurs. Some of these devices are external and some are internally used. Due the moderate risk
factors and intended uses, Class II medical devices and biomaterials require more strict regulations than Class I medical devices,
with more stringent reviews of the technical documentation. Examples of Class II devices are gastroscope and magnetic resonance
imaging (MRI) equipment. Class III medical devices and biomaterials are determined to be high risk. These devices and materials
are used in extremely invasive procedures and life-sustaining devices. They can even be permanently implanted into the body. The
failure of Class III medical devices can cause major injury or even death. For this reason, they are subject to the most scrutiny for
approval. They must perform exactly the way they are specified without failure and that having them in the body will not harm the
body. To do this, Class III devices are tested in animals and clinical trials to be completely sure of their safety and efficacy. Examples
of Class III devices are heart valve, pacemaker and hip implant. As expected, the higher the risk, the higher amount of scrutiny is
given to the devices before they pass the regulatory process.
There are development and manufacturing regulations as well as premarket entry requirements. The development and
manufacturing requirements are mostly standard throughout the world but the premarket rules can vary depending on the country.
The easiest way for a medical device or biomaterial to get approved is to effectively compare it to one that has previously been
approved. If they are determined to be similar enough, the regulatory process may move quickly for the new device or biomaterial.
If not, tests and other work may be needed, which slows down the process. If a manufacturer wants to change a product slightly, this
may cause a need for a new review of the device or material. It is up to the manufacturers to prove the safety and efficacy of the
product that they are producing in order to pass the regulations. They must provide full documentation for the medical device
or biomaterial for gaining regulatory approval. The regulatory process is an ongoing and ever changing system with reviews and
adjustments going on all the time. Getting through the regulatory process can take a long time, which makes new medical devices
and biomaterials take a long time to get to clinical use.
Biomaterials in Action
Materials in Orthopedics
In orthopedics, biomaterials are generally chosen for their strength or for mimicking the structure and properties of bone. They are
also wanted for promoting the mineralization of tissue around the implants, which calls for bioactive materials. Many times, this
can mean the use of metals or ceramics. Calcium phosphate ceramics, particularly synthetic hydroxyapatite (HA), closely resemble
bone apatite and have been developed for bone tissue repair. They allow better bone growth in areas surrounding the ceramic
implant than any other material such as orthopedic metals. In some applications, there can be metal implants coated with a bioac-
tive ceramic material for the strength of metals and for the bioactivity (i.e., osteoconductivity) of calcium phosphates.
Metals for use in orthopedics include titanium and its alloys, cobalt’chromium alloys, and 316L stainless steel. Metals are usually
used for implants that require mechanical strength such as hip implants, bone screws, nails, pins, and fixation plates. Popular bio-
ceramics are bioactive glasses, glass-ceramics and calcium phosphates such as HA and b-tricalcium phosphate (b-TCP). Materials
such as silicon can be added to HA to enhance the biological property and promote bone growth. Bioactive bioceramics can be used
as coatings for metals and as bone defect fillers. Two “bio-stable” polymers, ultra-high molecular weight polyethylene (UHMWPE)
and poly(methyl methacrylate) (PMMA), are commonly used in hip joint replacements. UHMWPE is used for acetabulum cup
while PMMA is used to cement metal stem of the hip prosthesis to bone. Commonly used biodegradable polymers include
poly(ε-caprolactone) (PCL), polylactide (PLA), polyglycolide (PGA), poly(2-hydroxyethylmethacrylate) (PHEMA), collagen and
hyaluronic acid. These polymers are used for a variety of applications based on their properties. They can be used to repair bone
fractures, as substitutes for bone, ligament and cartilage repair, sutures, fixation plates, etc. that will degrade over time. Bioactive
composite, such as HA reinforced high-density polyethylene (HDPE), are also developed for bone tissue repair.
Materials in Wound Dressings

There are a variety of wounds, ranging from cuts and scrapes to ulcers and burns. This means wound dressings must be able to
perform different functions, depending on the type of wound. In general, wound dressings must stop the bleeding, keep the wound
clean and disinfected, absorb excess liquid and promote the proper gas and temperature levels in order to start and promote the
body’s healing process. The biomaterials used must also be able to be formed into sheets easily in order to function properly.
There are passive biomaterials that are used to just cover the wound and keep it clean. Traditional passive wound dressings are
made of woven fabrics and foams that assist healing by absorbing excess liquid and blocking outside sources of contamination.
Some wound dressings are able to promote the healing of wounds, allowing them to heal quicker and better, instead of just covering
them. Interactive dressings allow for the movement of substances such as water and oxygen to pass through them without allowing
contaminants to enter. These types of dressings are made of materials that can be made into thin films. Another type of wound
dressing that promotes healing is termed bioactive. These bioactives are made of materials that come from the body and are known
to aid in the healing processes. By bringing these materials to the wounds, the healing process can be sped up. These natural bioma-
terials are hemostatic agents. Combining multiple types of biomaterials into one wound dressing can allow it to effective cover
multiple functions needed by a certain type of wound.
Some common synthetic biomaterials used in wound dressings include PLA, PGA, PCL, poly(lactic-co-glycolic acid) (PLGA),
poly(ethylene glycol) (PEG), polyesters, and polycarbonates. These biomaterials are able to be formed into sheets and sponges
for covering wounds. Some commonly used natural polymers are proteins such as fibrinogen, thrombin, collagen, gelatin and
albumin and polysaccharides such as chitin, chitosan and cellulose. These materials have been able to be made into sheets, sponges,
gels, powders, and liquids. These hemostatic materials help in regenerating new tissue in the wound due to the fact they are naturally
present in wound healing or support the growth without inflammation. Cotton is commonly used in bandages and gauze. Some
polymers such as hyaluronic acid and methacrylates can be used in hydrogels that support wound healing. A combination of
different biomaterials can be helpful, depending on the need for healing and how each material interacts with the wound.
Materials in Dentistry
Biomaterials used in dentistry are very similar to those used in orthopedics, since teeth are similar to bone and are anchored in bone.
Dental biomaterials include metals, polymers, ceramics, and composites. Using these materials is to either prevent or fix problems,
and different from the application of most other biomaterials, some of dental biomaterials are visible and hence matching the color
of the surrounding tissue can be important.
Metals are often used as anchors in dental implants. They provide the strength needed to hold the implant in the bone under the
stresses that the mouth goes through. The metals include titanium and its alloys, cobalt-based alloys and stainless steel. Dental
crowns and bridges can also be made of metal. Gold crowns are common. Dental amalgam, which is composed of mercury, copper,
tin, zinc, and silver, is used as a filler material for cavities after tooth decay.
Bioceramics are common dental biomaterials. In some cases, they can be used as the anchors of implants as well. Some bio-
ceramics, for example, medical grade alumina, have the high strength needed in the mouth and have low thermal conductivity,
while exhibiting a color similar to natural teeth. Calcium phosphates including HA are also commonly used. HA can be coated
on metal implants to promote osteointegration with bone.
Polymers are used mostly as cements or fillers in dentistry. Cements or fillers start out as liquids and/or solid powders and are
hardened to hold two solids together or fill holes. Some harden on their own after mixing, while others need to be hardened, for
example, by UV light. They are usually made by mixing solid and liquid components. There are cements made of zinc phosphate,
zinc polycarboxylate, glass ionomers such as calcium and aluminum silicate, resins such as urethanes, and other types that can be
used for specific purposes.
All dental biomaterials must survive the harsh and fluctuating conditions of the mouth. They are hard and stable material that
work together in multiple ways. The metals, ceramics, polymers and composites work together and allow for integration with the
host tissue.
Materials in Ophthalmology
Biomaterials used in ophthalmology are contact lenses, optical implants such as artificial corneas, intraocular implants, glaucoma
drainage tubes, scleral buckling materials, adhesives for repair of perforations and ulcers, etc. They are generally soft polymers,
considering the eyes are made of soft and delicate tissues. Some of the main biomaterials used are PMMA, silicones, and hydrophilic
polymers in the form of hydrogels.
PMMA is used for hard contact lenses but it does not have great oxygen permeability. Hence it can cause problems if used for too
long. It is also used for artificial cornea and intraocular lens. This hard polymer allows the artificial corneas and lenses to retain their
shape and not to break down easily. The clear material allows for good optics as well. Silicones are used in the forms of a hard
rubber, soft rubber, sponge, and liquid with high viscosity. Silicone rubber is used in some cases for contact lenses. It has high
oxygen permeability but can cause the formation of deposits due to its hydrophobicity. Soft silicone rubber and sponge are
used in retinal detachment surgeries as scleral buckling materials.
Materials for Cardiovascular Devices

The cardiovascular system needs a variety of biomaterials. Biomaterials for heart and blood vessels have different requirements. A
common requirement for all biomaterials for the vascular system is that they must not react or cause a reaction in the presence of
blood since they are in constant contact with blood for their entire lifespan. The materials also need to withstand mechanical
stresses because of the heart pumping and constant blood flow.
Metals, carbons, ceramics, and polymers are common biomaterials for cardiovascular applications. Metals have high structural
integrity. They are used in structural parts in heart valves, pacemakers, and stents. Some of the main metals for these purposes are
stainless steel, cobalt’chromium alloys and titanium alloys. Nickel’titanium shape memory alloy (SMA, e.g., Nitinol) is used in
vascular stents owing to its shape memory. Metals are also used in electrodes and wires in pacemakers. Platinum alloys are used
in electrodes, and stainless steel and tantalum are used in sensors and wires braids. Carbons, especially graphite, are used in pyro-
lytic coatings of heart valve components. They are thromboresistant, have high lubricity and are resistant to wear. Sapphires have
been used in high speed blood pumps to reduce the amount of friction during rotation. Polymers such as polytetrafluorethylene
(PTFE) and polyester are used as small grafts and sutures. They help to repair tissue in blood vessels. Silicone has been used in arti-
ficial heart valves as tissues and cells generally do not attach to it. Biological materials are generally used as coatings for other bioma-
terials. All of the biomaterials used in the cardiovascular system must not cause thrombogenesis, must resist physical wear, and must
not break down over time.
Materials in Drug Delivery and Controlled Release

The goals of controlled drug delivery are to control the duration of release and the amount of drug released, to deliver the drug to
a specific part of the body, to get through tissue barriers, and to get through cellular barriers. Biomaterials used in drug delivery
must have right chemical compositions. Drugs can be delivered through the digestive systems, respiratory system, through skin or
muscles, intravenously, or through the respiratory system. Each delivery method needs a specific type of device or biomaterial to
deliver the drug effectively to the targeted area. The drug delivery methods are meant to control the amount of drug in the system
and where the drug is delivered to and even to overcome cellular barriers. Good delivery vehicles deliver the drug with an appro-
priate amount to the proper location by releasing the drug or breaking down themselves under the conditions of the targeted
sites.
Controlled release means controlling the rate at which a drug is released into the system, as opposed to having the drug delivered
into the system in full at one point in time. The main mechanisms for controlled release are diffusion, chemical reaction, and
solvent activation and transport. The diffusion mechanism uses a polymeric material that creates a reservoir for the drug, or a mate-
rial in which the drug can be uniformly distributed throughout. In the delivery through chemical reaction, the biomaterial making
the delivery vehicle degrades in the presence of water or other agent. Solvent delivery uses a material that can swell in the presence of
water to release the drug that has been locked in the capsule or by osmotic effect. Polymers are the main biomaterials for drug
delivery and controlled release purposes. On the other hand, ceramics such as mesoporous silica can also be used for controlled
delivery.
Hydrogels are good for diffusion-based controlled release because they swell when exposed to water or other biological fluids.
This allows for drugs to diffuse out of the expanded gels. They can also be used for solvent activation. Some hydrogels can respond
to environmental factors such as ionization. They can increase the amount of swelling due to responses to the environment, which
can allow the drug to be released more quickly or slowly. Specific locations in the body can be targeted by creating a hydrogel that
binds to a specific tissue.
Materials in Reconstructive Plastic Surgery and Cosmetic Surgery

Reconstructive plastic surgery has the goal of repairing defects for the sake of the damaged tissue appearing normal and hopefully
regaining its function. Cosmetic surgery has the goal of changing the appearance of a bodily feature. Since they are closely related to
each other and have the hope of creating structures that will hold shape and last in the body, they can use similar biomaterials to
change and fix the structure of body parts. The biomaterials used in reconstructive surgery are intended to provide mechanically
stable structures of the tissue while allowing native tissue to use it as a scaffold to regenerate its proper structure. Some of the mate-
rials are meant to degrade over time, while some integrate with the body. Much attention is paid to wound healing so as to prevent
the appearance of scars after surgery.
Most of the biomaterials for plastic surgery should be mechanically strong and chemically stable. There is also the reason for the
materials to have some bioactivity. If they are bioactive, they can integrate with the surrounding tissue, which means a better func-
tioning tissue repair that will heal to look normal. For support materials, PTFE, PMMA and titanium have been used but they do not
integrate well with the tissue due to low bioactivity. Therefore, for soft tissue, a biomaterial called acellular dermal matrix (ADM)
was developed. ADM integrates well with native tissue and allows for the healing process to occur through tissue regeneration rather
than scar tissue formation. This makes for better healing and less noticeable scarring. For hard tissue reconstruction as in craniofacial
reconstruction, calcium phosphates (HA and TCP) are often used. The bioactivity of these materials allows for good integration with
the bone. Some polymers are also used for facial defect repair. They include silicones, PGA, PLGA, PCL, and PMMA. These are used
for structures without being bioactive. Growth factors are commonly added for enhancing new tissue formation.
Materials in Bioelectrodes and Biosensors

Bioelectrodes are devices that send an electrical signal to the body. Biosensors are devices that detect certain biochemical signals and
convert them to electrical signals that can be measured. Biosensors are made of a transducer with a biologically active molecule. The
biologically active molecule responds to the biochemical signal and relays that response to the transducer. Bioelectrodes and sensors
can be on the body surface or implanted. Implanted electrodes and sensors have many more needs for compatibility than the
surface ones.
Bioelectrodes are commonly made of metals of high electrical conductivity. A common metal is stainless steel due to its low
reactivity when in contact with the body. In some cases, copper, gold or platinum is used. Copper needs to be coated in the
body as it reacts to the biochemical conditions in the body. Biosensors are made of a transducer with a biologically responsive mate-
rial. The biologically active material responds to a certain biochemical factor which it wants to detect. This reaction in turn causes
a slight change in the material, which also affects a piezoelectric material that makes up the transducer. The slight change in the
materials causes a small electrical signal to be transmitted and measured. This is how the device measures the amount of a substance
that is present. The transducers can be made of pH- or ion-selective electrodes, thermistors, optical fibers, or piezoelectric crystals.
The biologically active material is what really makes the biosensor function. Materials that have been used as the biologically active
materials include enzymes, antibodies, DNA, organelles, microorganisms, cells, etc. Each of them can be selected or modified to
react specifically to certain biochemical signals. Purified enzymes are commonly chosen because they are specific in their reaction
to the factors of interest. Biosensor materials must be able withstand bodily factors, such as temperature, pH, movement and chem-
ically reactive substances while still functioning.
There is a new trend in using nanomaterials as biosensors. They include graphene, carbon nanotubes, nanowires, quantum dots
and nanocomposites. They have structures that allow them to be modified to detect certain biological factors. Their sensitivity,
response time, stability and specificity make them promising materials for future biosensors.
Looking Into the Future

Cancer Theranostics
Cancer theranostics combine cancer diagnosis and therapy. Diagnostic and therapeutic substances can be incorporated in and
released from theranostics. The release can be stimulated by internal or external stimuli. Internal stimuli come from within the
body and include pH, redox potential, oxidative stress, enzymatic presence, temperature, etc. External stimuli come from outside
of the body and include light, ultrasound, magnetic field, etc. Using biomaterials that can respond to these factors have advantages.
Nanoparticles can be used to form theranostics. The nanoparticles can deliver diagnostic materials, such as dyes, that can be used
to image the target tumors. They can also deliver cancer drug to cancer cells. Metal nanoparticles are major biomaterials for cancer
theranostics. They include gold, silver, quantum dots, iron oxide, and other nanoparticles. There are magnetic metal nanoparticles
that can be tracked in the body and manipulated. Some nanoparticles can react to external stimuli, such as ultrasound, to kill the
cancer cells that they are attached to. There is a long list of polymers and other biomaterials that can be used in certain forms to react
to stimuli and release their cargo. These include modified PEG, PCL, PLGA, polyesters, and many others. The development of novel
nanoparticles and nanostructures will allow for better and more effective cancer theranostics in the future.
Bioprinting
Bioprinting uses additive manufacturing with biomaterials or biologics to create complex three-dimensional structures for regener-
ative medicine through the layer-by-layer additive processes. 3D bioprinting is able to create complex structures based on computer
designs. It can also create objects with personal anatomical structures on the basis of computer files of medical imaging. It therefore
uses a wide range of biomaterials for different types of applications. With a wide range of desired structures and materials to use,
there are a number of types of bioprinters. The three main types of bioprinting are based on inkjet, laser/light, or extrusion. Inkjet
bioprinting uses multiple mechanisms, such as thermal, piezoelectric actuator, laser-induced forward transfer, and pneumatic pres-
sure, to deposit tiny droplets of the biomaterial onto a substrate. These tiny droplets eventually build up into the desired shape.
Stereolithography/projection bioprinting uses lasers or other light sources such as UV light to project a 2D image on a photopoly-
merizable material. During the process, a stage lowers and the next image is projected, and the layers are polymerized on top of each
other, creating a 3D structure. Extrusion-based bioprinting uses mechanical forces, such as air pressure or a motor, to extrude mate-
rial through a nozzle in a specific pattern. The biomaterials used must be able to be extruded but still able to hold their shape once
printed. Each bioprinting technique has advantages and limitations and use different materials.
The biomaterials used in bioprinting are called bioinks. Bioinks are biocompatible materials that can contain cells and other
biological factors. For a bioink to work, it must be able to be bonded to the layers below and above and to be able to hold its shape.
The bioinks can have a wide variety of properties, depending on the desired characteristics of the human body tissue it is meant to
mimic. Bioinks are made of biomaterials that are either curable or are mechanically tough materials that harden on their own after
printing and soft biomaterials. The soft biomaterials are capable of supporting cell growth, whereas the hard materials generally are
not suitable for this. Hence the harder materials are usually used as support structures while the softer biomaterials support the cells.
The hard biomaterials are generally curable and self-hardening polymers, while the soft bioinks are synthetic or natural polymers
that are often formed into hydrogels. Some synthetic polymers used in bioprinting are PEG, and its acrylated version and PCL. PCL
is a hard polymer with a melting temperature suited for bioprinting. Some natural polymers that are commonly used for bioprinting
are hyaluronic acid and its methacrylated version, collagen, gelatin, and alginate. The soft natural and synthetic polymers can be
made into hydrogels for bioprinting, which also allows them to support cellular life and biologics. The fact that they can support
cells makes them particularly valuable for 3D bioprinting for tissue regeneration. Each of these biomaterials has different mechan-
ical and biological properties and it is important to use the right biomaterial for a specific purpose. The development of new bioinks
and optimizing current ones with cells and biologics will help to realize the potential of bioprinting in tissue engineering and regen-
erative medicine.
Organs-on-Chips
An organ-on-a-chip is a micro-scale system used for mimicking the human body environment. The goal for organ-on-a-chip is to
develop human tissue models for disease modeling and drug testing. They use microfluidics, along with cells, to imitate the phys-
iological and mechanical conditions experienced in the body. They can control the movement and behavior of materials and cells
by using channels, chambers, membranes, etc. The devices are manufactured using soft lithography and BioMEMS (Bio-
MicroElectroMechanical Systems), which allow for the micro scale details to be properly produced. These fabrication techniques
allow the use of different materials, including thermoplastics and thermoset polymers.
Most of the materials used to create organ-on-a-chip devices need to be optically clear for viewing and imaging purposes,
although whether they are stiff or flexible depends on the use of the device. The materials must also have the right chemistry
and reactivity so as to not improperly affect the system. Glass and silicone have been used as materials for microfluidic devices.
A commonly used soft, synthetic polymer is polydimethylsiloxane (PDMS). It is optically clear, easy to stretch and easy to fabricate
and has high oxygen permeability. Organ-on-a-chip systems that need to be mechanically stable can use thermoplastics such as
polystyrene. They are stiff materials with stable surface chemistries. Other synthetic polymers used in making organ-on-a-chip
systems are PMMA and polycarbonate. Natural materials, such as collagen, in the form of hydrogels have been used in organ-
on-a-chip systems to assist cell organization. In some cases, biodegradable materials are desired as scaffolds in the system. Materials
such as PLGA and polydioxanone (PDO) are thus used.
A major requirement for the materials used in organ-on-a-chip systems is that they need to be able to be manufactured with
small details. A major technique for manufacture is soft lithography, which normally uses PDMS as the material for chips. Hot
embossing and injection molding are also used to make devices from thermoplastics. 2D printing now appears promising for con-
structing organ-on-a-chip systems.
Acknowledgments
The authors thank members of their respective research group in the The University of Hong Kong and University of Nebraska Medical Center for
assistance in writing this book chapter. Min Wang thanks The University of Hong Kong, Hong Kong Research Grants Council and the National
Natural Science Foundation of China and Bin Duan thanks University of Nebraska and American Heart Association for funding their research in
biomaterials and tissue engineering.
Further Reading
Agrawal, P., Soni, S., Mittal, G., & Bhatnagar, A. (2014). Role of polymeric biomaterials as wound healing agents. The International Journal of Lower Extremity Wounds, 13,
180–190.
Callister, W. D., Jr., & Rethwisch, D. G. (2014). Materials science and engineeringdAn introduction (9th edn.). Hoboken, NJ: John Wiley & Sons.
D’Souza, S. F. (2001). Immobilization and stabilization of biomaterials for biosensor applications. Applied Biochemistry and Biotechnology, 96, 225–238.
Ebewele, R. O. (2000). Polymer science and technology. Boca Raton, FL: CRC Press.
Hench, L. L. (Ed.). (2013). An introduction to bioceramics (2nd edn.). Singapore: World Scientific.
Inamdar, N. K., & Borenstein, J. T. (2011). Microfluidic cell culture models for tissue engineering. Current Opinion in Biotechnology, 22, 681–689.
Kim, J. J., & Gregory, G. R. D. (2012). Applications of biomaterials in plastic surgery. Clinics in Plastic Surgery, 39, 359–376.
Kumar, S., Ahlawat, W., Kumar, R., & Dilbaghi, N. (2015). Graphene, carbon nanotubes, zinc oxide and gold as elite nanomaterials for fabrication of biosensors for healthcare.
Biosensors and Bioelectronics, 70, 498–503.
Langer, R., & Peppas, N. A. (2003). Advances in biomaterials, drug delivery, and bionanotechnology. AIChE Journal, 49, 2990–3006.
Lanza, R. P., et al. (Eds.). (2013). Principles of Tissue Engineering (4th edn.). Burlington, MA: Academic Press.
Mayet, N., Choonara, Y. E., Kumar, P., et al. (2014). A comprehensive review of advanced polymeric wound healing systems. Journal of Pharmaceutical Sciences, 103,
2211–2230.
Molli, R. G., Lombardi, A. V., Jr., Berend, K. R., Adams, J. B., & Sneller, M. A. (2012). A short tapered stem reduces intraoperative complications in primary total hip arthroplasty.
Clinical Orthopaedics and Related Research, 470, 450–461.
Navarro, M., Michiardi, A., Castaño, O., & Planell, J. A. (2008). Biomaterials in orthopaedics. Journal of the Royal Society Interface, 5, 1137–1158.
Ratner, B. D., et al. (Eds.). (2013). Biomaterials science: An introduction to materials in medicine (3rd edn.). Amsterdam: Elsevier.
Refojo, M. F. (1982). Current status of biomaterials in ophthalmology. Survey of Ophthalmology, 26, 257–265.
Skardal, A., & Atala, A. (2014). Biomaterials for integration with 3-D bioprinting. Annals of Biomedical Engineering, 43, 730–746.
Teo, A. J. T., Mishra, A., Park, I., et al. (2016). Polymeric biomaterials for medical implants and devices. ACS Biomaterials Science & Engineering, 2, 454–472.
Von Recum, A. (Ed.). (1998). Handbook of biomaterials evaluation: Scientific, technical, and clinical testing of implant materials (2nd edn.). Boca Raton, FL: CRC Press.
Wang, Y., Shim, M. S., Levinson, N. S., Sung, H. W., & Xia, Y. (2014). Stimuli-responsive materials for controlled release of theranostic agents. Advanced Functional Materials,
24(27), 4206–4220.
Nano-Biomaterials and their Applications
Mian Wang, Northeastern University, Boston, MA, United States
Thomas J Webster, Northeastern University, Boston, MA, United States; and Wenzhou Medical University, Wenzhou, China
Introduction 153
Nano-Hydroxyapatite (nHA) 153
Advantage of the nHA Nanostructure 154
nHA Synthesis 154
Biomedical Applications of HA Composites 154
Drug delivery 155
Tissue engineering 155
Carbon Nanotubes (CNTs) 156
Advantages and Disadvantages of CNTs 156
Biomedical Applications (Fabrications and Improvements) 156
Metal Nanomaterials 157
Tissue Regeneration of Metal Nanomaterials 157
Drug Delivery Using Metal Nanomaterials 158
Polymer Hydrogels 158
Drug Encapsulation and Delivery 158
Tissue Engineering 159
Protein Based Nanomaterials 159
Drug Delivery 159
Antibacterial Applications 160
Conclusions 160
References 161
Further Reading 161
Introduction
The utilization of biomaterials in medicine is not new. The first applications of biomaterials happened in ancient Egypt and Greece,
where they used biomaterials such as corals, shells, ivory, stone, wood, and some metals like gold and silver as implants for the
skeletal system (Piao et al., 2008). The Chinese were the first to use a dental amalgam to repair decayed teeth. More and more
biomaterial applications have emerged since then.
Recent studies have focused on the use of small scale biomaterials into a growing number of biomedical applications (Kang
et al., 2014). The discovery and manipulation of innovative nanomaterials, such as nano-hydroxyapatite, carbon nanotubes
(CNTs), metallic nanomaterials, protein-based nanomaterials, and other nanostructured materials are becoming increasingly
common in biomedical applications in general. Nanomaterials for different pharmaceutical applications, like tissue engineering,
drug or DNA delivery, drug formulations, dentistry, and medicine have all been made. In this article, we review some known
methods for obtaining nanomaterials with multiple properties for numerous biomedical applications. We then demonstrate the
multifunctionalization of nanomaterials and the improvement in medicine from those modifications, as well as emerging
applications.
Nano-Hydroxyapatite (nHA)
Biomimetic nanohydroxyapatite [nHA, Ca10(PO4)6(OH)2] has received much attention in bone regeneration, owing to their simi-
larity in chemical composition to that of natural bone mineral. Bone is a complex composite with a porous structure consisting of
inorganic and organic components. The inorganic part is mainly hydroxyapatite, which is the hydroxyl end member of the
complex apatite group. Therefore, nHA plays an essential role in bone defects and related diseases, which can significantly affect
the quality of life. In the US alone, the number of orthopedic procedures has more than doubled, from 138,700 in 2000–310,800
in 2010 (Thein-Han and Misra, 2009). The number of hip replacements performed in the US has continuously increased, and the
procedure has become more common in younger people. Traditional orthopedic implants use various metals such as titanium and
its alloys. However, these conventional implants face a lot of problems, such as corrosion, mechanical issues, and biocompatibility
issues.

154 Biomaterials: Science and engineering j Nano-Biomaterials and their Applications
To achieve better bone cell adhesion and osseointegration, hydroxyapatite has been considered as the primary material which
can create bioactive and biomimetic interfaces with traditional implants. Another reason that nHA has been widely used in bone
regeneration is that nHA has a high surface to volume ratio and high surface energy giving nHA an ability to serve as a reinforcement
in biomaterial composites. Studies have revealed that nHA biomaterials possess enhance bioactivity and mechanical properties to
conventional, or micron structured, hydroxyapatite. These reasons allow nHA to form a promising osteoconductive bond between
native bone and implant replacements.
Advantage of the nHA Nanostructure

Natural hydroxyapatite is usually found in the form of Ca10(PO4)6(OH)2 with a Ca/P ratio of 1.67. Although other ratios of calcium
phosphates exist, the ratio of 1.67 is the most thermodynamically stable and hence is the most used calcium phosphate material in
bone regeneration studies. nHA with fine crystals apparently improved mechanical properties of composites when compared with
microHA or noncrystalline HA. In natural bone, nHA plays a vital role in reinforcing mechanical strength. Besides, type I collagen
fibers serves as the extracellular matrix providing support for cell adhesion and proliferation (Shen et al., 2016). Therefore, two
components of bone enable its strong properties and allow for new bone formation.
nHA has been proven to have a positive impact on cellular behavior such as osteoblast cell adhesion, proliferation, and bone
formation when compared with large sized hydroxyapatite (micro and macro) (Shen et al., 2016). In addition, studies have indi-
cated the importance of nHA in in vivo studies, indicating constant bone formation when using nHA. Webster et al. published
a series of reports that the surface roughness of implants created by adding nHA had a positive effect on cell proliferation and oste-
ogenic differentiation (Wang et al., 2017). During this process, cellular markers of cell adhesion, proliferation, and differentiation,
such as alkaline phosphatase synthesis and calcium deposition, were observed in greater amounts on a nanostructured hydroxyap-
atite based surface than on amorphous and micro hydroxyapatite surface. Protein adsorption on nHA, which leads to initial cell
attachment, was also found to be higher than standard hydroxyapatite according to those studies. The same group also indicated
that whereby tartrate-resistant acid phosphatase synthesis (TRAP, a phenotypic marker for osteoclasts which express this enzyme)
and resorption pitting, which are primary activities of osteoclasts was significantly greater for cells exposed to nanostructured
surfaces (compared to a hydroxyapatite sample with a surface area greater than 100 nm).
nHA Synthesis
It is possible to synthesize hydroxyapatite with a wide variety of methods, such as wet chemistry, hydrothermal, mechanical–chem-
ical synthesis, plasma spraying, and sol-gel. Generally, there are two main synthesis ways: wet chemistry and high-temperature
methods. For this section, we will introduce two typical methods, wet chemistry and hydrothermal methods.
Wet chemistry is a precipitation method which depends on a solution environment of calcium and phosphorus ions to precip-
itate onto an exposed surface. It is a wet method that enables calcium phosphate precipitation. The advantage of the wet chemistry
method is that it is cheaper and a more accessible process when compared with other hydroxyapatite synthesis methods. However,
the disadvantage of this technology also exists in this traditional approach. The size of HA particles in the wet chemical method
usually varies significantly, or are not evenly dispersed. To improve dispersion, multiple methods have been developed. For
example, ammonium hydroxide is added to control pH at a slow rate under stirring. The precipitation process is slow and crystal-
linity is controlled by a temperature change. Generally, the higher the temperature the better the crystallinity. An alternative method
is by adjusting the pH value of the solution that the samples are exposed to, resulting in the control of the calcium phosphate phase
and morphology. For instance, amorphous calcium phosphates are considered to form from pH 5 to 12. If the pH range is narrowed
to 5.5–7, hydroxyapatite will develop best with an even morphology. Another method for forming hydroxyapatite uses an ionic
solution which mimics human blood plasma. Simulated body fluid was developed by Kokubo and Takadama (2008) and was
used for hydroxyapatite formation. The process is thought to occur through dissolution, renucleation, crystal growth and finally
transformation to hydroxyapatite. According to this method, hydroxyapatite forms in a very uniform and well-dispersed way.
The hydrothermal method of hydroxyapatite synthesis is required for high pressure, high-temperature processes to form a crystalline
phase. Hydroxyapatite particles are generated from the reaction of calcium carbonate and diammonium hydrogen phosphate at
a temperature range from 200 to 300 C and pressures around 1–2 kbar. Particle sizes synthesized by this method are about
200 nm to 1 mm in a needle-like morphology. Temperature changes in a small range (30 C) will not influence particle sizes too
much according to Jiang et al. studies (Chen et al., 2016).
Biomedical Applications of HA Composites

Because of structural advantages, high surface to volume ratios, unique physical and chemical properties, hydroxyapatite has been
used for a multiple of biomedical applications. In addition, the surface properties of nHA make it easy to modify, possess excellent
biocompatibility and noninflammatory responses, which are all important for numerous biomedical applications.
Biocompatibility and nontoxic materials are essential requirements for nanocarriers used in drug delivery biomedical applica-
tions. To be a qualified nanocarrier, an easily modified surface with covalently immobilized ligands can provide controlled release
of proteins or drugs. There are two main groups of using hydroxyapatite as nanocarriers: one is the conjugation of a drug or protein
Biomaterials: Science and engineering j Nano-Biomaterials and their Applications 155
with a covalent linkage, while the other is with physical interactions. Covalent linkage can be realized by conjugating amino or
hydroxyl functional groups on the surface with linker groups such as iodoacetyls, maleimides, and the bifunctional linker pyridyl
disulfide. One of the advantages of those linkages is a sustainable release of drugs or proteins. On the other hand, physical inter-
actions, such as hydrophobic/hydrophilic and electrostatic, can also lead to the coupling of drug molecules with the surfaces of
hydroxyapatite (Pàmies and Stoddart, 2013). However, a cationic polymer which is usually used as a physical linker may bring
toxicity, which is a big challenge for its utilization in physical interactions. To overcome such constraints, the modification of poly-
mer linkers has been investigated to reduce cytotoxicity and enhance cellular uptake.
Drug delivery
Hydroxyapatite has a powerful ability to absorb proteins on its surface. One of the positive aspects of nHA as a drug carrier molecule
is its strong ability to adsorb proteins on the surface. Studies have demonstrated the various proteins such as bovine serum albumin
(BSA), hemoglobin, and cytochrome were used with HA for multiple purposes. In addition, studies have also revealed the release of
BSA from an HA carrier at different pH conditions. There are two stages of HA-conjugated protein release: the initial stage is a fast
release via desorption followed by a slow release stage because of crystal dissolution. Chitosan and HA have an ability to form
a stable partnership as a drug delivery carrier due to the surface charge properties of those two items. A negatively charged HA
core plays a vital role in attracting positively charged chitosan. One study from Venkatesan et al. showed that HA-chitosan nano-
particles were used to sustain the release of Celecoxib for antitumor research (Venkatesan et al., 2016). Less than 15% of drugs were
released by a burst effect of the adsorbed drugs, while over 10% of the drug remained after sustained released for 2 weeks. More
interesting applications of HA as a drug delivery carrier have been done. Targeted drug delivery and targeted drug therapy are
able to transport the designed drug to the affected sites of a body for a better therapeutic effect. A lot of nHA nanoparticles are
designed for targeted delivery via conjugation with iron oxides. Investigations have also been performed on magnetic nHA nano-
particles based on other dopants. Tran et al. mentioned that HA-coated iron oxide nanoparticles were able to increase osteoblast
functions (Tran et al., 2010). They synthesized HA coated Fe3O4 nanorods which had a saturation magnetization (Ms) of
35 emu/g revealing good osteoblast activity when cultured with osteoblasts.
Tissue engineering
The performance of HA materials in different applications mainly rely on their chemical composition, particle size, crystal
morphologies, and aggregation. Since they have similar morphology and functions to natural bone composition, HA materials,
especially nHA, have been widely used in bone regeneration, as well as other tissue regeneration studies. Bioceramics or
biopolymer composites are the best approach to make the artificial bone material with the required properties. One of the
most widely used materials is chitosan and its composite biomaterials for orthopedic tissue engineering. Chitosan is able to
be processed into numerous forms and used as bone graft substitutes, such as nanofibers, microspheres, membranes, and 3D scaf-
folds. nHA/chitosan composites are widely studied for bone graft implants and have proven to be excellent in bone tissue engi-
neering, since it has excellent biodegradable, pore-forming ability, and antibacterial properties. The addition of HA to chitosan
will further enhance mechanical properties and be able to mimic the natural structure of bone. Wang et al. reported on
a composite of HA/chitosan, which was prepared by a freeze-drying method, and used for bone regeneration (Wang et al.,
2014). Nanocomposite scaffolds with 20% wt HA had the ideal porous structure with a pore size ranging from 100 to
500 mm. Bone cell attachment and proliferation on the scaffold indicated that the nHA/chitosan is nontoxic and has good cyto-
compatibility. Collagen is the main component of bone that possesses a fibrous structure with a diameter of 50–500 nm.
Numbers studies on nHA/collagen have been undertaken for bone tissue engineering. Collagen has been reported to have several
kinds of negatively or positively charged groups as well as uncharged but polar groups on its surface. Based on those groups,
charged sites on the surface play an important role to impact the nucleation of the HA crystals on collagen membranes through
chemical interactions. nHA/collagen composite scaffolds displayed homogeneous interconnected macroporous structures which
have proper mechanical properties to natural bone.
PLA is another widely used synthetic polymer in bone tissue engineering because of its biodegradability and excellent biocom-
patibility. nHA/PLA composites prepared by phase separation methods showed excellent osteoconductive, osteoinductive and
mechanical properties. The porosity of scaffolds, prepared by a solid–liquid phase separation method, was up to 85% with
a pore diameter of around 64–175 mm.
Among those, the size of HA is not only related to the synthesis methods, but also has a crucial influence on cell response. The
Webster group has demonstrated that it is beneficial if the surface roughness of synthetic hydroxyapatite is in the nanoscale. In
particular, a nanoscale topography appears to have a positive effect on cell proliferation and osteoblastic differentiation. A
nano-roughened hydroxyapatite surface was superior to a less roughened one for positive bone cell responses and therefore better
osseointegration. More specifically, osteoblast adhesion, proliferation and cell differentiation markers, such as alkaline phosphatase
synthesis and calcium deposition, were significantly greater on nanoscale topographical surfaces than on a standard HA sample
surface with a roughness greater than 100 nm. Wang et al. fabricated nanoscale, microscale, and amorphous HA/chitosan scaffolds
via a freeze-drying method used for a bone regeneration study (Wang et al., 2014). Nanosized HA was proven to impact cell attach-
ment, proliferation, and differentiation for bone generation, when compared to microscale and amorphous phases. The reasons
behind such improvements are still largely under investigation. However, a common hypothesis is that protein adsorption such
as vitronectin, fibronectin, and bioactivity on surfaces or particles with nanoscale features is different from that on conventional
microscaled surfaces.
Carbon Nanotubes (CNTs)
Carbon nanotubes (CNTs) or carbon-based nanotubular structures have received much attention since the 1990s. They are hollow
cylinders formed by one (single-walled CNTs, SWCNTs) or several (multiwalled CNTs, MWCNTs) layers of graphene. CNTs can be
fabricated via a variety of methods, including chemical vapor deposition (CVD) and arc-discharge, and they have garnered high
interest in many different fields due to their unique mechanical, chemical, and electrical properties (Fig. 1).
Advantages and Disadvantages of CNTs

Progress in nanomedicine and using nanomaterials in biomedical applications have lately allowed us to develop multifunctional
nanosystems consisting of disease diagnosis, targeting, and treatment, all in one. Currently available nano drug delivery systems
include nanoparticles, liposomes, dendrimers, carbon nanotubes, nanoshells, and quantum dots. The primary purpose for the
development and application of those nanocarriers are for a better therapeutic response as well as low cytotoxicity. Among all nano-
materials, carbon nanotubes are receiving more and more attention for more effective and safe drug delivery. The advantages of
carbon nanotubes include high cell membrane permeability, excellent electrical, optical, thermal and mechanical properties,
ultra-lightweight and ultra-high surface area, photoluminescence property, photoacoustic effect, and pH-dependent unloading
of loaded materials. Meanwhile, there are some disadvantages of carbon nanotubes in biomedical applications, such as nonbiode-
gradable, aggregation and bundling phenomenon, some toxicity, accumulation in the liver, and that they are insoluble in organic
and inorganic solvents.
Biomedical Applications (Fabrications and Improvements)

The high aspect ratio of CNTs is a feature to apply CNTs for diverse biomedical applications. They have captured a lot of attention as
other nanoscale materials because of their nanometric structure and remarkable list of excellent properties that encourage their
exploitation for promising applications. Significant progress has been made to overcome many limitations such as aqueous solu-
bility and cytotoxicity for better applications in biomedical fields. Functionalized CNTs have been widely used in imaging, targeting
drug delivery, gene delivery, biosensing, gene delivery, and tissue engineering. The following section demonstrates the possible
potential of CNTs in imaging, targeted delivery, tissue engineering, and diagnostics and their future potential for biomedical
applications.
To overcome barriers like cytotoxicity and dispersibility, functionalization of CNTs provides a possible solution. Functionaliza-
tion of CNTs can not only enhance their solubility but also provide more functional sites to conjugate genes, drugs, ligands, and
agents. For example, PEG-conjugated CNTs helped to impede opsonization in vivo, as well as prevented reticular endothelial system
(RES) uptake. PEGylation is the most effective method to enhance in vivo properties of CNTs, and low cytotoxicity. Covalent func-
tionalization of CNTs showed better dispersion. Before covalent functionalization, a carboxylic acid group on the CNTs surface
must be activated using reagents like thionyl chloride, oxalyl chloride or N-hydroxysuccinimide in order to obtain highly reactive
intermediate groups for stable covalent bonding.
CNTs offer many benefits in various applications like tissue engineering, targeted drug delivery, imaging and diagnosis, and
photothermal therapy. The bone grafting process requires a bone mimetic scaffold that is associated with the natural bone healing
process. An improved healing process may be possible with CNTs assisting new bone formation. CNTs have been investigated to
Fig. 1 Representation of using a SWNT as a nanocarrier for targeted drug delivery of Doxorubicin (Dox) and a schematic of SWNT transportation
into a cell. Reprinted from Meng, L., Zhang, X., Lu, Q., Fei, Z. and Dyson, P. J. (2012). Single walled carbon nanotubes as drug delivery vehicles:
Targeting doxorubicin to tumors. Biomaterials 33, 1689–1698 (Copyright © 2012, with permission from Elsevier).
replace collagen in scaffolds for bone growth. CNTs are also used for gene therapy and stem cell differentiation, since CNTs have
sufficient contractility for muscle tissue regeneration. PEGylation of CNTs makes them stealth to prevent white blood cells from an
inflammatory response on CNTs, allowing them to circulate in the bloodstream for a sustained duration of time. Surface engi-
neered CNTs have emerged as a nano delivery carrier and imaging for disease treatment and health monitoring. Meanwhile,
due to extravagant mechanical properties, CNTs could also be embedded into materials as a nanocomposite for tissue engineering
applications. The elasticity, mechanical properties, conductivity, and toughness are significantly promoted by the addition of CNTs.
CNTs are not only able to facilitate structural reinforcement of a composite, but also enhance cellular performance. CNTs have
the capability to absorb light in the NIR region, which results in state jumping of CNTs. During this process, heat generated by
a photothermal process will be released in the targeted site. The photothermal agents in an in vivo tumor could induce thermal
destruction of cancer cells with sufficient CNT concentrations. Targeted CNTs have been able to spare cells from damage. CNTs
have also been used as a contrast agent in imaging and recognition of cancerous cells owing to their remarkable optical properties.
Fluorescent agent conjugated CNTs are utilized as a radio-opaque substance providing an image of the desired in vivo organs. De La
Zerda et al. revealed the potential use of RGD peptide conjugated SWCNTs in photoacoustic imaging (De la Zerda et al., 2008).
Welsher et al. investigated the use of antibody conjugated SWCNTs for probing cell surface receptors such as NIR fluorescent labels
(Welsher et al., 2008).
CNTs could enhance cell specificity by conjugating antibodies to CNTs, resulting in better internalizing within mammalian cells.
Meanwhile, CNTs have not only enhanced drug delivery, but also reduced cytotoxicity which has been found in an Amphotericin
nanotube study. In addition, an Amphotericin nanotube study indicated increased antifungal efficacy and reduced toxicity to
mammalian cells when compared to amphotericin antifungal study without nanotubes. Upon conjugation with peptides, it may
be possible to be used as a vaccine delivery since they have excellent structures for vaccine delivery. With the advancements in molec-
ular dynamic simulations, as well as unique structure, CNTs have potential use as a gene delivery tool. The ability of CNTs to trans-
port DNA across cell membranes is used in gene therapy. During DNA therapy process, CNTs could conjugate with DNA or can load
DNA inside tubes. It has been found that gene therapy with b-galactosidase marker gene nanotubes showed higher expression
compared to the transfer of naked DNA. Another application of CNT surfaces is the incorporation with carboxylic and ammonium
groups which makes them more suitable for the delivery of peptides, nucleic acids, and other molecules. Traditional CNTs are water-
insoluble forms which result in high in vitro toxicity compared with PEGlated CNTs which could increase water dispersible. It has
been proved that the extent of toxicity decreases with special functionalization. However, functionalization of CNTs has a negative
impact on the elimination of nanotubes. SWNTs have a high renal uptake. Whereas modest liver uptake as compared to single-
walled nanotubes with conjugation to a monoclonal antibody has higher liver uptake and lower renal uptake. In addition, it
was also reported that carbon nanotubes, except acetylated ones, when conjugated with peptides produced a higher immunological
reaction when compared to free peptides. Therefore, owing to the excellent properties of CNTs such as high surface area to volume
aspect ratio, high mechanical strength, high electrical conductivity, ultra-lightweight, and high thermal conductivity, multiple
advantages over other drug delivery carriers exist for CNTs.
Metal Nanomaterials
Traditional grafting techniques for tissue regeneration are being replaced by tissue engineering approaches of using 3D biomimetic
materials. Of these biomaterials, metals have the highest mechanical strength, as well as very good performance in biocompatible
properties. For example, lots of metal nanoparticles play an essential role in bone formation and regeneration, since they have an
inherent ability to promote osseointegration and osteoconductivity. Besides, a lot of metal nanoparticles could possess antimicro-
bial activity. This section will review multiple metal nanoparticles, such as Zn, Ti, Zr, B, Sr, Mg, Ag, and Cu along with their signif-
icant applications in the biomedical field, including tissue engineering, drug delivery, and antimicrobial studies.
Tissue Regeneration of Metal Nanomaterials

Metal nanoparticles have been used in biomedical applications for many years due to many attractive properties, especially for use
in dental implant and orthopedic surgery. Their great strength, ductility, and durability provide excellent properties as a bone substi-
tution. Several studies have shown that the incorporation of HA nanoparticles and metallic nanoparticles such as titanium and iron
oxide could increase collagen and calcium deposition by osteoblast cells, as well as promoting stem cell differentiation and bone
formation. It has been hypothesized that the nanostructural topographical properties generated by the incorporation of nanoma-
terials play a critical role in the improved biological performance of nanocomposites rather than the chemistry. Indeed, natural
tissue is also a nanostructured material, consisting of collagen fibrils and proteins with dimensions in the 100 nm size or less.
Although metal nanoparticles showed excellent biomedical applications in multiple aspects, metal ions release may have toxicity
and induce some complications in the clinics. Numerous studies have been completed in vitro to study the cytotoxicity of multiple
metal nanoparticles. Stainless steel contains more than one kind of metal element and may have more potential safety problems.
Some alloys may generate genotoxicity of medical implants, such as TiAlV and CoCrMo. The Cu, Zn, Mg, Ag, Al may have some
exceptional properties as essential elements in the human body. For instance, Zn can improve DNA synthesis, enzyme activity,
and hormonal activity. On the other hand, these metal elements also can lead to cytotoxicity when only used in high doses.
Different metal ions have different mechanisms resulting in cytotoxicity.
Drug Delivery Using Metal Nanomaterials

Recent studies have focused on antiinfection studies of metallic nanoparticles. Infection-induced by bacteria has existed over the
past several decades, even though sterilization technology and surgical equipment cleaning has improved. Antibiotics have been
used for many years for antibacterial purposes both in vivo and in vitro, as well as sterilization of biomedical equipment. Although
sterilization techniques significantly reduce the chance of infection, new technology is still needed to improve resisting bacterial
colonization. Passive strategies including surface modification and drug delivery of antibiotics have been proven to reduce the
chance of bacterial infection. One of the most direct approaches for improving the efficacy of implant materials is to deliver anti-
biotics at the implant from a surface coating. Another current strategy for the development of improved antibacterial biomaterials is
via surface modification, such as changing surface roughness and decorating with nanomaterials. Researchers filled nanoparticles
like silver (Ag), titanium (Ti), zinc (Zn), zinc oxide (ZnO) and zirconia (ZrO2) to further decrease bacterial adhesion of implant
materials. The results suggested that nanoparticle combined coatings significantly improved the antibacterial performance relative
to conventional Ti and Ti nanotube implant materials (Fig. 2). Puckett et al. determined decreased adhesion and growth of Staph-
ylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa on nanorough Ti generated by electron beam evaporation and
nanotubular and nanotextured Ti produced by two anodization methods compared to traditional Ti. Silver(Ag) is known for its
broad-spectrum antibiotic activity against Gram-negative and Gram-positive bacteria, fungi, chlamydia, mycoplasma and certain
viruses, especially antibiotic-resistant strains. There are many implant surface modifications using Ag as the antibacterial agent.
At proper doses, it is possible to fabricate coatings with long-term antibacterial characteristic by introducing and controlling Ag
release. Zinc (Zn) has been reported to act broadly against a wide range of bacterial adhesion. Notably, the antibacterial properties
of Ag have been known for centuries. The chemical nature of silver affords antibacterial activity in multiple ways. Karlsson et al.
showed that CuO particles were much more toxic than the Cu ions since CuO particles had more potential to cause DNA damage
and oxidative lesions (Karlsson et al., 2008).
Polymer Hydrogels
Many improvements to implants and cell interfaces have been made which could be pivotal in translating current polymer or hydro-
gel coating technologies into the nanoscale. It will benefit long-term implants as well as have a positive effect on an antiinflamma-
tory body response and poor tissue integration. Towards this end, a lot of effort has been made to look into implant-cell interactions
and to investigate the possibilities for controlling cell fate, such as adhesion, proliferation, and migration through the nanoscale as
well as managed drug delivery.
Drug Encapsulation and Delivery

In addition to traditional implants, nanosurface coatings also hold significant benefits over conventional metallic or inorganic semi-
conductor electrodes as functional molecules, such as extracellular matrix (ECM) molecules or antiinflammatory molecules, can be
easily incorporated via simple adsorption, entrapment, covalent binding or the doping process to encourage neural attachment and
to reduce inflammatory responses.
In recent years, it has been recognized that cells within all tissues are surrounded by a three-dimensional matrix called the ECM,
which provides structural support for cellular constituents and initiates critical biochemical and biomechanical dialogues between
Fig. 2 Surface SEM images of NT-TiO2, NT-Ag, and NT-Sr-Ags (Upper row); Cross-sectional SEM images of the samples (Bottom row). Reprinted
from Chen, Y. et al. (2017). Antibacterial, osteogenic, and angiogenic activities of SrTiO3 nanotubes embedded with Ag2O nanoparticles. Materials
Science and Engineering: C 75, 1049–1058 (Copyright © 2017, with permission from Elsevier).
various cellular components that eventually regulate cell proliferation, differentiation, migration, and apoptosis. One of the essen-
tial strategies for understanding and controlling processes that determine cell-material interfacing is to recreate the microenviron-
ment cells experience in native biological settings. In the context of neural tissues, the intricate 3D structure of neural tissues also
plays a huge role in determining neural functions. Motivated by these advances, there’s a trend in converting 2D CP surfaces into
functional 3D structures with the intent to further promote tissue integration. In general, a 3D construct of conducting polymers can
be created via two primary methods: CPs can be deposited onto nonconducting 3D–structured materials, such as electrospun fibers
or 3D–printed scaffold, or a 3D construct can be fabricated directly with a CP material. Although it is still at its early stage, results
indicate that porosity, morphology, mechanical properties and mechanical stability in these 3D scaffolding could be tunable; excel-
lent neuronal cell adhesion, proliferation, and alignment can be achieved; and antiinflammatory drugs can be precisely released
upon electrical stimulation. Taken together, this represents a promising way to construct biomimetic and biologically active coat-
ings, which will facilitate the development of stable long-term implants with better tissue integration and reduced foreign body
responses.
Tissue Engineering
One of the most heavily investigated set of molecules to improve the biological responses of nanorough coatings is the ECM due to
their well-known role in regulating cell attachment, proliferation, differentiation, migration, and apoptosis. It has been demon-
strated that scaffolds containing small, degradable polymer beads that release nerve growth factors (NGF) to mimic the chemical
microenvironment of developing tissue were found to improve the viability of fetal neural cells transplanted into rat brains, and
a later study further illustrated that this improvement in biological responses is in a dose-dependent manner. Additionally, the
discovery of adhesive domains derived from ECM proteins, notably the amino acid sequence Arg-Gly-Asp (RGD), has enabled
the development of synthetic nanomaterials that can modulate cell adhesion. Inspired by these advances, the incorporation of
ECM molecules with a nanosurface and their subsequent release has been one of the leading strategies to improve the biological
responses of nanoscaled coatings. For example, oligopeptides, such as RGD and YIGSR, have been introduced into PEDOT coatings
to form a nanoscale surface, and has been shown to increase cell adhesion in vitro, to be able to establish strong connections with
the neuronal structure and thereby improve recordings at the coated sites. Results showed that cell survival, attachment, extension,
and differentiation were indeed improved on a nanoscaled surface, suggesting the potential to help establish stable nanomaterials
and cell interfaces.
Protein Based Nanomaterials
Peptide amphiphiles (PAs) are another nanomaterial which receives more and more attention for biomedical applications. PA is
composed of a hydrophobic portion and a hydrophilic portion which meditate self-assembled supramolecular structures. Due
to its biocompatibility and biodegradability, a number of PA and its functionalized derivates has promising applications in drug
delivery, tissue engineering, and antibacterial studies.
Drug Delivery
Due to excellent biodegradability, biodegradability, and efficient cell membrane permeability, PAs have exceptional advantages
over liposomes and polymeric drug carriers. PAs are designed to target tumor cells via enhancing the permeability and retention
(EPR) effect of tumors. Self-assembled PA would be encapsulated by a tumor cell by being passively engulfed by tumors. Chen
et al. developed a novel PA of 15 nm micelles loaded doxorubicin (DOX) with a high loading efficiency, long-circulation and in vivo
stability (Chen et al., 2011) (Fig. 3). In addition, studies demonstrated that nanoparticles modified with cationic charged peptide
sequences conjugated with RGD showed excellent selectivity of the tumor. Chang et al. investigated amphiphilic peptide nanopar-
ticles (APNPs) with arginine-rich and RGD peptide sequences in their hydrophilic head groups (Chang et al., 2015). APNPs encap-
sulated with curcumin showed excellent antitumor selectivity. APNPs were able to self-assemble into 15–20 nm diameter
nanospheres in water, but disassemble at a pH below 4, which can be beneficial for pH-sensitive drug carriers. With pH-
sensitive properties, self-assembly at an acidic environment allows curcumin to be encapsulated, and when the pH went back to
pH 7, the APNPs could reassemble and release curcumin. The curcumin-loaded APNPs were easily encapsulated into MG-63 oste-
osarcoma cells compared with normal human osteoblast cells within 2 hours. Low water solubility plays a vital role in the process of
internalization of drug loaded nanoparticles into a cell membrane. In the first 24 h, curcumin-loaded APNPs decreased 85% of the
MG-63 osteosarcoma cell viability, which was remarkably lower than osteoblast cell viability. This novel nanoparticle indicated that
the APNP peptide sequence was selective towards osteosarcoma cells.
Tissue Engineering
Self-assembled PA nanomaterials have promising potential for tissue regeneration. The ECM is a microenvironment containing
insoluble macromolecules and soluble bioactive factors, which can regulate the behavior and functions of cells. Based on a firm
understanding of cellular interactions with the ECM, peptide sequences are capable of targeting cell receptors to promote cell
Fig. 3 A schematic of micelles consisted of amphiphilic triple-helix peptide-PEG conjugates for Dox delivery: (A) The short peptide with PEG chains
attached to the middle and the C-terminus; (B) The neighbored amphiphiles formed into subunits and further self-assembled into micelles (C); (D)
The TEM image of the self-assembled micelles. Reprinted from Chen, J. X. et al. (2011). Construction of surfactant-like tetra-tail amphiphilic peptide
with RGD ligand for encapsulation of porphyrin for photodynamic therapy. Biomaterials 32, 1678–1684 (Copyright © 2015, with permission from
Elsevier); Chen, Y. et al. (2017). Antibacterial, osteogenic, and angiogenic activities of SrTiO3 nanotubes embedded with Ag2O nanoparticles. Materials
Science and Engineering: C 75, 1049–1058.
functions. PAs modified with bioactive sequences can activate cell receptors and facilitate targeted cell adhesion, proliferation, and
activation of endogenous regeneration mechanisms of diseased tissue. A self-assembled PA system with a BMP-2 binding sequence,
NH2-TSPHVPYGGS-COOH, was designed and studied by Lee et al. (2013). The osteogenesis effects of the PA nanofibers loaded
with BMP-2 was evaluated by the level of alkaline phosphatase (ALP) activity, which is an indicator for osteoblast differentiation
from C2C12 premyoblasts. In addition, Hartgerink et al. designed a nanostructured scaffold formed by PA nanofibers, which con-
tained a phosphorylated serine residue that can interact strongly with calcium ions (Hartgerink et al., 2002). Those nanofiber
matrices showed excellent bone cell adhesion, proliferation, and bone formation.
The nanostructured PA systems have also been revealed to have a promising potential in angiogenesis promotion. The self-
assembled nanostructured PA system can keep a low growth factor level, such as vascular endothelial growth factor (VEGF) and
fibroblast growth factor-2 (FGF-2), which is a key factor in restoring blood flow during tissue regeneration and wound healing.
Conjugating with growth factors onto the self-assembled nanostructures of PAs can stabilize their biological conformation and
enhance binding strength for interactions with cell receptors. Those nanofibers had a strong affinity with loaded molecules, which
can increase local retention in the microenvironment. For example, the FGF-2 loaded PA heparin-binding nanofibers displayed
controlled-release over 10 days with only 57.1% of the growth factor released. The heparin-binding nanofibers combined with
growth factors significantly promoted rat cornea neovascularization in vivo.
Antibacterial Applications
Antimicrobial peptides (AMPs) are usually short peptides with a 2–9 positive net charge, which increased the interaction between
bacterial membrane and peptides. As a result, amphipathic conformations could stabilize the peptide into secondary structures such
as a-helix, b-sheet and loop structures are important to AMP activity. The cationic domain can induce the interaction with
a membrane surface while the hydrophobic domain would then drive the peptide insertion into the hydrocarbon chain membrane
core. A difference from traditional antibiotics, AMPs bind and penetrate into bacterial cell membranes, causing membrane disrup-
tion, membrane lysis and eventually cell death. More importantly, cationic AMPs can disrupt cell membranes especially for bacteria
membranes which consist of a large number of anionic phospholipids. However, mammalian cell membranes which consist of
more zwitterionic phospholipids, have a low association ability of interaction with cationic AMPs. Therefore, a conjugation with
hydrophobic domains would enhance the activity of amphiphilic AMPs. For example, self-assembly of amphiphilic peptides would
promote the interactions with bacterial cell membranes by increasing the local cationic charge. Cell-penetrating TAT peptides and
six arginine residues (R6) were shown to have a strong antimicrobial property. Compared with conventional antibiotics, these
peptide-based nanoparticles were superior to penicillin G in killing Gram-positive Bacillus subtilis, and had a lower hemolytic
effects than amphotericin B. AMPs have attracted more and more attention because of this selectivity between bacteria and mamma-
lian cells. Some antimicrobial peptides are already in clinical and commercial applications. For these peptides, it is possible to differ-
entiate between those structural features that contribute to the specificity of initial membrane binding of the bacterial membrane.
Multiple parameters would optimize the design of novel AMPs. Those parameters that should be taken into consideration include
production costs, toxicity against normal healthy cells, and degradation rate. Recombinant DNA techniques could make biosyn-
thesis feasible but the peptides are usually damaged by the bacteria used to produce them. Although there were early hopes that
bacteria would not easily develop resistance to AMPs, it is clear that some strains of bacteria already have.
Conclusions
Owing to the success of the rapid development of technologies for the synthesis of multiple nanomaterials during the past decades,
investigators currently have at their disposal an enormous diversity of available nanomaterials with required parameters with
respect of size, shape, structure, electrical and strength properties. Moreover, the question that is now on the agenda is the biomed-
ical applications of those nanomaterials. From the standpoint of biomedical applications, much significance was held by the devel-
opment of efficient technologies for nanomaterials belonging to various classes. This article reviewed multiple nanomaterials
including nano-hydroxyapatite, CNTs, metallic nanomaterials, protein-based nanomaterials, and nanostructured polymer hydro-
gels, as well as multiple biomedical applications, such as drug or DNA delivery, tissue engineering, bioimaging, and diagnosis.
Nevertheless, the results here describe the way to use nanomaterials in many biomedical applications and more complex systems;
the challenge is now to transfer nanomaterial technology to in vivo applications, and to develop next-generation devices able to
impact biomedical performance in a controlled fashion efficiently.
References
Chang, R., Sun, L., & Webster, T. J. (2015). Selective inhibition of MG-63 osteosarcoma cell proliferation induced by curcumin-loaded self-assembled arginine-rich-rgd nano-
spheres. International Journal of Nanomedicine, 10, 3351–3365.
Chen, J. X., et al. (2011). Construction of surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy. Biomaterials, 32,
1678–1684.
Chen, Z., et al. (2016). Osteoimmunomodulation for the development of advanced bone biomaterials. Materials Today, 19, 304–321.
De la Zerda, A., et al. (2008). Carbon nanotubes as photoacoustic molecular imaging agents in living mice. Nature Nanotechnology, 3, 557–562.
Hartgerink, J. D., Beniash, E., & Stupp, S. I. (2002). Peptide-amphiphile nanofibers: A versatile scaffold for the preparation of self-assembling materials. Proceedings of the National
Academy of Sciences, 99, 5133–5138.
Kang, I. K., Ito, Y., & Kwon, O. H. (2014). Nano/microfabrication of biomaterials. BioMed Research International, 2014.
Karlsson, H. L., Cronholm, P., Gustafsson, J., & Möller, L. (2008). Copper oxide nanoparticles are highly toxic: A comparison between metal oxide nanoparticles and carbon
nanotubes. Chemical Research in Toxicology, 21, 1726–1732.
Kokubo, T., & Takadama, H. (2008). Handbook of Biomineralization: Biological Aspects and Structure Formation (vol. 3, pp. 97–109).
Lee, S. S., et al. (2013). Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds. Biomaterials, 34, 452–459.
Pàmies, P., & Stoddart, A. (2013). Materials for drug delivery. Nature Materials, 12, 957.
Piao, Y., Burns, A., Kim, J., Wiesner, U., & Hyeon, T. (2008). Designed fabrication of silica-based nanostructured particle systems for nanomedicine applications. Advanced
Functional Materials, 18, 3745–3758.
Shen, X., et al. (2016). Sequential and sustained release of SDF-1 and BMP-2 from silk fibroin-nanohydroxyapatite scaffold for the enhancement of bone regeneration. Biomaterials,
106, 205–216.
Thein-Han, W. W., & Misra, R. D. K. (2009). Biomimetic chitosan-nanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomaterialia, 5, 1182–1197.
Tran, N., Pareta, R., Taylor, E., & Webster, T. J. (2010). Iron oxide nanoparticles: Novel drug delivery materials for treating bone diseases. Thermec 2009 Suppl., 89–91, 411.
Venkatesan, J., Jayakumar, R., Anil, S., & Kim, S. K. (2016). Nanocomposites for musculoskeletal tissue regeneration (pp. 161–174). https://doi.org/10.1016/B978-1-78242-452-
9.00007-8.
Wang, M., et al. (2014). Design of biomimetic and bioactive cold plasma-modified nanostructured scaffolds for enhanced osteogenic differentiation of bone marrow-derived
mesenchymal stem cells. Tissue Engineering. Part A, 20, 1060–1071.
Wang, M., Geilich, B. M., Keidar, M., & Webster, T. J. (2017). Killing malignant melanoma cells with protoporphyrin IX-loaded polymersome-mediated photodynamic therapy and
cold atmospheric plasma. International Journal of Nanomedicine, 12, 4117–4127.
Welsher, K., Liu, Z., Daranciang, D., & Dai, H. (2008). Selective probing and imaging of cells with single walled carbon nanotubes as near-infrared fluorescent molecules. Nano
Letters, 8, 586–590.
Further Reading
Chen, Y., et al. (2017). Antibacterial, osteogenic, and angiogenic activities of SrTiO3nanotubes embedded with Ag2O nanoparticles. Materials Science and Engineering: C, 75,
1049–1058.
Meng, L., Zhang, X., Lu, Q., Fei, Z., & Dyson, P. J. (2012). Single walled carbon nanotubes as drug delivery vehicles: Targeting doxorubicin to tumors. Biomaterials, 33,
1689–1698.
Natural Biopolymers for Biomedical Applications
Natalia Davidenko, Ruth Cameron, and Serena Best, University of Cambridge, Cambridge, United Kingdom
Introduction 162
Definition, Classification, Sources 162
Requirements, Advantages and Disadvantages of Natural Polymers 163
Demand and Areas of Application 163
Properties and Applications 164
Polysaccharides 164
Glucans 166
Seaweeds-derived polysaccharides 166
Chitin-chitosan 167
Mammalian glycosaminoglycans 168
Proteins 169
Combination of Polysaccharides and Proteins 172
Examples of Commercially Available TE Biomedical Implants and Wound Healing Products 175
Further Reading 176
Glossary
Antiangiogenic properties The ability of substance to stop the growth of tumors and progression of cancers by limiting the
pathologic formation of new blood vessels (angiogenesis).
Anticoagulant activity The ability of substances to prevent or reduce coagulation of blood, prolonging the clotting time.
Bioactivity A beneficial or adverse effect of a substance on living tissue (organism).
Biocompatibility The ability of a material to perform with an appropriate host response in a specific situation.
Biodegradability The capacity of being decomposed by biological agents, especially bacteria.
Hemostatic activity An act to arrest bleeding or hemorrhage.
Immune response The reaction of the cells and fluids of the body to the presence of a substance which is not recognized as
a constituent of the body itself.
Immunogenicity The ability to provoke an immune response in the body of a human or animal.
Self-assembly A process in which a disordered system of preexisting components forms an organized structure or pattern as
a consequence of specific, local interactions among the components themselves, without external direction.
Thrombogenicity The tendency of a material in contact with the blood to produce a thrombus, or clot.
Introduction
Definition, Classification, Sources
Biological structures are a source of inspiration for solving challenges in material science and medicine. Nature has developed, with
comparatively few base substances, a range of materials with remarkable diversity of functional properties and biological activities.
The term “natural” has always been used to refer to something that is present in- or produced by nature (rather than being artificial
or man-made) and is often synonymous with something which is good, pure or healthy. Throughout history, humans have relied
on natural biological materials such as wool, leather, silk, and cellulose.
Natural biopolymers may be defined as biological macromolecules that are produced by living organisms and that can be used
in/as biomedical materials. In general, they are composed of repeating structural units which are linked together to form long chains
or branched networks. The simple building blocks are called monomers, while the more complicated building units are sometimes
referred to as “repeat units.”
There are three main classes of natural biopolymers, categorized according to the monomeric units used and the structure of the
biopolymer formed:
(1) Proteins (include polypeptides) which are macromolecules composed of a-amino acids;
(2) Polysaccharides, which are linear or branch-bonded polymeric carbohydrate structures;
(3) Polynucleotides (nuclei acids DNA and RNA), which are polymers composed of 13 or more nucleotide structural units.

Biomaterials: Science and engineering j Natural Biopolymers for Biomedical Applications 163
Natural polymers, such as nucleic acids and proteins, carry and manipulate essential biological information, while others, like poly-
saccharides,dthat is nature’s family of sugarsdprovide fuel for cell activity and serve as structural elements in living systems.
Biopolymers, playing a central role in the natural world, can come from many forms of life and at any point or level of the Evolu-
tionary Tree. Interestingly, the form of lifedbe it simple or highly organized- does not define their usefulness in the biomedical area.
The natural sources of biopolymers include (1) microorganisms, (2) plants, or (3) animal tissues and can originate from aerial,
terrestrial, or marine living organisms. For example, microorganisms including bacteria, smuts, nests, yeasts, molds, fungi, and
many other forms of what is considered to be a primitive life can be very valuable to provide an enormous variety of polymeric
biomolecules with excellent structural and biochemical characteristics. These biopolymers include polysaccharides (cellulose,
dextran, chitin, hyaluronic acid, etc.) and proteins (silk, keratin, etc.). Plants have also, over time, been an extremely valuable
and renewable source of both polysaccharides (cellulose, starch, alginate, carrageenan, etc.) and proteins (soy, zein, wheat gluten,
etc.). Animals, whether highly developed or poorly developed, whether they live on land, in the sea, or in the air can provide natural
structures with strong potential in biomedical field. Examples of these structures include glycosaminoglycans (chitin, heparin, hya-
luronic acid, etc.), proteoglycans and proteins (collagen, elastin, gelatin, heparin, etc.) as well as deoxyribonucleic acid, the genetic
material originated from all living creatures.
Requirements, Advantages and Disadvantages of Natural Polymers

Any substance to be used in- or as a biomedical material, needs to fulfill some important requirements which derive from the defi-
nition of a biomaterial as “a substance (other than a drug) synthetic or natural in origin, that can be used as a system or as a part of
a system that treats, augments, or replaces any tissue, organ or function of the body.” As such, an ideal biomaterial is one that is
biocompatible, biodegradable and nontoxic or immunogenic. It is also extremely convenient that this material can be extracted
or processed at reasonable cost and in different forms, and that it provides a possibility to be functionalized, if required, with bioac-
tive chemicals. Remarkably, many naturally derived biopolymers are inherently biodegradable, biocompatible and nontoxic. In
addition, some of them carry specific protein binding sites and other biochemical signals that may assist in tissue healing, regen-
eration and integration. These properties make them favorable contenders for biomedical applications in different fields of
medicine.
However, natural biopolymers, as many other natural materials, can be associated with several problems. These include evoking
immunogenic reaction after being implanted; the batch to batch variability in molecular structure associated especially with animal
sourcing, and the tendency to denature or decompose at temperatures below their melting point. Another major problem yet to be
overcome with natural starting materials is their propensity for calcification (the abnormal deposition of calcium in a body tissue)
and they are also prom to bio-deterioration. Some important advantages and disadvantages of natural biopolymers are summarized
in Table 1. It should be stressed that current advances in extraction, purification and characterization of proteins and polysaccha-
rides from different natural sources provide effective solutions to some of these drawbacks and contribute to the development of
a diversity of new biomaterial platforms.
Demand and Areas of Application

The use of natural products as biomaterials is not a new area of science. The field is extremely multidisciplinary encompassing
elements of medicine, biology, chemistry, tissue engineering, and materials science. The demand for biocompatible, biodegradable,
and functional structures is constantly increasing and this is a tendency which is expected to continue in the future. For example, in
the US the demand is currently growing at a compound annual rate of 6.2% reaching $7.12 billion in 2018. Broadly, the main area
of applications of natural biopolymers include (1) implantable devices, (2) wound management products, (3) controlled drug
delivery systems, and (4) tissue engineering scaffolds. Reviews have appeared recently describing the structure, properties, classifi-
cation and potential use of natural biopolymers in different biomedical fields including orthopedics, ophthalmology, dentistry, soft
and cardiovascular tissue regenerations, among others. Some commercially available biopolymers are relatively expensive but their
application in specialized biomedical fields can justify their costs. Improvements in technologies for their extraction and processing
have expanded the use and incorporation of natural biopolymers as biomaterials; especially as drug-delivery vehicles and tissue
Table 1 Advantages and disadvantages of natural biopolymers
Advantages Disadvantages
➢Biocompatible ➢May elicit immunological reaction

➢Nontoxic, do not elicit a foreign body response ➢High natural lot-to lot variability
➢Possess biological functionality on both macroscopic and molecular levels ➢Structurally more complex than synthetic
➢Biodegradable via natural enzymes materials
➢Degradation kinetics can be adjusted by modification including cross- ➢Technological manipulation may be more
linking elaborate
➢Can be obtained at reasonable cost ➢Propensity for calcification
164 Biomaterials: Science and engineering j Natural Biopolymers for Biomedical Applications
engineering scaffolds. These and other applications of the natural biopolymer-based materials in biomedicine are described in
following sections. A comprehensive overview of the most important members of each family of natural biopolymer (according
to classification) is given and includes proteins and polysaccharides. Polynucleotides are mainly employed in molecular biology
principally for diagnosis and monitoring of hereditary diseases, in forensic science and in parentage testing for the analysis of
genetic fingerprints for DNA profiling, etc. These areas are not of direct interest in the biomaterial field and, as such, are out of scope
of this article.
Properties and Applications

Polysaccharides
Polysaccharides are families of natural sugar polymers that serve two main functions in living systems: for energy storage and as
extracellular structural elements of plant or animals. Polysaccharides comprise 75% of all organic material on earth. They are
also known as glycans, the term which is defined by IUPAC as synonym to polysaccharide, meaning “compounds consisting of
a large number of monosaccharides linked by glycosidic bonds.” The exact placement of the linkage can vary, and the orientation
of the associating functional groups may result in a- and b-glycosidic bonds. Polysaccharides may be linear (cellulose, chitin and
hyaluronan) or branched (starch, dextran) and may also differ in ring size (furanose or pyranose), absolute configuration (D- or L-)
and in the chemical identity of substitutes (hydroxyl, sulfate, carboxylate, amine, etc.). Examples of polysaccharides relevant to the
biomaterial fields include cellulose (plant and bacterial), starch, dextran, alginate, carrageenan, chitin/chitosan, pullulan, agar,
pectin, hyaluronic acid (hyaluronan), chondroitin sulfate, and heparin. Some of these polysaccharides and their structural units
are given in Figs. 1 and 2. The numbering in the polysaccharide nomenclature indicates the location of the linking carbon in
the ring.
Glycans are mainly of two types: those made up of one type of monomer unit (homo-polysaccharides) or those composed by
two or more types of monosaccharide residues (hetero-polysaccharides). Most homo-polysaccharides store fuel (starch) or are extra-
cellular organizational elements (cellulose and chitin) while hetero-polysaccharides (hyaluronan and chondroitin sulfate) typically
provide cellular support for microorganisms and animal tissues.
Fig. 1 Polysaccharides: Structures of glucans and seaweed-glycans.

Fig. 2 Polysaccharides: Structures of glycosaminoglycans (GAGs).
The term glycan should not be confused with that of glucan which specifically defines a polysaccharide comprising D-glucose
monomers meaning that not all glycans are glucans. For example, cellulose, composed of b-1,4-linked D-glucose is glucan
(Fig. 1) while chitin, comprised by b-1,4-linked N-acetyl-D-glucosamine monomeric unit (Fig. 2), is glycan, but not glucan.
Polysaccharides, as the other classes of biopolymers, may be derived from different natural sources including microorganisms,
plants and animals. They may be classified a nonmammalian and mammalian according to/depending on their origin. The most
abundant nonmammalian polysaccharides include cellulose, starch, chitin, alginate and dextran which can be extracted and puri-
fied using relatively simple procedures originating large quantities of material at low cost. This economic advantage coupled with
their low immunogenicity makes these natural polymers attractive for biomedical applications. Isolation and purification of
mammalian polysaccharides, such as chondroitin sulfate, hyaluronan and heparin, are more difficult than those of the nonmam-
malian polysaccharides, but they exhibit important biological functionality, including specific binding with multiple proteins,
which has motivated interest in their use in biomedical application.
Many naturally-derived polysaccharides exhibit properties which make them ideally suited for bio encapsulation technology and
tissue engineering. These properties include a unique gelation ability, high water binding capacity, biodegradability and similarity
to extracellular matrices (ECM) as well as the possibility to be easily modified and processed into adequate forms (micro /nano-
particles, films, hydrogels, and 3D scaffolds). Unfortunately, there are concerns about their purity and pathogen content especially
when extracted from sources such as algae, insects, bacterial expression, and/or mammalian tissues. Different approaches have been
developed to address these concerns; some of them are mentioned in this article.
Polysaccharides are polyelectrolytes, macromolecules that possess a relatively large number of functional groups which either are
charged, or under suitable conditions can become charged. In fact, their polyelectrolyte nature is one of the most important char-
acteristics of this class of biopolymers. The interaction between oppositely charged polyelectrolytes (by reversible electrostatic and
dipole–dipole bonding or/and hydrogen and hydrophobic bond formation) leads to development of polyelectrolyte complexes
(PEC). The great advantage of these complexes is that they can generally be formed by a simple procedure consisting of simulta-
neous mixing of the corresponding polyelectrolytes in solution without need for any additional chemicals as covalent cross-
linkers. As such these complexes are generally nontoxic, well-tolerated and biocompatible; very important characteristics for their
use as/in biomaterials. Furthermore, the stability and other important features of these PECs may be adjusted and modified in many
ways, for example, by changing the density of charges, degree of ionization, pH of reaction medium, concentration of polyelectro-
lytes, distribution of ionic groups, molecular weight, mixing ratio, order of reacting polyelectrolytes and drying process. Recently,
polysaccharides-based PECs have been extensively investigated for biomedical applications, such as drug encapsulation and
controlled delivery, DNA-binding, enzyme immobilization, tissue engineering and biosensor.
A brief description of the structure, properties and areas of biomedical applications of selected nonmammalian and mammalian
polysaccharides, relevant to the biomedical field, are reviewed as follows under specific headings. This selection includes:
(a) Glucans: cellulose (linear b-glucan from both plant and bacterial sources), starch (branched a-glucan of plant origin), and
dextran (branched a-glucan of bacterial origin).
(b) Seaweeds-derived polysaccharides: alginate and carrageenan, both of nonmammalian origin and of anionic nature but with
different functional groups in the saccharide rings.
(c) Chitosan: a unique positively charged nonmammalian glycosaminoglycan (GAG) with important characteristics for
biomedical use.
(d) Mammalian glycosaminoglycans: hyaluronan, chondroitin sulfate, and heparin, all highly used in numerous biomedical
applications.
Glucans
Cellulose, starch and dextran are all composed of D-glucose monomers but differ in the length of chains; the type of the linking units
and in the degree of branching; which define their diverse properties and applications.
Cellulose may be of plant or bacterial origin. Both possess the same molecular formula, composed of repeated D-glucose building
blocks (Fig. 1). They are linear homo-polymers characterized by their high hydrophilicity, biodegradability and broad chemical
modifying capacity. Plant cellulose is the most abundant organic compound on Earth used for more than 150 years as a chemical
raw material. The bacterial form may be produced by certain bacteria (Acetobacter xylinum), algae and fungi. Due to differences in
their origin, these celluloses significantly differ in their properties and architectural characteristics. Plant cellulose generally forms
a tough, mesh-like bulkwork structure and contains impurities such as hemicellulose or lignin, while microbial cellulose, with ultra-
fine micro-fibril architecture, is more chemically pure, more crystalline and more porous. The bacterial form has higher swelling
capacity and hydrophilicity as well as a greater tensile strength than its plant-derived counterpart. Furthermore, bacterial cellulose
can be produced on a variety of substrates and can be grown to virtually any shape due to the high moldability during formation.
Cellulose, independent of its origin, is degraded by microbial enzymes. Animal cells cannot cleave the b-(1 / 4)-bond between the
two glucose moieties, thus, cellulose degradation in tissues takes place by a slow nonenzymatic hydrolysis and therefore cellulose
can be regarded as an almost stable matrix. Both types of cellulose and their derivatives are well-tolerated by most tissues and cells
and as such cellulose-based nontoxic biocompatible materials offer numerous possibilities in medical fields. For example, cellulose
sponges have been used widely in experimental surgery as they do not affect the healing process, but act as a chemoattractant
inducing cells involved in the repair process to migrate towards them. Cellulose-based biomaterials are also used as coatings for
drugs, supports for immobilized enzymes, artificial kidney membranes, in wound care and as implant materials and scaffolds in
tissue engineering.
Starch, a branched a-glucan, is one of the most abundant and cheap polysaccharides. Natural starch occurs in a granular form,
and it is a principal carbohydrate storage product of plants. It is found in a variety of botanic sources such as cereals (maize, wheat,
rice, cassava, etc.) and tuber plants (e.g., potatoes). The content of amylose and amylopectin in starch is largely dependent on the
source. Most often, starch consists of about 30% amylose, a linear a-(1–4) glucan, and 70% amylopectin, a highly branched version.
Starch is a highly biodegradable polymer as it can be broken down into its constituent sugars by enzymes (known as amylases)
which are found in plants and in animals, including human saliva and pancreas secretions. Starch is insoluble in cold water, but
it is very hygroscopic and binds water reversibly. It is biodegradable, inexpensive, and as such can play an important role in the
medical field. The unique physicochemical and functional characteristics of starches such as swelling power, solubility, and capacity
of gel formation, rheological characteristics, mechanical behavior and enzymatic digestibility make them potentially useful for
a wide variety of biomedical applications ranging from bone cements, drug carriers for controlled release to tissue engineered
cellular supports.
Dextran is a nonmammalian glucan. This polysaccharide is expressed by certain lactic acid bacteria, the best-known being
Leuconostoc mesenteroides and Streptococcus mutans. The structure of dextran consists of linear (1 / 6)-a-D-glucose, with branches
extending mainly from (1 / 3) and occasionally from (1 / 4) or (1 / 2) positions. Dextran is highly water-soluble and easily
functionalized through its reactive hydroxyl chemistries. Characterization of many types of dextran have indicated that branching,
average molecular weight and molecular weight distributions can vary widely depending on the conditions and strain of bacteria
used for expression. In the early 1940s, dextran was investigated as a blood plasma replacement and has since become of interest as
a biodegradable and biocompatible material. Dextran biodegradation occurs through natural enzymatic splitting of saccharide
bonds by dextran-1, 6-glucosidase found in spleen, liver, lungs, kidneys, brain, and muscle tissue as well as by dextranases expressed
by bacteria in the colon. Dextran lacks nonspecific cell binding and resists protein adsorption, characteristics which have increased
interest in its use as a biomaterial. Like other nonmammalian glucans, the relatively low cost and availability of dextran as well as its
hydroxyl functionality for chemical modification has enlarged the utilization of dextran in the field of polysaccharide-based bioma-
terials. For example, dextran has recently been investigated as an alternative to polyethylene glycol for low protein-binding and
cell-resistant coatings on biomaterial surfaces. It has been shown that the multivalent properties of dextran are advantageous
when high-density surface immobilization of biologically active molecules to low protein-binding surface coatings is desired.
Seaweeds-derived polysaccharides
Seaweed-originating nonmammalian polysaccharides have been used extensively over decades for biomedical and biological appli-
cations including tissue engineering, drug delivery, wound healing and biosensor being alginate and carrageenan among the most
widely employed resources.
Alginates represent a whole family of linear copolymers containing blocks of alternating b-D-mannuronic acid and a-L-guluronic
acid residues with (1 / 4)-linkages, displaying carboxylic acid functionality at the C5 residue. Alginates are hydrophilic non
mammalian polysaccharides commercially extracted from marine brown algae (Phaeophyceae). They can also be produced by soil
bacteria such as Azotobacter and Pseudomonas. Depending on source and processing, alginates have broad distributions in molecular
weights (from 10 to 1000 kDa) and chemical structures. Bacterial alginate has more defined chemical composition and physical
characteristics than its seaweed-derived equivalent. In the unmodified form, alginates are not enzymatically degradable in mammals
and do not support cellular adhesion. Nevertheless, alginates have been extensively investigated for many biomedical applications,
due to their biocompatibility, low toxicity, relatively low cost, and mild gelation by forming ionic complexes with divalent cations
such as calcium. Over decades, sodium alginate has been indispensable part of dental practice as a cost-effective and simple impres-
sion material. More recently, alginates have been used for coencapsulation of proteins and growth factors, such as vascular endo-
thelial growth factor. The experimental procedure is very simple and consists of the simultaneous addition of alginate and protein
solutions into calcium chloride solutions. Alginate, in form of hydrogels has been particularly attractive in wound healing, drug
delivery, and tissue engineering applications. Alginate-based wound dressings maintain a physiologically moist microenvironment,
minimize bacterial infection and facilitate wound healing. The lack of cellular adhesion has been addressed by covalent association
to its structure of different cellular adhesive peptides and proteins. As negatively charged polyelectrolyte, alginate can interact elec-
trostatically with the positively charged macromolecules, for example chitosan, forming PECs. These PECs possess many improved
physicochemical properties and advanced biomedical applications, especially for controlled drug delivery, in comparison with algi-
nate calcium gels. These alginate-chitosan delivery systems, in the form of nanoparticles, microspheres and hydrogel beads, have
been used to encapsulate small drugs, including anticancer, ocular, asthma and pulmonary drugs, antiinflammatory medications,
etc. In form of membranes and films alginate-base PECs have been employed to encapsulate biological macromolecules (proteins,
peptides, and nucleotides) for implantable drug delivery systems.
The term carrageenan defines a naturally occurring biopolymer family of linear sulfated polysaccharides, extracted from red
marine algae (Rhodophyta). The main chain of the carrageenan molecule is composed of D-galactose and D-anhydrogalactose units
linked by glycosidic bonds. The properties of carrageenan are defined by its structural characteristics. Depending upon sources and
the extraction methods, they are generally of three types: kappa (k), iota (ι), and lambda (l), mainly differing in number of the
sulfate groups per disaccharide repeating unit and the presence of the 3,6-anhydro bridges. As a result, these carrageenans show
different linear charge density and solubility, both of which increase with sulfate group content (k < ι < l). Among the three types
of carrageenan, only k- and ι-varieties possess gelling ability, with the former being firm and rigid while the latter being soft and
elastic. Different strategies have been applied to exploit carrageenans biocompatibility and attractive physicochemical properties
including special gelling mechanisms, strong water absorption capacity and abundant functional groups. Due to the presence of
the ester sulfate groups, carrageenan molecule is highly negatively charged and has the ability to interact with positively charged
molecules forming PECs. Carrageenan incorporation into drug delivery matrices increases drug loading and drug solubility,
enabling the release of orally administrated medications in zero-order or in a significantly prolonged period. Other features of
carrageenan-based formulations, such as pH-sensitivity and adhesive properties, contribute to its broad application in pH /
temperature-sensitive delivery systems in response to physiological environments. Some promising results have been achieved
using carrageenan-containing tissue engineering devices due to their capacity to induce important osteogenic, adipogenic, and
chondrogenic differentiation in stem cells. However, it has been shown that carrageenan may provoke some adverse effects on
the biological system such as unwanted immune responses and inhibition of blood coagulation. As such there is a pressing demand
for a careful and comprehensive analysis of all carrageenan properties for a better development and a safer use of this biopolymer in
biology and medicine.
Chitin-chitosan
Chitin, a homopolymer of b (1 / 4) linked N-acetyl-D-glucosamine, is the second most abundant biopolymer in nature. It is
commonly found in the exoskeletons of crustacean and insects as well as in the cell walls of fungi. Because of its insolubility in
aqueous media, most chitin applications are based on its deacetylated form, chitosan, which occurs rarely in nature, but can be effi-
ciently obtained by extensive deacetylation of chitin. The resultant chitosan consists of b (1 / 4) linked glucosamine and N-acetyl-
glucosamine, randomly or block distributed throughout the biopolymer chain, depending on the preparation method and source
material. N-deacetylation not only increases the aqueous solubility of the polymer but also provides reactive primary amines for
further chemical modifications. The microstructure of chitosan and its solubility are influenced strongly by the characteristics of
the deacetylation procedure: chitosan is soluble when obtained under homogeneous conditions and insoluble when heterogeneous
deacetylation is carried out. The molecular weight and degree of deacetylation are important parameters determining chitosan prop-
erties and applications. Chitosan itself possesses hemostatic, antimicrobial, antiviral and antitumoral activities. Over recent decades
it has been one of the most popular naturally-derived biopolymers in biomedical field due to its biocompatibility (approved for
human use), excellent biodegradability (degrades by human enzymes, such as lysozyme and lipase), low toxicity, wound healing
properties, strong bactericidal effect as well as abundant availability and low production cost. Many chitosan-based formulations
have been investigated for wound-dressing materials, drug encapsulation and drug-delivery devises. As a unique positively charged
polysaccharide, chitosan can form PECs by electrostatic interactions with many negatively charged natural biopolymers, for
example anionic nonmammalian (alginate, carrageenan, cellulose, etc.) and mammalian (hyaluronan, chondroitin sulfate, etc.)
polysaccharides; essential components of ECM (such as proteoglycans and proteins) and other negatively-charged biomolecules
(growth factors, nucleic acids, cytokines, etc.). Biomaterials fabricated from chitosan-based polyelectrolyte complexes in forms
including nanoparticles, microspheres, beads, tablets, gels, films, and membranes, are especially popular in drug encapsulation
applications. Recently, increasing attention has been paid to chitosan-based biomaterials in tissue-engineering approaches
particularly in epithelial and soft tissues repair and regeneration. Both types of tissues play an important role in supporting anatom-
ical structures and physiological functions of living organisms. Chitosan-based matrices have been investigated, for example, to
repair body surface linings, for regeneration of connective tissue, and to support nerve connection and vascular growth. It has
been reported that chitosan may be beneficial in regulating many tissue regeneration functions including preservation of cellular
phenotype, binding and enhancement of bioactive factors. Chitosan also assists in controlling gene expression and in synthesis
and deposition of tissue-specific extracellular matrix. Although chitosan itself is nonadhesive to cells it can be efficiently modified
by attaching to its structure of cell-adhesive polymers such as peptides, proteins and mammalian GAGs. This modification results in
providing important cell-recognition sites to otherwise inert chitosan-based formulations. The choice of chitosan as a base compo-
nent for many tissue supports is determined by the fact that its biological, physical and chemical properties can be modified,
controlled and engineered by multiple ways and under mild conditions. The novel use of chitosan-based scaffolds in promoting
regeneration of various tissues demonstrates that these materials may serve as promising substrates for a great number of future
applications.
Mammalian glycosaminoglycans
Mammalian glycosaminoglycans are complex polysaccharides that play important roles in cell growth, differentiation, morphogen-
esis and migration. They can be classified as nonsulfated, such as hyaluronic acid (HA), and sulfated, such as heparin, chondroitin
sulfate, dermatan sulfate, and keratin sulfate. Many GAGs, (except from hyaluronic acid) are generally attached to a central protein
forming proteoglycans. Due to their polyelectrolyte nature, GAGs possess high water absorption capacity. Their chemical versatility
and biological performance make them attractive building blocks for great variety of biomaterials aimed especially at tissue engi-
neering applications.
Hyaluronic acid (HA), also known as hyaluronan or hyaluronate, is a natural, nontoxic, biocompatible and biodegradable
mammalian polysaccharide composed by 2-acetamide-2-deoxy-a-D-glucose and b-D-gluconic acid residues linked by alternate
(1 / 3) and (1 / 4) glycoside bonding. Hyaluronan, a unique nonsulfated mammalian glycosaminoglycan, is found naturally
in the extracellular matrix of skin, cartilage, vitreous body of the eye, and other body tissue. It also occurs as an extracellular poly-
saccharide in a variety of bacteria. HA has a high capacity of lubrication, water sorption, and water retention, and influences several
cellular functions such as cell migration, adhesion, and proliferation. For biomedical applications it is produced mainly via micro-
bial fermentation, to reduce the risk of crosspieces viruses, infection, and contamination. HA clinical uses include dermal filling,
viscosupplementation in deteriorated joints and ophthalmic surgical aids. For such applications, HA has the advantage of being
biocompatible and safe, inducing minimal foreign body reaction. Owing to its important physiological role in tissue repair, HA
is also used for wound healing. HA can be chemically modified in two different ways: crosslinking or conjugation. Due to the
high density of negative charges, hyaluronan can form PECs with different cationic biopolymers among which chitosan is one
of the most explored. HA-based PECs in forms including nano- and micro-particles, films, membranes, hydrogels and porous
matrices have been investigated for drug delivery systems and tissue engineering. Hyaluronan itself has been indicated to impact
cell–cell and cell–substrate interactions, to aid in the organization of proteoglycans and to promote angiogenesis. This range of
properties together with its high lubrication capacity and low cytotoxicity have motivated the widespread use of HA in ophthalmic
surgery, arthritis treatment, in vocal fold repair, wound repair and healing, antiinflammatory materials, drug delivery, and tissue
engineering scaffolds, especially for soft tissue repair and regeneration.
Chondroitin sulfate (CS), is a sulfated mammalian polysaccharide, widely distributed as one of the most physiologically impor-
tant GAGs in ECMs and at cell surfaces. It is mainly found attached to proteins as proteoglycan in connective tissues, working as
a structural component, or on cell surface and basement membranes, functioning as a receptor. CS is composed of repeating disac-
charide units of D-glucuronic acid and N-acetyl galactosamine sulfate at either 4- or 6-positions. Commercially available CS is ob-
tained via extraction and purification from sources including shark and whale cartilage, and bovine or porcine tissues. CS is
a polyelectrolyte with strong negative charges and as such it could efficiently interact with proteins and different positively charged
polyelectrolytes (for example chitosan), forming PECs and other associates. This provides multiple ways for modification and
tailoring of physicochemical, mechanical and biological properties of CS-based biomaterials. CS, as an ECM component, plays
important roles in modulating the stability, activity, release, and spatial localization of growth factors. It also contributes to wound
healing, tissue morphogenesis, hemostasis and cell division in vivo. Its biodegradability, biocompatibility, high versatility and
availability make CS a promising candidate for biomedical applications. The ability of CS to induce the synthesis of cartilage-
specific markers, to inhibit cartilage destruction processes and to stimulate the cartilage formation explains its wide use in cartilage
engineering platforms. Other TE applications of CS include repair and regeneration of damaged bone, skin and neural tissues. In
spite of its potential as biomaterial there are some concerns associated with the quality differences and properties variability of
different samples of chondroitin sulfate due the fact that it is extracted frim living organisms. Recent technological advancements
in extraction and purification methods have significantly contributed towards overcoming these potential drawbacks, increasing
clinical utility of CS-base formulations.
Heparin and heparin sulfate are both sulfated heterogeneous linear mammalian glycosaminoglycans composed by a or b (1 / 4)
linked uronic acids (90% a-L-iduronic acid, 10% b-D-glucuronic acid) and a-D-glucosamine residues, containing a heterogeneous
mixture of carboxylic acids, hydroxyls, sulfates, and amine functional groups. Heparin is only produced in mast cells, where it is
cleaved from the core protein (serglycin), while heparin sulfate is found in most tissues where it remains attached to the core protein
forming proteoglycan. Both play an important role in many biological processes. Commercial production of heparin from mam-
mallian tissues such as porcine or bovine intestinal mucosa involves complicated extraction and purification processes which add to
heterogeneity in chemical structure and molecular weight of naturally-derived heparin. The most important characteristics of
heparin, for biomedical use, include its anticoagulant activity and ability to bind and stabilize growth factors preventing them
from denaturating and increasing the affinity of complex to cell receptors. As such, heparin integration to biomaterials was origi-
nally aimed at reducing the thrombogenicity of materials in contact with blood. Heparin also exhibits antiangiogenic and apoptotic
effects, characteristics which favored its use as a component in tumor-targeted delivery systems. The high negative charge of heparin
promotes its ionic interactions with proteins, such as growth factors, proteases, and chemokines, and other natural biomolecules.
For practical use heparin is often conjugated to naturally-derived hydrogels to provide sustained release of the anticoagulant, small
drugs and protein-based systems. Recent biomedical applications include heparin-containing nanoparticles and scaffolds for tissue
engineering, where heparin is generally associated with other polysaccharides (e.g., hyaluronan, chitosan) or proteins, including
collagen, gelatin, fibrin, etc. Heparin-based biomaterials in form of nanoparticles and scaffolds have been employed as cell carriers
in therapeutics for cancer treatments and in tissue engineering applications. Heparin-functionalized nanocarriers stimulate cell
differentiation and promote therapeutic efficacy of drugs by increasing the cellular uptake of the delivered molecules. Due to its
biocompatibility, low toxicity, benign gelation conditions and advantageous biological activities together with its relatively low
cost heparin incorporation in biomaterials is constantly growing and extending to new emerging applications.
Proteins
Proteins form a vast variety of natural tissues and organs from which they can be extracted and processed. Some of the most familiar
protein materials include wool, leather, silk, gelatin, and collagen. Proteins are large biological macromolecules composed of one or
more long chains of a-amino acids. Once linked in the protein chain, an individual amino acid is called a residue, and the linked
series of carbon, nitrogen, and oxygen atoms are known as the main chain or protein backbone. A linear chain of amino acid resi-
dues is called a polypeptide. Fig. 3 shows the structures of a-amino acid, the formation of the peptide bond in polypeptide chain
and the three-dimensional representation of protein structure. Proteins differ from one another primarily in their sequence of
a-amino acids, which is dictated by the nucleotide sequence of their genes. They are in fact unique biopolymers where the position
of the exact amino acid in the chain is determined by a specific template (DNA). Like other biological macromolecules (polysac-
charides and nucleic acids), proteins are essential parts of living organisms where they play a vital role in cell signaling, immune
responses, cell adhesion, and the cell cycle. Some of them are enzymes that catalyze biochemical reactions and are essential to
metabolism; the others have structural or mechanical functions, conferring stiffness and rigidity to biological components. Most
structural proteins such as collagen and elastin, for example, can be derived from ECM of mammalian connective tissue and carti-
lage where they are crucial components. Other important structural protein, keratin, is found in hard- or filamentous structures such
as hair, nails, feathers, hooves, and some animal shells. The great advantage of proteins is that they are structurally identical or very
similar to macromolecular substances which the biological environment is prepared to recognize and deal with metabolically. As
a result, the problems of toxicity and simulation of chronic inflammatory reaction are suppressed or highly reduced.
Over decades proteins have been viewed as potential resources for many biomedical platforms owing to their intrinsic ability to
perform very specific biochemical, mechanical and structural roles. ECM-derived structures, in particular, have become an obvious
Fig. 3 Structures of Proteins. Peptide chain is from https://commons.wikimedia.org/wiki/File:Peptide_bond.png by Webridge. Protein structure is
from https://commons.wikimedia.org/wiki/File:Proteinviews-1tim.png by Opabinia regalis.
starting point in the development of devices in different tissue engineering approaches due to their similarity in composition and
properties to native tissue. Most of them possess a defined, three-dimensional microstructure that supports cell-guided tissue forma-
tion providing appropriate binding motifs for cell attachment, proliferation and maintenance of phenotype. As such, the use of
collagen, albumin, gelatin, silk fibroin and keratin in the naturally-derived biomaterials has been increasing. Due to the importance
of proteins in the biomedical field, selected members of this class of natural biopolymers (namely, silk, keratin, elastin, and
collagen) are reviewed as follows under specific heading. Particular focus is directed towards collagen as this is the most significant
and widely used component in tissue engineered devices and other biomedical materials. Further details on its origin, type, and
structural diversity are provided and the results of our research group in developing collagen-based formulations, especially for
tissue engineering applications are presented.
Silk is a natural fibrous protein produced by Lepidoptera larvae such as silkworms, spiders, scorpions, mites, and flies. Silk fibers
are composed of at least two main proteins: sericin (globular protein) and fibroin (fibrous protein). This biopolymer has always
been a material of great fascination. Spiders can process silk protein into a material that has a tensile strength 16 times greater
than that of nylon and a very high degree of elasticity. Silk without sericin displays higher stability than others. Silk fibroin shows
excellent physical and chemical properties and for decades it has been used as a surgical suture material. As a biomaterial, silk can be
processed in different forms including fibers, films, membranes, gels, sponges, powders, and scaffolds. Due to its low toxicity,
biocompatibility, tunable biodegradation rate and remarkable mechanical characteristics silk, has been popular choice for a broad
range of tissue engineering applications. The unique mechanical properties of silk fibroin together with its ability to support the
differentiation of mesenchymal stem cells along the osteogenic lineage; predicates its use especially for bone tissue engineering.
Silk fibroin can be also combined synergistically with other biomaterials to form composites and can be chemically modified.
As such silk fibroin scaffolds can be tailored to specific applications including vascular prostheses, structural implants, nets, and
sutures among others.
The term “keratin” was initially referred to the broad category of insoluble proteins originated from hair, wool, horns, hooves and
nails. Mammalian keratins can be classified into two distinct groups, “hard” and “soft” keratins, based on their structure, function
and regulation. By their origin they can be divided in epithelial and hair keratins both containing nonhelical domains and central
alpha-helical domain. Hair keratins are richer in cysteine residues in their nonhelical domains and thus form tougher and more
durable structures. Advances in the extraction, purification, and characterization of keratin proteins from hair and wool fibers
have led to the development of numerous keratin based biomaterial platforms. Like many naturally-derived biomolecules, keratins
have intrinsic biological activity and biocompatibility. In addition, extracted keratins are capable of forming self-assembled struc-
tures that regulate cellular recognition and behavior. These qualities have directed its use in forms including powders, films, gels,
coatings, fibers, foams and scaffolds in wound healing, drug delivery and tissue engineering devices.
Elastin is an ECM protein that is known to provide elasticity to tissues and organs and allows many tissues in the body to resume
their shape after stretching or contracting. It is most abundant in connective tissues and other body structures where elasticity is of
major importance. Keratin comprises up to 70% of the dry weight in elastic ligaments, about 50% of large arteries, 30% of lung, and
2%–4% of skin tissues. The insoluble elastin is one of the most stable proteins with a half-life of about 70 years. Tropoelastin, the
precursor protein of elastin, and elastin-like peptides possess an important ability to self-assemble under physiological conditions.
Biomaterials based upon elastin and elastin-derived molecules are increasingly investigated for their application in tissue engi-
neering. This interest is fueled by the remarkable properties of this structural protein, such as elasticity, self-assembly, long-term
stability, and biological activity. Elastin can be applied in biomaterials in various forms, including insoluble elastin fibers, hydro-
lyzed soluble elastin, recombinant tropoelastin (or tropoelastin fragments) and as block copolymers of elastin. Furthermore,
repeated elastin-like sequences can be produced by synthetic or recombinant means and incorporated to biomaterials. Used alone
or in combination with other biopolymers, elastin-containing biomaterials have been suggested for applications including skin
substitutes, vascular grafts, heart valves and elastic cartilage.
Collagen is the most abundant of all proteins found in mammals, typically accounting for more than 30% of total body protein
mass. It forms a significant part of connective tissues such as bone, tendons, ligaments, blood vessels, nerves and skin. Collagen,
being a principal structural protein present in all vertebrates, comprises a family of genetically distinct molecules with a common
triple helix configuration of three polypeptide subunits, known as a-chains (Fig. 4). These triple helices form a molecule of tropo-
collagen, the basic building block of collagen. Tropocollagen molecules associate in a staggered fashion to produce collagen fibrils,
which are strengthened and stabilized mainly by enzymatically and nonenzymatically catalyzed covalent crosslinks. The extent of
these crosslinks is age-dependent and tissue-specific. To date 28 types of collagen have been identified and described. Variations in
collagen type are due to differences in the assembly of the polypeptide subunits, lengths of the helix, interruptions and terminations
of the collagenous helical domains. The best known and the most abundant are fibrillar collagens I, II and III, each containing
different affinity cell-recognition motifs that support cellular activity, mainly through interaction with cell-surface integrins
a1b1, a2b1, a10b1, and a11b1. In biological systems all these collagens have important functions: Col I, a major ECM component,
accomplishes both structural and cell adhesive roles in many vital organs and tissues while Col II is the chief element in articular
cartilage (approximately 60% of the dry weight of this tissue) and Col III is an important component of reticular fibers, where it is
commonly found alongside Col I.
For biomedical purposes, for decades, collagen, especially type I, in forms including gels, films, nanoparticles, microspheres,
membranes and scaffolds has been the principal biomaterial of choice. Its use is based on characteristics such biocompatibility,
biodegradability, low immunogenicity as well as on its ability to form strong fibers with high tensile strength. In addition, collagen
displays numerous triple-helical cell binding motifs, GFOGER being a major and most important ligand of collagen I.
Fig. 4 Chemical structure of collagen (showing Gly-Hyp-Pro repeats, left), and the triple-helical arrangement of collagen peptides (right). Repro-
duced from Goodsell, D. RCSB Protein Data Bank. 2000. www.rcsb.org/pdb/101/motm.do?momID¼4.
In tissue engineering applications, collagen is often used in combination with other biomolecules such as proteins and polysac-
charides. Collagen-containing scaffolds are biologically active and typically promote cell adhesion and growth. Furthermore, they
are biodegradable and, over time, allow host cells to produce their own ECM and replace the degraded scaffold. Along with these
attractive physical and biological attributes collagen, especially type I, is abundant, easy to purify at reasonable cost and to modify.
Furthermore, it can be easily processed in different forms and shapes which additionally broaden its use in tissue engineering
constructs, wound management products and drug delivery strategies. For example, in the form of nanoparticles collagenous formu-
lations have been employed for gene delivery; as mini-pellets and tablets for protein release, in the form of thin films as shields in
ophthalmology and as sponges for burn and wound healing products. Collagen gels, in combination with liposomes, have been
successfully used as a controlling material for transdermal delivery and as matrices for cell culture systems.
However, fabricating biomaterial devices from naturally-derived collagens with suitable stability and reproducible structures
presents some challenges. One of these concerns is the poor mechanical properties of collagenous matrices for TE applications,
which limits their use in different biological environments and especially in load bearing situations (e.g., orthopedic). It should
be stressed, that in its native state collagen possesses multiple inter- and intramolecular cross-links, which provide strength and
durability to their matrices. However, extraction and the purifying methods applied to process it into useful forms markedly reduce
collagen cross-linking density with a consequent impact on material properties and biological response. To overcome this limita-
tion, collagen-based scaffolds are usually crosslinked by different chemical (aldehydes, isocyanates, carbodiimides, etc.) and phys-
ical (ultraviolet irradiation, dehydrothermal treatments, etc.) procedures. Among them, the chemical treatment based on
carbodiimide hydrochloride (EDC) in the presence of succinimide constitutes one of the most successful methods for stabilization
of collagenous biomaterials in TE applications. This treatment is highly efficient, nontoxic (cross-linkers do not take part in linkage),
and the resultant by-products can be easily removed by washing. However, EDC-promoted bonding presents a significant drawback
as it uses free primary amino groups (on lysine residues) and carboxylate anions (on glutamate or aspartate residues) for crosslink-
ing. Many of these amino acid side chains, for example the acidic E of GFOGER, are essential components of cell binding sites on
collagen-type materials and so EDC-promoted crosslinking can impinge on bioactivity of collagenous formulations. This should be
addressed for the successful use of collagen-based scaffolds as templates for tissue regeneration.
Over recent years, intensive research in our Center has been directed to development nano-and macro-particulate systems for
controlled drug-delivery, and for production of tailor-made three-dimensional collagen-based scaffolds. The latter 3D matrices
have been mainly designed for (1) in vivo use as 3D engineered environments for regenerative medicine and (2) as models of
natural tissue for in vitro applications. A controlled freeze drying experimental approach has been employed to obtained highly
porous, interconnected sponges of collagen (mainly type I) alone or in combination with glycosaminoglycans (chondroitin sulfate,
hyaluronic acid, heparin, and chitosan) and other proteins (gelatin, elastin, fibrinogen, and peptides). These scaffolds have been
directed mainly to repair and replacement of bone, tendon, cartilage, ligament, skin, nerve and myocardial tissues. Recent
in vitro applications have included cell therapies to aid in the culture of megakaryocytes for in vitro production of platelets
from adult stem cells and in vitro models of mammary glands to assist in drug screening for breast cancer. These in vitro models
of mammary glands have been developed to reduce the use of animals in testing and to assist in the development of personalized
medicine initiatives through tailored assays of breast biopsy material.
To achieve a desirable biological performance from 3D engineered matrices, several important parameters have been finely
tuned. These include the nature and availability of cell binding ligands, the material (swelling profiles, degradation rates, etc.)
and the mechanical properties of these scaffolds as well as their morphology and spatial characteristics. To accomplish this goal,
the scientific base of freeze-drying technology has been broadened, different treatments collagenous formulations have been
applied and appropriate biomolecules have been incorporated into collagenous matrices. By applying compositional, positional
and mechanical controls at the developing stage we have obtained collagen-based cellular supports with varying local biochemical,
spatial and mechanical environments, closely matching those of natural tissues. Furthermore, 3D structures with controlled and
complex pore orientation that closely mimic many normal multioriented tissue arrangements have been produced recently. Fig. 5
shows some examples of different scaffold morphologies achieved by a combination of freeze-drying technique with molding tech-
nology, which has been designed to induce uniaxial or multidirectional temperature gradients in collagen slurries during scaffold
fabrication stage. The attachment to collagen-based scaffolds of novel peptides designed to control and guide cell behavior has
opened avenues for successful restoration of their biological activity after crosslinking treatment. A range of characterization meth-
odologies have been developed to assess the contribution of the important structural determinants of the biological performance of
collagen-based materials. These include the control of porosity, interconnectivity, percolation diameter, pore size and morphology
together with adjustment of swelling properties, dissolution characteristics, structural stability as well as mechanical control at both
a local and macroscopic level to provide mechanical cues for cell activity. The research is continuing to achieve the ultimate goal of
producing systems with structural and functional properties of native tissues for novel emerging approaches.
Combination of Polysaccharides and Proteins

The integration and extensive use of natural polymers as biomaterials can be significantly expanded by combination of different
biopolymers in the same formulation. Typically, each class of biopolymers employed in the fabrication of biomaterials has specific
advantages and disadvantages. The development of composite materials comprised of different components, frequently produces
a desired synergistic effect, so enhancing the material biological performance and practical use. As mentioned previously, while
describing design and properties of different biopolymers, the addition to biologically inactive nonmammalian polysaccharides
of GAGs or proteins (peptides) enables a biological recognition to be introduced to otherwise benign polymeric systems. As
such, the combination of nonmammalian polysaccharides with bioactive polymers has led to the design of many robust systems
while maintaining the low cost and low immunogenicity of the material with specific receptor responses and cell stimulation.
Combination of proteins and peptides with the natural polysaccharides can be achieved via versatile and efficient protocols to
provide biologically active composite materials that can be recognized by enzymes or cells. The precise amino-acid sequence
and structural conformations of these peptides and proteins dictate high affinity binding constants and efficient assembly compared
to those of polysaccharides alone. This leads to more specific control over biomaterial design and properties. Taken together, the
Fig. 5 Examples of different morphologies of collagen scaffolds. Degree and type of anisotropy in the microstructures of the scaffolds were ach-
ieved by imposing temperature gradients, uniaxial or multidirectional, during the phase of crystallization of water in collagen suspensions, using
molding technology. Images from Cambridge Centre for Medical Materials, University of Cambridge, UK.
Table 2 Examples of commercially available biomedical implants based on naturally-derived biopolymers
Tissue Product Composition Use/Form
Skin Apligraf, Organogenesis Lower layer of human fibroblasts and bovine collagen, upper Leg ulcers/Sheet
layer of keratinocytes
Biomaterials: Science and engineering j Natural Biopolymers for Biomedical Applications

ICX-SKN, Intercytex Allogenic fibroblasts and human collagen with additional layer of Burns and acute wounds/Sheet
keratinocytes
Integra dermal regeneration template, Integra lifesciences Porous bovine collagen crosslinked with chondroitin-6-sulfate Burns/Sheet
with upper layer of silicon
Integra flowable wound matrix, Integra lifesciences Granulated bovine collagen crosslinked with chondroitin-6- Ulcers/Gel
sulfate
Xelma, Molnlycke ECM protein (amelogenins) inpropylene glycol alginate carrier
Hyalograft 3-DTM (fidia advanced biopolymers) Human fibroblasts on a laser-microperforated membrane of Epidermal skin substitute/Membrane
benzyl hyaluronate
Laserskin™ (fidia advanced biopolymers) Human keratinocytes on alaser-micro perforated membrane of Epidermal skin substitute/Membrane
benzyl hyaluronate
INFUSE bone graft, medtronic Bovine type I collagen sponges Spinal fusion/Solid
Bone OP-1, Stryker Bovine type I collagen Bone injury/Paste
Vitoss Scaffold FOAM, Orthovita Porous foam of bovine type I collagen Bone injury/Foam
Bioset IC, pioneer surgical Composite of human demineralized bone matrix with type I Bone injury/Paste
collagen
FortrOss, pioneer surgical Composite of nanocrystalline hydroxyapatite and porcine Bone injury/Paste
collagen and dextran co-polymer
Regenafil, regeneration technologies/exatech Human mineralized bone matrix in porcine gelatin carrier Bone injury/Paste
Orthoss® Collagen and Geistlich Bio-Oss® Collagen, Composite of highly purified natural bone mineral combined Orthopedic bone regeneration/Porous block
Geistlich Pharma AG with native collagen
Cartilage Synvisc, genzyme Hyaluronic acid (Hylan GF-20 and Hylan B) from chicken combs Synovial fluid replacement/Gel
CaReS, arthro kinetics Rat-tail type I collagen matrix Articular cartilage injury/3D disc
Menaflex, regenbiologics Bovine type I collagen with hyaluronic acid and GAGs Meniscus cartilage injury
Hyalograft C autograft, Fidia advanced biopolymers HYAFF (esterified derivative of hyaluronic acid) scaffold Articular cartilage injury/Mesh
MACI, genzyme unreg Type I collagen membrane Articular cartilage injury/Sheet
Chondro-Gide®, Geistlich pharma AG Bilayer collagen matrix Cartilage regeneration/Porous block
Blood vessel VascuGel, pervasis Gelfoam porcine gelatin foam sponges Vessel reconstruction/Tubular
Nerve NeuraGen, integra Bovine type I collagen nerve conduits Nerve injury/Tubular
(Continued)
173
174
Biomaterials: Science and engineering j Natural Biopolymers for Biomedical Applications
Table 2 Examples of commercially available biomedical implants based on naturally-derived biopolymersdcont'd
Tissue Product Composition Use/Form
Pancreas Islet sheet, Cerco medical Alginate sheet with encapsulated pancreatic islets (pig or Diabetes mellitus/Sheet
human)
Amcyte/ReNeuron Alginate/poly-L-lysine encapsulated human pancreatic islets Diabetes mellitus/Beads
Novocell Alginate/PEG encapsulated human pancreatic islets Diabetes mellitus/Beads
Dura mater Durepair® Dura regeneration matrix, medtronic 3D matrix of type I and type III collagen from fetal bovine tissue Dural draft substitute/3D matrix
Duraform dural graft implant, depuy Collagen- based biocompatible graft Dural draft substitute
Biodesign® Dural graft, cook biomedical Collagen-based scaffold Dural substitute
Duramatrix, Collagen Matrix Inc. Matrix from highly purified type I collagen Dural substitute/Membrane matrix
Enter cutaneous Biodesign fistula plug, cook medical Collagen scaffold Fistula filler/Scaffold
fistula
Abdominal wall Permacol surgical implant (Covidien) Cross-linked porcine collagen for enhanced durability Hernia supports/Mesh
Peripheral nerve Neuragen nerve guide, Integra): Bovine type I collagen and GAG conduit and collagen matrix Peripheral nerve conduit/Conduit, porous matrix
Tendon/ligament Collagen tendon sheet, Rotation Medical Inc. Resorbable type I collagen matrix Tendon protector/Matrix
GraftJacket® regenerative tissue matrix, wright medical Noncross-linked human dermal collagen scaffold Support, protection, and reinforcement of tendon and
ligamentous tissue/Scaffold
Various Extracel, glycosan biosystems Crosslinkable bacterial hyaluronic acid, bovine and porcine Examples: cartilage, bone, vocal fold TE/Gel, sheet,
gelatin, porcine heparin and PEG derivatives tubular
Miromatrix biological mesh, miromatrix medical Inc. Comprised primarily of collagen type I Soft tissue repair/Mesh
Geistlich Bio-Gide® and geistlich BioeOss pen, geistlich Collagen membrane Oral tissue regeneration/Membrane
pharma AG
Geistlich Mucograft® and geistlich Mucograft®Seal, Geistlich Collagen 3D matrix Soft tissue regeneration/Spongy or compact blocks
Pharma AG
development of polysaccharide–protein composites has permitted the production of increasingly diverse and useful materials with
tailored biological responses and chemical and mechanical properties to better mimic the properties of the natural extracellular
matrix. This has led to important new classes of environmentally sensitive materials with response to the biological environment
and stimuli, that can further increase the efficacy of materials as tissue replacements.
Examples of Commercially Available TE Biomedical Implants and Wound Healing Products

The history of the development and marketing of TE biomedical implants has shown that a medically effective product is not in
itself sufficient to ensure commercial success. Apart from the obvious scientific, medical and human benefits, for it to reach the clinic
it needs to bring a clear financial reward to those who invested in translating a new technology to the clinic. As such, for material to
be able to compete successfully in the market a combination of clinical performance, marketing and cost-effectiveness should be
effectively achieved. This explains why so many promising formulations have not been converted into commercially offered prod-
ucts. Nevertheless, there are a growing number of TE implantable biomaterials, based on naturally-derived biopolymers, which are
already approved and commercially available as therapies for soft and hard tissues. These tissues include skin, cartilage, bone, blood
vessel, pancreas, nerve, abdominal wall, tendon, and ligament. A nonexhaustive set of examples of biomedical implants, based on
naturally-derived biopolymers that are currently on the market is presented in Table 2.
With the advancement in technology, more than 3000 products have been developed to treat different types of wounds by
targeting various aspects of the healing process. Based on the cause and type of wound, numerous products are available in
the market, among them being bioactive dressings, a modern generation of wound healing materials. They are generally
composed of naturally-derived biopolymers such as collagen, hyaluronic acid, chitosan, alginate, and elastin, and are avail-
able in different forms including gels, pads, particles, pastes, powders, sheets, or solutions (more than 900 products). Some
examples of the commercially existing wound healing products, based on naturally-derived biopolymers, are shown in
Table 3.
Table 3 A nonexhaustive set of examples of commercially available wound dressing products based on natural biopolymers
Principal components Product and manufacturer Form
Alginate (calcium/sodium) AlgiDERM (Bard), AlgiSite(Smith & Nephew, Inc.), Algosteril (Johnson Films, patches, membranes, foams
& Johnson), CarraSorb H (Carrington), CURASORB and CURASORB
Zinc (Kendall), Dermacea (Sherwood-Davis & Geck) FyBron (B.
Braun), Gentell (Gentell), Hyperion Advanced (Hyperion), Alginate
Dressing (Medical, Inc.), KALTOSTAT (ConvaTec), KALGINATE
(DeRoyal), Maxorb (Medline), PolyMem (Ferris Mfg.), Restore
(Hollister), SORBSAN (Dow Hickam), SeaSorb (Coloplast Sween
Corp.), Tegagen HG and Tegagen HI (3 M Health), etc.
Carboxymethyl cellulose, Granuflex™ (Conva Tec), Comfeel™ (QuickCompany), Tegasorb™ Thin films, sheets
gelatin and pectin (AliMed), WALKER® (Compeed), etc.
Chitosan HemCon (Medical Technologies, Inc.), HemCon Bandage (Cath Lab Films, sheets
Digest, SP Services UK Ltd., Chitoderm® plus (Trusetal, POmedic,
etc.) etc
Bacterial cellulose Biofill® (Cellulose Solution LLC.), Bioprocess® and XCell® (Medline Membrane, films
Industries, Inc.), etc.
Hyaluronic acid Exm. Hyiodine (H&R Healthcare), IDRA® hydrogel (Trusetal), Viscose gel, pads
Hyalomatrix KC Wound Dressing (Laserskin), etc.
Collagen BIOPAD™(Angelini Pharma, Inc.), Helix3® Bioactive Collagen (Amerx Sponges, thin films, sheets, powders,
Health Care Corp.), Stimulen™ Collagen Powder (Southwest sprays, gels, ropes, porous pads
Technologies, Inc.), Cutimed® Epiona (BSN medical, Inc.),
CellerateRX® Gel and CellerateRX® Powder (Wound Care
Innovations, LLC), DeRoyal C-Pro® 3D (DeRoyal), Gentell Collagen
(Gentell Wound and Skin Care), Puracol® Plus MicroScaffold™
Collagen (Medline Industries, Inc.), Simpurity™ Collagen Pad and
Simpurity™ Collagen Powder (Safe n’ Simple), Stimulen™ Collagen
Gel (Southwest Technologies, Inc.), SkinTemp™ II Dressings Human
(BioSciences, Inc.), Triple Helix Collagen Dressing (MPM Medical,
Inc.), etc.
Collagen, alginate FIBRACOL™ Plus (KCIdAn Acelity Company), etc. Sheets
Collagen, alginate, cellulose DermaCol™ (DermaRite Industries, LLC), ColActive® Plus (Covalon 3D pads
and EDTA Technologies, Ltd.),etc.
Collagen, matrix metalloproteinases BIOSTEP* Collagen Matrix (Smith & Nephew, Inc.) 3D pads
Collagen, cellulose PROMOGRAN® Matrix (KCIdAn Acelity Company),etc. Pads, sheets
Collagen, ECM proteins Endoform Dermal Template™ (Hollister IncorporateddWound Care) Sheets
Concluding Remarks
A variety of biomaterials has been developed from biopolymers using approaches inspired by nature. While major advances in the
use of natural biopolymers have taken place with increasing numbers of products entering the market and into clinical trials, signif-
icant research is still required. Currently increasing number of technologies are entering the clinical and commercial fields with
more ongoing research dedicated to the development of sophisticated biomimetic biomaterials with added levels of complexity
and stimuli-responsive properties. New and expanding research in this area demonstrates just how multidisciplinary the field of
biomaterials has become and while the challenges are huge, the opportunities for improving human health in a whole variety
of areas are immense. Advances in these areas will facilitate the development of new generations of biopolymer-based platforms
with controlled function, offering expanded options for guiding cellular behavior for applications in tissue replacement and other
areas. Recent technological progresses and growing knowledge in biochemistry, molecular biology, and bioengineering have added
to the optimization of biological performance of biopolymer-based formulations, expanding significantly, the areas available for
emerging applications.
Further Reading
Cen, L., Liu, W., Cui, L., Zhang, W., & Cao, Y. (2008). Collagen tissue engineering: Development of novel biomaterials and applications. Pediatric Research, 63, 492–496.
Daamen, W. F., Veerkamp, J. H., van Hest, J. C. M., & van Kuppevelt, T. H. (2007). Elastin as a biomaterial for tissue engineering. Biomaterials, 28, 4378–4398.
Davidenko, N., Schuster, C. F., Bax, D. V., et al. (2015). Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics. Acta Biomaterialia, 25, 131–142.
Klemm, D., Heublein, B., Fink, H.-P., & Angew, A. B. (2005). Cellulose: Fascinating biopolymer and sustainable raw material. Angewandte Chemie International Edition, 44,
3358–3393.
Kogan, G., Soltes, L., Stern, R., & Gemeiner, P. (2007). Hyaluronic acid: A natural biopolymer with a broad range of biomedical and industrial applications. Biotechnology Letters,
29, 17–25.
Koha, L.-D., Cheng, Y., Tenga, C.-P., et al. (2015). Structures, mechanical properties and applications of silk fibroin materials. Progress in Polymer Science, 46, 86–110.
Kwoni, H. J., & Han, Y. (2016). Chondroitin sulfate based biomaterials for tissue engineering. Turkish Journal of Biology, 40, 290–299.
Lee, K. Y., & Mooney, D. J. (2012). Alginate: Properties and biomedical applications. Progress in Polymer Science, 37, 106–126.
Liang, Y., & Kiick, K. L. (2014). Heparin-functionalized polymeric biomaterials in tissue engineering and drug delivery applications. Acta Biomaterialia, 10, 1588–1600.
Liu, J., Zhan, X., Wan, J., Wang, Y., & Wang, C. (2015). Review for carrageenan-based pharmaceutical biomaterials: Favourable physical features versus adverse biological effects.
Carbohydrate Polymers, 121, 27–36.
Rouse, J. G., & Van Dyke, M. E. (2010). A review of keratin-based biomaterials for biomedical applications. Materials, 3, 999–1014.
Yang, T.-L. (2011). Chitin-based materials in tissue engineering: Applications in soft tissue and epithelial organ. International Journal of Molecular Sciences, 12, 1936–1963.
Baldwin, A. D., & Kiick, K. (2010). Polysaccharide-modified synthetic polymeric biomaterials. Biopolymers, 94, 128–140.
Coimbra, P., Ferreira, P., Alves, P., & Gil, M. H. (2014). 1. Polysaccharide-based polyelectrolyte complexes and polyelectrolyte multilayers for biomedical applications. Carbo-
hydrates Applications in Medicine, 2014, 1–29.
Dash, M., Chiellini, F., Ottenbrite, R. M., & Chiellini, E. (2011). ChitosandA versatile semi-synthetic polymer in biomedical applications. Progress in Polymer Science, 36,
981–1014.
Gagner, J. E., Kim, W., & Chaikof, E. L. (2014). Designing protein-based biomaterials for medical applications. Acta Biomaterialia, 10, 1542–1557.
Ige, O. O., Umoru, L. E., & Aribo, S. (2012). Natural products: A minefield of biomaterials. ISRN Materials Science, 2012, 1–20. https://doi.org/10.5402/2012/983062.
Lee, C. H., Singla, A., & Lee, Y. (2001). Biomedical applications of collagen. International Journal of Pharmaceutics, 221, 1–22.
Kaplan, D. I. (Ed.). (1998). Biopolymers from renewable resources. Verlag Berlin Heidelberg New York: Springer.
Luo, Y., & Wang, Q. (2014). Recent development of chitosan-based polyelectrolyte complexes with natural polysaccharides for drug delivery. International Journal of Biological
Macromolecules, 64, 353–367.
Reis, R. (Ed.). (2008). Natural-based polymers for biomedical applications. Cambridge England: Woodhead Publishing Limited.
Ricard-Blum, S. (2011). The collagen family. Cold Spring Harbor Perspectives in Biology, 3, 1–19.
Polymeric Coatings and Their Fabrication for Medical Devices
Dimitrios A Lamprou, University of Kent, Canterbury, United Kingdom
Nikolaos Scoutaris, Steven A Ross, and Dionysios Douroumis, University of Greenwich, Greenwich, United Kingdom
Introduction 177
Applications 177
Antimicrobial 177
Drug-Eluting Coating 178
Stents 178
Microneedles 179
Coatings for Osseointegration 179
Coating to Improve the Mechanical Properties 180
Technology 180
Spray Coating 180
Pulsed Laser Deposition 181
Chemical Vapor Deposition 182
Sputter Coating 182
Inkjet Printing 184
Immobilization of Molecules Onto Implant’s Surface 185
Layer by Layer Coating (LbL) 185
Conclusion 186
References 186
Further Reading 187
Introduction
According to U.S. Food Drug Administration (FDA), medical implants are devices or tissues that are placed inside or on the surface
of the body. Many implants are prosthetics, intended to replace missing body parts. Other implants deliver medication, monitor
body functions, or provide support to organs and tissues (U.S. food and Drug Administration, 2015).
The implant surface is critical for determining the integration of the implant with the human body. This can range from enzyme
inhibition, whether specific or unspecific, to how antibodies and albumin may act when stored in glass containers, the biocompat-
ibility of hip replacements or how blood proteins may react with stents. These interactions can be either useful (improved biocom-
patibility) or detrimental (thrombosis and toxicity) so it is important to understand how and why they occur. Appropriate coatings
are advantageous and often essential for the acceptance and smooth functioning of the implant. The coating on medical devices can
help to reduce friction in the body to improve the placement of the implant and also minimize irritation and inflammation. It can
improve the biocompatibility inhibiting the formation of scar tissue surrounding implanted devices, the chance of infection related
to the implanted device, and encouraging the growth of tissues to help the healing process.
Applying a coating to a device that is placed in the body is a very critical process. The coating must be uniform, covering the
complete surface which often consists of a complex structure and eliminating the bringing across the structure. Nowadays,
numerous technologies have been developed in order to achieve a thin coating on medical devices. These include spray coating
to deposit a thin film, physical vapor deposition (PVD) to transfer a solid source to a surface film, chemical vapor deposition
(CVD) for a surface reaction to create film, surface polymerization to create a film from a monomer vapor, and inkjet placement
of coating via impingement of tiny droplets.
Applications
Antimicrobial
All implanted medical devices, from transient, easily inserted, and retrieved contact lenses, urinary catheters, and endotracheal
tubes, to more permanently surgically implanted cardiac valves, embolic coils, vascular grafts, hip, knee and shoulder joints, pace-
makers, coronary stents, and plastic surgery augmentation devices suffer from recognized risks of “device-related” or “implant-
associated” infection (Wu and Grainger, 2006). In order to reduce the incidence of device-related infections two main strategies
have been implemented: first, antiadhesive biomaterials using physicochemical surface modification methods such as nondrug con-
taining coating and second direct incorporation of drugs into or onto the medical device either immobilized or released (Wu and
Grainger, 2006).

178 Biomaterials: Science and Engineering j Polymeric Coatings and Their Fabrication for Medical Devices
There have been several antimicrobial surfaces described in the literature, including antibiotic coatings such as cefazolin min-
ocycline–rifampin, teicoplanin, and vancomycin which have been tested on biomaterials. Alternative antimicrobial coatings
such as salicylic acid, quaternary ammonium compounds, and chlorhexidine have also been trialed. In this concept, polymers
which are used as carriers for the antibiotics are deposited on implant’s surface. Harris et al. (2006) used various polymers including
(poly(D,L-lactide) (PDLLA), politerefate (PTF), calcium phosphate/anodic plasma-chemical treatment (CaP/APC), polyurethane
(PU), and polyvinylpyrollidone (PVP) on titanium surfaces with and without chlorhexidine diacetate (CHA) to investigate their
cytocompatibility for Staphylococcus aureus, Staphylococcus epidermidis, and human telomerase reverse transcriptase (hTERT) fibro-
blasts. The study showed that the release kinetics varied from slow (over 200 h) to burst release: PDLLA > PTF > PU > CaP/
APC ¼ PVP. This study showed that PDLLA and PTF have the best potential as coatings on implants for drug delivery, as they
were cytocompatible to hTERT fibroblasts, eluted CHA effectively, and passed mechanical testing. The actual release kinetics of
PDLLA and PTF are important, as the amount of CHA present should remain above the minimal inhibitory concentration value
for a limited time before disappearing completely.
Silver antimicrobial agents have also been utilized as an alternative strategy for reducing bacterial adhesion and preventing bio-
film formation. Silver (Ag) is a potent bactericide and has attracted increasing attention due to a host of additional benefits such as
a broad antibacterial spectrum including antibiotic-resistant bacteria, noncytotoxicity at proper doses, satisfactory stability, and
a smaller possibility to develop resistant strains. However, the use of silver must be undertaken with caution, since a concentra-
tion-dependent toxicity has been demonstrated. Panácek et al. (2006) assessed the suitability of a mouse spermatogonial stem
cell line as an in vitro model to evaluate the toxicity of silver resulting that concentrations of silver nanoparticles between 5 and
10 mg/mL induced necrosis or apoptosis of mouse spermatogonial stem cells. Apart from silver, other promising metals for antimi-
crobial coating are Copper (Cu) and Zinc (Zn).
Besides conventional antibiotics, different types of alternative antimicrobial coatings for medical devices have been proposed
including antimicrobial peptides (AMP) and polymers. Polymers have been introduced in order to overcome problems associated
with the low-molecular-weight antimicrobial agents, such as toxicity to the environment and short-term antimicrobial ability,
where antimicrobial functional groups can be introduced into polymer molecules. The use of antimicrobial polymers offers promise
for enhancing the efficacy of some existing antimicrobial agents and minimizing the environmental problems accompanying
conventional antimicrobial agents, by reducing the residual toxicity of the agents, increasing their efficiency and selectivity, and pro-
longing the agents lifetime. For instance, Hoffman et al. applied poly(ethylene glycol-stat-propylene glycol) prepolymers (Star
PEG). Taken together, for Star-PEG-covered substrates demonstrated a profound reduction of various blood–biomaterial interac-
tions compared to noncoated substrates. A number of polymers that have been applied to influence the amount and/or conforma-
tion of adsorbed proteins, preventing bacterial adhesion and biofilm formation, such as poly(hydroxyethylmethacrylate),
poly(methacrylic acid), and polyurethanes.
Recently, antimicrobial peptides (AMP) have been applied to the implant’s surface in order to prevent colonization of bioma-
terials as they display long-term stability, even though the sterilization process, and have a low cytotoxic profile. Because of their
physicochemical characteristics, which involve their highly cationic character and tendency to adopt amphipathic structures,
AMPs have the tendency to associate with negatively charged microbial surfaces and membranes. These peptides offer several attrac-
tive advantages: they exhibit bactericidal, fungicidal, viricidal, and tumoricidal properties, they act at a very low concentration, and
they are less likely to promote bacterial resistance. Hence, AMPs’ coating on implants has been developed to reduce bacterial strains
of Gram-positive (S. aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria.
Drug-Eluting Coating
Stents
Stents are small expandable tubes used to treat coronary heart diseases. A stent is implanted using a procedure called percutaneous
coronary intervention (PCI), also known as coronary angioplasty. PCI restores blood flow through narrow or blocked arteries. A
stent helps support the inner wall of the artery in the months or years after PCI. Biocompatibility of the stents materials is important
to minimize inflammation and to allow endothelial cell growth to continue as normal. The first generation of stents was made of
bare metal. Although bare-metal stents almost eliminated the risk of the artery collapsing, they only modestly reduced the risk of
restenosis. About 25% of all coronary arteries treated with bare-metal stents would close up again, usually within about 6 months.
Stents are normally made of a metal like tantalum or an alloy such as 316L stainless steel. While these materials typically show
reasonable biocompatibility, coating stents with polymer or ceramic layers has the potential to improve the properties further.
Therefore, new platforms for drug-eluting stents have been developed in order to eliminate the risk of restenosis. Coatings can
either be passive providing an improved tissue interface or active to elute drugs which can control neointimal growth, restenosis,
and inflammation which would narrow the lumen and may give rise to dangerous thrombi. There are currently stents are made of
biodegradable polymers for drug elution. Polymeric coating of metallic stents also reduces the risk of toxicity from metal released
through corrosion. Drug-eluting stents consist of the metallic platform, a polymer, and the antiproliferative agent. In drug-eluting
stents, the polymer is used as a reservoir which controls the drug release, whereas the drug is used to inhibit the endothelial cells to
proliferate inhibiting restenosis. Employing a spray-coating technique Bege et al. (2012) coated rotating stents with a poly(ethylene
carbonate) (PEC) solution of 0.8% (w/w) in dichloromethane with or without paclitaxel. Other drugs that were used for stent
coating are rapamycin, tacrolimus, everolimus, zotarolimus, and biolimus. Moreover, a wide range of polymers can be permanent,
such as poly-n-butyl methacrylate (PBMA) and polyethylene-co-vinyl acetate (PEVA), which is used in cypherÒ stents developed by
Biomaterials: Science and Engineering j Polymeric Coatings and Their Fabrication for Medical Devices 179
Cordis. Most modern polymer coatings are bioerodible and degraded by hydrolysis and are eventually metabolized to water and
carbon dioxide, examples include polyglycolic acid, poly-L-lactid acid grades, and PLGA-PEG block polymers. Drugs and polymers
can be deposited on stent’s surface by using various techniques including dip coating, spray coating, inkjet printing, and layer by
layer.
Microneedles
Microneedles is a novel approach for transdermal delivery of drug substances, vaccines or macromolecules which cannot be
administered orally due to their poor absorption or enzymatic degradation in the gastrointestinal tract and liver. The aim of
microneedles is to create large pathways of microscope dimensions using an array of microscopic needles attached to a metal
or polymer base. Regarding metal microneedles, drug/excipient compositions are coated both sides of each needle and delivered
transdermally by piercing the skin. Currently, microneedles are coated using the dip-coating approaches. A dip-coating process
typically involves dipping and withdrawing an object from a coating solution, after which a continuous liquid film adheres
and dries on the object’s surface, leaving behind a uniform coating. However, conventional dip-coating methods have difficulty
controlling the material deposition of specified sections of micron-dimensioned structures, especially when those structures are
closely spaced. McGrath et al. (2011) spray coated microneedle patches using a conventional film-coating process and investi-
gated two film polymeric-coating materials, hydroxypropylmethylcellulose and carboxymethylcellulose; these could be poten-
tially loaded for intradermal drug and vaccine delivery. However, the main disadvantage of the latter technology is the loss of
material. Therefore, recently inkjet printing technology has been applied to deposit single droplets of the coating solution directly
onto the microneedles.
Coatings for Osseointegration

Titanium (Ti) and its alloys are the most commonly used metallic materials for medical implants in orthopedic and dental appli-
cations, due to their low density, high strength, nontoxicity, and excellent corrosion resistance. However, there have been reports on
inflammatory reaction around these implants as a result of the creation of an avascular fibrous tissue that encapsulated the implants.
Therefore, a hydroxyapatite layer is deposited on the metal alloy to assist the osseointegration of these implants with surrounding
tissues. Synthetic hydroxyapatite [Ca10(PO4)6(OH)2, HA] has been extensively used in a number of dental, orthopedic, and other
medical applications, due to its similarity in chemical composition and crystal structure with bone mineral. It has been shown that
hydroxyapatite stimulates the expression of bone-related mRNAs and proteins in osteoblasts. Several techniques have been used to
create the HA coating on metallic implants, such as plasma spraying process, thermal spraying, sputter coating, pulsed laser abla-
tion, dip coating, sol–gel, electrophoretic deposition (EPD), ion-beam-assisted-deposition, and hot isostatic pressing. A number of
approaches toward improving the integration rates of HA with bone have included the incorporation of biological entities such as
growth factors, proteins and cells, into the HA implant (Mankani et al., 2006).
Other strategies to improve the osteoinduction of embedded biomolecules are the coating on the surface of the implant with
biomolecules. Large proteins or glycosaminoglycans such as collagen and chondroitin sulfate provide a biomimetic coating on
the surface of an implant which can improve integration once implanted in the body. In order to manage efficient loading of
biomolecules onto the implant surface and to modulate the release of such molecules in a controlled manner which in turn can
promote osseointegration hydrogel coatings, layer-by-layer (LBL) coating techniques have been developed.
In hydrogel coatings, orthopedic implants are immersed in hydrogel solutions that contain biomolecules of interest. The
implant is then removed and air dried to allow adsorption of molecules onto the surface of the implant. Given its ease of appli-
cation, hydrogel coatings have been applied to coat various orthopedic implants with a broad range of biomolecules including
growth factors, viruses, and peptides. Hydrogels are appealing candidates for the development of scaffolds for mineralization.
The intrinsic elasticity and water retention ability of synthetic hydrogels resemble those of collagen, and their porosity may
be controlled by various techniques. Another important feature of hydrogels is that they can be assembled in a three-dimensional
form, displaying multiple functional domains through copolymerization of different monomers. Hence, proteins regulating
mineral growth, and biological epitopes such as the tripeptide RGD that promote cellular adhesion have been applied to collagen
hydrogel and poly(ethylene glycol) hydrogels. Other hydrogel coatings involve the utilization 2-hydroxyethylmethacrylate
(HEMA) and HEMA copolymerized with a phosphate-containing monomer, 2-methacryloyloxyethyl phosphate (MOEP), where
P(HEMA–MOEP) coating was to entrap and release rhVEGF which showed its potential to enhance implant site calcification and
angiogenesis.
Moreover, coating metal surfaces with suitably functionalized/modified polymer films capable of improving implant osteointe-
gration and at the same time inhibiting metal substrate corrosion is another useful strategy. Such as polymers include poly(bis(tri-
fluoroethoxy)phosphazene), pyrrole, poly(methyl methacrylate), and poly(acrylic acid).
In LBL approach, the adsorption of biomolecules onto the implants’ surface occurs by introducing a surface charge to the
implant surface by plasma etching or modifying with molecules such as heparin. The charge is achieved by dipping implants repeat-
edly in polyelectrolyte solutions with opposite charges. The LBL technique has been used for the deposition of growth factors on
a variety of implant surfaces. Polycaprolactone/b-tricalcium phosphate (PCL/b-TCP) scaffolds coated with bone morphogenetic
protein 2 (BMP-2) via an LBL approach led to enhanced ectopic bone formation in the rat hindlimb tissue.
Coating to Improve the Mechanical Properties

The most common coating material which is currently used to improve the mechanical properties of the implant is the diamond-
like carbon (DLC) coating. DLC is an amorphous carbon biomaterial that in recent years has demonstrated its coating potential due
to its high hardness, low frictional coefficient, high wear and corrosion resistance, chemical inertness, high electrical resistivity,
infrared-transparency, high refractive index, and excellent smoothness. All these properties match well with the criteria of a safe
biomaterial for applications in orthopedic, cardiovascular, contact lenses, or dentistry. Also, it has been shown that DLC has better
biocompatibility and wear resistance than stainless steel, titanium, and Ti alloys. Moreover, when DLC coatings are applied to
implant’s abutment’s screws, they demonstrate more resistance to an applied force than without the coating.
The most common method used for coating DLC is plasma-enhanced CVD (PECVD). In addition to PECVD-coating process,
other technologies which were used to impart the implant with desirable coating properties include pulsed layer deposition, sput-
tering, bipolar type plasma-based ion implantation and deposition (PBII&D), and more rarely cathodic arc deposition. In the
PBII&D processing, the glow discharge plasma is formed near the target surface by applying a positive pulse voltage directly to
the target, and the plasma is omnidirectionally implanted into (or deposited on) the target surface by a subsequent negative
high pulse voltage.
Technology
Spray Coating
Spray coating is a technology that has been successfully used for various purposes such as for drug release, antibacterial, and
osseointegration coating by depositing various active agents such as polymers and drugs on implantable medical devices, including
catheters, stents, drug-eluting balloons, pacemakers, heart valves, sensors, surgical implant coatings, and orthopedic implant coat-
ings. There are various technologies which can be characterized as spray coating. Among the most prevalent is the ultrasonic spray
nozzle. According to this, the coating solution is atomized into a fine mist spray using high frequency sound vibrations. Piezoelec-
tric transducers convert the electrical input into mechanical energy in the form of vibrations, which create capillary waves in the
liquid when introduced into the nozzle. For the liquid to atomize, the vibrational amplitude of the atomizing surface must be care-
fully controlled. Below the critical amplitude, the energy is insufficient to produce atomized drops. If the amplitude is excessively
high, the liquid is ripped apart, and large “chunks” of fluid are ejected, a condition known as cavitation. Only within a narrow band
of input power is the amplitude ideal for producing the nozzle’s characteristic fine, low velocity mist.
Ultrasonic spray nozzle has been extensively used to coat stents. The operating requirements in this application call for flow rates
on the order of 20–100 mL/min, spray diameters in the range of 0.5–2 mm, and tiny median drop diameters. In this method, the
stent is placed on a mandrel that is attached to a rotating shaft. The nozzle is mounted above the stent. The sprayed liquid consists of
the polymer/drug system dissolved in a suitable organic solvent. Typically, high vapor pressure solvents are used, so that drying
occurs quickly. Repeated traverses, coupled with low flow rates, produce the best coatings and maximum material transfer efficiency.
By varying rotational speed, the distance of the spray from the stent, and the number of traverses a process can be optimized. Specif-
ically, poly (D,L-lactic-co-glycolic acid) (PLGA) was used as a drug carrier to generate two types of stents loaded with different concen-
trations of sirolimus, and curcumin-eluting PLGA coatings were fabricated on the surface of 316L stainless steel stents by an
ultrasonic spray method. An interesting study from Choi et al. showed that sirolimus (SRL) release from the biodegradable
(PLGA) matrix is affected by the organic solvents due to the interaction between SRL and PLGA and the different surface topography
(Choi et al., 2014).
Another spray coating technique is the use of aerosol. For aerosol generation, gas is passed through a loose powder contained in
a chamber, thereby producing a fluidized bed. Driven by a pressure difference the aerosolized particles are transported from the
aerosol chamber through a nozzle to the deposition chamber. The particles in the focused jet collide with the substrate at high
speed. Hahn et al. used aerosol deposition on AZ31 Mg alloy samples, sprayed in a vacuum, with a specially mixed HA–chitosan
powder at room temperature. The nozzle was placed vertically opposite the sample, which allows rotation in the x- and y-directions,
but not in the z-direction (Hahn et al., 2011). Moreover, HA and 4-hexylresorcinol (4-HR), a well-known antiseptic, were success-
fully coated onto a titanium surface.
A spray-coating technique which is widely used for coating of orthopedic implants with hydroxyapatite are the thermal spray
techniques. These are coating processes in which melted (or heated) materials are sprayed onto a surface. The “feedstock” (coating
precursor) is heated by electrical (plasma or arc) or chemical means (combustion flame). Another technique is called high velocity
oxygen fuel coatings (HVOF). In HVOF spraying, fuel and oxygen are pressed into a combustion chamber in a continuous flow,
producing a jet of combustion products at extremely high speed. Powder particles injected into this gas stream are accelerated to
a very high velocity. Fusion is obtained by the kinetic impact of the coating particles, rather than by their increased temperature.
This process is carried out in an ambient atmosphere. HVOF has been applied in order to coat implants with HA with titanium
addition. The titanium was found to improve the Young’s modulus, fracture toughness, and shear strength of HVOF-sprayed
HA-based coatings. However, the increase of titanium content from 10 to 20 vol% induces a small decrease in Young’s modulus.
Moreover, the influence of particle temperatures and velocities has been investigated, and it was observed that they have linear corre-
lation with the phase composition, Vickers microhardness, and the residual stress.
One of the most promising suspension spray technology is the high-velocity suspension flame spraying (HVSFS) process devel-
oped at the Institute for Manufacturing Technologies of Ceramic Components and Composites. HVSFS is a new approach for
spraying micron, submicron, and nanoparticles with hypersonic speed by feeding a suspension directly into the combustion
chamber of a HVOF torch. The aim in mind is to achieve dense coatings with an improved microstructuredprobably reaching
the nanoscale, from which superior physical properties are expected. Compared to the alternative approach, that is, using agglom-
erated (nano- and micron-sized) powders, direct spraying of suspensions shows much higher flexibility in combining and process-
ing different materials and is far less expensive. Several suspensions consisting of an organic solvent and a solid phase composed of
a micron or a nanopowder have been prepared and HVSFS sprayed, examples include suspensions containing oxide nanopowders
of titanium oxide (n-TiO2), chromium(III) oxide (n-Cr2O3), yttrium-stabilized zirconia (n-YSZ), and n-hydroxyapatite (n-HA).
Different process parameters such as gas flow, spray distance, air-fuel ratio, and electric arc current can affect the coating properties.
Hence Gadow et al. (2010) prepared thermally sprayed hydroxyapatite coatings by using three different thermal coating technol-
ogies HVOF, APS, and HVSFS. They found that HVSFS HA coatings have the most refined microstructure regarding the microstruc-
ture of the deposited coatings. Also, the main effects of the different processes on the adhesive behavior were reviewed. The pull-off
performance of the HVSFS–HA coatings strongly depends on the dispersive medium and the oxygen flow rate, for HVOF–HA coat-
ings, it is the spray distance and for the APS–HA coatings, the electric arc current is the most influencing parameter. Moreover, APS
has been applied in order to produce amorphous HAP. It was found that three factors strongly influence the formation of the amor-
phous phase; dehydroxylation of the molten particle during flight, the cooling rate as it impinges onto the metal substrate, and the
substrate temperature. Crystalline regions were identified as unmelted particles and elongated recrystallized areas. Amorphous
phase regions vary throughout the coating but are more commonly found at the coating-substrate interface, that is, the areas
decrease toward the surface of the coating.
Pulsed Laser Deposition

Pulsed laser deposition (PLD) is a PVD process, carried out in a vacuum system, which shares some process characteristics shared
with molecular beam epitaxy and some with sputter deposition. In PLD, a pulsed laser is focused onto a target of the material to be
deposited. For sufficient high laser energy density, each laser pulse vaporizes or ablates a small amount of the material creating
a plasma plume. The ablated material is ejected from the target in a highly forward-directed plume. The ablation plume provides
the material flux for film growth. The film growth process depends greatly on the parameters of the condensing plasma fluxes
(density, spatial distribution, particle energy, ionization degree, etc.), as well as on the thermodynamic parameters of the substrate
surface, such as temperature, density of adsorption sites, activation energy of surface desorption and diffusion, etc.
PLD is one of the most promising coating technique for hydroxyapatite providing excellent quality, and high performance coat-
ings. Pure crystalline hydroxyapatite (HA) films with thicknesses of ranging from 0.5 to 5 mm have been deposited on titanium
substrate using the PLD technique (Fig. 1). Experimental results indicate that the structure and properties of the PLD-HA films
varied with deposition parameters. Torrisi and Setola (1993) have obtained a smooth, uniform PLD-HA film on a titanium
substrate at 400 C. When deposited on a room temperature substrate, however, the coating had a granular-uniform mixed
morphology. Jelinek et al. (1995) have also obtained a uniform laser-deposited HA film on a titanium substrate at intermediate
temperatures (200–400 C). When deposited on a higher temperature (600–760 C) substrate, the coating roughness increased
and inhomogeneity and buckling were observed. Moreover, Zeng and Lacefield (2000) showed that the Ca/P ratios of HA powder
and targets were larger than 1.67. Ca/P of HA ratios of coatings varied with the deposition conditions. Specifically, coatings depos-
ited at room temperature under vacuum had higher Ca/P ratios than the HA target material. Coating occurred at room temperature
but with the presence of an ambient gas had a Ca/P ratio closer to that of the target, whereas coating at elevated temperature with an
Fig. 1 SEM micrograph of the cross section of a PLD coating, showing a dense HA film. Reproduced with permission from Dinda, G.P., Shin, J.
and Mazumder, J. (2009). Pulsed laser deposition of hydroxyapatite thin films on Ti–6Al–4V: Effect of heat treatment on structure and properties.
Acta Biomaterialia 5(5), 1821–1830.
ambient gas had a Ca/P ratio that was in between the Ca/P ratios for specimens deposited under the other two conditions. Cotell
et al. (1992) have reported that the amorphous PLD-HA films deposited at room temperature were demonstrated calcium defi-
ciency, displaying Ca/P ratios between 1.2 and 1.6. However, when deposited at 400–700 C crystalline HA films with Ca/P ratios
ranging from 1.6 to 3.6 were obtained. Although the substrate temperature was perhaps the most critical parameter, variations in
Ca/P ratio of as much as 60% were noted, even when deposited at the same substrate temperature.
Chemical Vapor Deposition

In CVD, there is no universal equipment, but each piece of CVD equipment is individually tailored for specific coating materials,
substrate geometry, etc., whether it is used for research and development or commercial production. In general, the CVD equipment
consists of three main components: the chemical vapor precursor supply system, a CVD reactor, and the effluent gas handling
system. The role of the chemical vapor precursor system is to generate vapor precursors and then deliver to the reactor. A CVD
reactor consists of a reaction chamber equipped with a load lock for the transport and placement of the substrate into the chamber,
a substrate holder, and a heating system with temperature control. The primary function of the CVD reactor is used to heat the
substrate to the deposition temperature. The CVD reactor can either be a hot-wall or cold-wall reactor. A hot-wall reactor uses
a heated furnace into which the substrates are placed for indirect heating, whereas in the cold wall reactor, only the substrate is
heated and the wall of the reactor is cold. Finally, the effluent gas handling system consists of a neutralizing part for the exhaust
gases, and/or a vacuum system to provide the required reduced pressure for CVD processing, that performs at low pressure or
high vacuum during deposition. The main function of the effluent gas handling system is to remove the hazardous by-product
and the toxic unreacted precursor safely. The unreacted precursors and corrosive by-products such as HCl are neutralized or trapped
using a liquid nitrogen trap to prevent these gases from entering the rotary or diffusion pump, which can cause damage to the pump.
In terms of medical implants, CVD is suitable for titanium nitride, titanium carbonitride, and aluminium oxide, although many
other materials may be deposited by CVD as well.
The main parameters of CVD that affect the quality of coating are temperature, pressure, reactant gas concentration, and total gas
flow, which require accurate control and monitoring. Specifically, the temperature at which the coating is deposited is a critical
factor, as it controls both the thermodynamics and the kinetics of the coating process. The deposition temperature must be achieved
and maintained in order for the reaction to occur on the substrate and not in the gas phase, and with an appropriate microstructure.
Moreover, CVD processes are carried out from atmospheric pressure to high vacuum. At atmospheric pressure, the growth processes
are often considered to be “transport controlled.”
The main disadvantage of CVD is that the heat input can result in damage to temperature-sensitive substrates and so alternative
forms of energy input have been developed which allow deposition at lower temperatures. One way of reducing growth tempera-
tures is to use plasma-assisted or PECVD. With this technique, deposition can occur at very low temperatures, even close to ambient,
since electrical energy rather than thermal energy is used to initiate homogeneous reactions for the production of chemically active
ions and radicals that can participate in heterogeneous reactions, which, in turn, lead to layer formation on the substrate. In PECVD
processes, deposition is achieved by introducing reactant gases between parallel electrodesda grounded electrode and an
RF-energized electrode. The capacitive coupling between the electrodes excites the reactant gases into a plasma, which induces
a chemical reaction resulting in the reaction product being deposited on the substrate. The substrate, which is placed on the
grounded electrode, is typically heated to 250–350 C, depending on the specific film requirements. In comparison, CVD requires
600–800 C. PECVD has been applied extensively to deposit DLC films producing a smooth surface morphology exhibiting high
hardness and elastic modulus (200 GPa) and good adhesion to Ti–6Al–4V substrate. Also, other biocompatible coatings for
implants involve the deposition of SiOx, TiOx, and SiO–TiO mixed oxide films and TiN and TiAlN coated on Ti Surface to enhance
the osteoblast’s proliferation (Fig. 2). Finally, multilayers with extension to layer thicknesses in the nanometer range of TiN/Ti–B–N
multilayers have been deposited in an industrially sized deposition chamber to optimize mechanical and tribological properties.
Sputter Coating
In sputter coating, a gaseous plasma is created for the purpose of ion bombardment of the source material known as the target. The
source material is eroded by the arriving ions via energy transfer and is ejected in the form of neutral particlesdeither individual
atoms, clusters of atoms, or molecules. As these neutral particles are ejected, they will travel in a straight line unless they come into
contact with something, such as other particles or a nearby surface. If a “substrate” is placed in the path of these ejected particles, it
will be coated with a thin film of the source material.
Sputter coating has been used extensively to coat hydroxyapatite and other CaP materials. However, it has been reported that it
would produce coatings whose chemistry was different upon deposition, than the bulk target. Specifically, the surface Ca/P ratio for
the sputtered coatings has been reported to range from 1.6 to 2.6. Normally, the coating coming from sputtering technique is
uniform and dense, where the thickness can be as small as 10 nm depending on the deposition time. Furthermore, it was found
that CaP deposition can improve the osseointegration even in osteoporotic conditions (Fig. 3). Also, it has been applied to deposit
zinc oxide to control and prevent the spreading and persistence of bacterial infections.
EPD is a technique that exploits the movement of charged particles in suspension in the presence of electric field. This electric
field enables the consolidation of particles onto any shaped substrate. Two groups of parameters determine the characteristics of this
process: (i) those related to the suspension and (ii) those related to the process including the physical parameters such as the
Fig. 2 Scanning electron microscopy (SEM) images of MG63 cells cultured on (A) Ti, (B) TiN, and (C) TiAlN-coated surfaces after 24 h of cultures.
Reproduced with permission from Jang, H.W., et al. (2011). Surface characteristics and osteoblast cell response on TiN- and TiAlN-coated Ti implant.
Biomedical Engineering Letters 1(2), 99–107.
Fig. 3 Histological images present bone features around coated and noncoated implants in osteoporotic vs. healthy rats. In osteoporosis (A and B)
more bone shows in direct contact with coated implants but disconnected from the preexisting bone compared to healthy (C and D). Bone covering
noncoated implant surface is thin and sparse, and a layer of fibrous tissues could be always detected (Scale bar ¼ 500 mm at 10 objective magnifi-
cation). Reproduced with permission from Alghamdi, H.S., et al. (2013). Osteogenicity of titanium implants coated with calcium phosphate or
collagen type-I in osteoporotic rats. Biomaterials 34(15), 3747–3757.
electrical nature of the electrodes and the electrical conditions (voltage/intensity relationship, deposition time, etc.). The main
parameters that affect the coating quality are the suspension properties (which are related to particle size), dielectric constant of
the liquid, conductivity and viscosity of the suspension, zeta potential, and stability of suspension. Other parameters involve the
deposition time, the applied voltage, concentration of solid in suspension, and the conductivity of substrate.
Nowadays, even though plasma spraying is currently the only commercial process for fabricating HA coatings on metallic
implants. EPD has been suggested to deposit HA onto implants. Usually, the electrodeposition is carried out from a bath containing
low concentrations of Ca(NO3)2 and NH4H2PO4 at pH 6 by cathodic polarization. The advantages of EDP include control over the
composition and structure of the coating, and the ability to coat irregular surfaces efficiently.
EPD has been applied chitosan/silk fibroin to deposit composite coatings onto titanium substrates which were recommended
for protein stability and cell viability. The silk fibroin content in the coatings increased proportionally with the increase of the silk
fibroin in the electrophoretic solutions, whereas the shear and tensile bond strength of the coatings to titanium substrates increased
with the increasing silk fibroin content. The in vitro biological tests indicated that chitosan/silk fibroin composite coatings had
better cellular affinity than pure chitosan coatings. Moreover, colloidal suspensions of lysozyme and silver nanoparticles were elec-
trophoretically deposited onto the surface of stainless steel surgical blades and needles. This antimicrobial coating on medical
instruments combines the bacteriolytic activity of lysozyme and the biocidal properties of silver nanoparticles. Also, EPD has
been applied to coat stents with curcumin-loaded PLGA nanoparticles producing uniform surface morphology.
Inkjet Printing
Inkjet printing encompasses a set of material deposition technologies which are dispersed in a single, or in a system of solvents and
are ejected onto a substrate through a nozzle. The advantages of inkjet printing compared to other conventional coating techniques
are that it is an entirely automated process, provides accurate and precise control over the droplet size which in turn minimizes
material losses, allows the design of various deposition patterns, and reduces the contamination of the deposited material.
Inkjet printing can operate either in continuous (CIJ) or drop on demand (DOD) modes. In CIJ mode the printer is operated in
a continuous stream mode at high speeds which results in low printing quality, and it is mainly used for high-speed graphical appli-
cations. When using DOD mode, the droplets of a solution are jetted only according to the requirements and are perfectly located
onto a predetermined position. Depending on the technology, inkjet printing is categorized into thermal, piezoelectric, acoustic, or
electrostatic inkjet. An excellent paper which describes these technologies was published in 1998 (Le, 1998). Thermal ink-jetting
(TIJ) and piezoelectric ink-jetting (PIJ) are the predominant technologies, whereas acoustic and electrostatic ink-jetting are still
in the development stages. In thermal inkjet printing, the ink is heated, by means of a thin film resistor of heater which is activated
by an electrical pulse and thus creating a rapidly expanding vapor bubble which in turn ejects the ink from the nozzle. Conversely,
the piezoelectric dispensers rely on the deformation of a piezoelectric material. Specifically, when a voltage is applied to the piezo
element, it changes its shape to a preordained direction, causing a sudden volume change which creates pressure waves resulting in
a drop being ejected from the orifice. Most of the research nowadays rely on PIJ technique due to its flexibility regarding the ink
compatibility and the higher range in the operation frequency over the TIJ. In thermal inkjet printing, the degradation of the
compounds due to the applied heat is possible. However, this has not been proved as the heat lasts for a few milliseconds and hence
it does not affect the stability of the compounds.
The most important properties of the liquid dispensers that implement a piezoelectric response are viscosity, surface tension, and
density. These fluid properties influence the drop formation mechanism and subsequent the drop size at a given voltage. Also, these
properties can provide information about the impact of the droplet into the substrate. Hence, Jang et al. (2009) investigated the
printability of the fluids by applying the inverse (Z) of the Ohnesorge number (Oh) which relates to the viscosity, surface tension,
and density of the fluid. They have experimentally defined the printable range as 4 < Z < 14 by considering characteristics such as
single droplet formability, positional accuracy, and maximum allowable jetting frequency. This range of Z corresponds to low
viscosity fluid. Yang et al. (2006) showed that a smaller value of surface tension represents a weaker cohesive which leads to a slender
liquid ligament with a relatively longer break-up length. The shapes of the liquid and tail droplet tend to be round for the cases of
high surface tension. It also takes more time for contraction of a spherical droplet of the liquid with lower surface tension.
The predominant factor that determines the size and volume of the droplet is the size of the dispenser’s nozzle. For a given
dispenser, the volume of the droplet is directly dependent on the voltage and the duration of the signal that is applied. Hence,
the droplet velocity and volume are found to show a linear relation with driving voltage but show a more complicated and periodic
behavior with changing frequency and pulse width. Reis et al. (2005) showed that droplet velocity exhibits a maximum as a function
of pulse width, which remains unchanged when driving voltage amplitude increased. This factor can be proved critical to control the
deposited amount. Also, due to the low surface tension of organic solvents, the voltage increase can result to air bubble formation
inside the dispenser. Finally, during the printing process, droplet satellites can be formed which are undesirable as it can create non-
precise and nonhomogenous coating. Many methods are being developed to eliminate these droplet satellites which are related to
the wave propagating in the dispenser and the amplitudes and pulse durations (Tsai and Hwang, 2008).
Due to the controllable and reproducible nature of the droplets in the jet stream and the ability to direct the stream to exact
locations on the device surfaces, inkjet printing technology has shown its potency to applications where high-resolution coating
is required. Tarcha et al. (2007) programmed target deliveries of 100 mg of the drug, a typical dose for a small stent, into cuvettes,
which demonstrated standard deviation of 0.6 mg per stent. According to the coating pattern, the dispenser moves longitudinally
across the stent and is then positioned in such a way that the stream of reagent droplets is tangent to the cylinder swept out by
the stent as it is rotated. Jetting on coated uncut stent tubes exhibited 100% capture efficiency with a (137 1.8 mg), while contin-
uous jetting on actual stents presented efficiencies up to 91% with coefficients of variation as low as 2%. Also, Scoutaris et al. (2015)
implemented inkjet printing technology to apply biodegradable and biocompatible polymeric coatings of PDLLA with the antipro-
liferative drugs, simvastatin and paclitaxel, on coronary metal stents. Finally, Gill and Prausnitz (2007) developed a new coating
methodology based on inkjet printing whereby metal microneedles are coated with varies molecules such as calcein, an anticancer
drug, and insulin protein. In terms of medical implants, inkjet printing has been applied to create rifampicin as antibiotic and
calcium eluting as a means of preventing the formation of biofilm colonies and facilitating osteogenic cell development on ortho-
pedic implant surfaces. The micropatterns consisted of a periodic array of circular dots.
Fig. 4 Schematic diagram of BMP-2 protein attachment to TI surface by initial introducing of (11-hydroxyundecyl)phosphonic acid. Reproduced
with permission from Adden, N. et al. (2006). Phosphonic acid monolayers for binding of bioactive molecules to titanium surfaces. Langmuir 22(19),
8197–8204.
Immobilization of Molecules Onto Implant’s Surface

Generally, active pharmaceutical ingredients are deposited onto the implant’s surface. However, this approach failed to achieve
delivery of a sustained and efficient dosage over a relatively prolonged period of time. Vancomycin has been successfully attached
to titanium and proven to be bactericidal to S. aureus and S. epidermidis. Nevertheless, the immobilization of molecules on implant’s
surface is extremely beneficial in the case of depositing AMP. In general, the surfaces of metals are derivatized into reactive groups,
then biomolecules are conjugated to the surface by reacting with these groups. One of the most prevalent methods is the silanization
of the titanium implant resulting into a terminal-maleimide or into aldehyde groups where RGD-containing peptides proteins such
alkaline phosphatase and albumin were bound on the surface, respectively. Immobilization of Arg-Gly-Asp (RGD) containing
peptides has received significant interest because RGD is the essential sequence mediating cell adhesion in many extracellular matrix
proteins. Other strategies involve functionalized PLL-g-PEG polymer adsorbed as a monolayer on a negatively charged metal oxide
surface (e.g., titanium oxide). The PLL backbone is positively charged at pH 7.4 due to its amine groups, and the PEG chains are
exposed to the fluid as a brush. The peptide sequence arginine–glycine–aspartic acid (RGD) is covalently attached to a fraction
of the PEG chains. Another strategy to attach RGD peptide involves the dopamine’s polymerization onto implant’s surface and
subsequently immobilized of the RGD sequence. Also, BMP-2 protein has been successfully attached to titanium by introducing
of monolayers of (11-hydroxyundecyl)phosphonic acid and (12-carboxydodecyl)phosphonic acid molecules produced by a simple
dipping process. The terminal functional groups on these monolayers were activated (carbonyldiimidazole for hydroxyl groups and
N-hydroxysuccinimide for carboxyl groups) to bind amine-containing molecules (Fig. 4). In a recent study, BMP-2 was encapsu-
lated in chitosan coatings on functionalized Ti surfaces. Slow release of the protein enhanced the osteoinductivity of the implant.
Layer by Layer Coating (LbL)

LBL coating involves the dipping of surfaces repeatedly in polyelectrolyte solutions with opposite charges. LBL has several applica-
tions on the biomaterial area as it enables the nanometer level control of the composition of a thin film, and the generation of
highly sophisticated, tailor-made coating compositions. The LBL technique has been used for the deposition of growth factors
on a variety of implant surfaces. Hence, BMP-2, which are capable of directing the host tissue response to create bone from native
progenitor cells, has been coated on PCL/b-TCP scaffolds. In another work, osteogenic rhBMP-2 (recombinant human bone
morphogenetic protein-2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios
in a degradable [poly(b-amino ester)/polyanion/growth factor/polyanion] LBL tetralayer repeat architecture were promoted the
differentiation cascade in MC3T3-E1S4 preosteoblasts, while rhVEGF165 upregulated HUVEC proliferation and accelerated closure
of a scratch in HUVEC cell cultures in a dose-dependent manner (Shah et al., 2011).
To improve the surface biocompatibility of titanium films, chitosan (Chi) and gelatin (Gel) were used leading to the formation
of multilayers on the titanium thin film surfaces. The film growth was initialized by deposition of one layer of positively charged
poly(ethyleneimine) (PEI). Then the thin film was formed by the alternate deposition of negatively charged Gel and positively
charged Chi utilizing electrostatic interactions. The results showed that the cells’ proliferation and viability had been increased
due to the coating (Fig. 5). Moreover, chitosan combined with heparin which is oppositely charged polymer electrolyte form a stent
coating through a LBL self-assembly method in order to promote reendothelialization.
Fig. 5 Focal laser scanning microscopy images of osteoblasts adhered to (A) original titanium and (B) LBL-modified (PEI/Gel/(Chi/Gel)3) titanium
surfaces after 24 h incubation. Initial cell seeding density was 5000 cells/cm2. Bar ¼ 20 mm. Osteoblasts attached to LBL-modified titanium film dis-
played more bundles (arrow) of actin microfilaments than that of the control sample. Reproduced with permission from Cai, K., et al. (2005).
Polysaccharide-protein surface modification of titanium via a layer-by-layer technique: Characterization and cell behaviour aspects. Biomaterials
26(30), 5960–5971.
Finally, PLGA microneedles were coated by using a spray LBL technique with DNA encapsulated lipid-modified PLGA nanopar-
ticles. PLGA microneedle arrays were immersed in solutions of polycationic protamine sulfate and polyanionic poly(4-styrene
sulfate). Additional multilayers were then deposited through alternating deposition of polycationic polymer-1 and polyanionic
plasmid DNA or PLGA NP to give pDNA or PLGA NP-coated arrays, respectively.
Polyelectrolyte LBL thin films can be assembled with nanometer scale control over spatial architecture and morphology, which
can control the release of multiple types of biomolecules, but this approach is not widely adopted. The reason for this, according to
Goodman et al. (2013), is that a few hundred layers are required to avoid a burst release of the biomolecules, rendering the LBL
method labor intensive, costly, and may lead to batch-to-batch variability. Also, LBL-coating process is typically performed using
acidic solution for efficient loading. However, this approach is not considered to be biomolecule friendly.
Conclusion
The performance of implantable medical devices is dictated by the surface of which it is implanted. Therefore, the implant’s surface
has to be modified in order to avoid any inflammatory response during implantation, for local drug delivery and to improve the
biocompatibility. Moreover, the coating must be homogenous without cracks covering most of the surface. Modern technologies
have been developed for the purpose of combating this problem. These techniques involve solvent-based deposition methods
such as spray coating and inkjet printing, plasma-coating techniques such as PECVD and HVOF and techniques which take advan-
tage the chemistry of the surface including the LBL method. Each of these technique offers a unique approach to deposit materials
on implant’s surface rendering the implantation of medical devices a low-risk clinical operation.
References
Bege, N., et al. (2012). Drug eluting stents based on poly(ethylene carbonate): Optimization of the stent coating process. European Journal of Pharmaceutics and Biopharmaceutics,
80(3), 562–570.
Choi, J., et al. (2014). Effect of solvent on drug release and a spray-coated matrix of a Sirolimus-eluting stent coated with poly(lactic-co-glycolic acid). Langmuir, 30(33), 10098–
10106.
Cotell, C. M., et al. (1992). Pulsed laser deposition of hydroxylapatite thin films on Ti–6Al–4V. Journal of Applied Biomaterials, 3(2), 87–93.
Gadow, R., Killinger, A., & Stiegler, N. (2010). Hydroxyapatite coatings for biomedical applications deposited by different thermal spray techniques. Surface and Coatings Tech-
nology, 205(4), 1157–1164.
Gill, H. S., & Prausnitz, M. R. (2007). Coated microneedles for transdermal delivery. Journal of Controlled Release, 117(2), 227–237.
Goodman, S. B., et al. (2013). The future of biologic coatings for orthopaedic implants. Biomaterials, 34(13), 3174–3183.
Hahn, B.-D., et al. (2011). Aerosol deposition of hydroxyapatite–chitosan composite coatings on biodegradable magnesium alloy. Surface and Coatings Technology, 205(8),
3112–3118.
Harris, L. G., et al. (2006). Bacteria and cell cytocompatibility studies on coated medical grade titanium surfaces. Journal of Biomedical Materials Research. Part A, 78A(1), 50–58.
Jang, D., Kim, D., & Moon, J. (2009). Influence of fluid physical properties on ink-jet printability. Langmuir, 25(5), 2629–2635.
Jelinek, M., et al. (1995). Effect of processing parameters on the properties of hydroxylapatite films grown by pulsed laser deposition. Thin Solid Films, 257(1), 125–129.
Le, H. P. (1998). Progress and trends in ink-jet printing technology. Journal of Imaging Science and Technology, 42(1), 49–62.
Mankani, M. H., et al. (2006). Canine cranial reconstruction using autologous bone marrow stromal cells. The American Journal of Pathology, 168(2), 542–550.
McGrath, M. G., et al. (2011). Determination of parameters for successful spray coating of silicon microneedle arrays. International Journal of Pharmaceutics, 415(1–2), 140–149.
Panácek, A., et al. (2006). Silver colloid nanoparticles: Synthesis, characterization, and their antibacterial activity. The Journal of Physical Chemistry B, 110(33), 16248–16253.
Reis, N., Ainsley, C., & Derby, B. (2005). Ink-jet delivery of particle suspensions by piezoelectric droplet ejectors. Journal of Applied Physics, 97(9). https://doi.org/10.1063/
1.1888026.
Scoutaris, N., et al. (2015). Development and biological evaluation of inkjet printed drug coatings on intravascular stent. Molecular Pharmaceutics, 13(1), 125–133.
Shah, N. J., et al. (2011). Tunable dual growth factor delivery from polyelectrolyte multilayer films. Biomaterials, 32(26), 6183–6193.
Tarcha, P. J., et al. (2007). The application of ink-jet technology for the coating and loading of drug-eluting stents. Annals of Biomedical Engineering, 35(10), 1791–1799.
Torrisi, L., & Setola, R. (1993). Thermally assisted hydroxyapatite obtained by pulsed-laser deposition on titanium substrates. Thin Solid Films, 227(1), 32–36.
Tsai, M.-H., & Hwang, W.-S. (2008). Effects of pulse voltage on the droplet formation of alcohol and ethylene glycol in a piezoelectric inkjet printing process with bipolar pulse.
Materials Transactions, 49(2), 331–338.
U.S. food and Drug Administration, Implants and prosthetics. 2015; Available from: http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ImplantsandProsthetics/.
Wu, P., & Grainger, D. W. (2006). Drug/device combinations for local drug therapies and infection prophylaxis. Biomaterials, 27(11), 2450–2467.
Yang, A., Cheng, C., & Lin, C. (2006). Investigation of droplet-ejection characteristics for a piezoelectric inkjet printhead. Proceedings of the Institution of Mechanical Engineers, Part
C: Journal of Mechanical Engineering Science, 220(4), 435–445.
Zeng, H., & Lacefield, W. R. (2000). XPS, EDX and FTIR analysis of pulsed laser deposited calcium phosphate bioceramic coatings: The effects of various process parameters.
Biomaterials, 21(1), 23–30.
Further Reading
Adden, N., et al. (2006). Phosphonic acid monolayers for binding of bioactive molecules to titanium surfaces. Langmuir, 22(19), 8197–8204.
Alghamdi, H. S., et al. (2013). Osteogenicity of titanium implants coated with calcium phosphate or collagen type-I in osteoporotic rats. Biomaterials, 34(15), 3747–3757.
Cai, K., et al. (2005). Polysaccharide-protein surface modification of titanium via a layer-by-layer technique: Characterization and cell behaviour aspects. Biomaterials, 26(30),
5960–5971.
Dinda, G. P., Shin, J., & Mazumder, J. (2009). Pulsed laser deposition of hydroxyapatite thin films on Ti–6Al–4V: Effect of heat treatment on structure and properties. Acta
Biomaterialia, 5(5), 1821–1830.
Jang, H. W., et al. (2011). Surface characteristics and osteoblast cell response on TiN- and -coated Ti implant. Biomedical Engineering Letters, 1(2), 99–107.
Porous Biomaterials and Scaffolds for Tissue Engineering
Liliana Liverani, University of Erlangen-Nuremberg, Erlangen, Germany
Vincenzo Guarino, National Research Council of Italy, Naples, Italy
Vincenzo La Carrubba, University of Palermo, Palermo, Italy
Aldo R Boccaccini, University of Erlangen-Nuremberg, Erlangen, Germany
Tissue Engineering and Scaffolds Requirements 188

Porous Scaffolds Fabrication Techniques 189
Electrospun Scaffolds 189
Principle/Mechanism at the Basis of the Technique 189
Advantages and Limitations of the Techniques 191
In Vitro/In Vivo/Clinical Applications 191
Polymeric Foams and Scaffolds 192
Advantages and Limitations of the Technique 192
Polymeric foams produced by TIPS 192
Polymeric membranes produced by DIPS 193
In Vitro Applications 193
Inorganic Scaffolds 193
Inorganic Porous Scaffold Fabrication Techniques 194
Scaffolds Fabricated by Additive Manufacturing 194
Advantages and Limitations of the Technique 195
Biomaterials printing 195
Tissue and organ printing 196
In Vitro/In Vivo Clinical Applications 197
Integration of Different Scaffolds Fabrication Techniques 198
Conclusions and Future Perspectives 199
Acknowledgments 199
References 199
Tissue Engineering and Scaffolds Requirements
Tissue engineering is an emerging field proposing a novel approach for repairing, improving, and restoring tissue and organ func-
tion. Its definition started in the 1990s and evolved through the last decades (Langer and Vacanti, 1993; Vacanti and Langer, 1999;
O’Brien, 2011). Tissue engineering approach aims to overcome all the limits presented by the current treatments for tissue replace-
ment, like autografts, homografts, or xenografts. In fact, this approach is based on the use of functional three-dimensional scaffolds
fabricated by using biodegradable biomaterials, having the function to support, promote, and enhance the new tissue formation.
Scaffolds could be fabricated by using polymeric, ceramic, metallic, and composite materials suitable for different tissue target of
the regeneration and should be characterized by the properties briefly reported as follows. Biocompatibility is a property related to
the host tissue response, and it was introduced as fundamental properties of biomaterials in the 1940s regarding implantable
medical devices. The first definitions considered biocompatible less reactive or inert materials, while the evolution of the definition
implies a suitable and appropriate host response, related to the specific anatomical site. Recently, in the last years, a definition of
biocompatibility specifically addressed to scaffolds was stated by Williams (2008), and it refers to the scaffold’s ability to “support
the appropriate cellular activity, including the facilitation of molecular and mechanical signaling systems, in order to optimize
tissue regeneration, without eliciting any undesirable local or systemic responses in the eventual host” (Williams, 2008). Biodegrad-
ability is another pillar of scaffold characteristics; in fact, the kinetic of the scaffold degradation should be synergic with the new
tissue formation, allowing an initial support from mechanical and morphological point of view, suitable for cell adhesion, migra-
tion, and colonization of the scaffolds, supported eventually also by biomolecules release. Degradation rate and progressively
decrease in mechanical properties are strictly related to the selection of the appropriate materials, combined also with the scaffold
surface functionalization with biomolecules or growth factors that enhance the new tissue formation (Hutmacher, 2001).
Scaffold mechanical properties play a pivotal role in an effective scaffold design and should be tailored according to the tissue
target of the regeneration. In fact, the scaffolds should retain the desired shape, facilitating the implant in vivo and should have
similar or comparable mechanical strength of the healthy native tissue surrounding the implant. Scaffold mechanical and structural
integrity should not be lost before the complete neotissue formation (Karande et al., 2014).

Biomaterials: Science and engineering j Porous Biomaterials and Scaffolds for Tissue Engineering 189
Scaffold surface properties are fundamental to improve and enhance cell response, adhesion, and migration. The functionaliza-
tion of scaffold surface and the related release of biomolecules could be enhanced by appropriate surface scaffold morphologies,
like fibrous structure (Karande et al., 2014).
Porosity and pore interconnectivity, finalized to cell migration, oxygen and nutrient permeation and transport, and metabolite
removal to improve tissue vascularization and new tissue formation, are also fundamental for the regeneration of all the tissues.
Appropriate scaffold porosity and pore interconnectivity should be compromised with decreased or suboptimal scaffold mechan-
ical properties and stability (Loh and Choong, 2013). In particular, recently, many research works have been focused on the fabri-
cation of scaffolds with controlled three-dimensional architecture to enhance the biomimetic approach including the fabrication of
scaffolds able to mimic the native healthy tissue in terms not only of composition but also in particular of morphology, three-
dimensional structure, and therefore cell response (Hollister, 2005). Usually, referring to scaffold pore size, it is not commonly
applied the classification introduced by IUPAC (Everett, 1972), which defined the pores exceeding 50 nm as macropores, the
one not exceeding 2 nm as micropores, and the intermediate size between 2 and 50 nm as mesopores. Considering the average
cell dimensions and that the pore size needed to fulfill the scaffold requirements explained earlier, this classification seems to
not report an appropriate range of pore size suitable for tissue engineering applications. In order to ensure all these functional
requirements, the selection of the appropriate biomaterials is fundamental. In particular, it is possible to fabricate polymeric,
ceramic, metallic, and composite scaffolds. Fabrication techniques and scaffold properties regarding polymeric, ceramic, and their
composite scaffolds are analyzed more in details in the next paragraphs. Metallic scaffolds are not suitable for applications in soft
tissue regeneration, and they are mainly use for hard tissue regeneration as bone tissue engineering, in particular for load-bearing
areas, because of metal fatigue resistance and high compressive strength. There are some limits and disadvantages in the use of
metals for scaffold fabrication in particular for the obtainment of controlled internal scaffold architecture with conventional
processes. In particular, the limited number of biodegradable metals (mostly used are magnesium (Mg) (Yazdimamaghani
et al., 2017), iron (Fe) (Mohd Daud et al., 2014), and their alloys) and the metal degradation rate in the human body are pivotal
parameters to take into account during the scaffold design and fabrication (Wang et al., 2016). In vitro and in vivo characterizations,
by using animal models, are fundamental to assess cell–scaffold interactions and their capability to enhance tissue regeneration
(Peric et al., 2015).
Porous Scaffolds Fabrication Techniques
As reported in the previous paragraph, porosity is a key scaffold characteristic. In particular, the pore size and interconnectivity play
a pivotal role in triggering cell–scaffold interactions. Several scaffold fabrication techniques could be used for the obtainment of
porous scaffolds; the main characteristics of these techniques are summarized in Table 1.
Salt-leaching technique is based on the use of a porogen material (i.e., salt crystals), which is added to the polymeric solution
and subsequently removed by dissolution of the porogen in a proper solvent. The pore size is related to the size of the porogen used
(Aramwit et al., 2015). In the gas foaming process, the gas is used as porogen for the formation of porous structure (Mikos and
Temenoff, 2000), and it is possible to remove the leaching step during the fabrication process. As a drawback, it is not possible
to control the pore size and interconnectivity, but it is possible to combine this technique with other scaffold fabrication techniques
to improve the control on these two key parameters (Joshi et al., 2015). The scaffold fabrication techniques based on the phase
separation, electrospinning, additive manufacturing, and foam replica method processes are described in details in the following
paragraphs. In the freeze-drying technique, the formation of pores is based on the sublimation of the frozen water into the gas
phase. The porosity and pore size are strictly related to the process parameters (like gradient of temperature and time of the process)
and solution parameters (like water-to-polymer ratio and viscosity of the solution) (Zhang et al., 2014).
There are different techniques for the measurement and the evaluation of scaffold porosity and pore size. The total porosity,
expressed in percentage, is often calculated starting from the bulk density of the scaffold, and it is related to the amount of pores
presented in the scaffolds. Physical characterization could be used for the assessment of the porosity as gravimetric method, mercury
porosimetry, gas adsorption analysis, liquid displacement method, etc. Also, imaging techniques (i.e., scanning electron microscopy
analysis and microcomputed tomography imaging) could be exploited for the evaluation of scaffold porosity and pore size (Loh
and Choong, 2013).
Electrospun Scaffolds
Principle/Mechanism at the Basis of the Technique
The electrospinning technique is based on the application of a high electric potential between two electrodes of opposite polarities.
This high voltage is able to overcome the surface tension inside a polymeric solution, or melt, allowing the complete evaporation of
the solvent and the formation of fibrous structure. This technique is widely used for the fabrication of nano- and microfibrous scaf-
folds, resembling the morphology of the extracellular matrix (ECM) (Teo et al., 2011).
The standard setup is composed of a high-voltage generator, a syringe pump able to control the solution flow rate, a syringe with
a metallic needle, and a grounded fiber collector. There are three categories of parameters affecting the process and the properties of
the obtained electrospun fibers: parameters related to the solution properties (i.e., polymer concentration, solvent system,
190
Biomaterials: Science and engineering j Porous Biomaterials and Scaffolds for Tissue Engineering
Table 1 Summary of the characteristics of the different scaffold fabrication techniques
Scaffold fabrication
technique Advantages Disadvantages Range of pore size (mm) Applications (tissue target of regeneration)
Salt leaching - Pore size related to porogen particle size - Not control on pore interconnectivity Pore size related to porogen particle size Many fields of applications, in particular
- Versatile in combination with other scaffold - Low mechanical properties respect to the (usually in the range of 100–500) related to other scaffold techniques, like
fabrication techniques, in particular to native tissues (not suitable for hard tissue wound healing (Aramwit et al., 2015), bone
improve the pore interconnectivity regeneration) tissue engineering (Sadiasa et al., 2014)
- Possible residual of porogen or toxic
solvents not suitable for biomedical
applications
Gas foaming - Avoid the leaching step in the scaffold - Not control on pore size and on pore Pore size up to 100 Many fields of applications, in particular
fabrication process interconnectivity related to other scaffold techniques, like
- Reduced use of solvents - Low mechanical properties respect to the skin tissue engineering (Poursamar et al.,
native tissues (not suitable for hard tissue 2016)
regeneration)
Phase separation Avoid the leaching step in the scaffold - Addition of organic solvents could inhibits 1–100 Bone tissue engineering (Rezabeigi et al.,
fabrication process the incorporation of biomolecules during the 2016), vascular tissue engineering (Pavia
process et al., 2013)
- Small pore size not suitable for all the
application
- Low mechanical properties respect to the
native tissues (not suitable for hard tissue
regeneration)
Freeze drying High temperature and leaching step not - Longtime processing 50–200 Cartilage tissue engineering (Vishwanath
required during the process - Low mechanical properties respect to the et al., 2016); osteochondral tissue
native tissues (not suitable for hard tissue engineering (Levingstone et al., 2016)
regeneration)
Additive Control of the geometry, pore size, and - Constrains in the process of some 100–500 Bone tissue engineering (Wang et al., 2016),
manufacturing interconnectivity polymer/biomaterials cartilage tissue engineering (Jessop et al.,
2016), organ printing (Murr, 2016)
Electrospinning Control over fiber diameter and related - Limited mechanical properties From 1 to 500 Mainly soft tissue engineering applications
openings between the fibers - Required the integration with other and interface tissue engineering if combined
techniques for the fabrication of bulk 3-D with other scaffold fabrication techniques
scaffolds (O’Connor and McGuinness, 2016)
Foam replica Control over the pore size and pore - High values of porosity but reduced Pore size related to the foam (usually Bone tissue engineering (Miguez-Pacheco
method interconnectivity, related to the selected mechanical properties in the range of 100–600) et al., 2016; Baino et al., 2016)
sacrificial foam
conductivity, viscosity and type (solution vs. melt electrospinning) of the solution, its volatility, dielectric effect, and surface
tension), electrospinning process parameters (i.e., applied voltage, feed rate of polymeric solution, its temperature, capillary-to-
collector distance, effect and type of collector, needle diameter, and configuration of the nozzle), and environmental properties
(like temperature and relative humidity) (Putti et al., 2015).
The fabrication of porous electrospun scaffolds and in particular the obtainment of macroporosities in the electrospun mats has
been the focus of many researches in the last years (Rnjak-Kovacina and Weiss, 2011). In particular, besides the combination with
other scaffold fabrication techniques, it is not often possible to achieve a pore size and pore interconnectivity appropriate for allow-
ing cell infiltration inside the scaffold structure (Rnjak-Kovacina and Weiss, 2011).
Advantages and Limitations of the Techniques

The electrospun mats have a fibrillar structure resembling the morphology of the native extracellular matrix (ECM), having the
advantage of representing a suitable example of biomimetic approach in scaffold fabrication (Woods and Flanagan, 2014),
enhanced also by the possibility to obtain aligned fibers resembling the native tissue anisotropy. In fact, this technique is versatile
because it allows the use and the processability of a huge number of natural and synthetic polymers and their blends, the possibility
to select different type of solvents (Shenoy et al., 2005), with particular focus in the recent research works on the use of benign
solvents and on green electrospinning (Sun et al., 2010). In particular, the use of polymeric blends of natural and synthetic polymers
could be interesting to trigger the polymer degradation rate, as showed in Fig. 1, in which electrospun mats obtained from a blend of
poly (ε-caprolactone) (PCL) and zein showed fiber structure degradation after 1 day and 1 week of immersion in phosphate-
buffered saline (PBS) solution, demonstrating an increased degradation rate respect to the neat PCL. The control over the fiber
degradation rate is relevant to improve cell adhesion and enhance the new tissue growth and also for the release of drugs or particles
embedded in the polymeric solution.
It is also possible to obtain composite electrospun fibers by adding a different phase in the polymer matrix; in particular, the
most commonly used have been hydroxyapatite particles (Liverani et al., 2014), bioactive glasses particles (Liverani and Boccaccini,
2016; Gönen et al., 2016; Lepry et al., 2016), and ß-tricalcium phosphate particles (Erisken et al., 2008) relevant and suitable for
bone tissue engineering applications (Liverani et al., 2016). Particularly, relevant results were obtained in terms of preservation of
bioactive glass particle bioactivity, even if embedded in polymeric electrospun fibers (Liverani and Boccaccini, 2016; Gönen et al.,
2016; Lepry et al., 2016). It is also possible to obtain different fiber functionalizations with biomolecules, like growth factors (Li
et al., 2006; Fu et al., 2008b). As reported in the previous section, the obtainment of an appropriate pore size and effective scaffold
pore interconnectivity in the electrospun mats could be challenging, and different techniques have been used to increase the pore
size, like the increase of the fiber diameter, the use of different fiber collectors, wet electrospinning, the use of a sacrificial polymer to
add in blend before the electrospinning, the addition of surfactant in the solution before the electrospinning, cryogenic electrospin-
ning, and the combination with salt leaching and gas foaming (Rnjak-Kovacina and Weiss, 2011).
In Vitro/In Vivo/Clinical Applications

Many studies have been focused on the investigation of cell–electrospun fiber interactions, in particular for the evaluation of the
effect of fiber alignment (Jin et al., 2017). Those studies about the applications of 3-D structure and gradient morphologies are
particularly relevant, in particular for bone (Obata et al., 2013), cartilage (Chen et al., 2016b), and vascular tissue engineering
(Ju et al., 2010).
Even if in vitro tests play a fundamental role in the evaluation of scaffold performance in terms of cell response and new tissue
formation, in vivo studies represent the gold standard in the preclinical step before human clinical trials. During the design of an
in vivo experiment is pivotal to select the proper animal model considering the tissue target of the regeneration (i.e., in the case of
vascular or bone tissue (Rocco et al., 2014; Peric et al., 2015)). For what concerns the clinical applications, there are several electro-
spun products already available on the market, like coronary balloon expandable stent system (Papyrus, Biotronik AG and Bioweb,
Zeus Inc.), vascular access graft (AVflo, Nicast Ltd.), dural patch (ReDura, Medprin Regenerative Medical Technologies Co. Ltd.), and
synthetic bone (Rebossis, Ortho ReBirth Co. Ltd.).
Fig. 1 SEM micrographs of electrospun mats obtained started from a blend of PCL and zein: as-spun (A) after 1 day of immersion in PBS solution
(B) and after 1 week of immersion in PBS solution (electrospun mats fabricated and characterized by the authors, unpublished data).
192 Biomaterials: Science and engineering j Porous Biomaterials and Scaffolds for Tissue Engineering
Polymeric Foams and Scaffolds

The largest part of porous structure is produced by techniques based on liquid–liquid phase separation of polymer solutions (van de
Witte et al., 1996a), which still seem to be the most promising, flexible, and tunable route for the preparation of 3-D interconnected
polymeric porous structures, owing to its versatility and ease of operation and to the vast latitude of achievable morphologies. It has
been shown by several attempts in the last few years that the thermodynamics of phase separation plays a crucial role in determining
the final structure (Matsuyama et al., 2000). Depending on the use of a thermal or diffusive driving force, the technique is often
called thermally or diffusion-induced phase separation (TIPS or DIPS, respectively) (Akki et al., 1999). Along decades, many
research groups have studied the thermodynamic and kinetic properties of polymer solutions (Akki et al., 1999; Han, 2000), in
order to achieve a better control in membrane and foam production processes. The most widely model system studied is a ternary
solution composed of a polymer, a solvent, and a nonsolvent, which has the function of promoting phase separation. The typical
mechanism of phase separation of polymer solutions is liquid–liquid demixing leading to a polymer-rich and a polymer-lean phase
(Han, 2000), according to a binodal demixing or to a spinodal decomposition or to a combination of both. The former, charac-
terized by nucleation and growth, takes place inside the metastable region (between the binodal and the spinodal curve), while
the second takes place inside the spinodal region (unstable). The morphology developed during a phase separation process is there-
fore crucially affected by the particular path followed in the thermodynamic diagram.
If the prevailing mechanism is nucleation and growth, a porous structure with poorly interconnected cells is obtained, while
spinodal decomposition gives rise to an interconnected network (Gong et al., 2006). The growth and coalescence of the
polymer-lean phase leads to a porous structure according to phase separation path, which depends upon the utilization of a binary
system (polymer/solvent) and/or of a ternary system (polymer/solvent/nonsolvent). After polymer solidification and solvent
removal from the demixed solution, the space that was occupied by the solvent will create voids (pores).
By changing the polymer concentration, the solvent, the cooling rate, and the final temperature, it was then possible to vary the
phase separation path leading to scaffolds exhibiting different micro- and macrostructures and final properties (Ho et al., 2004).
Among the polymers employed for preparing scaffolds via phase separation routes, poly-L-lactide (PLLA) occupies a predominant
role. With this respect, van de Witte et al. (1996b) studied the influence of solid–liquid demixing, liquid–liquid demixing, and vitri-
fication on PLLA membrane morphology obtained via immersion precipitation. Chloroform and methanol were used as solvent
and nonsolvent, respectively. In a different study, the same research group applied the relations between phase diagram and
membrane morphology for the ternary PLLA–chloroform–methanol system to PLLA–dioxane–methanol, PLLA–dioxane–water,
and other ternary systems (van de Witte et al., 1996c).
Advantages and Limitations of the Technique

Polymeric foams produced by TIPS
Thermally induced phase separation (TIPS) is a widely used technique to prepare scaffolds for tissue engineering purposes (Gong
et al., 2006) and porous membranes (Tanaka and Lloyd, 2004). It involves cooling a homogeneous polymeric solution to a temper-
ature where the single-phase system becomes thermodynamically unstable and spontaneously separates into a polymer-rich and
a polymer-lean phase. Generally, the system used is a ternary solution of a polymer, a solvent, and a nonsolvent. Thermodynamic
diagrams provide information about a possible phase separation process, depending on temperature, pressure, and composition.
Moreover, composition of separated phases and nucleating phase(s) is determined by thermodynamics. The dense, microporous,
macroporous nucleating phase, in its turn, determines the type of microstructure obtained. Therefore, knowing the phase diagram
of a given system can aid scientists in choosing the appropriate processing conditions, to obtain the desired microstructure. Never-
theless, the construction of a phase diagram for a ternary polymeric system is not straightforward. Experiments for the determina-
tion of tie lines are time-consuming. Moreover, as commercial polymers are polydisperse, phase diagrams lose their deterministic
accountability. A more straightforward way to get experimental information about phase separation is the determination of cloud-
point curves (Barton and McHugh, 1999); however, this method is not fully quantitative. A more precise way to carry out turbidity
measurement is to adopt light transmission detection methods (Bulte et al., 1996). Besides thermodynamic properties, even kinetic
features must be considered in the design of a TIPS protocol. Phase separation kinetics depends on temperature, composition, cool-
ing rate, and demixing time. Each parameter affects the final morphology of porous structure (Nunes and Inoue, 1996). An overview
of the morphology exhibited by the foams allows one to infer that a multilevel hierarchical structure sets in, like often it occurs in
complex processing procedures inducing structure gradients, for example, injection molding of polymers.
As a matter of fact, at least three hierarchically ordered structural levels might be recognized (Pavia et al., 2008): a “macroporos-
ity” level, with pores characterized by dimensions up to 10 mm, depending upon the experimental conditions (viz., demixing
temperature and time); an “interconnection” level, whose effectiveness relies on the number of interconnecting channels per
pore (with a characteristic size of the order of ca 10 mm); and a “microporosity” level, appearing when very long residence times
in the metastable region are applied. The foams prepared at lower a demixing temperatures (25 C) present a constant pore size
independent of demixing time, whereas at higher demixing temperatures (30 and 35 C), a classical sigmoidal shape is observed,
with a final plateau level depending upon the demixing temperature (in the range of 40–100 mm). Pore size results can be inter-
preted on the basis of a balance between nucleation and growth processes. A slight variant of the aforementioned approach consists
in the use of an innovative experimental apparatus able to imposedvia Peltier cellsddifferent thermal histories on two sample
surfaces, thus giving the opportunity to produce nonhomogeneous foams. Although the underlying physical principles are the same
regarding phase separation in homogeneously cooled polymer solutions, this technique allows the fabrication of foams with pore
size monotonously varying along the thickness within a single sample, thus obtaining a material with features not accessible via
a “classical” TIPS approach (Mannella et al., 2015). This type of scaffold structure has promising tissue engineering applications
wherever a hierarchical architecture involving morphological variations in space is required, for example, in bone tissue repair.
Polymeric membranes produced by DIPS

The production of polymeric membranes via diffusion-induced phase separation (DIPS) has been widely studied and applied for
a number of model systems. The main steps of membrane formation through DIPS protocol are the casting of a polymer solution
on a support and the immersion into a coagulation bath, usually composed of a nonsolvent. The mutual exchange of solvent and
nonsolvent will modify the casted solution composition, inducing phase separation when the equilibrium curve is crossed. The
liquid–liquid phase separation is responsible for the formation of a porous membrane, and it can occur via nucleation and growth
and/or spinodal decomposition mechanisms. Local composition determines the demixing mechanism and the amounts of sepa-
rated phases, according to its location in phase diagram. As a general rule, the immersion in a coagulation bath composed only
of nonsolvent will produce a porous membrane but with a dense skin; this was related to a rapid increase of polymer concentration
at the interface, owing to a fast outward solvent diffusion, which induces crystallization. This fact has been inferred by mass transfer
modeling, providing an estimate of concentration profiles inside the polymeric film (Reuvers et al., 1987). Several experimental
studies regarding to the formation of PLLA membranes by immersion precipitation have been reported in literature (Zoppi
et al., 1999), where the effect of different solvents and nonsolvents, casting and coagulation bath concentration, and temperature
were investigated to control the membrane morphology. However, some points, related to the external surface morphology and the
influence of drying step, need to be further investigated and clarified. As a matter of fact, membranes prepared via DIPS ordinarily
show a completely closed and nonporous external surface, owing to a local polymer concentration increase (Bulte, 1996). This fact
reduces sensibly the overall membrane permeability, thus affecting the choice of potential applications. A number of solutions to
this limitation were investigated, for example, employing a coagulation bath composed of both nonsolvent and solvent (Wijmans
et al., 1983) or multiple coagulation baths (Yang et al., 2007) adopting a sacrificial layer approach (Li et al., 2008) or wetting both
surfaces with a modified spinneret (He et al., 2003). Furthermore, the influence of desiccation step, that is, of solvent removal on the
morphology of external surface needs to be accounted for. In typical DIPS practice, the membrane is washed or dried immediately
after the immersion step; however, researchers often neglect the effects of this last operation on the membrane microstructure. As
a matter of fact, during solvent removal, the system composition will vary; thus, an influence on membrane morphology can be
reasonably expected (Montesanto et al., 2015).
In Vitro Applications
Porous and biodegradable PLLA scaffolds for vascular tissue engineering applications, with a vessel-like shape, can be produced by
a two-step experimental protocol, including a dip coating of a viscous polymer solution around a nylon fiber and a diffusion-
induced phase separation (DIPS) by immersion into an antisolvent bath (Pavia et al., 2013). The first step (dip-coating bath,
PLLA/dioxane solution) allows the production of the tubular structure of the vascular graft, whereas the second (DIPS) is respon-
sible for the interesting level of porosity and for the surface microporosity as potentially ensuring nutrient and metabolites exchange
during blood flow. The as-prepared scaffold can be utilized either as vascular grafts or embedded into a porous scaffold for the
regeneration of an injured tissue as a pseudoperipheral circulatory system, with the aim of promoting a rapid vascularization of
the whole tissue-engineered construct (Pavia et al., 2013). A cell culture inside the vessel-like scaffolds was carried out, by using
endothelial cells (EC), which are the solely components of capillary bed and the first to form during embryonic development. After
21 days, the internal lumen of the scaffold is totally covered by EC, which have organized themselves into a well-differentiated
vessel structure. Cells shown to form stable cell–cell interactions and spindle membrane protrusion, characteristic of mesenchymal
endothelial phenotype, were not observed. These results suggest that the scaffold produced could be usefully employed in vascular
tissue engineering applications (Pavia et al., 2013). In another research study, mouse mesoangioblasts (A6) were seeded onto bidi-
mensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA) prepared according to the protocol
described in (Carfì-Pavia et al., 2009), in the presence or absence of a type I collagen coating. Results show that PLLA films allow
direct cell adhesion and represent an optimal support for cell growth (Carfì-Pavia et al., 2009).
Inorganic Scaffolds
Inorganic biomaterials, like hydroxyapatite, other calcium phosphate phases, apatite–wollastonite glass ceramics, and bioactive
glasses, have been commonly used for tissue engineering applications, in particular for bone-tissue-related applications (Bose
et al., 2012), because of the similarity with the mineral phase of native bone, but they could also find other applications in contact
with soft tissues (Miguez-Pacheco et al., 2015). The use of the aforementioned inorganic biomaterials is suitable for tissue engi-
neering applications because of their bioactivity, defined as the property of the materials, like bioactive glasses and bioactive glass
ceramic, to form interfacial bonding with tissues (Cao and Hench, 1996). In healthy native hard tissue, the mechanism of apatite
biomineralization occurs as a self-remodeling process leaded by bone cells and proteins, but it also possible to detect mineralization
on the surface on bioactive inorganic materials, like crystalline calcium phosphate and bioactive glasses, acting as an interface for the
integration with tissues (Kim et al., 2004). The use of bioactive glasses is also valuable for tissue engineering applications because of
their angiogenic, osteogenic, and antibacterial properties (Hoppe et al., 2011; Jones et al., 2006). Despite these properties, these
inorganic biomaterials showed also low mechanical strength and low fracture toughness, limiting their applications in load-
bearing tissue regeneration (Gerhardt and Boccaccini, 2010).
Inorganic Porous Scaffold Fabrication Techniques

Different scaffold fabrication techniques could be used for the production of inorganic porous scaffolds, like foam replica method,
foaming techniques, freeze casting, and additive manufacturing. It is also possible to incorporate the inorganic particles in a poly-
meric matrix and subsequently process it to fabricate porous composite scaffolds by using other techniques, as electrospinning or
TIPS. A brief description of these techniques is reported in this section. The foam replica method, used for scaffold fabrication since
2006 (Chen et al., 2006) is based on the use of a sacrificial porous template, like polyurethane foam, for the obtainment of a positive
replica of this template. In details, the bioactive glass particles were dispersed in a slurry with a binder, that is, polyvinyl alcohol
(PVA); then, the template foam is immersed in the slurry and coated with it; during a heat treatment, the sacrificial foam is burned
out, and the bioactive glass is sintered at high temperature, an example of the scaffold morphology that it is possible to obtain is
reported in Fig. 2.
The application of this technique is not limited to a specific bioactive glass composition, allowing the fabrication of scaffolds
with different type of bioactive glasses (Chen et al., 2006; Fu et al., 2008a) and also enhancing the scaffold functionalities, by using
ion-doped bioactive glasses (Hoppe et al., 2013). The obtained scaffolds usually could reach total porosity values up to 80%–90%;
that value plays a crucial role in inducing in vivo vascularization, as reported by Arkudas et al. (2013), but the obtainment of this
high porosity implies scaffolds’ low mechanical properties. In order to reinforce the scaffold structure, different solutions were
proposed, as the use of different type of sacrificial templates with different porosities, in order to compromise a decrease the total
porosity value and an increase in the mechanical properties (Boccardi et al., 2015), or it is possible to perform polymeric coating to
enhance the scaffold mechanical properties, for example, by using gelatin (Desimone et al., 2013) or blend of natural and synthetic
polymers (Fereshteh et al., 2015).
Other porous scaffold fabrication processes are the foaming techniques. These approaches are based on the formation of pore
and based on the introduction of gas (Jones and Hench, 2003), with the limit of the lack of control in the pore size or in the use of
surfactants inside a bioactive glass slurry, before the aging and the sintering process (Sepulveda et al., 2002). Few research works are
focused on the use of freeze drying for the fabrication of inorganic porous scaffolds; in fact, this technique is consolidated for the
fabrication of polymeric or composite (polymeric scaffolds with incorporated inorganic phase). The use of this technique has still
the limit in the pore size that it will be possible to obtain; in fact, it is not possible to obtain pore bigger than 100 mm (Mallick, 2009;
Song et al., 2006).
Interesting and promising results were obtained from in vivo studies about inorganic porous scaffolds (Fu et al., 2010; Living-
ston et al., 2002), even if there are not still available clinical applications of these porous scaffolds.
Scaffolds Fabricated by Additive Manufacturing

In the last years, additive manufacturing (AM) technologies have imposed themselves as the most appropriate method for the
production of ordered 3-D structures to be successfully used as instructive scaffolds for tissue and organ regeneration. They are
Fig. 2 SEM micrographs of 45S5 bioactive glass-based porous scaffold (A) and higher magnification of the wall of the pore (B) (porous scaffolds
fabricated and characterized from the authors, unpublished data).
generally based on layer-by-layer fabrication strategies that allow manufacturing solid platforms with complex shapes and micro-
structures from three-dimensional (3-D) model data, showing high degree of automation, good accuracy, and reproducibility.
Therefore, they allow finely controlling morphological patterns to exert all the structural functionalities required to support viability
of cells within the 3-D printed structure. In the last 10 years, several studies have widely demonstrated the enormous potential of
AM technologies to design tailor-made scaffolds to properly guide cell activities for the regeneration of different kinds of soft and
hard tissues (Billiet et al., 2012). Strong efforts have been spent to improve current technology based on the AM principia to further
enhance the control of morphological features (i.e., pore size distribution, pore volume, and pore interconnectivity, anisotropy) or
to implement less invasive processing routes able to more easily manipulate recently synthesized biomaterials with biological func-
tionalities (i.e., modified proteins and polysaccharides, smart polymers, and new bioactive hydrogels) (Forbes and Rosenthal,
2014). Three-dimensional platforms fabricated by properly adapted AM technologies are also successfully emerging as 3-D
in vitro models able to bridge traditional cell culture and in vivo modeling; indeed, they have been used to predict relevant aspects
of in vivo behavior, only traditionally assessed by animal implants and/or human trials (Khademhosseini et al., 2006). Besides, it is
well-known that traditionally used in vitro 2-D models show significant limitations to recapitulate the complex tissue microenvi-
ronment due to the innate tendency of cells to mimic in vivo behavior when grown in 3-D conditions (Fischbach et al., 2007). In
this context, novel approaches based on 3-D bioprinting can combine main advantages of consolidated rapid prototyping (RP)
techniques with innovative biofunctionalization strategies, thus providing much more physiologically relevant information about
organogenesis, disease progression, and molecular release onto specific targets.
Advantages and Limitations of the Technique

To date, a large variety of 3-D printing-based techniques have been implemented to design 3-D scaffolds for the replacement of
tissues and organs. Each technique presents benefits and disadvantages in terms of feasibility, material processability, strut resolu-
tion, and productivity. A first classification may be attempted based on the working principle, that is, inkjet, laser-assisted tech-
niques, electrically assisted induced atomization, pneumatic or screw extrusion, and stereolithographic techniques. Other
classifications of AM technologies mainly refer to the implementation of custom-made setups to realize complex geometries during
the printing process (Onuh and Yusuf, 1999) with the support of tailor-made dispensing devices (i.e., laser-, nozzle-, or printer-
based systems) (Billiet et al., 2012). As for biomedical applications, an alternative but useful classification is between AM and
cell-friendly AM technologies depending upon (a) the processing of biomaterials in the form of 3-D bioinspired scaffolds or
(b) the creation of 3-D tissue/organ analogues by the combination of biomaterials with living cells (i.e., bioinks), as summarized
in Fig. 3.
Biomaterials printing
In the class of AM technologies to process biomaterials, it may be included traditional 3-D printing technology used to create 3-D
ordered structures made of ceramic, metal, plastic, and polymers (synthetic or natural ones) via layer-by-layer approach. This
process is commonly guided by 3-D modeling softwaredthat is, computer-aided design (CAD) or computer tomography (CT)
scan imagesdable to physically reproduce the CAD/CAM sketch with the support of automatic printing equipment. Their working
principle may drastically vary as a function of selected materials and desired structural/functional features by the application of
different driving forces, that is, UV or laser light, electrostatic, piezoelectric, pressure, thermal, thermodynamic, or magnetic
ones. Among them, most commonly used technologies to fabricate layer-by-layer structures are based on ultraviolet (UV) light pho-
topolymerization. They include stereolithography (SLA) (Skoog et al., 2014), digital light processing (DLP) (Wallace et al., 2014),
and selective laser sintering (SLS) (Ko et al., 2007) that reproduce complex designs by fast processing and high resolution. These
methods allow fabricating 3-D scaffolds by hardening a photopolymer resin under the controlled exposure to UV light or another
similar power source. In these cases, much attention is required for the selection of cytocompatible photoinitiators to minimize
damaging effects on cell membrane, protein, and nucleic acids, ascribable to the formation of free radicals that may potentially
restrict their use in tissue engineering applications (Hutmacher et al., 2004). In tissue engineering, another relevant benefit of photo-
polymerization mainly concerns the suitability to fabricate hydrogel-like scaffolds. In the past, all the main photopolymerization
techniques generally worked by a bottom-up scheme; it means that the object was built from a fabrication support just below the
resin surface. Subsequent layers were cured on the top of the previous layers by irradiation from earlier (Liska et al., 2007). Only
recently, alternative approaches based on top-down setups are emerging with enormous success (Melchels et al., 2010). Focusing on
the fabrication of polymer or composite scaffolds, the most commonly used technologies are based on extrusion processes such as
fused deposition modeling (FDM) (Drummer et al., 2012). In this case, thermoplastic polymers are extruded from the 3-D printer
head under melting conditions until forming thin filaments that can create 3-D architectures by adding layer by layer (Guarino et al.,
2012). FDM allows for an accurate control over scaffold morphological properties, despite their use is restricted in the case of poly-
mer melt with relatively high viscosity that can be hardly extruded through small diameter nozzle. Independently, upon the intrinsic
viscosity of melt polymer solutions, fiber diameter cannot achieve average size down to 100 mm. Hence, similar technologies have
been properly revisited to process polymer solution or slurry (i.e., 3-D colloidal inkjet printing) in order to drastically scale down
strut resolution, that is, about 0.5–10 mm as average diameter. However, the generation of clinically relevant bioinspired systems
from such colloidal inks shows relevant problems in terms of regulatory perspective, which drastically limit their use for medical
applications (Smay et al., 2002).
Biomaterial printing Tissue/Organ printing
• Selective laser sintering (SLS)

Powder based
• 3D printing (3DP)
• Stereolithography (SLA) • Laser assisted bioprinting (LaBP)

Light based
• Digital light processing (DLP) • Biological Laser Printing (BioLP)
• Fused deposition modeling (FDM) • Bioextrusion

• Precise extrusion deposition (PED) Melt based • Bioplotter
• Melt electrospinning writing (MEW)
• Pressure assisted mycrosyringe (PAM) • Thermal/piezoelectric ink-jet

• Low temperature assisted manufacturing (LDM) Solution based
• Electrofluidodynamic ink-jet printing (EIP)
• Robocasting
AM Technologies
Fig. 3 Classification of AM technologies for biomaterials and cell printing.
Tissue and organ printing

Recent discoveries in medical and materials science have enabled for the development of new AM technologies to design biocom-
patible materials in concert with cells to form complex 3-D functional biohybrid tissues that better address strong need of regen-
erative medicine to ex novo fabricate tissues and organs suitable for transplantation. Compared with biomaterial printing strategies,
this approach involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and
technical improvements related to the sensitivities of living cells and the construction of tissues (Murphy and Atala, 2014). To date,
biological AM technologies have already been used for the generation and transplantation of several tissues (i.e., bone, vascular
grafts, trachea, myocardium, skin, and cartilage). More recently, they are forcefully emerging also for the development of high-
throughput 3-D bioprinted tissue models for research, drug discovery, and toxicology (Oskui et al., 2016).
The working principle is based on a layer-by-layer precise positioning of biological materials, biochemicals, and living cells, with
spatial control of the placement of functional components, into a three-dimensional arrangement. There are several AM approaches
to combine cells and biomaterials into hierarchically ordered 3-D constructs with biological and mechanical properties suitable for
clinical restoration of tissue functions. They may provide (a) the manufacturing of identical replica of cellular and extracellular
components of tissues/organs (Ingber et al., 2006), (b) the reproduction of biological tissues by self-assembling embryonic organs
as a guide for mature organ development (Kasza et al., 2007), or (c) the fabrication of mini-tissue building blocks as smallest struc-
tural and functional components to be assembled in macrotissues with biologically inspired design and organization (Mironov
et al., 2009). In particular, it is possible to roughly distinguish between bioprinting systems able to deposit a continuous material
bead (dispensing systems) or multiple materials in short interrupted or defined spaces (dropping systems) to form the 3-D struc-
ture, as reported in Fig. 4.
The former ones commonly involve the use of microextrusion or inkjet bioprinters properly set to process biological matter at
safe body conditions (i.e., temperature). As for microextrusion technologies, working principle is generally based on robotically
controlled extrusion of materials by a properly designed head that allows dispensing biological matter in the form of continuous
beads rather than liquid droplets. Small beads of bioslurry or bioink are deposited layer by layer in two dimensions onto the stage,
under specific instructions imparted by a CAD–CAM system. A myriad of bioinks are compatible with microextrusion printers,
Fig. 4 Schematic summary of AM technologiesdexploiting specific drawing forcesdfor 3-D cell printing.
including hydrogels, biocompatible copolymers, and cell spheroids (Peltola et al., 2008). The most common methods to extrude
biological materials via 3-D bioprinting are driven by pneumatic or mechanical (piston or screw) forces (Cohen et al., 2006). Anal-
ogously, inkjet printing technologies may allow directly printing spatially well-organized patterns by drop-to-drop deposition of
cells and biomaterial slurries for the realization of bioinspired microstructures variously encoded with bioactive molecules to
address specific cell behaviors. Among AM technologies, inkjet techniques are the most commonly used in 3-D tissue/organ printing
for their increasing resolution, precision, and speed in comparison with other ones. The working principle is based on the appli-
cation of thermal and/or piezoelectric forces able to form droplets with controlled sizes via actuation mechanism (Boland et al.,
2006). More in general, the most part of current printing technologies are constrained by several limitations related to (a) the
ink viscositydup to 10 cP excessive force may be required to eject dropsd(b) clogging of small size nozzles, and (c) the generation
of pattern smaller than the nozzle size. More recently, many researchers are focusing on electrohydrodynamic inkjet printing (EIP)
based on the administration of electric field to polymeric solution or slurry. The use of electric field as driving force to draw micro-
patterns by physical additive mass flow is promoting the development of a new set of bioprinting techniques to form continuous
patterning, drop-on-demand printing, and thin-film deposition (i.e., electrospray) as a function of the ejection mode of liquid from
the nozzle (Jayasinghe et al., 2006).
In Vitro/In Vivo Clinical Applications

Despite the fact that a certain number of AM processes and 3-D scaffold products have already been approved by regulatory bodies
for (routine) clinical use, an insufficient number of studies have been deeply characterized though extensive in vitro and in vivo
studies and obtained results referred to a restricted number of biomaterials. Indeed, there is a still limited availability of materials
really compatible with AM processes. Indeed, traditionally used biomaterials cannot be processed by AM techniques, while the best-
performing materials in AM machines, in terms of accuracy and functionality, are often not biocompatible or do not exhibit the
required biodegradation behavior for the regeneration of specific tissues. The development of novel biomaterials or bioinks is
one of the most important challenges for the future of AM technologies in biomedical field. Future outlook of advanced AM tech-
nologies still remains the design and fabrication of products processed by a rational printing of cells and biomacromolecules
derived from native extracellular matrix (ECM), in combination with unexplored biomaterials (i.e., bioinks and powders), in order
to generate in vitro and/or in vivo tissue analogue structures. In this case, the choice of bioink may be further limited by stringent
conditions related to the combination of printing materials with cells. Recent approaches based on dropping strategies (i.e., inkjet or
laser-assisted bioprinting) are brightly overcoming these limitation by encapsulating cells in biodegradable hydrogels to mimic
a tissue-like environment (Bertassoni et al., 2014). In this case, the peculiar characteristics of hydrogels can protect inner cells
from the shear force generated during the printing process, maintaining cell biofunctions (i.e., self-renewal ability and multilineage
differentiation potency in the case of stem cells) without penalizing basic key features of AM technologies (i.e., high resolution and
accuracy in single-cell deposition). Moreover, multiple bioinks and cell types can be also distributed within 3-D printed scaffolds to
guide tissue generation processes, thus enabling regeneration of more complex tissue structures (Rutz et al., 2015). By newly imple-
mented AM technologies, it will be possible to design and rapidly fabricate mechanically stable, functional, human-scale tissues
such as the mandible, calvarial bone, cartilage, and skeletal muscle by simultaneously plotting cell-laden material with biodegrad-
able porous structures to assure an efficient transport of nutrient transport and the required exchange of metabolites.
Integration of Different Scaffolds Fabrication Techniques
The integration of different scaffold fabrication techniques allows to overcome the limitations of the single techniques, obtaining
scaffolds with enhanced properties in terms of morphology, compositional and porosity gradients, functionalization with biomol-
ecules, and appropriate mechanical properties related to the tissue target of regeneration. Usually, this combination of different
techniques results in particularly suitable for interface tissue engineering applications, aiming to repair or regenerate the functions
of diseased or damaged zones at the interface of different tissue types (also called “interface tissues”) (Seidi et al., 2011). In this
section, few examples of synergic integration of different techniques are reported. Mi et al. (Mi et al., 2016) reported the successful
integration of electrospinning technique and TIPS. Interesting results were reported by Biswas et al. (2017), reporting the integration
of mechanical foaming process with TIPS to control pore formation inside scaffolds. In order to achieve highly performance porous
structures using an automated stage, melt electrospinning writing (MEW) based on the combination of electrospinning with larger-
scale AM is recently emerging as the most interesting AM technology to realize ordered 3-D architectures to be used as scaffolds for
hard tissue engineering applications. This technique is based on the interaction of melt polymers by electrostatic forces (i.e., melt
electrospinning) and is supported by the use of automated machines able to mechanically control spinneret translation along x–y-
axis, in order to realize fiber dispensing systems to directly write polymers in the form of 3-D scaffolds with controllable architec-
tures and patterns (Brown et al., 2011). MEWs allow for the deposition of polymeric filaments with average diameters down to
10 mm, by a fine and synergic control of extrusion rate and spinnerets/collectors speeds, until to form a reproducible three-
dimensional (3-D) lattice with characteristic dimensions suitable for cell and tissue colonization (Farrugia et al., 2013). Using
this approach, scaffold architectures can be readily created without the need for organic solvents, combining specific benefits of
melt extrusion-based AM methods and electrospinning in a unique way (Dalton et al., 2013). Accordingly, some authors also
attempted to combine solution and melt electrospinning to reproduce a random nanofiber texturing onto surfaces of ordered fila-
ment meshes for the fabrication of a hybrid vascular graft with interesting results in terms of cell adhesion and structural recognition
(Centola et al., 2010). Moreover, through an accurate manipulation of the melt electrospinning process parameters (i.e., spinneret
diameter, voltage, and collector distance), different struts with peculiar fiber sizes can be processed by using a large set of biodegrad-
able and/or bioactive materials, for the fabrication of tailor-made scaffolds for tissue engineering use. To date, poly(ε-caprolactone)
(PCL) is the most frequently processed polymer by MEW due to its low melting pointdabout 63 Cdin combination with
well-known biodegradation properties and acceptable in vivo host response (Brown et al., 2012). Recently, it has been readily copo-
lymerized with other cyclic monomers such as lactide and trimethylene carbonate to generate new substrates with different biodeg-
radation properties and biological response. More recently, photo-cross-linkable poly(L-lactide-co-ε-caprolactone-co-acryloyl
carbonate) copolymers (poly(LLA-ε-CL-AC)) have been processed by MEW followed by UV irradiation at room temperature to
realize 3-D scaffolds without relevant alteration of surface structure but a significant improvement of mechanical stiffness under
dynamic loads to be successfully used in bone and connective tissue regeneration (Chen et al., 2016a). For instance, melt electro-
spinning writing (MEW)drising from the combination of FDM and electrospinningdhas demonstrated to be a computer-aided
electrofluidodynamic printing technique able to rationally design and fabricate fibrous scaffolds with thinner fiber sizesddown
to 50 mm as diameter. MEWs have been successfully used to process thermoplastics like poly(ε-caprolactone) or photo-cross-link-
able biopolymer poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) to obtain 3-D scaffolds with improved mechanical prop-
erties for hard tissue engineering (TE) applications. Besides, melt electrospinning allows bridging the gap between nanoscale
production methods with insufficient fiber deposition control (i.e., electrospinning) and resolution-limited AM methodologies.
Several studies have demonstrated that large meshes generated by MEW may more efficiently contribute to 3-D cell invasiveness,
with relevant improvements respect to electrospun scaffolds, frequently perceived by cells as 2-D structures with limited cell pene-
tration (Simonet et al., 2007) or, respect to FDM processed scaffolds with not efficient cell-to-cell and cell-to-material interactions,
due to too much larger pores (> 100 mm). Moreover, polymers approved by Food and Drug Administration (FDA) such as PCL can
be processed through basic MEW in their pure form without the use of toxic solvents (Hutmacher and Dalton, 2011), thus mini-
mizing postproduction costs. In terms of functional graded scaffolds, a recent and innovative approach is based on the “4-D bio-
printing,” considering the variable time as a fourth dimension, relevant for the scaffold property evaluation and functionalities. In
fact, in this approach, the fact that living cells colonized the scaffolds and dynamically interact with it induces an evolution in the
scaffold properties (Gao et al., 2016).
Conclusions and Future Perspectives
The main challenge for the regeneration, restoration, or replacement of defective or injured functional living organs and tissues is to
design 3-D scaffolds with specific functions tailor-made for specific targets. In particular, they should provide internal pathways for
cell attachment and migration, promoting the exchange of specific growth factors and waste products and preserving their struc-
tural/mechanical properties during ex novo tissue formation. In order to achieve these functions, they have to present a highly inter-
connected porous structure able to create a friendly microenvironment for cells from chemical and morphological point of view. To
reach this goal, many scaffold fabrication techniques could be exploited and also combined, as reported in this contribution.
In the next future, it could be possible to design innovative implantable devices with more efficiently control of drug adminis-
tration for innovative therapeutic uses.
Acknowledgments
Liliana Liverani acknowledges funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-
Curie grant agreement No. 657264.
References
Akki, R., Desai, P., & Abhiraman, A. S. (1999). A framework for morphological evolution vis-à-vis phase transitions in polymer solutions. Journal of Applied Polymer Science, 73(8),
1343–1355.
Aramwit, P., et al. (2015). A green salt-leaching technique to produce sericin/PVA/glycerin scaffolds with distinguished characteristics for wound-dressing applications. Journal of
Biomedical Materials Research Part B: Applied Biomaterials, 103(4), 915–924. Available from: http://doi.wiley.com/10.1002/jbm.b.33264 [accessed 27 January 2017].
Arkudas, A., et al. (2013). Evaluation of angiogenesis of bioactive glass in the arteriovenous loop model. Tissue Engineering Part C: Methods, 19(6), 479–486. Available from: http://
online.liebertpub.com/doi/abs/10.1089/ten.tec.2012.0572 [accessed 3 February 2017].
Baino, F., et al. (2016). Using porous bioceramic scaffolds to model healthy and osteoporotic bone. Journal of the European Ceramic Society, 36(9), 2175–2182.
Barton, B. F., & McHugh, A. J. (1999). Kinetics of thermally induced phase separation in ternary polymer solutions. I. Modeling of phase separation dynamics. Journal of Polymer
Science Part B: Polymer Physics, 37(13), 1449–1460. Available from: http://doi.wiley.com/10.1002/%28SICI%291099-0488%2819990701%2937%3A13%3C1449%3A%
3AAID-POLB11%3E3.0.CO%3B2-T [accessed 3 February 2017].
Bertassoni, L. E., et al. (2014). Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels. Biofabrication, 6(2), 24105. Available from: http://www.ncbi.nlm.nih.gov/
pubmed/24695367 [accessed 3 February 2017].
Billiet, T., et al. (2012). A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials, 33(26), 6020–6041. Available from: http://www.ncbi.
nlm.nih.gov/pubmed/22681979 [accessed 3 February 2017].
Biswas, D. P., et al. (2017). Combining mechanical foaming and thermally induced phase separation to generate chitosan scaffolds for soft tissue engineering. Journal of
Biomaterials Science, Polymer Edition, 28(2), 207–226. Available from: https://www.tandfonline.com/doi/full/10.1080/09205063.2016.1262164 [accessed 31 January 2017].
Boccardi, E., et al. (2015). Characterisation of bioglass based foams developed via replication of natural marine sponges. Advances in Applied Ceramics, 114(Suppl. 1), S56–S62.
Available from: https://www.tandfonline.com/doi/full/10.1179/1743676115Y.0000000036 [accessed 3 February 2017].
Boland, T., et al. (2006). Application of inkjet printing to tissue engineering. Biotechnology Journal, 1(9), 910–917. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16941443
[accessed 3 February 2017].
Bose, S., Roy, M., & Bandyopadhyay, A. (2012). Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology, 30(10), 546–554.
Brown, T. D., et al. (2012). Design and fabrication of tubular scaffolds via direct writing in a melt electrospinning mode. Biointerphases, 7(1), 13. Available from: http://www.ncbi.
Brown, T. D., Dalton, P. D., & Hutmacher, D. W. (2011). Direct writing by way of melt electrospinning. Advanced Materials, 23(47), 5651–5657. Available from: http://www.ncbi.
Bulte, A. (1996). Diffusion induced phase separation with crystallizable nylons. I. Mass transfer processes for nylon 4,6. Journal of Membrane Science, 121(1), 37–49. Available
from: http://linkinghub.elsevier.com/retrieve/pii/0376738896001639 [accessed 3 February 2017].
Bulte, A. M. W., et al. (1996). Equilibrium thermodynamics of the ternary membrane-forming system nylon, formic acid and water. Polymer, 37(9), 1647–1655. Available from:
http://linkinghub.elsevier.com/retrieve/pii/0032386196837141 [accessed 3 February 2017].
Cao, W., & Hench, L. L. (1996). Bioactive materials. Ceramics International, 22(6), 493–507.
Carfì-Pavia, F., et al. (2009). Porous poly (L-lactic acid) scaffolds are optimal substrates for internal colonization by A6 mesoangioblasts and immunocytochemical analyses. Journal
of Biosciences, 34(6), 873–879. Available from: http://link.springer.com/10.1007/s12038-009-0101-8 [accessed 3 February 2017].
Centola, M., et al. (2010). Combining electrospinning and fused deposition modeling for the fabrication of a hybrid vascular graft. Biofabrication, 2(1), 14102. Available from: http://
www.ncbi.nlm.nih.gov/pubmed/20811117 [accessed 3 February 2017].
Chen, F., et al. (2016a). Additive manufacturing of a photo-cross-linkable polymer via direct melt electrospinning writing for producing high strength structures. Biomacromolecules,
17(1), 208–214. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26620885 [accessed 3 February 2017].
Chen, Q. Z., Thompson, I. D., & Boccaccini, A. R. (2006). 45S5 bioglass®-derived glass–ceramic scaffolds for bone tissue engineering. Biomaterials, 27(11), 2414–2425.
Chen, W., et al. (2016b). Superabsorbent 3D scaffold based on electrospun nanofibers for cartilage tissue engineering. ACS Applied Materials & Interfaces, 8(37), 24415–24425.
Available from: http://pubs.acs.org/doi/abs/10.1021/acsami.6b06825 [accessed 31 January 2017].
Cohen, D. L., et al. (2006). Direct freeform fabrication of seeded hydrogels in arbitrary geometries. Tissue Engineering, 12(5), 1325–1335. Available from: http://www.ncbi.nlm.nih.
gov/pubmed/16771645 [accessed 3 February 2017].
Dalton, P. D., et al. (2013). Electrospinning and additive manufacturing: Converging technologies. Biomaterials Science, 1(2), 171–185. Available from: http://xlink.rsc.org/?
DOI¼C2BM00039C [accessed 3 February 2017].
Desimone, D., et al. (2013). Biosilicate®–gelatine bone scaffolds by the foam replica technique: Development and characterization. Science and Technology of Advanced Materials,
14(4), 45008. Available from: http://www.tandfonline.com/doi/full/10.1088/1468-6996/14/4/045008 [accessed 3 February 2017].
Drummer, D., Cifuentes-Cuéllar, S., & Rietzel, D. (2012). Suitability of PLA/TCP for fused deposition modeling. Rapid Prototyping Journal, 18(6), 500–507. Available from: http://
www.emeraldinsight.com/doi/10.1108/13552541211272045 [accessed 3 February 2017].
Erisken, C., Kalyon, D. M., & Wang, H. (2008). Functionally graded electrospun polycaprolactone and b-tricalcium phosphate nanocomposites for tissue engineering applications.
Biomaterials, 29(30), 4065–4073.
Everett, D. H. (1972). International Union of Pure and Applied Chemistry manual of symbol and terminology for physicochemical quantities and units appendix definitions, terminology
and symbols in colloid and surface chemistry part I. Pure and Applied Chemistry, 31(4), 579–638.
Farrugia, B. L., et al. (2013). Dermal fibroblast infiltration of poly(ε-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode. Biofabrication, 5(2), 25001.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/23443534 [accessed 3 February 2017].
Fereshteh, Z., et al. (2015). The effect of coating type on mechanical properties and controlled drug release of PCL/zein coated 45S5 bioactive glass scaffolds for bone tissue
engineering. Materials Science and Engineering: C, 54, 50–60.
Fischbach, C., et al. (2007). Engineering tumors with 3D scaffolds. Nature Methods, 4(10), 855–860. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17767164 [accessed 3
February 2017].
Forbes, S. J., & Rosenthal, N. (2014). Preparing the ground for tissue regeneration: From mechanism to therapy. Nature Medicine, 20(8), 857–869. Available from: http://www.ncbi.
Fu, Q., et al. (2010). Vivo evaluation of 13-93 bioactive glass scaffolds with trabecular and oriented microstructures in a subcutaneous rat implantation model. Journal of Biomedical
Materials Research Part A, 95A(1), 235–244. Available from: http://doi.wiley.com/10.1002/jbm.a.32827 [accessed 3 February 2017].
Fu, Q., et al. (2008a). Mechanical and in vitro performance of 13–93 bioactive glass scaffolds prepared by a polymer foam replication technique. Acta Biomaterialia, 4(6),
1854–1864.
Fu, Y.-C., et al. (2008b). Optimized bone regeneration based on sustained release from three-dimensional fibrous PLGA/HAp composite scaffolds loaded with BMP-2. Biotechnology
and Bioengineering, 99(4), 996–1006. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17879301 [accessed 25 April 2016].
Gao, B., et al. (2016). 4D bioprinting for biomedical applications. Trends in Biotechnology, 34(9), 746–756.
Gerhardt, L.-C., & Boccaccini, A. R. (2010). Bioactive glass and glass-ceramic scaffolds for bone tissue engineering. Materials, 3(7), 3867–3910. Available from: http://www.mdpi.
com/1996-1944/3/7/3867/ [accessed 2 February 2017].
Gönen, S.Ö., Taygun, M. E., & Küçükbayrak, S. (2016). Fabrication of bioactive glass containing nanocomposite fiber Mats for bone tissue engineering applications. Composite
Structure, 138, 96–106. Available from: https://doi.org/10.1016/j.compstruct.2015.11.033.
Gong, Y., et al. (2006). Specially elaborated thermally induced phase separation to fabricate poly(L-lactic acid) scaffolds with ultra large pores and good interconnectivity. Journal of
Applied Polymer Science, 101(5), 3336–3342. Available from: http://doi.wiley.com/10.1002/app.23931 [accessed 1 February 2017].
Guarino, V., et al. (2012). Bio-inspired composite and cell instructive platforms for bone regeneration. International Materials Reviews, 57(5), 256–275. Available from: http://www.
tandfonline.com/doi/full/10.1179/0950660812Z.00000000021 [accessed 3 February 2017].
Han, M.-J. (2000). Biodegradable membranes for the controlled release of progesterone. 1. Characterization of membrane morphologies coagulated from PLGA/progesterone/DMF
solutions. Journal of Applied Polymer Science, 75(1), 60–67. Available from: http://doi.wiley.com/10.1002/%28SICI%291097-4628%2820000103%2975%3A1%3C60%3A%
3AAID-APP7%3E3.0.CO%3B2-8 [accessed 1 February 2017].
He, T., Mulder, M. H. V., & Wessling, M. (2003). Preparation of porous hollow fiber membranes with a triple-orifice spinneret. Journal of Applied Polymer Science, 87(13), 2151–
2157. Available from: http://doi.wiley.com/10.1002/app.11591 [accessed 3 February 2017].
Ho, M.-H., et al. (2004). Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials, 25(1), 129–138. Available from: http://www.ncbi.nlm.
nih.gov/pubmed/14580916 [accessed 1 February 2017].
Hollister, S. J. (2005). Porous scaffold design for tissue engineering. Nature Materials, 4(7), 518–524. Available from: http://www.nature.com/doifinder/10.1038/nmat1421
[accessed 25 January 2017].
Hoppe, A., et al. (2013). In vitro reactivity of cu doped 45S5 Bioglass® derived scaffolds for bone tissue engineering. Journal of Materials Chemistry B, 1(41), 5659. Available from:
http://xlink.rsc.org/?DOI¼c3tb21007c [accessed 3 February 2017].
Hoppe, A., Güldal, N. S., & Boccaccini, A. R. (2011). A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials,
32(11), 2757–2774. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0142961211000056.
Hutmacher, D. (2001). Scaffold design and fabrication technologies for engineering tissuesdState of the art and future perspectives. Journal of Biomaterials Science, Polymer
Edition, 12(1), 107–124.
Hutmacher, D. W., & Dalton, P. D. (2011). Melt electrospinning. Chemistry - An Asian Journal, 6(1), 44–56. Available from: http://doi.wiley.com/10.1002/asia.201000436
Hutmacher, D. W., Sittinger, M., & Risbud, M. V. (2004). Scaffold-based tissue engineering: Rationale for computer-aided design and solid free-form fabrication systems. Trends in
Biotechnology, 22(7), 354–362. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15245908 [accessed 3 February 2017].
Ingber, D. E., et al. (2006). Tissue engineering and developmental biology: Going biomimetic. Tissue Engineering, 12(12), 3265–3283. Available from: http://www.ncbi.nlm.nih.gov/
Jayasinghe, S. N., Qureshi, A. N., & Eagles, P. A. M. (2006). Electrohydrodynamic jet processing: An advanced electric-field-driven jetting phenomenon for processing living cells.
Small, 2(2), 216–219. Available from: http://doi.wiley.com/10.1002/smll.200500291 [accessed 3 February 2017].
Jessop, Z. M., et al. (2016). Combining regenerative medicine strategies to provide durable reconstructive options: Auricular cartilage tissue engineering. Stem Cell Research &
Therapy, 7, 19.
Jin, L., et al. (2017). Engineering 3D aligned nanofibers for regulation of cell growth behavior (p. 1600448). Macromolecular Materials and Engineering. Available from: http://doi.
wiley.com/10.1002/mame.201600448 [accessed 31 January 2017].
Jones, J. R., et al. (2006). Controlling ion release from bioactive glass foam scaffolds with antibacterial properties. Journal of Materials Science: Materials in Medicine, 17(11), 989–
996. Available from: http://link.springer.com/10.1007/s10856-006-0434-x [accessed 3 February 2017].
Jones, J. R., & Hench, L. L. (2003). Regeneration of trabecular bone using porous ceramics. Current Opinion in Solid State and Materials Science, 7(4), 301–307.
Joshi, M. K., et al. (2015). Multi-layered macroporous three-dimensional nanofibrous scaffold via a novel gas foaming technique. Chemical Engineering Journal, 275, 79–88.
Ju, Y. M., et al. (2010). Bilayered scaffold for engineering cellularized blood vessels. Biomaterials, 31(15), 4313–4321. Available from: http://www.ncbi.nlm.nih.gov/pubmed/
20188414 [accessed 31 January 2017].
Karande, T. S., Kaufmann, J. J. M., & Agrawal, C. M. (2014). Functions and requirements of synthetic scaffolds in tissue engineering. In C. T. Laurencin, & L. S. Nair (Eds.),
Nanotechnology and Regenerative Engineering: The Scaffold (pp. 63–102). CRC Press.
Kasza, K. E., et al. (2007). The cell as a material. Current Opinion in Cell Biology, 19(1), 101–107. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17174543 [accessed 3
February 2017].
Khademhosseini, A., et al. (2006). Microscale technologies for tissue engineering and biology. Proceedings of the National Academy of Sciences of the United States of America,
103(8), 2480–2487. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16477028 [accessed 3 February 2017].
Kim, H.-M., et al. (2004). The mechanism of biomineralization of bone-like apatite on synthetic hydroxyapatite: An in vitro assessment. Journal of the Royal Society, Interface, 1(1),
17–22. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16849149 [accessed 2 February 2017].
Ko, S. H., et al. (2007). All-inkjet-printed flexible electronics fabrication on a polymer substrate by low-temperature high-resolution selective laser sintering of metal nanoparticles.
Nanotechnology, 18(34), 345202. Available from: http://stacks.iop.org/0957-4484/18/i¼34/a¼345202?key¼crossref.9e338b772eb19964ed575dbb1f725cb0 [accessed 3
February 2017].
Langer, R., & Vacanti, J. P. (1993). ARTICLES tissue engineering. Science, 260(May), 920–926.
Lepry, W. C., et al. (2016). Acellular bioactivity of sol–gel derived borate glass-Polycaprolactone electrospun scaffolds biomedical glasses. Biomedical Glasses, 2, 88–98.
Levingstone, T. J., et al. (2016). Multi-layered collagen-based scaffolds for osteochondral defect repair in rabbits. Acta Biomaterialia, 32, 149–160.
Li, C., et al. (2006). Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials, 27(16), 3115–3124.
Li, X.-M., et al. (2008). A sacrificial-layer approach to prepare microfiltration membranes. Journal of Membrane Science, 320(1–2), 1–7.
Liska, R., et al. (2007). Photopolymers for rapid prototyping. Journal of Coatings Technology and Research, 4(4), 505–510. Available from: http://link.springer.com/10.1007/
s11998-007-9059-3 [accessed 3 February 2017].
Liverani, L., et al. (2014). Electrospinning of hydroxyapatite-chitosan nanofibers for tissue engineering applications. Asia-Pacific Journal of Chemical Engineering, 9(3).
Liverani, L., & Boccaccini, A. R. (2016). Versatile production of poly(epsilon-caprolactone) fibers by electrospinning using benign solvents. Nanomaterials, 6(4).
Liverani, L., Roether, J. A., & Boccaccini, A. R. (2016). Nanofiber composites in bone tissue engineering. In M. Ramalingam, & S. Ramakrishna (Eds.), Nanofiber Composite
Materials for Biomedical Applications.
Livingston, T., Ducheyne, P., & Garino, J. (2002). In vivo evaluation of a bioactive scaffold for bone tissue engineering. Journal of Biomedical Materials Research, 62(1), 1–13.
Available from: http://www.ncbi.nlm.nih.gov/pubmed/12124781 [accessed 3 February 2017].
Loh, Q. L., & Choong, C. (2013). Three-dimensional scaffolds for tissue engineering applications: Role of porosity and pore size. Tissue Engineering Part B: Reviews, 19(6), 485–
502. Available from: http://online.liebertpub.com/doi/abs/10.1089/ten.teb.2012.0437 [accessed 25 January 2017].
Mallick, K. K. (2009). Freeze casting of porous bioactive glass and bioceramics. Journal of the American Ceramic Society, 92(s1), S85–S94. Available from: http://doi.wiley.com/10.
1111/j.1551-2916.2008.02784.x [accessed 3 February 2017].
Mannella, G. A., et al. (2015). Preparation of polymeric foams with a pore size gradient via thermally induced phase separation (TIPS). Materials Letters, 160, 31–33.
Matsuyama, H., et al. (2000). Kinetic studies of thermally induced phase separation in polymer-diluent system. Journal of Applied Polymer Science, 76(7), 1028–1036. Available
from: http://doi.wiley.com/10.1002/%28SICI%291097-4628%2820000516%2976%3A7%3C1028%3A%3AAID-APP6%3E3.0.CO%3B2-Z [accessed 1 February 2017].
Melchels, F. P. W., Feijen, J., & Grijpma, D. W. (2010). A review on stereolithography and its applications in biomedical engineering. Biomaterials, 31(24), 6121–6130. Available
from: http://www.ncbi.nlm.nih.gov/pubmed/20478613 [accessed 3 February 2017].
Mi, H.-Y., et al. (2016). Approaches to fabricating multiple-layered vascular scaffolds using hybrid electrospinning and thermally induced phase separation methods. Industrial &
Engineering Chemistry Research, 55(4), 882–892. Available from: http://pubs.acs.org/doi/abs/10.1021/acs.iecr.5b03462 [accessed 13 January 2017].
Miguez-Pacheco, V., et al. (2016). Development and characterization of lithium-releasing silicate bioactive glasses and their scaffolds for bone repair. Journal of Non-Crystalline
Solids, 432, pp, 65–72.
Miguez-Pacheco, V., Hench, L. L., & Boccaccini, A. R. (2015). Bioactive glasses beyond bone and teeth: Emerging applications in contact with soft tissues. Acta Biomaterialia, 13,
1–15. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25462853 [accessed 2 February 2017].
Mikos, A. G., & Temenoff, J. S. (2000). Formation of highly porous biodegradable scaffolds for tissue engineering. Electronic Journal of Biotechnology, 3(2). Available from: http://
www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/427 [accessed 27 January 2017].
Mironov, V., et al. (2009). Organ printing: Tissue spheroids as building blocks. Biomaterials, 30(12), 2164–2174. Available from: http://www.ncbi.nlm.nih.gov/pubmed/19176247
Mohd Daud, N., et al. (2014). Degradation and in vitro cell–material interaction studies on hydroxyapatite-coated biodegradable porous iron for hard tissue scaffolds. Journal of
Orthopaedic Translation, 2(4), 177–184.
Montesanto, S., et al. (2015). Coagulation bath composition and desiccation environment as tuning parameters to prepare skinless membranes via diffusion induced phase
separation. Journal of Applied Polymer Science, 132(26). Available from: http://doi.wiley.com/10.1002/app.42151 [accessed 3 February 2017].
Murphy, S. V., & Atala, A. (2014). 3D bioprinting of tissues and organs. Nature Biotechnology, 32(8), 773–785. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25093879
Murr, L. E. (2016). Frontiers of 3D printing/additive manufacturing: From human organs to aircraft fabrication. Journal of Materials Science & Technology, 32(10), 987–995.
Nunes, S. P., & Inoue, T. (1996). Evidence for spinodal decomposition and nucleation and growth mechanisms during membrane formation. Journal of Membrane Science, 111(1),
93–103.
O’Brien, F. J. (2011). Biomaterials & scaffolds for tissue engineering. Materials Today, 14(3), 88–95.
O’Connor, R. A., & McGuinness, G. B. (2016). Electrospun nanofibre bundles and yarns for tissue engineering applications: A review. Proceedings of the Institution of Mechanical
Engineers, Part H: Journal of Engineering in Medicine, 230(11), 987–998. Available from: http://journals.sagepub.com/doi/10.1177/0954411916656664 [accessed 31
January 2017].
Obata, A., et al. (2013). Cotton wool-like poly(lactic acid)/vaterite composite scaffolds releasing soluble silica for bone tissue engineering. Journal of Materials Science: Materials in
Medicine, 24(7), 1649–1658. Available from: http://link.springer.com/10.1007/s10856-013-4930-5 [accessed 31 January 2017].
Onuh, S. O., & Yusuf, Y. Y. (1999). Rapid prototyping technology: Applications and benefits for rapid product development. Journal of Intelligent Manufacturing, 10(3/4), 301–311.
Available from: http://link.springer.com/10.1023/A:1008956126775 [accessed 3 February 2017].
Oskui, S. M., et al. (2016). Assessing and reducing the toxicity of 3D-printed parts. Environmental Science & Technology Letters, 3(1), 1–6. Available from: http://pubs.acs.org/doi/
abs/10.1021/acs.estlett.5b00249 [accessed 3 February 2017].
Pavia, F. C., et al. (2013). Poly-left-lactic acid tubular scaffolds via diffusion induced phase separation: Control of morphology. Polymer Engineering & Science, 53(2), 431–442.
Available from: http://doi.wiley.com/10.1002/pen.23273 [accessed 31 January 2017].
Pavia, F. C., et al. (2008). Polymeric scaffolds prepared via thermally induced phase separation: Tuning of structure and morphology. Journal of Biomedical Materials Research Part
A, 86A(2), 459–466. Available from: http://www.ncbi.nlm.nih.gov/pubmed/17975822 [accessed 3 February 2017].
Peltola, S. M., et al. (2008). A review of rapid prototyping techniques for tissue engineering purposes. Annals of Medicine, 40(4), 268–280. Available from: http://www.ncbi.nlm.nih.
gov/pubmed/18428020 [accessed 3 February 2017].
Peric, M., et al. (2015). The rational use of animal models in the evaluation of novel bone regenerative therapies. Bone, 70, 73–86.
Poursamar, S. A., et al. (2016). Potential application of gelatin scaffolds prepared through in situ gas foaming in skin tissue engineering. International Journal of Polymeric Materials
and Polymeric Biomaterials, 65(6), 315–322. Available from: http://www.tandfonline.com/doi/full/10.1080/00914037.2015.1119688 [accessed 27 January 2017].
Putti, M., et al. (2015). Electrospinning poly(ε-caprolactone) under controlled environmental conditions: Influence on fiber morphology and orientation. Polymer, 63, 189–195.
Available from: http://linkinghub.elsevier.com/retrieve/pii/S0032386115002347.
Reuvers, A. J., van den Berg, J. W. A., & Smolders, C. A. (1987). Formation of membranes by means of immersion precipitation. Journal of Membrane Science, 34(1), 45–65.
Available from: http://linkinghub.elsevier.com/retrieve/pii/S0376738800800204 [accessed 3 February 2017].
Rezabeigi, E., Wood-Adams, P. M., & Drew, R. A. L. (2016). Morphological examination of highly porous polylactic acid/Bioglass® scaffolds produced via nonsolvent induced phase
separation. Journal of Biomedical Materials Research Part B: Applied Biomaterials. Available from: http://doi.wiley.com/10.1002/jbm.b.33784 [accessed 31 January 2017].
Rnjak-Kovacina, J., & Weiss, A. S. (2011). Increasing the pore size of electrospun scaffolds. Tissue Engineering Part B: Reviews, 17(5), 365–372. Available from: http://www.ncbi.
nlm.nih.gov/pubmed/21815802 [accessed 27 January 2017].
Rocco, K. A., et al. (2014). In vivo applications of electrospun tissue-engineered vascular grafts: A review. Tissue Engineering Part B: Reviews, 20(6), 628–640. Available from:
http://online.liebertpub.com/doi/abs/10.1089/ten.teb.2014.0123 [accessed 31 January 2017].
Rutz, A. L., et al. (2015). A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels. Advanced Materials, 27(9), 1607–1614. Available from: http://www.ncbi.
Sadiasa, A., Nguyen, T. H., & Lee, B.-T. (2014). In vitro and in vivo evaluation of porous PCL-PLLA 3D polymer scaffolds fabricated via salt leaching method for bone tissue
engineering applications. Journal of Biomaterials Science, Polymer Edition, 25(2), 150–167. Available from: http://www.tandfonline.com/doi/abs/10.1080/09205063.2013.
846633 [accessed 27 January 2017].
Seidi, A., et al. (2011). Gradient biomaterials for soft-to-hard interface tissue engineering. Acta Biomaterialia, 7(4), 1441–1451. Available from: http://www.ncbi.nlm.nih.gov/
Sepulveda, P., Jones, J. R., & Hench, L. L. (2002). Bioactive sol-gel foams for tissue repair. Journal of Biomedical Materials Research, 59(2), 340–348. Available from: http://www.
ncbi.nlm.nih.gov/pubmed/11745571 [accessed 3 February 2017].
Shenoy, S. L., et al. (2005). Role of chain entanglements on fiber formation during electrospinning of polymer solutions: Good solvent, non-specific polymer-polymer interaction limit.
Polymer, 46(10), 3372–3384.
Simonet, M., et al. (2007). Ultraporous 3D polymer meshes by low-temperature electrospinning: Use of ice crystals as a removable void template. Polymer Engineering & Science,
47(12), 2020–2026. Available from: http://doi.wiley.com/10.1002/pen.20914 [accessed 25 April 2016].
Skoog, S. A., Goering, P. L., & Narayan, R. J. (2014). Stereolithography in tissue engineering. Journal of Materials Science: Materials in Medicine, 25(3), 845–856. Available from:
http://www.ncbi.nlm.nih.gov/pubmed/24306145 [accessed 3 February 2017].
Smay, J. E., et al. (2002). Directed colloidal assembly of 3D periodic structures. Advanced Materials, 14(18), 1279–1283. Available from: http://doi.wiley.com/10.1002/1521-
4095%2820020916%2914%3A18%3C1279%3A%3AAID-ADMA1279%3E3.0.CO%3B2-A [accessed 3 February 2017].
Song, J.-H., et al. (2006). Fabrication of a porous bioactive glass–ceramic using room-temperature freeze casting. Journal of the American Ceramic Society, 89(8), 2649–2653.
Available from: http://doi.wiley.com/10.1111/j.1551-2916.2006.01092.x [accessed 3 February 2017].
Sun, J., et al. (2010). Nanofibers by green electrospinning of aqueous suspensions of biodegradable block copolyesters for applications in medicine, pharmacy and agriculture.
Macromolecular Rapid Communications, 31(23), 2077–2083.
Tanaka, T., & Lloyd, D. R. (2004). Formation of poly(l-lactic acid) microfiltration membranes via thermally induced phase separation. Journal of Membrane Science, 238(1), 65–73.
Teo, W.-E., Inai, R., & Ramakrishna, S. (2011). Technological advances in electrospinning of nanofibers. Science and Technology of Advanced Materials, 12(1), 13002.
Vacanti, J. P., & Langer, R. (1999). Tissue engineering: The design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet, 354,
SI32–I34.
Vishwanath, V., Pramanik, K., & Biswas, A. (2016). Optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application.
Journal of Biomaterials Science, Polymer Edition, 27(7), 657–674. Available from: http://www.tandfonline.com/doi/full/10.1080/09205063.2016.1148303 [accessed 31
January 2017].
Wallace, J., et al. (2014). Validating continuous digital light processing (cDLP) additive manufacturing accuracy and tissue engineering utility of a dye-initiator package. Bio-
fabrication, 6(1), 15003. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24429508 [accessed 3 February 2017].
Wang, X., et al. (2016). Topological design and additive manufacturing of porous metals for bone scaffolds and orthopaedic implants: A review. Biomaterials, 83, 127–141.
Wijmans, J. G., Baaij, J. P. B., & Smolders, C. A. (1983). The mechanism of formation of microporous or skinned membranes produced by immersion precipitation. Journal of
Membrane Science, 14(3), 263–274. Available from: http://linkinghub.elsevier.com/retrieve/pii/0376738883800052 [accessed 3 February 2017].
Williams, D. F. (2008). On the mechanisms of biocompatibility. Biomaterials, 29(20), 2941–2953.
van de Witte, P., Esselbrugge, H., Dijkstra, P. J., et al. (1996a). A morphological study of membranes obtained from the systems polylactide-dioxane-methanol, polylactide-dioxane-
water and polylactide-N-methyl pyrrolidone-water. Journal of Polymer Science Part B: Polymer Physics, 34(15), 2569–2578. Available from: http://doi.wiley.com/10.1002/%
28SICI%291099-0488%2819961115%2934%3A15%3C2569%3A%3AAID-POLB4%3E3.0.CO%3B2-O [accessed 1 February 2017].
van de Witte, P., Dijkstra, P. J., et al. (1996b). Phase separation processes in polymer solutions in relation to membrane formation. Journal of Membrane Science, 117(1), 1–31.
van de Witte, P., Esselbrugge, H., Dijkstra, P. J., et al. (1996c). Phase transitions during membrane formation of polylactides. I. A morphological study of membranes obtained from
the system polylactide-chloroform-methanol. Journal of Membrane Science, 113(2), 223–236. Available from: http://linkinghub.elsevier.com/retrieve/pii/0376738895000682
Woods, I., & Flanagan, T. C. (2014). Electrospinning of biomimetic scaffolds for tissue-engineered vascular grafts: Threading the path. Expert Review of Cardiovascular Therapy,
12(7), 815–832. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24903895 [accessed 31 January 2017].
Yang, Q., Chung, T.-S., & Santoso, Y. E. (2007). Tailoring pore size and pore size distribution of kidney dialysis hollow fiber membranes via dual-bath coagulation approach. Journal
of Membrane Science, 290(1), 153–163.
Yazdimamaghani, M., et al. (2017). Porous magnesium-based scaffolds for tissue engineering. Materials Science and Engineering: C, 71, 1253–1266.
Zhang, Q., et al. (2014). Pore size effect of collagen scaffolds on cartilage regeneration. Acta Biomaterialia, 10(5), 2005–2013.
Zoppi, R., et al. (1999). Porous poly(l-lactide) films obtained by immersion precipitation process: Morphology, phase separation and culture of VERO cells. Polymer, 40(12),
3275–3289.
Preparation and Properties of Coatings and Thin Films on Metal Implants
Zhong Li and Khiam Aik Khor, Nanyang Technological University, Singapore
Introduction 204
Preparation of Coatings and Thin Films on Metal Implants 204
Commonly Used Metallic Implant Materials 204
Physical Methods for Preparing Coatings and Thin Films 205
Thermal spraying 205
Glow discharge plasma 206
Ion implantation and deposition 206
Physical vapor deposition 206
Chemical Methods for Preparing Coatings and Thin Films 206
Electrochemical methods 206
Chemical vapor deposition 207
Sol–gel processes 207
Biomimetic modifications 207
Properties of Coatings and Thin Films on Metal Implants 207
Desirable Surface Functionalities of Metallic Implants 207
Osteoconductive and Osteogenic Coatings 207
Corrosion-Resistant Coatings 208
Wear-Resistant Coatings 209
Hemocompatible Coatings 209
Antibacterial Coatings 210
References 211
Glossary
Angioplasty Procedure with a balloon-tipped catheter to enlarge a narrowing in a blood vessel, especially a coronary artery.
Bioactivity The specific biological response at the interface of a material that results in the formation of a bond, usually through
a carbonated hydroxyapatite layer, between the tissues and the material.
Biocompatibility The ability of a biomaterial to perform its desired function without eliciting any undesirable local or systemic
effects in the recipient.
Biodegradability/bioresorbability The disintegration of materials by biological means.
Biomaterial A natural or synthetic substance that has been adapted and is used to direct, by control of interactions with
components of living systems, the course of any therapeutic or diagnostic procedure.
Hemocompatibility An implanted material’s compatibility with blood.
Osseointegration The direct structural and functional connection between living bone and the surface of an implant.
Osteoconductivity The supply of a surface along which bone migrates.
Osteogenesis The development and formation of the bone.
Osteoinductivity The process that induces osteogenesis by stimulating immature cells into preosteoblasts.
Osteolysis An active resorption of bone matrix by osteoclasts, cells that function in the breakdown and resorption of bone
tissue.
Osteosynthesis A surgical procedure used to reduce and internally fix a bone fracture with implantable devices.
Stress-shielding effect Reduction in the density of the bones around an implant site caused by the implant’s higher Young’s
modulus that reduced mechanical loads on the bones.
Thrombosis Formation or the presence of a blood clot within a blood vessel.

204 Biomaterials: Science and engineering j Preparation and Properties of Coatings and Thin Films on Metal Implants
Introduction
The surgical use of metallic implants can be traced back to the 16th century (Gotman, 1997). However, it was not until the intro-
duction of Lister’s aseptic surgery techniques in the 1860s that the steep increase in the use of metals and alloys in implant surgery
was realized (Lister, 1867). Since then, metallic biomaterials have played a predominant role in orthopedic surgery.
The unmet clinical need for human hard tissue repair has been the primary driving force for the development of metal implants.
This unmet need gap is being widened by the world’s rapidly aging population. A wide range of materials, including metals, poly-
mers, and ceramics, have been developed and used for reconstructive surgery of hard tissue. Among them, metallic biomaterials fall
into a most attractive category because of their excellent mechanical properties such as high ultimate tensile strength, good damage
tolerance, ductility, formability, and machinability.
The early medical uses of metal implants were mainly in bone contacting and replacement applications, such as the internal
fixation of fractured long bones. Their applications were later extended to dental implants and cardiovascular stents. More recently,
increasing research interest has been drawn to the study on biodegradable magnesium-based implants (Hornberger et al., 2012).
Metallic biomaterials are currently being utilized in a vast number of permanent (such as total joint replacements) and temporary
implants (such as bone plates, pins, and screws).
Corrosion behavior is a key consideration for the selection of metallic implant materials because it fundamentally determines
the implants’ long-term biocompatibility. Furthermore, in load-bearing applications, they have to possess sufficient strength, frac-
ture toughness, and wear resistance to sustain cyclic loading and present minimal implant-bone modulus mismatch to mitigate the
stress-shielding effect. While the bulk properties are primarily responsible for the biomechanical requirements, the corrosion and
wear resistance, which constitutes a major disadvantage of metal implants, is more dependent on the surface characteristics. The
surface properties of metal implants play a vital role in their interactions with cells and are strongly correlated with their attachment
to the surrounding tissue. Therefore, tuning the physiochemical niche through surface modification of metallic implants has long
been employed to enhance their corrosion and wear resistance and hence biocompatibility and, at the same time, retain their meri-
torious bulk attributes.
The surface modification of metal implants is also warranted by the relentless pursuit of improved functionality in various
medical practices. Biocompatibility and functionality stand for two sets of properties that regulate the performance of an implant
material. Most metallic biomaterials are classified as first-generation biomaterials, meaning they are bioinert (Hench and Polak,
2002). Therefore, most of them are surrounded by a fibrous foreign body capsule (FBC) and do not elicit active biological responses
in the human body because of their inherent bioinertness. This may be considered acceptable when they are only sought to match
the anatomical form of the diseased tissue and provide mechanical support and stabilization. Nevertheless, for an increasing
number of biomedical applications, the implant materials are expected to evoke and elicit desired responses from the body.
This has resulted in the growing popularity of the clinical use of second- and third-generation biomaterials, either in their bulk
form or as coatings or thin films on a substrate. Second-generation biomaterials are either bioactive or bioresorbable (e.g., Bio-
glassÒ, poly(lactic-co-glycolic acid)), and third-generation ones are both bioactive and bioresorbable (e.g., hydroxyapatite/collagen
composite) (Murugan and Ramakrishna, 2005). The “economical” use of such materials by coating them on metallic implants can
significantly diversify and augment the biological aspects of the implant surfaces, such as osseointegration and suppression of
adverse effects (e.g., FBC formation, thrombogenicity, and implant infection) (Goodman et al., 2013). The ultimate goal is to facil-
itate the restoration of full tissue and organ function.
Two general approaches for the surface modification of metal implants are (1) mechanically/physically altering the atoms in
their existing forms and (2) fabricating a surface coating. Typical examples of the first approach are machining, grinding, polishing,
grit blasting, and beading. They can be used to achieve specific roughness, topography, and surface energy. They are also frequently
employed as pretreatment steps for subsequent coating deposition, mostly for the removal of surface contamination and strength-
ened interfacial bonding. This section focuses on the surface modification of metal implants through the fabrication of coatings and
thin films. Emphasis will be attached to the creation and evaluation of the produced coatings in the context of intended clinical
applications of the metal implants.
Preparation of Coatings and Thin Films on Metal Implants

Commonly Used Metallic Implant Materials
The selection of metallic biomaterials is essentially dependent on their corrosion resistance and toxicity. Commonly used metallic
implant materials include stainless steel, titanium alloys, cobalt-based alloys, and magnesium alloys (Chen and Thouas, 2015).
Stainless steel is a generic term referring to iron-based alloys with at least 10.5 wt% Cr. It is the minimum amount required for
the formation of a protective passivation chromium oxide film. The types of stainless steel frequently used in medicine include 316,
316L, 420, and 440. Stainless steel has relatively low corrosion resistance compared with Ti- and Co-based alloys but incurs lower
production cost and has higher availability. It is the predominant stem material in total hip replacements. It has also been widely
used in temporary/transient implants such as fracture plates, bone nails, and screws. Their service life ranges from several months to
several years. Major issues associated with stainless steel implants are corrosion-induced toxicity and device degradation, as well as
allergic responses of surrounding tissues caused by wear debris.
Biomaterials: Science and engineering j Preparation and Properties of Coatings and Thin Films on Metal Implants 205
Compared with stainless steel, Ti- and Co-based alloys possess higher chemical stability and are more preferred in long-term and
permanent implants. Commercially pure Ti and Ti6Al4V are among the most frequently used Ti-based implant materials. At room
temperature, a 3–7 nm thick oxide film (mainly composed of TiO2) is generated on the surface of Ti-based materials when they are
exposed to the atmosphere. The strong passivation and repassivation ability renders them highly corrosion-resistant. Besides, they
have smaller density and Young’s modulus and larger specific strength than most metallic biomaterials. Ti and its alloys have been
successfully used in permanent load-bearing implants such as total joint replacements and dental implants, in addition to various
osteosynthesis applications (e.g., bone plates, nails, screws, and craniofacial implants). Outstanding issues facing such applications
are mainly the insufficient bending fatigue strength and low wear and abarasion resistance of Ti materials (Fu et al., 1998).
Ti and Ti alloys have also been frequently used in cardiovascular applications such as prosthetic heart valves, cardiovascular
stents, and protective cases in pacemakers. One of the major challenges facing these applications is the formation of blood clots
on device surfaces. Particularly, NiTi alloy is one of the most popular materials in angioplasty by virtue of its special shape-
memory effect (Cragg et al., 1983). This effect refers to the restoration of the predefined shape of a material by heating it. A number
of alloys, such as Cu–Zn, Au–Cd, and NiTi alloys, exhibit shape-memory effect, but NiTi is the most attractive and has been most
widely used. When utilized as a self-expandable stent, the implant is usually inserted as a thin wire; upon being triggered by the
body temperature, it dilates and returns to its preset shape. Besides vascular stents, gastrointestinal stents made from NiTi alloy
have also achieved commendable clinical success. It should be noted that the biocompatibility of NiTi alloy is still controversial
because of the potential toxicity of released Ni ions.
Cobalt alloys utilized in medical implants usually refer to CoCrMo alloys. The alloying element Cr imparts to them a high corro-
sion resistance, which is comparable with that of Ti materials. The wear resistance of Co-based alloys is superior to that of Ti alloys
because of their higher hardness. CoCrMo alloys are therefore the most frequently used metallic materials in joint-bearing systems.
By contrast, they are less attractive as stem materials because of the stress-shielding effect caused by their high Young’s modulus. The
release of Co, Cr, and Ni ions from permanent implants is also of concern.
Mg alloys are classified as third-generation biomaterials. Such materials are designed to provide temporary mechanical support
and would be gradually degraded and eventually replaced by native host tissues. The complete degradation eliminates the need to
remove them with a second procedure when their function is no longer required. Mg alloys are advantageous over many other
metallic implant materials because their density and Young’s modulus are similar to those of human bone. Their major drawback
is the poor corrosion resistance and resultant hydrogen evolution and alkaline pH shift. The tribological properties of Mg alloys also
need to be improved to mitigate the adverse effects caused by wear particles. Mg alloys have been more widely used in cardiovas-
cular stents than in orthopedic implants (Witte, 2010).
To enhance the performance of the aforementioned metallic implant materials, a myriad of techniques have been employed to
apply coatings and thin films on them. They can be generally classified into physical and chemical methods. Based on the produc-
tion mechanism, the coatings can be broadly categorized into two types, conversion coatings and deposited coatings (Hornberger
et al., 2012). While deposited coatings have a wider compositional range, in conversion coatings coating delamination is less of
a problem. In terms of chemical composition, metal, ceramic, polymer/biomolecule, and inorganic–organic hybrid coatings
have been produced onto metal implants.
Physical Methods for Preparing Coatings and Thin Films

Physical processes exploited for coating preparation are characterized by the supply of high thermal, electric, and kinetic energy. This
generic term does not necessarily exclude methods involving chemical reactions.
Thermal spraying
In thermal spraying processes, particulate materials are injected into a high-temperature heat source where they are thermally melted
and energetically propelled to a substrate on which the droplets flatten and stick. They are capable of depositing thick coatings (up
to several millimeters) over a large area at high deposition rates. In addition, thermal spraying processes allow versatile control of
the coatings’ microstructural and chemical characteristics and are suitable for substrates in complex shapes. Common thermal spray-
ing processes include plasma spraying, high-velocity oxygen-fuel spraying (HVOF), flame spraying, wire arc spraying, cold spraying,
and warm spraying, with the first two being most frequently used for modifying the surface of metal implants.
Plasma refers to partially ionized gas comprising free radicals, ions, photons, and electrons. Plasma spraying processes make use
of electric energy to ionize the gas and create plasma. The plasma jet can offer very high temperatures (typically 12,000 K at the core)
and energy densities. Plasma spraying is thus widely employed to prepare ceramic coatings. There are two types of plasma spraying
processes, atmospheric plasma spraying and vacuum plasma spraying. Fig. 1A presents the typical surface morphology of a HA
coating deposited by atmospheric plasma spraying.
The HVOF technique generates a supersonic flame through the combustion of fuel gas (e.g., hydrogen, propane, propylene, and
acetylene) in oxygen under high pressure (Khor et al., 2003). The powder is axially fed into the jet by an inert carrier gas, heated, and
accelerated in the flame, whose temperature and velocity are typically > 1000 m/s and < 3000 C.
The properties and performance of thermally sprayed coatings depend strongly on the process parameters. These include power
setting, atmosphere, gas combination and flow rate, powder feedstock, standoff distance, substrate temperature and angle, and cool-
ing rate.
Fig. 1 (A) Scanning electron microscopy (SEM) image of a plasma-sprayed HA coating on Ti6Al4V. (B) SEM image of commercially pure Ti treated
by MAO in phosphoric acid. (C) Atomic force microscopy image of diamond-like carbon (DLC) on stainless steel prepared by chemical vapor
deposition (CVD).
Glow discharge plasma

Glow discharge plasma is formed when the voltage applied on a low-pressure gas exceeds its breakdown voltage and causes its ioni-
zation. The workpiece immersed in glow discharge plasma is bombarded by electrons and ions, leading to rearrangement and sput-
tering of the surface atoms. The two main glow discharge plasma sources are magnetron discharge and radio-frequency (RF)
discharge. Glow discharge plasma is frequently used to remove surface contamination and increase surface energy of biomaterials.
It has also been used to generate nitride, carbonitride, and oxynitride coatings on Ti alloys (Sobiecki et al., 2001).
Ion implantation and deposition

Ion implantation and deposition processes employ ions with high energy and speed to bombard the substrate surface. It is one of
the most widely and successfully used surface modification techniques for Ti alloys. It can be classified into two broad categories,
conventional beamline ion implantation and plasma-immersion ion implantation (PIII). The former is a line-of-sight technique
and thus not suitable for objects with complex geometry. This restriction can be circumvented by PIII, where the negatively biased
workpiece is immersed in a plasma sheath and ions are accelerated normally to its surface. Plasma-immersion ion implantation and
deposition (PIIID) is the current pinnacle of ion implantation technology. It is a hybrid process that combines ion implantation and
deposition and can generate not only a thick coating obtainable in PIII but also an atomically intermixed layer between the substrate
and the coating that leads to high bonding strength (Sun et al., 2011).
Physical vapor deposition

Physical vapor deposition (PVD) refers to a variety of vacuum deposition methods. Physical processes such as sputtering and evap-
oration are used in PVD to generate a vapor, in the form of atoms, molecules, or ions, of the coating material supplied from a target.
They are then transported to and deposited on the substrate surface, resulting in coating formation. In PVD processes, the substrate
temperature is substantially lower than the melting temperature of the target material, making it feasible to coat temperature-
sensitive materials.
Examples of commonly used PVD processes include evaporative deposition, ion plating, pulsed laser deposition, and sputter
deposition. Compared with evaporating, sputtering is more suitable for target materials that are difficult to deposit by evaporation,
such as ceramics and refractory metals (Thian et al., 2006). In addition, coatings prepared by sputtering usually have a better
bonding strength to the substrate than those deposited by evaporation.
Chemical Methods for Preparing Coatings and Thin Films

Surface modification of metal implants by most chemical methods involves chemical reactions at the interface between the implant
and a gas/liquid phase. Coatings produced by these processes can be conversion coatings or deposited coatings.
Electrochemical methods
Anodic oxidation, or anodization, is a most widely used electrochemical method for the surface modification of Ti- and Mg-based
metallic implants. In this process, the workpiece is immersed in an electrolyte and acts as the anode of an electric circuit. The DC
electric field drives the migration of oxygen and metal ions in the electrolyte. The process can be regarded as the facilitation and
enhancement of the naturally occurring oxidation process on the metal surface. The oxide film formed is harder and more corro-
sion- and wear-resistant than the substrate and has high adhesive strength.
As a special type of anodization, microarc oxidation (MAO) occurs when the anodizing voltage applied is higher than the break-
down voltage of the oxide film. Microdischarges, intense plasma, and high local temperature and pressure are generated on the
sample surface in the electrolyte. The representative surface morphology of MAO-treated Ti is shown in Fig. 1B.
Chemical vapor deposition

While PVD employs physical forces to deposit the coating, CVD uses chemical processes. The volatile chemical precursors are
injected into a chamber, where they react with or decompose on the heated substrate to form a nonvolatile compound. The by-
products and unreacted precursor gases are exhausted out of the chamber by gas flow. CVD usually produces films with higher
uniformity than PVD and is particularly suitable for elaborately shaped objects. Thin films with high purity can be deposited by
CVD as impurities can be removed with ease from gaseous precursors by distillation.
Sol–gel processes
A sol is a colloidal suspension of solid particles in a continuous liquid, while a gel contains an integrated solid network. The conver-
sion of small molecules into solid materials is realized in sol–gel processes through chemical reactions. The earliest commercial
application of sol–gel technology was in the preparation of coatings and thin films because of its versatility and simplicity. Sol-
gel processes have been widely used to deposit thin ceramic coatings such as calcium phosphates (CaP), silica, and titania. One
of the drawbacks is coating shrinkage upon drying, which may lead to fracture due to large internal stress. Sol–gel-derived coatings
are deposited coatings.
Biomimetic modifications
Naturally occurring biochemical processes offer a wealth of inspiration for biomaterial design. Biomimetic surfaces are developed
by imitating properties or processes found in physiological systems to induce specific cell and tissue response. Biomimetic surface
modifications are typically realized by creating nature-inspired structural and topographical features and through the immobiliza-
tion/anchoring of proteins, peptides, growth factors, or certain polymers (Chen et al., 2006a).
Properties of Coatings and Thin Films on Metal Implants

Desirable Surface Functionalities of Metallic Implants
Generally, the surface of metallic implants is expected to be highly corrosion-resistant and biocompatible. The corrosion of metallic
implants causes the release of metal ions that may be necessary in trace amounts but can turn toxic at high concentrations. This
relates to the essence of their biocompatibility. The biocompatibility of 17 elemental metals has been investigated in a recent study
(Zhang et al., 2017). It should be noted that the lower pH value of inflamed tissues, like those frequently found at surgical sites,
expedites the corrosion process. In addition, the pH value and oxygen concentration, which strongly influence the corrosion
behavior, differ in different parts of our body.
Wear resistance is another important consideration for many metallic implants. Wear debris generated at the bearing surfaces
attracts macrophages and tends to kill them subsequently. It causes severe acidification of the microenvironment and facilitates
the corrosion process. This in turn leads to the formation of more debris and promotes third-body wear. Besides corrosion, cyclic
stress can also complicate the wear process as it results in faster materials fatigue than static fixed loading.
It is critical for load-bearing implants to achieve efficient tissue integration. Most metallic biomaterials are intrinsically bioinert,
and hence, they are surrounded by a FBC in vivo. The strength of this intervening soft-tissue bonding is far from sufficient. By modi-
fying the implant surfaces with a bioactive coating, rapid fixation, fast integration, and strong bonding can be realized.
The surface of many implants is also required to possess antibacterial properties because the surrounding tissues at the implant
site are susceptible to bacterial infection due to the compromised host immune ability. The purpose is to retard or prevent biofilm
formation at the implant/tissue interface in the race between host cells and bacteria to colonize the implant surface. Antibacterial
coatings on metallic implants have been actively researched and clinically used. Ideal coatings should be able to tackle various bacte-
rial species and at the same time not hamper the tissue integration ability.
The surface of blood-contacting implantable devices has to be hemocompatible. For example, the surface of coronary artery
stents should ideally induce no platelet activation and adhesion, thereby minimizing the occurrence of thrombosis or restenosis
(renarrowing of the arteries). Both organic and inorganic coatings have been developed to prevent blood coagulation. While organic
coatings allow more versatile chemical functionalization, inorganic ones usually possess higher stability and durability.
Osteoconductive and Osteogenic Coatings

Osteoconductive and osteogenic coatings facilitate implant anchorage. They are especially important for cementless implantation
that is widely used on young patients. Direct chemical bonds can be formed between bioactive materials and living human tissues.
As a typical bioactive ceramic from the family of CaP, hydroxyapatite (HA) is the most popular coating material on metallic
implants by virtue of its close chemical resemblance to the inorganic part of human hard tissue. After HA-coated devices are
implanted, the partial dissolution of the coating causes the saturation of the surrounding solution and the precipitation of a carbon-
ated HA layer. Collagen is then incorporated in this layer, and bone remodeling occurs in the areas of load transfer. Generally,
ceramic materials are chemically stable and corrosion-resistant but brittle. The insufficient fracture toughness prevents bulk HA
from being used in major load-bearing applications. Ti and its alloys are frequently used substrates for HA coatings, because
they have a lower density, stronger bonding strength to the coating, and closer thermal expansion coefficient to HA compared
with many other metals.
Fig. 2 Histological images of HA-coated steel K-wire. (A) Physiological new bone formation (arrow) starting from the endosteal part of the cortex
(A) (magnification, 10 ). (B) Detailed histology of the marked region reveals deposition of uncalcified bone matrix (arrowhead) by a layer of osteo-
blasts (arrows) that subsequently become osteocytes (double arrows) after being embedded by mineralized bone in rabbit (objective, 100). Repro-
duced from Fig. 6 of Alt, V., Bitschnau, A., Österling, J., Sewing, A., Meyer, C., Kraus, R., Meissner, S.A., Wenisch, S., Domann, E. and Schnettler, R.
(2006). The effects of combined gentamicin–hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a rabbit
infection prophylaxis model, Biomaterials 27, 4627–4634.
Thermal spraying is one of the most commonly used methods to deposit HA coatings. Plasma-sprayed HA coatings have been
clinically used in orthopedics and dentistry since the 1980s (Geesink, 1989). Typical applications include dental implants, femoral
stems, bone screws and pins, and total knee arthroplasty. Most short- and medium-term clinical studies have shown faster and
stronger fixation of HA-coated bone plates, screws, pins, intramedullary nails, dental implants, and femoral stems. Fig. 2 shows
the histological images of HA-based coatings for a cementless metallic prosthesis implanted in the intramedullary canal of rabbit
tibia. The deposition of uncalcified bone matrix and its mineralization could be clearly observed 28 days after implantation (Alt
et al., 2006).
The major concerns about plasma-sprayed HA coatings are their degradation, resorption, and debonding in physiological envi-
ronments. Coatings with low crystallinity usually suffer from coating debonding due to their fast dissolution, although they can
bring about faster implant fixation (Cheang and Khor, 1996). On the other hand, coatings with higher crystallinity and stability
would usually comprise more unmelted or partially melted particles (see Fig. 1A). They thus have weaker coating/substrate inter-
faces and interparticle cohesive strength. Another important reason for controlling coating crystallinity is that the fast dissolution of
coatings with low crystallinity and the weak interparticle bonding (for many coatings with high crystallinity) can both lead to the
generation of disintegrated HA debris. This may accelerate third-body wear and result in osteolysis. In biomedical applications, the
required degree of crystallinity is 65%–70% to ensure satisfactory long-term performance of HA coatings. Post-spraying heat treat-
ment can be utilized to increase the coatings’ crystallinity (Yu et al., 2003).
CaP represent the most widely used osteoconductive and osteogenic coatings. To achieve higher bonding strength to the
substrate, other bioactive ceramics such as calcium silicate and BioglassÒ have also been plasma-sprayed (Liu et al., 2002). However,
such coatings also face the challenge of rapid coating degradation. To address these issues, increasing research interest has been
drawn to the fabrication of gradient coating (e.g., HA and tricalcium phosphate) and HA-containing composite coatings
(Li et al., 2002).
Other methods that have been employed to prepare osteoconductive and osteogenic coatings on metallic implants include RF
sputtering (Thian et al., 2006), sol–gel spin coating or dip coating (Wang et al., 2007), chemical treatment (e.g., H2O2, acid, and
alkali) (Wang et al., 2003), anodic oxidation (Yang et al., 2004), and biomimetic procedures (Habibovic et al., 2002). To further
assist tissue ingrowth and integration, growth factors, bioactive molecules, and DNA can be incorporated in such coatings (Bose and
Tarafder, 2012).
Corrosion-Resistant Coatings
While CaP coatings on Ti alloys are primarily intended for increased bioactivity and have been clinically used, such ceramics are
deposited on Mg and its alloys mainly to improve their corrosion resistance, and currently no Mg-based medical implants are
commercially available. Their fast and localized corrosion is the major limitation to their clinical applications (even as biodegrad-
able implants). Therefore, the research work on the surface modification of Mg and its alloys has been focused on realizing their
delayed and controlled in vivo degradation.
Conversion coatings have been prepared by a variety of processes on Mg and its alloys. The natural oxide layer on Mg-based
materials is vulnerable and provides very limited protection. This layer can be thickened by using thermal, hydrothermal, or alkali
treatment (Lorenz et al., 2009). CaP coatings are commonly prepared on Mg and its alloys by immersion in solutions containing
calcium and phosphate ions such as simulated body fluid (SBF) (Gray-Munro and Strong, 2009). However, satisfactory results have
yet to be obtained due to crack formation and poor coating adhesion. Similar immersion procedures can also be used to fabricate
fluoride-containing thin films.
MAO is frequently and commercially utilized to enhance the corrosion and wear resistance of Mg-based materials through the
formation of a stable, hard ceramic coating. Higher corrosion resistance and hence improved cell adhesion have been observed for
a MAO-treated Mg–Ca alloy compared with the uncoated samples (Gu et al., 2011). By tuning the electrolyte composition, it is
possible to produce coatings containing CaP and fluoride by MAO.
In addition, MAO is widely used as a pretreatment for the preparation of polymeric deposited coatings (Zhang and Kang, 2014).
Such composite coatings are expected to possess increased corrosion resistance and enhanced adhesion strength. Most deposited
coatings on Mg and Mg alloys are fabricated by wet chemical procedures as their high chemical reactivity makes many high-
energy physical methods inappropriate. Frequently used processes for preparing deposited coatings include electrodeposition,
dipping, immersion, and sol–gel methods. Coatings produced by such processes can be not only corrosion-resistant but also highly
functional. They have thus been actively investigated for their potential in drug delivery, immobilization of biomolecules (such as
growth factors, enzymes, and peptide sequences), and enhanced osteointegration (Di Mario et al., 2004). Nevertheless, the primary
focus is to obtain long-term uniform degradation at an appropriate rate.
Diamond-like carbon, which was developed in the 1970s, has been successfully used to improve the corrosion and wear resis-
tance, biocompatibility, and hemocompatibility of metallic implants. DLC is a hydrogenated amorphous carbon and contains
various quantities of sp2 and sp3 carbon bonds. It possesses bioinertness, low friction coefficient, and higher hardness and stiffness
than most ceramics. A variety of methods have been developed to deposit DLC coatings, such as CVD, ion beam deposition, PIIID,
magnetron sputtering, pulsed laser ablation, and filtered cathodic arc deposition (Dearnaley and Arps, 2005). Fig. 1C presents the
typical surface topography of a DLC coating prepared by CVD. Although DLC coating is able to effectively retard the corrosion of Mg
and its alloys, the nondegradability and durability make it not suitable for biodegradable Mg-based implants. Nevertheless, DLC
has been widely deposited on metallic implants designed for long-term applications and led to commendable outcomes. For
instance, experimental results have shown that with a DLC coating, the corrosion rates of stainless steel, Ti6Al4V and CoCrMo
in 10 wt% HCl solution, could be decreased by a factor of 10,000–15,000 (Lappalainen et al., 1998).
While the corrosion behavior of metals can be effectively improved through surface modification, the intrinsic corrosion resis-
tance of the bulk material plays a fundament role. For example, the purification of Mg and incorporation of suitable alloying
elements can both substantially enhance its corrosion resistance.
Wear-Resistant Coatings
The excessive wear of acetabulum is a major challenge facing total hip and knee replacements, where Ti alloys, CoCrMo alloys, and
stainless steels are widely used. It leads to joint loosening and the generation of wear debris that can cause osteolysis. The wear resis-
tance of Mg and its alloys also needs to be enhanced to use them in load-bearing medical implants. Such problems can be alleviated
by depositing a wear-resistant coating with high hardness and a low friction coefficient.
Oxide ceramics such as Al2O3, ZrO2, and TiO2 have excellent corrosion and wear resistance. They have been thermally sprayed to
enhance the tribological properties of Ti6Al4V and stainless steel (Cizek et al., 2013; Chen et al., 2002). For the same purpose,
thermal spraying has also been used to prepare metal-matrix or ceramic-matrix composite coatings (Khun et al., 2015).
Another very successful approach is the use of ion implantation. For coatings and thin films prepared by ion implantation,
surface delamination is seldom an issue because of the graded composition and obscure coating/substrate interface. Ions commonly
implanted into metallic implant materials include oxygen, nitrogen, carbon, calcium, and sodium ions. Among them, nitrogen ions
are frequently implanted to improve the wear, corrosion, and fatigue resistance of Ti and Ti alloys. It was found that the (Ti, O, N)/Ti
composite coating prepared by PIIID could augment not only the mechanical properties and wear resistance but also the biocom-
patibility of NiTi shape-memory alloy (Sun et al., 2012). Similarly, nitrided and carbonitrided surface layers on Ti alpha alloy fabri-
cated by glow discharge plasma showed enhanced wear and corrosion resistance and increased fatigue strength (Sobiecki et al.,
2001).
As mentioned earlier, DLC holds great promise as a wear-resistant coating material. Compared with pyrolytic carbon, DLC can
be produced at lower temperatures and is thus more suitable for heat-sensitive substrates such as acetabular cups lined with
ultrahigh-molecular-weight polyethylene (UHMWPE) or polytetrafluoroethylene. It has been found that the frictional behavior
and wear resistance of a UHMWPE/CoCrMo sliding pair in SBF could be significantly improved by coating both sliding surfaces
with DLC (Sheeja et al., 2005). It is worth mentioning that the surface roughness of the DLC coating strongly influences the
wear rate of the counterface, especially in orthopedic applications with a sliding surface made from UHMWPE.
It was pointed out earlier that the poor wear resistance is an obvious drawback for Mg and Mg alloys used as implant materials.
Their tribological properties can also be enhanced by MAO. It was found in sliding wear tests that the mass loss of AZ91D Mg alloy
in Hanks’ solution could be reduced by one-third after MAO treatment (Zhang et al., 2007).
Hemocompatible Coatings
Besides biocompatibility, hemocompatibility is another crucial consideration for biomaterials used in blood-contacting implants. A
wide range of surface modification strategies have been employed to achieve hemocompatible surfaces. Certain organic coatings can
render device surfaces hemocompatible through biochemical reactions at a molecular level. However, they are inferior to inorganic
coatings in terms of stability and durability and therefore have been much less used.
DLC is a well-known hemocompatible coating material, which should be attributed to their bioinertness and smoothness
(smooth DLC surface shown in Fig. 1C). Conventionally, artificial heart valves are made from low-temperature isotropic (LTI)
carbon that risks fracture-induced failure. DLC shows comparably good hemocompatibility to LTI carbon, and the replacement
of LTI carbon with DLC in the heart valves has been commercially successful. DLC has also been coated onto metallic cardiovascular
stents. The results from many in vitro and in vivo tests proved that DLC could obviously suppress platelet adhesion, activation, and
aggregation and decrease their area coverage on cardiovascular implants. Furthermore, the reduced metal ion release as a result of
the DLC coating may further contribute to the device’s biocompatibility and hemocompatibility (Gutensohn et al., 2000). When
DLC is deposited on needles and medical guidewires, it brings about an additional advantage as the low friction coefficient helps
reduce the friction force.
Another group of inorganic hemocompatible materials are ceramics such as titanium oxides and titanium nitrides. Implantation
of oxygen ions has been used to enhance the hemocompatibility of Ti by thickening the surface oxide layer. Ti–O thin films man-
ufactured by PIII exhibited superior thromboresistant properties than LTI carbon in both in vitro and in vivo experiments (Yang
et al., 2002). The coimplantation of nitrogen and oxygen ions was carried out by PIIID, and the incorporation of nitrogen in the
coating was found to be able to further improve its blood compatibility (Tsyganov et al., 2005).
While the above techniques emphasize the passivation of a surface, there has been relentless research work targeting active inhi-
bition of platelets. This includes the use of drug-eluting coatings and the growth of endothelial cells on implant surfaces. Endothe-
lial cells form the interior lining of blood vessels, and their anticoagulant mechanisms are attracting increasing research interest
(Werner et al., 2007).
Antibacterial Coatings
Antibacterial coatings are deposited on device surfaces to mitigate implant-associated infection. They can be either passive or active,
depending on whether antibacterial agents are locally delivered.
Passive coatings can hinder the bacterial attachment and/or kill bacteria upon contact. The physical and chemical characteristics
of the coating, such as surface roughness, wettability, and conductivity, have strong influences on the bacterial behavior (Li et al.,
2016). By creating a crystalline anatase-enriched surface on Ti through anodization and heat treatment, the attachment of three
bacterial strains could be markedly reduced, while the soft- and hard-tissue cellular response was not negatively influenced (Del
Curto et al., 2005). Ti surfaces coated with certain polymers, such as poly(ethylene glycol) and poly(methacrylic acid), have also
shown the ability to inhibit bacterial adhesion (Harris et al., 2004; Zhang et al., 2008). Passive coatings are preferable as they induce
no bacterial resistance development, but their antibacterial efficacy is limited and varies for different bacterial strains.
Active coatings release antibacterial agents, such as antibiotics, bioactive molecules, and inorganic antimicrobial agents, to the
surrounding tissues. A number of antibiotics have been incorporated in bioceramic or biodegradable polymer coatings, such as
gentamicin, amoxicillin, carbenicillin, cephalothin, cefamandole, vancomycin, and tobramycin (Stigter et al., 2004). Although
they possess a broad antibacterial spectrum, it is difficult to achieve optimum release kinetics with minimum harmful effects on
cellular functions and tissue integration. Besides, potential drug resistance of bacteria is another problem.
The development of pathogen resistance is less of an issue for coatings containing nonantibiotic organic antibacterial agents
(Tan et al., 2014). These include chloroxylenol, chlorhexidine, poly(hexamethylene biguanide), chitosan, and hyaluronic acid
(Campbell et al., 2000). Again, it is challenging to realize controlled and sustained release of such molecules. Convincing
in vivo evidence has yet to be obtained to unambiguously prove their cytocompatibility and efficacy. In addition, since antibiotics
and organic antibacterial agents are both heat-sensitive, they cannot be loaded in the coatings through high-energy process routes
such as thermal spraying.
Active coatings can also be loaded with inorganic antimicrobial agents, among which Ag is the most commonly used. Silver
possesses a broad antibacterial spectrum and high biocompatibility. It is not prone to antibiotic resistance and can be introduced
by well-established methods such as PIIID and thermal spraying (Sanpo et al., 2009a). Compared with pure HA coating on Ti
substrate, Ag-doped HA coating showed remarkably enhanced antibacterial ability without compromising the coating’s cytocom-
patibility (Chen et al., 2006b). Nevertheless, further research is needed to understand the possible long-term tissue toxicity and its
exact antibacterial mechanisms. Other inorganic antibacterial agents include Cu, F, Ca, N, and Zn (Sanpo et al., 2009b). Like Ag,
they can be introduced to a wide variety of biomaterials including ceramics, polymers, metals, and DLC.
To realize widespread clinical applications of antibacterial coatings requires more information on their in vivo performance.
Since host immune ability plays a paramount role in infection prevention, future studies may be directed to antimicrobial coatings
with the capability to facilitate tissue integration and enhance host defense ability.
Concluding Remarks
This section broadly presented the preparation techniques and properties of coatings and thin films on metallic implants. While the
different coating characteristics were discussed separately, it is of vital importance to take into account the whole range of properties
and functionalities holistically and comprehensively in coating design and manufacture. The clinical application of a coating tech-
nology for metallic implants can only become realistic when it possesses high reproducibility and satisfies the stringent
requirements in the aforementioned interrelated aspects. This calls for effective communication between researchers, clinicians, and
manufacturers.
Furthermore, coatings on metallic implants not only present a new physiochemical niche but also alter the topography and
mechanical properties. The latter two factors also need to be scrutinized as they can elicit significant influences on the surrounding
tissues. To encourage natural interactions between the coating surface and host cells, one promising method is to mimic the exqui-
site topographic features of extracellular matrix. The highest level of biocompatibility is realized when the body cannot distinguish
the implant material from its own. The concept of biomimetic fabrication of medical implants has thus been embraced by many
researchers.
In biological evaluations of deposited coatings and thin films, it is crucial to keep in mind that the in vitro conditions are usually
far less aggressive and complicated, both mechanically and biochemically, than the in vivo environment. This warrants necessary
in vivo tests to bridge the knowledge gaps in this aspect.
While researchers have been persistently seeking to extend the functionality of coatings and thin films, it is critical to keep the
complex coating-induced immune responses under control. On top of other desirable properties, an ideal coating should be able to
enhance the host immune ability and promote the body’s self-healing.
Finally, an increasing fraction of patients are outliving the service longevity of their implants. Therefore, extending metallic
implants’ service life is a critical consideration for their coating design. Alternatively, biodegradable metallic implants, which
may be produced from Mg alloys, can be developed to allow their complete adsorption and replacement by newly grown tissues.
References
Alt, V., Bitschnau, A., Österling, J., Sewing, A., Meyer, C., Kraus, R., Meissner, S. A., Wenisch, S., Domann, E., & Schnettler, R. (2006). The effects of combined gentamicin–
hydroxyapatite coating for cementless joint prostheses on the reduction of infection rates in a Rabbit infection prophylaxis model. Biomaterials, 27, 4627–4634.
Bose, S., & Tarafder, S. (2012). Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: A review. Acta Biomaterialia, 8, 1401–1421.
Campbell, A. A., Song, L., Li, X. S., Nelson, B. J., Bottoni, C., Brooks, D. E., & DeJong, E. S. (2000). Development, characterization, and anti-microbial efficacy of hydroxyapatite-
chlorhexidine coatings produced by surface-induced mineralization. Journal of Biomedical Materials Research, 53, 400–407.
Cheang, P., & Khor, K. A. (1996). Addressing processing problems associated with plasma spraying of hydroxyapatite coatings. Biomaterials, 17, 537–544.
Chen, Q., & Thouas, G. A. (2015). Metallic implant biomaterials. Materials Science & Engineering R: Reports, 87, 1–57.
Chen, H., Zhang, Y., & Ding, C. (2002). Tribological properties of nanostructured zirconia coatings deposited by plasma spraying. Wear, 253, 885–893.
Chen, H., Tang, Z., Liu, J., Sun, K., Chang, S. R., Peters, M. C., Mansfield, J. F., Czajka-Jakubowska, A., & Clarkson, B. H. (2006a). Acellular synthesis of a human enamel-like
microstructure. Advanced Materials, 18, 1846–1851.
Chen, W., Liu, Y., Courtney, H. S., Bettenga, M., Agrawal, C. M., Bumgardner, J. D., & Ong, J. L. (2006b). In vitro anti-bacterial and biological properties of magnetron co-sputtered
silver-containing hydroxyapatite coating. Biomaterials, 27, 5512–5517.
Cizek, J., Khor, K. A., & Dlouhy, I. (2013). In-flight temperature and velocity of powder particles of plasma-sprayed TiO2. Journal of Thermal Spray Technology, 22, 1320–1327.
Cragg, A., Lund, G., Rysavy, J., Castaneda, F., Castaneda-Zuniga, W., & Amplatz, K. (1983). Nonsurgical placement of arterial endoprostheses: A new technique using nitinol wire.
Radiology, 147, 261–263.
Dearnaley, G., & Arps, J. H. (2005). Biomedical applications of diamond-like carbon (DLC) coatings: a review. Surface and Coatings Technology, 200, 2518–2524.
Del Curto, B., Brunella, M., Giordano, C., Pedeferri, M., Valtulina, V., Visai, L., & Cigada, A. (2005). Decreased bacterial adhesion to surface-treated titanium. International Journal of
Artificial Organs, 28, 718–730.
Di Mario, C., Griffiths, H., Goktekin, O., Peeters, N., Verbist, J., Bosiers, M., Deloose, K., Heublein, B., Rohde, R., & Kasese, V. (2004). Drug-eluting bioabsorbable magnesium stent.
Journal of Interventional Cardiology, 17, 391–395.
Fu, Y. Q., Batchelor, A. W., Wang, Y., & Khor, K. A. (1998). Fretting wear behaviors of thermal sprayed hydroxyapatite (HA) coating under unlubricated conditions. Wear, 217,
132–139.
Geesink, R. (1989). Experimental and clinical experience with hydroxyapatite-coated hip implants. Orthopedics, 12, 1239–1242.
Goodman, S. B., Yao, Z., Keeney, M., & Yang, F. (2013). The future of biologic coatings for orthopaedic implants. Biomaterials, 34, 3174–3183.
Gotman, I. (1997). Characteristics of metals used in implants. Journal of Endourology, 11, 383–389.
Gray-Munro, J. E., & Strong, M. (2009). The mechanism of deposition of calcium phosphate coatings from solution onto magnesium alloy AZ31. Journal of Biomedical Materials
Research. Part A, 90A, 339–350.
Gu, X. N., Li, N., Zhou, W. R., Zheng, Y. F., Zhao, X., Cai, Q. Z., & Ruan, L. (2011). Corrosion resistance and surface biocompatibility of a microarc oxidation coating on a Mg–Ca
alloy. Acta Biomaterialia, 7, 1880–1889.
Gutensohn, K., Beythien, C., Bau, J., Fenner, T., Grewe, P., Koester, R., Padmanaban, K., & Kuehnl, P. (2000). In vitro analyses of diamond-like carbon coated stents: Reduction of
metal ion release, platelet activation, and thrombogenicity. Thrombosis Research, 99, 577–585.
Habibovic, P., Barrère, F., Van Blitterswijk, C. A., de Groot, K., & Layrolle, P. (2002). Biomimetic hydroxyapatite coating on metal implants. Journal of the American Ceramic Society,
85, 517–522.
Harris, L. G., Tosatti, S., Wieland, M., Textor, M., & Richards, R. G. (2004). Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-
functionalized poly(l-lysine)-grafted-poly(ethylene glycol) copolymers. Biomaterials, 25, 4135–4148.
Hench, L. L., & Polak, J. M. (2002). Third-generation biomedical materials. Science, 295, 1014–1017.
Hornberger, H., Virtanen, S., & Boccaccini, A. R. (2012). Biomedical coatings on magnesium alloysdA review. Acta Biomaterialia, 8, 2442–2455.
Khor, K. A., Li, H., & Cheang, P. (2003). Processing–microstructure–property relations in HVOF sprayed calcium phosphate based bioceramic coatings. Biomaterials, 24,
2233–2243.
Khun, N. W., Li, R. T., Loke, K., & Khor, K. A. (2015). Effects of Al-Cr-Fe Quasicrystal content on Tribological properties of cold-sprayed titanium composite coatings. Tribology
Transactions, 58, 616–624.
Lappalainen, R., Anttila, A., & Heinonen, H. (1998). Diamond coated total hip replacements. Clinical Orthopaedics and Related Research, 352, 118–127.
Li, H., Khor, K. A., & Cheang, P. (2002). Titanium dioxide reinforced hydroxyapatite coatings deposited by high velocity oxy-fuel (HVOF) spray. Biomaterials, 23, 85–91.
Li, Z., Tang, X.-Z., Zhu, W., Thompson, B. C., Huang, M., Yang, J., Hu, X., & Khor, K. A. (2016). Single-step process toward achieving superhydrophobic reduced graphene oxide.
ACS Applied Materials & Interfaces, 8, 10985–10994.
Lister, J. (1867). On the antiseptic principle in the practice of surgery. The Lancet, 90, 353–356.
Liu, X., Tao, S., & Ding, C. (2002). Bioactivity of plasma sprayed dicalcium silicate coatings. Biomaterials, 23, 963–968.
Lorenz, C., Brunner, J. G., Kollmannsberger, P., Jaafar, L., Fabry, B., & Virtanen, S. (2009). Effect of surface pre-treatments on biocompatibility of magnesium. Acta Biomaterialia, 5,
2783–2789.
Murugan, R., & Ramakrishna, S. (2005). Development of nanocomposites for bone grafting. Composites Science and Technology, 65, 2385–2406.
Sanpo, N., Tan, M. L., Cheang, P., & Khor, K. (2009a). Antibacterial property of cold-sprayed HA-Ag/PEEK coating. Journal of Thermal Spray Technology, 18, 10–15.
Sanpo, N., Ang, S. M., Cheang, P., & Khor, K. (2009b). Antibacterial property of cold sprayed chitosan-Cu/Al coating. Journal of Thermal Spray Technology, 18, 600–608.
Sheeja, D., Tay, B. K., & Nung, L. N. (2005). Tribological characterization of surface modified UHMWPE against DLC-coated Co–Cr–Mo. Surface and Coatings Technology, 190,
231–237.
Sobiecki, J. R., Wierzchon, T., & Rudnicki, J. (2001). The influence of glow discharge nitriding, oxynitriding and carbonitriding on surface modification of Ti–1Al–1Mn titanium alloy.
Vacuum, 64, 41–46.
Stigter, M., Bezemer, J., de Groot, K., & Layrolle, P. (2004). Incorporation of different antibiotics into carbonated hydroxyapatite coatings on titanium implants, release and antibiotic
efficacy. Journal of Controlled Release, 99, 127–137.
Sun, T., Wang, L. P., & Wang, M. (2011). (Ti, O)/Ti and (Ti, O, N)/Ti composite coatings fabricated via PIIID for the medical application of NiTi shape memory alloy. Journal of
Biomedical Materials Research. Part B, Applied Biomaterials, 96B, 249–260.
Sun, T., Wang, L.-P., Wang, M., Tong, H.-W., & Lu, W. W. (2012). PIIID-formed (Ti, O)/Ti, (Ti, N)/Ti and (Ti, O, N)/Ti coatings on NiTi shape memory alloy for medical applications.
Materials Science and Engineering: C, 32, 1469–1479.
Tan, X. W., Goh, T. W., Saraswathi, P., Nyein, C. L., Setiawan, M., Riau, A., Lakshminarayanan, R., Liu, S., Tan, D., & Beuerman, R. W. (2014). Effectiveness of antimicrobial
peptide immobilization for preventing perioperative cornea implant-associated bacterial infection. Antimicrobial Agents and Chemotherapy, 58, 5229–5238.
Thian, E. S., Huang, J., Best, S. M., Barber, Z. H., Brooks, R. A., Rushton, N., & Bonfield, W. (2006). The response of osteoblasts to nanocrystalline silicon-substituted
hydroxyapatite thin films. Biomaterials, 27, 2692–2698.
Tsyganov, I., Maitz, M. F., Wieser, E., Richter, E., & Reuther, H. (2005). Correlation between blood compatibility and physical surface properties of titanium-based coatings. Surface
and Coatings Technology, 200, 1041–1044.
Wang, C. X., Wang, M., & Zhou, X. (2003). Nucleation and growth of apatite on chemically treated titanium alloy: An electrochemical impedance spectroscopy study. Biomaterials,
24, 3069–3077.
Wang, Y., Zhang, S., Zeng, X., Ma, L. L., Weng, W., Yan, W., & Qian, M. (2007). Osteoblastic cell response on fluoridated hydroxyapatite coatings. Acta Biomaterialia, 3, 191–197.
Werner, C., Maitz, M. F., & Sperling, C. (2007). Current strategies towards hemocompatible coatings. Journal of Materials Chemistry, 17, 3376–3384.
Witte, F. (2010). The history of biodegradable magnesium implants: a review. Acta Biomaterialia, 6, 1680–1692.
Yang, P., Huang, N., Leng, Y. X., Chen, J. Y., Sun, H., Wang, J., Chen, F., & Chu, P. K. (2002). In vivo study of Ti–O thin film fabricated by PIII. Surface and Coatings Technology,
156, 284–288.
Yang, B., Uchida, M., Kim, H.-M., Zhang, X., & Kokubo, T. (2004). Preparation of bioactive titanium metal via anodic oxidation treatment. Biomaterials, 25, 1003–1010.
Yu, L. G., Khor, K. A., Li, H., & Cheang, P. (2003). Effect of spark plasma sintering on the microstructure and in vitro behavior of plasma sprayed HA coatings. Biomaterials, 24,
2695–2705.
Zhang, J., & Kang, Z. (2014). Effect of different liquid–solid contact models on the corrosion resistance of superhydrophobic magnesium surfaces. Corrosion Science, 87, 452–459.
Zhang, X. P., Zhao, Z. P., Wu, F. M., Wang, Y. L., & Wu, J. (2007). Corrosion and wear resistance of AZ91D magnesium alloy with and without microarc oxidation coating in Hank’s
solution. Journal of Materials Science, 42, 8523–8528.
Zhang, F., Zhang, Z., Zhu, X., Kang, E.-T., & Neoh, K.-G. (2008). Silk-functionalized titanium surfaces for enhancing osteoblast functions and reducing bacterial adhesion.
Biomaterials, 29, 4751–4759.
Zhang, D., Wong, C. S., Wen, C., & Li, Y. (2017). Cellular responses of osteoblast-like cells to 17 elemental metals. Journal of Biomedical Materials Research. Part A, 105,
148–158.
Titanium Alloys
Mitsuo Niinomi, Tohoku University, Sendai, Japan; Osaka University, Osaka, Japan; Meijo University, Nagoya, Japan; and Nagoya
University, Nagoya, Japan
Introduction 213
Nontoxic Ti Alloys 213
Low Modulus Ti Alloys 214
Low Young’s Modulus b-Type Ti Alloys With Changeable Young’s Modulus and for Reconstructive Implants 215
Improvement of Mechanical Properties of Low Modulus b-Type Titanium Alloy for Biomedical Applications 217
Improvement of Static Strength by Severe Cold Working 217
Improvement of Dynamic Strength by Thermomechanical Treatment 217
Improvement of Dynamic Strength Using O as Alloying Element 218
Effect of Low Young’s Modulus on Stress Shielding 220
Summary 222
References 223
Further Reading 224
Introduction
Biomaterials are grouped into biotolerant, bioinert, and bioactive materials from the pattern of osteogenesis as listed in Table 1
(Niwa, 2004). With regards to biocompatibility, biotolerant materials are superior to bioinert materials, which are in turn less
than bioactive materials. Some representative metallic biomaterials are austenitic stainless steel (mainly SUS 316L), Vitallium
(Co–Cr–Mo alloys), and titanium (Ti) and its alloys. As shown in Table 1, Ti and its alloys are classified as bioinert materials.
On the other hand, SUS 316L and Co–Cr–Mo alloys are grouped into the biotolerant category. Therefore, the biocompatibility
of Ti and its alloys is better than that of SUS 316L and Co–Cr–Mo alloys. Ti is regarded as a nontoxic and nonallergenic element
based on cell viability characterization (Steinemann, 1980; Kawahara et al., 1963; Yamamoto et al., 1988) and rate of metallic
allergy of pure metals (Uggowitzer et al., 1997; Niinomi, 2000). Ti and its alloys also present fewer imaging artifacts, which results
in clear MRI (magnetic resonance imaging) images (Radzi et al., 2014). Furthermore, Ti alloys exhibit high corrosion resistance,
high specific strength meaning a light weight and an high strength, and excellent balance of strength and ductility. For these reasons,
Ti alloys are receiving significant attention as biomaterials for constructing implants that replace failed hard tissues as cortical bone
(Fig. 1; Nakano, 2010).
In the present article, from the view point of biomedical applications, review of the latest research on nontoxic Ti alloys, low
Young’s modulus b-type Ti alloys, and low Young’s modulus b-type Ti alloys with changeable Young’s modulus and for reconstruc-
tive implants is presented. Further, improvements in the mechanical properties of low Young’s modulus b-type Ti alloys for biomed-
ical applications and the effect of low Young’s modulus on stress shielding are described.
Nontoxic Ti Alloys
The earliest used Ti-biomaterial was pure Ti. Lately, an alloy of Ti has also come into use: Ti–6Al–4V ELI (extra low interstitial) (mass
%). There are four grades for pure Ti: grades 1 through 4, where the concentration of impurities such as nitrogen (N), iron (Fe), and
oxygen (O) increases with increasing grade number. The strength of pure Ti increases with impurity concentration, while the
ductility decreases. Pure Ti and Ti–6Al–4V ELI have been standardized as ASTM F-67-13 (ASTM International, 2016a) and F136-
13 (ASTM International, 2016b). Additionally, Ti–6Al–4V has been standardized as ASTM F1108-14 (ASTM International,
Table 1 Biocompatibility of various biomaterials judged by patterns of osteogenesis
Pattern of osteogenesis Biomaterials
Intervend osteogenesis Stainless steel, Vitallium, PMMA (polymethyl Biotolerant materials

methacrylate)
Contact osteogenesis Titanium, titanium alloys, carbon, alumina, zirconia, Bioinert materials
titania, TiN, Si3N4
Bonding osteogenesis Bioglass, ceravital, tricalcium phosphate, hydroxyapatite, Bioactive materials
A-W glass ceramic

214 Biomaterials: Science and engineering j Titanium Alloys
Artificial shoulder joints Artificial dental implants
Spinal fixation Artificial elbow

devices joints
Artificial hip joints
Artificial finger joints
Bone fixation devices

Artificial knee joints
Artificial ankle
joints
Fig. 1 Various kinds of implants constructed with metallic biomaterials: any of them are made of titanium alloys.
2016c) for castings in surgical implants and as ASTM F1472-14 (ASTM International, 2016d) as a wrought alloy for the same appli-
cation. Pure Ti and Ti–6L–4V ELI continue to be the primary Ti-based materials for constructing implants, although they have been
diverted from general structural usage.
However, since the discovery of the toxicity of vanadium (V) which is a constitutional element of Ti–6Al–4V ELI, V-free Ti alloys
for biomedical applications such as Ti–5Al–2.5Fe (ISO, 1996), Ti–6Al–7Nb (ASTM International, 2016e), Ti–6Al–6Nb–1Ta (Nii-
nomi, 2002), and Ti–6Al–2Nb–1Ta (JISC, 2002a) have been developed. Since then, Al, a constitutional element of Ti–6L–4V ELI,
has also been pointed out as a harmful element for the human body, V- and Al-free Ti alloys such as Ti–15Sn–4Nb–2Ta–0.2Pd
(Okazaki et al., 1996), Ti–15Zr–4Nb–2Ta–0.2Pd (JIS T 7401-5) (JISC, 2002b), and Ti–4.5Al–6Nb–2Fe–2Mo (Ueda et al.,
2013). The latter has been modified super plasticity Ti–4.5Al–3V–2Fe–2Mo (SP-700) (Suzuki et al., 1999) for biomedical
applications.
Ti alloys for biomedical applications mentioned earlier are all (a þ b)-type Ti alloys except for pure Ti, which is an a-type Ti
alloy.
Low Modulus Ti Alloys
The development of Ti alloys for biomedical applications has been not only by the need for nontoxic elements but also nonaller-
genic elements, but for inhibiting stress shielding, which is the inhomogeneous stress transfer between the host bone and the
implant that leads to bone resorption and poor bone remodeling.
The high-risk elements for allergy are Ni, Cr, and Co. Based on the rate of allergy of pure metals reported in the field of dentistry,
Ni is the most restricted allergenic element in metallic biomaterials. The nontoxic components of Ti alloys for biomedical applica-
tions can be identified using cell viability testing results. From this data, Nb, Ta, Mo, Zr, etc. have been found to be appropriate
nontoxic and nonallergenic elements for alloying with Ti, judging from cell viability and rate of allergy of pure metals for designing
Ti alloys for biomedical applications.
Ti alloys are roughly grouped into a-, (a þ b)-, and b-type Ti alloys based on the constituent phases. The Young’s moduli of
b-type Ti alloys, where the b-phase with the body-centered cubic (bcc) structure is predominant, are expected to be lower than those
of a-type Ti alloys where the a-phase with the hexagonal close packed (hcp) structure is predominant, and (a þ b)-type Ti alloys
where a- and b-phases are mixed because the atomic density of the bcc structure is less than that of the hcp structure. Typically, Ti
alloys designed to be b-type Ti alloys, as b-type Ti alloys are usually composed of nontoxic and nonallergenic b stabilizing elements
(Nb, Ta, Mo, etc.) and exhibit Young’s moduli close to that of the cortical bone (approximately 10–30 GPa (Niinomi and Boehler,
2015)). It is noted that the Young’s moduli of representative metallic biomaterials such as Ti–6Al–4V ELI, SUS 316L, and Co–Cr–
Mo alloy are approximately 110, 180, and 210 GPa, respectively (Niinomi et al., 2012).
A number of b-type titanium alloys with low Young’s modulus for biomedical applications have been developed thus far. Typi-
cally, these alloys contain a large amount of b stabilizing elements such as Nb, Mo, and/or Ta, and a small amount of other elements
such as Zr, Sn, Hf, Si, and/or Fe (Niinomi et al., 2012).
Fig. 2 (Niinomi et al., 2012) shows the Young’s moduli of various low Young’s modulus b-type titanium alloys developed for
biomedical applications. The data shown includes samples subjected to various treatments and moduli measured by various
Biomaterials: Science and engineering j Titanium Alloys 215
Alloy, treatment, measuring method

Ti-6Al-4V ELI (Mill anealed, Unclear)
Ti-6Al-4V (Annealed, Unclear)
Ti-6Al-4V (Annealed,Ultrasonic method)
Ti-6Al-4V (Annealed, Nanoindenter)
Ti-20Nb (WQ: water quenching, NQ: liquid nitrogen cooling, AC: air cooling, FC: furnace cooling, Ultrasonic method)
Ti-13Nb-13Zr (ST, STA, Ultrasonic method)
Ti-13Nb-13Zr (ST, STA, AC, WQ+50-75%CW:cold working, Three point bending test)
Ti-27Nb-8Zr (ST: solution treatment, STA: aged after ST, Tensile test)
Ti-29Nb-13Ta (ST, STA, Tensile test)
Ti-22.5Nb-0.7Zr-2Ta-(0.5-2.5)O (ST, Tensile test)
Single cryatal of Ti-29Nb-13Ta-4.6Zr ((100), ST+ zone melting, Electromagnetic acoustic resonance)
Single cryatal of Ti-29Nb-13Ta-4.6Zr ((111)ST+ zone melting, Electromagnetic acoustic resonance)
Ti-23Nb-0.7Ta-2.0Zr-1.2O (ST, Dynamic mechanical analysis)
Ti-29Nb-13Ta-4.6Zr (ST, STA, CR: cold rolling, Resonance vibration method)
Ti-29Nb-13Ta-4.6Zr (0.3 mm dia. Wire, Cold drawing, Dynamic super micro hardness tester)
Ti-29Nb-13Ta-4.6Zr (1.0 mm dia. Wire, Cold drawing, Dynamic super micro hardness tester)
Ti-29Nb-13Ta-4.6Zr (0.1-0.7)O (ST, Resonance vibration method)
Ti-29Nb-13Ta-4.6Zr-(0-0.5)B (ST+ cold rolling, Resonance vibration method)
Ti-29Nb-13Ta-4.6Zr-(0.05-1.0))Y2O3 (ST + cold rolling, Resonance vibration method)
Ti-35Nb-(0-10)Ta-(0-10)Zr (ST, STA, Tensile test)
Ti-35Nb-2Ta-3Zr (in TD direction, ST,Tensile test)
Ti-35Nb-2Ta-3Zr (in 45 degree, ST,Tensile test)
Ti-35Nb-2Ta-3Zr (in RD direction, ST,Tensile test)
Ti-35.5Nb-7Zr-5Ta (TNZT) (ST,Transmission ultrasonic tequnique)
Ti-(22-35.5)Nb-(5-22)Ta-(4-7.2)Zr (ST, Transmission ultrasonic tequnique)
Single crystal Ti-36Nb-2Ta-3Zr-0.09O (ST + zone melting, Electromagnetic acoustic resonance)
Ti-35Nb-4Sn ( ST to sever cold rolling, Tensile test)
Ti-16Nb-13Ta-4Mo (ST, STA, Tensile test)
Ti-29Nb-13Ta-4Mo (ST, STA, Tensile test)
Ti-29Nb-13Ta-2Sn (ST, STA, Tensile test)
Ti-29Nb-13Ta-4.6Sn (ST, STA, Tensile test)
Ti-29Nb-13Ta-6Sn (ST, STA, Tensile test)
Ti-28Nb-13Zr-0.5Fe(TNZF) (ST, STA, Tensile test)
Ti-24Nb-4Zr-7.9Sn (Ti2448) (ST + cold rolled, Hot forged,Cold rolled, Annealed, Tensile test Resonance vibration method)
Ti-7.5Mo (ST, Unclear)

Ti-15Mo (ST, Tensile test)
Ti-(15-18)Mo (ST, Resonance vibration method)
Ti-12Mo-3Nb (ST, Nanoindenter)
Ti-12Mo-5Ta (ST, Ultrasonic method)
Ti-15Mo-5Zr-3Al (ST, STA, Ultrasonic method)
Ti-15Mo-2.8Nb-0.2Si (21RX) (ST, Tensile and compression)
Ti-16Nb-9.5Hf (Tiadyne) (STA, Unclear)
Ti-12Mo-6Zr-2Fe (TMZF) (Solution annealed, Tensile test)
Ti-(10-70)Ta (ST, Resonance vibtation method)

Ti-50Ta (ST, STA, Resonance vibration method)
Ti-30Zr-(1-3)Cr-(3-5)Mo (ST, Resonance vibration method)
Ti-(10-14)Cr (ST, Resonance vibration method)
0 100 200
Young’s modulus (GPa)
Fig. 2 Young’s moduli of representative b-type titanium alloys for biomedical applications with treatment and measuring method.
methods. The Young’s moduli of low Young’s modulus b-type titanium alloys that are subjected to solution treatment (ST) lie
between 40 and 80 GPa. These alloys show a single b-phase. However, in the specific case, where the entire sample is a single crystal
growth directions, the Young’s modulus is found to be as that of cortical bone. For example, a single crystal of the low Young’s
modulus b-type titanium alloy Ti–29Nb–13Ta–4.6Zr (TNTZ), growing in the h100i direction exhibits a lower Young’s modulus
(35 GPa) when compared with crystal growth in directions such as h111i and h110i (Tane et al., 2008). It is noted that tensile tests
appear to measure slightly lower values of Young’s modulus than the free-resonance or ultrasonic methods, while the dynamic
super microhardness test measures slightly higher values. The Young’s modulus of these alloys may be increased to 100 GPa
and over using processing techniques such as by applying an aging treatment.
Due to the high cost of elements such as Nb, Ta, Mo, and Zr, biomedical b-type titanium alloys with low Young’s moduli
composed of low cost elements such as Fe, Cr, Mn, Sn, and Al have also been developed. Some examples of low Young’s moduli
alloys with low cost elements are Ti–10Cr–Al (Hatanaka et al., 2010), Ti–Mn (Ikeda et al., 2009), Ti–Mn–Fe (Ikeda et al., 2012), Ti–
Mn–Al (Ikeda et al., 2010), Ti–Cr–Al (Ikeda and Sugano, 2005), Ti–Sn–Cr (Ashida et al., 2012), Ti–Cr–Sn–Zr (Murayama and
Sasaki, 2009), Ti–(Cr, Mn)–Sn (Kasano et al., 2010), and Ti–12Cr (Nakai et al., 2011).
Low Young’s Modulus b-Type Ti Alloys With Changeable Young’s Modulus and for Reconstructive Implants
Low Young’s modulus Ti alloys are considered to be suitable for rods used in spinal fixation devices. The flexibility of these alloys is
patient-friendly feature as it is expected to inhibit the damage to adjacent vertebra. However, during operations, the rods in spinal
fixation devices are bent by surgeons to match the physiological curvature of the patient’s spine as possible as closely as possible
(Steib et al., 2004). This necessitates that the return back of bent rod to its original shape, that is, springback, should be prevented.
In summary, the materials used in spinal fixation rods must have low Young’s modulus measured across the entire rod and minimal
springback. To satisfy both requirements, an increased Young’s modulus is required at the bent section of the rod. This demand can
be satisfied if the secondary phase with high Young’s modulus can be induced by bending, that is, deformation. It is known that the
deformation-induced a0 martensite and a00 martensite phases can decrease the Young’s modulus of Ti alloys, while the u-phase is
known to significantly influence the mechanical properties of b-type Ti alloys, and likely to increase the Young’s modulus. Using this
rationale, Ti–12Cr was developed (Nakai et al., 2011). Fig. 3 shows the change in Young’s moduli of b-type, alloys A, B, and C, and
Ti–12Cr subjected to solution treatment before and after deformation by cold rolling to a 10% reduction (CR), which simulates
α”
80 No phase α’ ω ω
transformation α” ω
oung’s modulus , E / GPa

β
TEM DF of allo
alloy B 60
after deformation
α’ TEM DF of Ti-12Cr
-12Cr
40 after deformation
CR
CR
CR
CR
ST
ST
ST
ST
Young
20
TEM DF of alloy A
after deformation
0
ST:: So
Solut
ution
on treatm
atment
nt β-type
-type β-type
-type β-type
-type
CR: CoCold rolli
lling
ng alloy A Ti-12Cr
-12Cr
alloy B alloy C
Fig. 3 Change in Young’s moduli of b-type alloy A, B, C and Ti–12Cr subjected to solution treatment (ST) indicating before deformation and cold
rolling by a 10% reduction (CR) indicating after deformation.
bending deformation of the spinal fixation rods. The deformation was confirmed using TEM diffraction patterns shown in this
figure. For b-type alloy A, no change in Young’s modulus is recognized as no deformation-induced secondary phase is formed.
For b-type alloys B and C, the Young’s moduli decrease after deformation due to the formation of deformation-induced
a00 martensite and a0 martensite, respectively. For Ti–12Cr, the Young’s modulus increases after deformation due to the formation
of the deformation-induced u-phase. Fig. 4 (Zhao et al., 2012a) shows springback of Ti–12Cr and TNTZ. It is noted that in TNTZ, no
deformation-induced phase is formed. The springback of Ti–12Cr is much smaller than that of TNTZ.
The enhancement in the Young’s modulus of the Ti–Cr-based alloys can be further increased by adding a small amount of
oxygen (O) to prevent the formation of the athermal u-phase and by controlling the Cr content. As a result, Ti–10Cr–0.2 and
Ti–9Cr–0.2 have been developed as Young’s modulus changeable b-type alloys for the rods in spinal fixation devices (Liu et al.,
2015, 2016).
Ti–17Mo (Zhao et al., 2012b), Ti–30Zr–7Mo (Zhao et al., 2011a), Ti–30Zr–5Cr (Zhao et al., 2011b), and Ti–30Zr–3Cr–3Mo
(Zhao et al., 2011b) were also developed as Young’s modulus changeable b-type Ti alloys with low Young’s modulus. Among those,
Ti–30Zr–7Mo, Ti–30Zr–5Cr, and Ti–30Zr–3Cr3Mo were developed for use in reconstructive implants. Reconstructive implants,
which are removable implants constructed using Ti alloys, should (a) not tightly fuse with bone and (b) have good biocompati-
bility. This is because, in some cases, certain internal fixation devices implanted into the bone marrow (femoral, tibial, or humeral
marrow) or those, which would otherwise be incorporated into the bone, need to be removed after surgery. These include the screws
used for bone plates fixation and implants used for children, which might have to be removed to address local symptoms such as
palpable hardware and wound dehiscence/exposure of hardware, or to enable athletes returning to contact sport (Kobayashi et al.,
2007). In these cases, the assimilation of the removable internal fixators with the bone (because of the precipitation of calcium
0.06
Ratio of springback per unit load,
0.05
0.04
R/(%N–1)
TiNTZ
0.03
0.02
0.01
Ti-12Cr
0
0 2 4 6 8 10 12 14
Applied strain (%)
Fig. 4 Spring-backs of Ti–12Cr and Ti–29Nb–13Ta–4.6ZrTNTZ.
phosphate) might cause refracture of the bone during the fixator-removal operation. The Ti alloys used in these fixators contain
a large amount of Zr because Zr has been reported to show the ability to prevent the precipitation of calcium phosphate (Kobayashi
et al., 2007; Tsutsumi et al., 2009). Further, some titanium alloys with Zr contents exceeding 25 mass% have also been reported to
prevent the formation of calcium phosphate, which is the main component of human bones as mentioned earlier (Tsutsumi et al.,
2009).
Improvement of Mechanical Properties of Low Modulus b-Type Titanium Alloy for Biomedical Applications
Fig. 5 shows the elastic modulus (Young’s modulus), tensile strength, and elongation of selected biomedical b-type titanium alloys
with a single b-phase. The Young’s modulus and elongation tend to increase and decrease with increasing tensile strength. In b-type
titanium alloys with a single b-phase and with relatively lower Young’s moduli such as Ti–35.3Nb–7.1Zr–5.1Ta and TNTZ, the
strengths are found to be poor. For these kinds of alloys, an improvement in strength, while maintaining a low Young’s modulus
and a good ductility, is required.
Improvement of Static Strength by Severe Cold Working

Static strength such as tensile strength can be improved significantly by severe cold working techniques such as cold rolling (Nii-
nomi, 2010), cold swaging (Niinomi et al., 2002a), and cold drawing (Niinomi et al., 2007), as well as by severe plastic deformation
such as high-pressure torsion (Yilmazer et al., 2013). By these methods, the tensile strength of TNTZ is increased to values equal to
or over that of Ti–6Al–4V ELI, while maintaining good ductility and a Young’s modulus equal to or lower than that of TNTZ sub-
jected only to solution treatment (approximately 60 GPa).
Improvement of Dynamic Strength by Thermomechanical Treatment

While static strength can be improved, it is difficult to increase dynamic strength such as fatigue strength by the earlier-mentioned
severe cold working process. To improve the dynamic strength, introducing a secondary phase is required. A short aging treatment to
introduce a small amount of u-phase in TNTZ, which significantly increases the strength and Young’s modulus of a b matrix is effec-
tive in also increasing fatigue strength to values similar to those of Ti–6Al–4V ELI, with good ductility and the Young’s modulus at
the level of approximately 75 GPa (Nakai et al., 2012).
Adding a small amount of ceramics such as TiB or Y2O3 is also effective in improving the fatigue strength to values similar to
those of Ti–6Al–4V ELI, while maintaining the Young’s modulus as same as that of TNTZ subjected only to ST (approximately
60 GPa (Song et al., 2010)).
Further improvement in fatigue strength can also be achieved by special thermomechanical treatment such as a direct aging treat-
ment after severe cold working, However, by this method, the Young’s modulus becomes relatively high (approximately order of
80 GPa, which is approximately the average Young’s moduli of low Young’s modulus b-type Ti alloy with single b-phase (Narita
et al., 2012)).
Fig. 5 Elastic modulus (Young’s modulus), tensile strength, and elongation of selected b-type titanium alloys for biomedical applications.
Fig. 6 (Narita et al., 2012) shows the special thermomechanical processes (CRA (cold rolling and aging) and CWA (cold swaging
and aging)) and general thermomechanical process (STA (solution treatment and aging)) applied to TNTZ. Fig. 7 (Narita et al.,
2012) shows the fatigue strengths (S–N curves) of TNTZ subjected to CRA and CWA, and STA along with those of Ti–6Al–4V
ELI (Ti-64). The fatigue strengths of TNTZ subjected to CRA and SWA are much greater than those of TNTZ subjected to STA
and Ti-64. The fatigue strength of TNTZ subjected to SWA is a little greater than that of TNTZ subjected to CRA. Evidently, the fatigue
strength can be improved significantly by special thermomechanical treatments such as SWA and CRA.
Improvement of Dynamic Strength Using O as Alloying Element

O is in general regarded as an impurity in Ti and its alloys, but currently O is being recognized as an effective alloying element to
improve mechanical properties of titanium alloys.
Fig. 8 (Liu et al., 2017a) shows the room temperature true stress–strain curves and the corresponding work-hardening rates of
TNTZ containing 0.14, 0.33, and 0.70 mass% O subjected to solution treatment (ST), which forms a single b-phase. These alloys are
(A)
STA Solution treatment :

1063K, 3.6ks
Coil Aging treatments:
Wire drawing βtr : 1013K
723K, 259.2 ks
(Area reduction
ratio: 16%) WQ
Rod
WQ
(B)
CRA Solution treament:
1063K, 3.6ks Aging treatments:
Cold caliber rolling 723K, 259.2 ks
βtr : 1013K
(Area reduction
Bar WQ ratio: 88%)
WQ
Rod
(C)
SWA
Aging treaments:
Cold swaging 723K, 259.2 ks
(Area reduction
Bar ratio: 91%)
WQ
βtr : β transus
WQ :Water quenching Rod
Fig. 6 Special thermomechanical processes (CRA and CWA) and general thermomechanical process (STA) for TNTZ.
1200
Maximum cyclic stress, σmax/ MPa
1000
800
600
400
STA
CRA
200 SWA
Ti-64
0
104 105 106 107
Number of cycles to failure, Nf
Fig. 7 Fatigue strength (S–N curves) of TNTZ subjected to special thermomechanical processes (CRA and CWA) and general thermomechanical
process (STA) for TNTZ shown in Fig. 7.
2000
Work-hardening rate, dσ/dε (MPa)

Work-hardening 0.1ST
rate curves 0.3ST
0.7ST
True stress, σ (MPa)

1500
Necking point
1000
500
True stress-strain curves

0
0.00 0.05 0.10 0.15 0.20 0.25 0.30
True strain, ε
Fig. 8 Room temperature true stress–strain curves and corresponding work-hardening rates of 0.1ST, 0.3ST, and 0.7ST.
referred to as TNTZ-0.14ST, TNTZ-0.33ST, and TNTZ-0.70-ST, respectively. The tensile strength and 0.2% proof stress of TNTZ
increases with increasing O content. On the other hand, however, the elongation decreases at an O content of 0.33 mass%, and
then increases at an O content of 0.70 mass%. This trend with O content is contradictory to those conventionally reported. Their
tensile strength and elongation of TNTZ-0.70ST can reach to approximately 1100 MPa and 20%, respectively. Both the tensile
strength and elongation of TNTZ-0.70ST are greater than those of Ti–6Al–4V ELI. An obvious yielding point can be recognized
in TNTZ-0.7ST, but such yielding cannot be observed in TNTZ-0.14ST and TNTZ-0.33ST. Double yielding, which is observed in
the case of TNTZ-0.14ST, is not observed in TNTZ-0.7ST. The work-hardening rate curve obtained for TNTZ-0.7ST, which shows
the highest work-hardening rate among the examined alloys, intersects with the true stress–strain curve at true strains higher
than 20%. This suggests that this alloy exhibits the greatest work-hardening effect among all the alloys, and undergoes late necking
under tension. The excellent strength and elongation of TNTZ-0.70ST have been attributed to the occurrence of slips with wavy or
polyline morphologies corresponding to cross slips with multiple directions in the same grain (Liu et al., 2017a).
The Young’s moduli of TNTZ-0.14, 0.33, and 0.70ST are shown in Fig. 9 (Liu et al., 2017a) along with that of Ti–6Al–4V ELI. The
Young’s moduli of TNTZ-0.14ST and TNTZ-0.33ST are lower than 65 GPa. The Young’s modulus of TNTZ-0.70ST is lower than
75 GPa, which is much lower than that of Ti–6Al–4V ELI, which shows a Young’s modulus of approximately 100–110 GPa.
The fatigue strength of TNTZ can be improved by adding a large amount of O as shown in Fig. 10 (Liu et al., 2017b). In this case,
deformation-induced martensite is formed. The width of the deformation-induced martensite decreases with increasing O content
120
110
100
Young's modulus, E/GPa
90 Ti-6Al-4V ELI (ASTM F136)

80
70
60
50
40
30
20
10
0
TN
TN
TN
TZ
TZ
T
Z-
-0
- 0.
0.
.7
14
33
0
ST
ST
ST
Fig. 9 Young’s moduli of TNTZ-0.14O, TNTZ-0.33O, and TNTZ-0.70O subjected to hot rolling followed by solution treatment respectively.
1000
900
Maximum cyclic stress,σmax/MPa

Fatigue limit range of Ti-6Al-4V ELI
800
700
600
500
400
300
200 0.7O
0.5O
100 0.3O
0.1O
0
103 104 105 106 107
Number of cycles to failure, N
Fig. 10 S–N curves of prepared 0.1O, 0.3O, 0.5O, and 0.7O alloys with fatigue limit range of Ti–6Al–4V ELI.
as shown in Fig. 11. Therefore, the increment in the fatigue strength of TNTZ with O content can be considered to be due to O solid
solution strengthening and microstructural refinement by deformation-induced martensite (Liu et al., 2017b).
It has been reported that a small amount of O addition also increases both the strength and elongation in (a þ b)-type Ti–4Cr as
shown in Fig. 12 (Kanga et al., 2014), which is a plot of the nominal stress–strain curve of Ti–4Cr and Ti–4Cr–0.2O subjected to an
aging treatment at 1023 K for 18.0 ks. In this case, a significant work hardening was observed due to twinning-induced plasticity
(TWIP) in the b-phase.
Effect of Low Young’s Modulus on Stress Shielding
It is important to evaluate the bonding ability and bone affinity, and the role of low Young’s modulus in preventing stress shielding
and bone remodeling using animal testing.
300
Width of martensite, nm
250
200
150
100
50
0
0.1 0.5 0.7
Oxygen content
Fig. 11 Width of martensite in TNTZ0.1O. TNTZ0.5O, and TNTZ0.7O.
1000
800
Nominal stress (MPa)
600
Ti-4Cr-0.2
400
Ti-4Cr
200
0
0 2 4 6 8 10 12
Nominal strain (%)
Fig. 12 Nominal stress–strain curve in aged Ti–4Cr and Ti–4Cr–0.2O alloy aged at 1023 K for 18.0 ks.
A study of the bonding ability and bone affinity of rods made of TNTZ subjected to aging treatment (TNTZAT) and solution-
treated TNTZ (TNTZST), as well as rods of pure Ti (CP-Ti), and Ti–6Al–4V ELI (Ti64), was carried out using these rods in canine
mandibular implant beds of 18-month-old, male beagle dogs (Edamatsu et al., 2015). Fig. 13 (Edamatsu et al., 2015) shows
the bone area ratio (BAR) and bone contact ratio (BCR) in the cancellous bone area 3 and 6 months after the implantation of
TNTZAT, TNTZST, CP-Ti, and Ti64 rods. Both BAR and BCR of TNTZ are greater than those of CP-Ti and Ti64, indicating that the
bonding ability and affinity of TNTZ are greater than those of CP-Ti and Ti64.
Furthermore, the effectiveness of the low Young’s moduli of the implants in inhibiting stress shielding, namely inhibiting bone
resorption and enhancing bone remodeling, were proved by evaluating bone fracture healing and bone remodeling caused by
implanting intramedullary rods and bone fracture fixation plates made of TNTZ, Ti-6Al-4V ELI (Ti-64), and SUS 316L into a fracture
model made in the tibia of Japanese white rabbits (Niinomi et al., 2002b; Sumitomo et al., 2008). In both cases, the low Young’s
modulus TNTZ was proved to be much more effective in inhibiting stress shielding.
Fig. 14 (Niinomi et al., 2006) shows the three-point bend strength (fracture load) of tibia of three Japanese white rabbits with
and without implants of intramedullary rods made of TNTZ, Ti–6Al–4V ELI, and SUS 316L. The three-point bending fracture
(A) (B)
70 70
∗ ∗ ∗ ∗
∗
60 ∗ ∗ 60 ∗
∗
50 50
∗
BCR (%)
BAR (%)
40 40
30 30
20 20
10 10
0 0
AT
ST
-Ti
AT
ST
-Ti
AT
ST
-Ti
AT
ST
-Ti
4
Ti6
Ti6
Ti6
Ti6
TZ
TZ
TZ
TZ
TZ
TZ
TZ
TZ
CP
CP
CP
CP
TN
TN
TN
TN
TN
TN
TN
TN
Alloys Alloys Alloys Alloys

3 Months 6 Months 3 Months 6 Months
Fig. 13 (A) Bone area ratio (BAR) and (B) bone contact ratio (BCR) in the cancellous bone area at (A) 3 and (B) 6 months after implantation of
TNTZAT, TNTZST, CP-Ti, and Ti64.
TNTZ Ti-6Al-4V ELI SUS316L

(n = 3) (n = 3) (n = 3)
kgf
80
+19.4%
60
–15.0%
–12.5%
–0.2%
+9.2%
+13.2% –8.5% –9.1%
40
died by +10.5%
disease at 43 weeks
20
Control Intramedullary Control Intramedullary Control Intramedullary
side rod side side rod side side rod side
Fig. 14 Three-point bend strength: fracture load.
Table 2 Analysis of BVF obtained from histological image analysis with

paired t-test in rabbit
BVF
Number of rabbits Ti2448 TiAlV
T1 0.479 0.329
T2 0.430 0.296
T3 0.420 0.327
T4 0.475 0.335
T5 0.484 0.342
Mean 0.458 0.326
SD 0.027 0.016
Paired t-test P 0.00019
(A) (B) (C)
A A
B B
Middle level
A B C
A A
B B
Fig. 15 CMRs (contact micro-radiographs) of cross sections of fracture models implanted with and without bone plates made of TNTZ at middle
and distal level at 48 weeks after implantation: (A) cross section of fracture model, (B) parts of , of (A), namely high magnification CMR of
branched parts of bones formed outer and inner sides of tibiae, and (C) cross sections of unimplanted tibiae.
strength of tibia of rabbits with implants of the intramedullary rods made of TNTZ is greater than those of Ti–6Al–4V ELI and SUS
316L, suggesting that the low Young’s modulus is effective in inhibiting bone absorption and promoting good bone remodeling.
A animal testing similar to those mentioned earlier was carried out using intramedullary rods made of Ti–6Al–4V ELI and Ti–
24Nb–4Zr–7.9Sn (Ti2448 (TNZS)), which have a Young’s modulus of approximately 42 GPa. These two intramedullary rods were
implanted into the tibiae of New Zealand white rabbits for 4 weeks. After this period, the rabbits were sacrificed to evaluate the bone
volume fraction (BVF) around each intramedullary rod (Guo et al., 2009). The results are listed in Table 2 (Guo et al., 2009). BVF in
the case of Ti2448 is greater than that of Ti–6Al–4V ELI (TiAlV). Therefore, here too it is found that low Young’ modulus is effective
in enhancing the bone formation.
Furthermore, Fig. 15 (Sumitomo et al., 2008) shows an increase in the diameter of the tibia and the double wall structure in the
intramedullary bone tissue, which was observed only for the case of the bone plate made of TNTZ (Narita et al., 2012). In this figure,
the inner wall bone structure indicates the original bone, that is, the remaining old bone, whereas the outer wall bone structure
indicates the newly formed bone. This bone remodeling is the direct result of using a bone plate with a low Young’s modulus.
Therefore, in implants made of the b-type Ti alloy with low Young’s modulus for biomedical applications, stress transfer between
the bone and the implant becomes homogeneous. The prevention of bone resorption and the good bone remodeling are achieved,
although this has been proved only at the stage of animal testing.
Summary
Biomedical Ti alloys with new concept have been developed and are currently being developed. However, their biofunctionalities
are still poor. To address this issue, surface modifications using biofunctional materials are performed on Ti alloys for biomedical
applications. However, such biofunctionalization of Ti alloys, for example, smart Ti alloys that change according to the mechanical
circumstances in the body should be further developed.
References
Ashida, S., Kyogaku, H., & Hosoda, H. (2012). Fabrication of Ti-Sn-Cr shape memory alloy by PM and its properties. Materials Science Forum, 706–709, 1943–1947.
ASTM International. (2016a). Designation F67-13: Standard specification for unalloyed titanium, for surgical implant applications (UNS R50250), UNS R50400, UNS R50550, UNS
R50700 (pp. pp. 49–54). West Conshohocken, PA: ASTM International.
ASTM International. (2016b). Designation F136-13: Standard specification for wrought titanium-6aluminum4aanadium ELI (extra low interstitial) alloy for surgical implant
applications (UNS R56401) (pp. pp. 70–74). West Conshohocken, PA: ASTM International.
ASTM International. (2016c). Designation F1108-14: Standard specification for wrought titanium-6aluminum4aanadium alloy casting for surgical implants (UNS R56406) (pp. pp.
419–423). West Conshohocken, PA: ASTM International.
ASTM International. (2016d). Designation F1472-14: Standard specification for wrought titanium-6aluminum4aanadium alloy for surgical implant applications (UNS R56400) (pp.
pp. 524–528). West Conshohocken, PA: ASTM International.
ASTM International. (2016e). F1295-11: Standard specification for wrought titanium-6aluminum-7niobium alloy for surgical implant applications (UNS R56700) (pp. pp. 478–483).
West Conshohocken, PA: ASTM International.
Edamatsu, H., Kawai, T., Matsui, K., Nakai, M., Takahashi, T., Echigo, S., Niinomi, M., & Kamakura, S. (2015). High bone bonding ability and high bone affinity of new low rigidity
b-type Ti-29Nb-13Ta-4.6Zr alloy as a dental implant. Journal of Dental and Oral Health. Open access 8 pages.
Guo, Z., Fu, J., Zhang, Y. Q., Hu, Y. Y., Wu, Z. G., Shi, L., Sha, M., Li, S. J., Hao, Y. L., & Yang, R. (2009). Early effect of Ti-24Nb-4Zr-7.9Sn intramedullary nails on fractured bone.
Materials Science and Engineering C, 29, 963–968.
Hatanaka, S., Ueda, M., Ikeda, M., & Niinomi, M. (2010). Isothermal aging behavior in Ti-10Cr-Al alloys for medical applications. Advanced Materials Research, 89–91, 232–237.
Ikeda, M., & Sugano, D. (2005). The effect of aluminum content on phase constitution and heat treatment behavior of Ti-Cr-Al alloys for healthcare applications. Materials Science
and Engineering C, 25, 377–381.
Ikeda, M., Ueda, M., Matsunaga, R., Ogawa, M., & Niinomi, M. (2009). Isothermal aging behavior of beta titanium-manganese alloys. Materials Transactions, 50, 2737–2743.
Ikeda, M., Ueda, M., Matsunaga, R., & Niinomi, M. (2010). Phase constitution and heat treatment behavior of Ti-7mass%Mn-Al alloys. Materials Science Forum, 654–656,
855–858.
Ikeda, M., Ueda, M., Kinoshita, T., Ogawa, M., & Niinomi, M. (2012). Influence of Fe content of Ti-Mn-Fe alloys on phase constitution and heat treatment behavior. Materials Science
Forum, 706–709, 1893–1898.
ISO. (1996). ISO 5832: Implant for surgery – Metallic materials – Part 10: Wrought titanium 5-aluminum 2.5-iron alloy. Geneva, Switzerland: ISO.
JISC. (2002a). JIS T7401-3: Titanium materials for surgical implant applications, Part 3: Wrought titanium 6-aluminium 2-niobium 1-tantalum alloy. Tokyo, Japan: JISC.
JISC. (2002b). JIS T7401-4: Titanium materials for surgical implant applications Part 4: Wrought titanium 15-zirconium 4-niobium 4-tantalum alloy. Tokyo, Japan: JISC.
Kanga, D.-S., Kogaa, N., Sakat, M., Nakad, N., Tsuchiyam, T., & Takaki, S. (2014). Enhanced work hardening by redistribution of oxygen in (a þ b)-type Ti–4Cr–0.2O alloys.
Materials Science and Engineering A, 606, 101–107.
Kasano, Y., Inamura, T., Kanetaka, H., Miyazaki, S., & Hosoda, H. (2010). Phase constitution and mechanical properties of Ti-(Cr, Mn)-Sn biomedical alloys. Materials Science
Forum, 654–656, 2118–2121.
Kawahara, H., Ochi, S., Tanetani, K., Kato, K., Isogai, M., Mizuno, Y., Yamamoto, H., & Yamaguchi, A. (1963). Biological test of dental materials, effect of pure metals upon the
mouse subcutaneous fibroblast, strain L cell in tissue culture. Journal of Japanese Society for Dental Materials and Devices, 4, 65–75.
Kobayashi, F., Ando, M., Tsutsumi, Y., Doi, H., Yoneyama, T., Kobayashi, M., & Hanawa, T. (2007). Inhibition effect of zirconium coating on calcium phosphate precipitation of
titanium to avoid assimilation with bone. Materials Transactions, 48, 301–306.
Liu, H., Niinomi, M., Nakai, M., & Cho, K. (2015). b-type titanium alloys for spinal fixation surgery with high Young’s modulus variability and good mechanical properties. Acta
Liu, H., Niinomi, M., & Nakai, M. (2016). Athermal and deformation-induced u-phase transformations in biomedical beta-type alloy Ti-9Cr-0.2O. Acta Materialia, 106, 162–170.
Liu, H., Niinomi, M., Nakai, M., Cong, X., Cho, K., Boehlert, C. J., & Khademi, V. (2017a). Abnormal deformation behavior of oxygen-added beta-type Ti-29Nb-13Ta-4.6Zr alloys for
biomedical applications. Metallurgical and Materials Transactions A, 48, 139–149.
Liu, H., Niinomi, M., Nakai, M., Obara, S., & Fujii, H. (2017b). Improved fatigue properties with maintaining low Young’s modulus achieved in biomedical beta-type titanium alloy by
oxygen addition. Materials Science and Engineering A, 704, 10–17.
Murayama, Y., & Sasaki, S. (2009). Mechanical properties of Ti-Cr-Sn-Zr alloys. Bulletin of Niigata Institute of Technology, 14, 1–8.
Nakai, M., Niinomi, M., Zhao, X. F., & Zhao, X. L. (2011). Self-adjustment of Young’s modulus in biomedical titanium alloy during orthopaedic operation. Materials Letters, 65,
688–690.
Nakai, M., Niinomi, M., & Oneda, T. (2012). Improvement in fatigue strength of biomedical b-type Ti–Nb–Ta–Zr alloy while maintaining low Young’s modulus through optimizing u-
phase precipitation. Metallurgical and Materials Transactions A, 43, 294–302.
Nakano, T. (2010). Mechanical characteristics and their evaluation methods. In T. Hanawa (Ed.), Metals for medicine (pp. 184–198). Sendai, Tokyo: Japan Institute for Materials.
Narita, K., Niinomi, M., Nakai, M., Hieda, J., & Oribe, K. (2012). Development of thermo-mechanical processing for fabricating highly durable b-type Ti-Nb-Ta-Zr rod for use in spinal
fixation devices. Journal of the Mechanical Behaviors of Biomedical Materials, 9, 207–216.
Niinomi, M. (2000). Development of high biocompatible titanium alloys. Functional Materials, 20, 36–44.
Niinomi, M. (2002). Recent research and development in titanium alloys for biomedical applications. Denki-seiko, 73, 113–120.
Niinomi, M. (2010). Tend and present state of titanium alloys with body centered structure for biomedical applications. Bulletin of The Iron and Steel Institute of Japan, 15,
661–670.
Niinomi, M., Boehler, C. (2015). Titanium alloys for biomedical applications. Niinomi, M., Narushima, T., Nakai, M. (eds.). Advances in metallic biomaterials, pp. 179–214. Berlin,
Heidelberg, Germany: Springer-Verlag Vol. 3(2015).
Niinomi, M., Fukui, H., Hattori, T., Kyo, K., & Suzuki, A. (2002a). Development of high biocompatible titanium alloy. Materia Japan, 41, 221–223.
Niinomi, M., Hattori, T., Morikawa, K., Kasuga, T., Suzuki, A., Fukui, H., & Niwa, S. (2002b). Development of low rigidity b-type titanium alloy for biomedical applications. Materials
Transactions, 43, 2970–2977.
Niinomi, M., Akahori, T., Nakai, M., Hattori, T., & Kasuga, T. (2006). Low modulus titanium alloys with multi functionalities for biomedical applications. Kinzoku, 77, 128–134.
Niinomi, M., Akahori, T., Katsura, S., Yamauchi, K., & Ogawa, M. (2007). Mechanical characteristics and microstructure of drawn wire of Ti-29Nb-13Ta-4.6Zr for biomedical
applications. Materials Science and Engineering C, 27, 154–161.
Niinomi, M., Nakai, M., & Hieda, J. (2012). Development of new metallic alloys for biomedical applications. Acta Biomaterialia, 8, 3888–3903.
Niwa, S. (2004). Recent trends and development of biomaterials for artificial bone. Materia Japan, 43, 186–192.
Okazaki, Y., Ito, Y., Ito, A., & Tateishi, T. (1996). New titanium alloys to be considered for medical implants. In S. A. Brown, & J. E. Lemons (Eds.), Medical applications of titanium
and its alloys (pp. 45–59). West Conshohocken, PA: ASTM. ASTM STP 1272.
Radzi, S., Cowin, G., Robinson, M., Pratap, J., Volp, A., Schuetz, M. A., Schmutz, B., & B. (2014). Metal artifacts from titanium and steel screws in CT, 1.5T and 3T MR images of
the tibial Pilon: A quantitative assessment in 3D. Quantitative Imaging in Medicine and Surgery, 4, 163–172.
Song, X., Niinomi, M., Tsutsumi, H., Akahori, T., Nakai, M., Yonezawa, S., & Wang, L. (2010). Effect of Y2O3 on mechanical properties of Ti-29Nb-13Ta-4.6Zr for biomedical
applications. Materials Science Forum, 654–656, 2142–2145.
Steib, J. P., Dumas, R., & Skalli, W. (2004). Surgical correction of scoliosis by in situ contouring: A detorsion analysis. Spine, 29, 193–199.
Steinemann, S. G. (1980). Corrosion of surgical implantsdIn vivo and in vitro tests. In G. D. Winter, J. L. Leray, & K. de Groot (Eds.), Evaluation of biomaterials (pp. 1–34). New
York: Wiley.
Sumitomo, N., Noritake, K., Hattori, T., Morikawa, K., Niwa, S., Sato, K., & Niinomi, M. (2008). Experiment study on fracture fixation with low rigidity titanium alloy – Plate fixation of
tibia fracture model in rabbit. Journal of Materials Science: Materials in Medicine, 19, 1581–1586.
Suzuki, N., Iizumi, H., & Ogawa, A. (1999). Super plastic formability of Ti-4.5%Al-3%V-2%Fe-2%Mo alloy. Journal of Japan Institute of Light Metals, 49, 363–367.
Tane, M., Akita, S., Nakano, T., Hagihara, K., Umakoshi, Y., Niinomi, M., & Nakajima, H. (2008). Peculiar elastic behavior of Ti-Nb-Ta-Zr single crystals. Acta Materialia, 56,
2856–2863.
Tsutsumi, Y., Nishimura, D., Doi, H., Nomura, N., & Hanawa, T. (2009). Difference in surface reactions between titanium and zirconium in Hanks’ solution to elucidate mechanism of
calcium phosphate formation on titanium using XPS and cathodic polarization. Materials Science and Engineering C, 29, 1702–1708.
Ueda, K., Nakaoka, S., & Narushima, T. (2013). b-grain refinement of a þ b-type Ti–4.5Al–6Nb–2Fe–2Mo alloy by using rare-earth-oxide precipitates. Materials Transactions, 54,
161–168.
Uggowitzer, P. J., Bähr, W.-F., & Speidel, M. O. (1997). Metal injection molding of nickel-free stainless steels. Advances in Powder Metallurgy and Particulate Materials, 3, 18.113–
18.121.
Yamamoto, A., Honma, R., Sumita, M., & M. (1988). Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells. Journal of Biomedical Materials
Research Part A, 30, 331–340.
Yilmazer, H., Niinomi, M., Nakai, M., Hieda, J., Todaka, Y., & Miyazaki, T. (2013). Mechanical properties of a medical b-type titanium alloy with specific microstructural evolution
through high pressure torsion. Materials Science and Engineering C, 33, 2499–2507.
Zhao, X. L., Niinomi, M., & Nakai, M. (2011a). Relationship between various deformation-induced products and mechanical properties in metastable Ti-30Zr-Mo alloys for biomedical
applications. Journal of the Mechanical Behavior of Biomedical Materials, 4, 2009–2016.
Zhao, X. L., Niinomi, M., Nakai, M., Miyamoto, G., & Furuhara, T. (2011b). Microstructures and mechanical properties of metastable Ti-30Zr-(Cr, Mo) alloys with changeable Young’s
modulus for spinal fixation applications. Acta Biomaterialia, 7, 3230–3236.
Zhao, X. F., Niinomi, M., Nakai, M., Hieda, J., Ishimoto, T., & Nakano, T. (2012a). Optimization of Cr content of metastable b-type Ti-Cr alloys with changeable Young’s modulus for
spinal fixation applications. Acta Biomaterialia, 8, 2392–2400.
Zhao, X. F., Niinomi, M., Nakai, M., & Hieda, J. (2012b). Beta-type Ti-Mo alloys with changeable Young’s modulus for spinal fixation applications. Acta Biomaterialia, 8, 1990–1997.
Further Reading
Ambrosioand, L., & Tanner, E. (2012). Biomaterials for spinal surgery. Sawston, Cambridge, England: Woodhead Publishinig Ltd.
Kuroda, D., Niinomi, M., Morinaga, M., Kato, Y., & Yashiro, T. (1998). Design and mechanical properties of new beta type titanium alloys for implant materials. Materials Science
and Engineering A, 243, 244–249.
Niinomi, M. (1998). Mechanical properties of biomedical titanium alloys. Materials Science and Engineering A, 243, 231–236.
Niinomi, M. (2003a). Fatigue performance and cyto-toxicity of low rigidity titanium alloy Ti–29Nb–13Ta–4.6Zr. Biomaterials, 16, 2673–2683.
Niinomi, M. (2003b). Recent research and development in titanium alloys for biomedical applications and healthcare goods. Science and Technology of Advanced Materials, 4,
445–454.
Niinomi, M. (2008a). Mechanical biocompatibilities of titanium alloys for biomedical applications. Journal of the Mechanical Behavior of Biomedical Materials, 1, 30–42.
Niinomi, M. (2008b). Biologically and mechanically biocompatible titanium alloys. Materials Transactions, 49, 2170–2178.
Niinomi, M. (2010). Fatigue failure of metallic biomaterials, metals for biomedical devices. Cambridge, England: Woodhead Publishing Ltd.
Niinomi, M. (2014). Biomedical implant devices fabricated from low Young’s modulus titanium alloys demonstrating high mechanical biocompatibility. Materials Matters, 9, 39–46.
Niinomi, M., & Akahori, T. (2010). Improvement of the fatigue life of titanium alloys for biomedical devices through microstructural control. Expert Review of Medical Devices, 7,
481–488.
Niinomi, M., Narushima, T., & Nakai, M. (2015a). Advances in metallic biomaterials, Part I: Tissues, materials and biological reactions. Wang, M. (series editor). In Springer Series
in Biomaterials Science and Engineering (Vol. 3). Berlin, Heidelberg, Germany: Springer.
Niinomi, M., Narushima, T., & Nakai, M. (2015b). Metallic biomaterials, Part II: Processing and applications. In Springer Series in Biomaterials Science and Engineering (Vol. 4).
Berlin, Heidelberg, Germany: Springer. Wang, M. (series editor).
Pignatello, R. (2011). Biomaterials science and engineering. Rijeka, Croatia: In Tech.
Ramalingam, M., & Kobayashi, H. (2010). Integrated materials for biomedical technology. Linkoping, Sweden: VBRI Press.
Sasaki, K., Suzuki, O., & Takahashi, N. (2017). Interface oral health science 2016: Innovative research on biosis–abiosis interface. Berlin, Heidelberg, Germany: Springer.
BIOMATERIALS: IN VITRO AND IN VIVO STUDIES OF BIOMATERIALS
Anatomy and Physiology for Biomaterials Research and Development

Inn Chuan Ng and Pornteera Pawijit, National University of Singapore, Singapore
Jordon Tan, Temasek Polytechnic, Singapore
Hanry Yu, National University of Singapore, Singapore; Agency for Science, Technology and Research (A*STAR), Singapore;
BioSyM, Singapore-MIT Alliance for Research and Technology, Singapore; and Nanfang Hospital, Southern Medical University,
Guangzhou, China
Biomaterials Interact With Biological Systems 226

Principles of Anatomy and Physiology 226
Anatomy 226
Levels of organization 226
Physiology 227
Processes of life 227
Homeostasis 227
Structure to Function 228
Organ Systems and Organs 228
The Circulatory System 228
Heart and blood vessels 229
Blood 229
Skeletal System 230
Bone 230
Cartilage and ligaments 230
Integumentary System 231
Epidermis 231
Dermis 231
Tissues 231
Cells 231
Structure of a Cell 232
Cell membrane 232
Organelles 232
Cytosol 233
Cytoskeleton 233
Cells Interact With Extracellular Environment (Input) 233
Cell–ECM interaction 233
Cell-soluble signals interaction 234
Cell–cell interaction 234
Signaling Cascade 234
Cell Response (Output) 235
Gene expression 235
Cell differentiation 235
Cell growth and division 235
Cell spreading and migration 236
Mechanistic Insights Into the Biomaterials Interaction With Biological Systems 236
Further Reading 236
Glossary
Adaptive immunity Specific resistance in which the body recognizes and memorizes antigens that it encounters. Response to
the particular antigens improves each time the body encounters the antigens again.
Anatomy A discipline of biology that studies the structure and organization of organisms.

226 Biomaterials: In vitro and in vivo studies of biomaterials j Anatomy and Physiology
Cell The most basic functional unit of life.

Cytokines Small proteins released by cells. They act as chemical mediators.
Gene expression A process in which genetic information is used to synthesize functional products. This process is explained by
the central dogma of molecular biology, which describes the flow of genetic information from DNA to RNA to protein. Gene
expression involves gene regulation, transcription of DNA into RNA, RNA processing, translation of RNA into protein, and
posttranslational modifications of protein. Epigenetic regulation of the genetic information is usually involved in regulating
gene expression.
Homeostasis The tendency of biological systems to maintain a relatively constant equilibrium in their internal environment.
Hormones Chemical mediators produced by specialized glands. They can be proteins, steroids, amino acid derivatives, or fatty
acid derivatives.
Immune response A reaction of the body to protect itself against substances that appear foreign.
Innate immunity General resistance to certain antigens. Response to the antigens is the same each time the body is exposed.
Organ A collection of tissues that form a distinct structural unit and carry out a specific function together.
Organ system A group of organs and tissues that perform similar functions.
Physiology A discipline of biology that studies the functions and processes of organisms.
Tissue A group of similar cells that carry out a specific function together.
Biomaterials Interact With Biological Systems
Biomaterials are materials that have been designed to interface with biological systems, for the treatment, augmentation, or replace-
ment of biological functions. Biomaterials and biological systems interact both ways. Biomaterials and any compounds released
from them may induce positive or negative responses from biological systems, from increase in wound healing and improvement
in biological functions to toxicity and activation of the immune response. Conversely, biological systems may modify the surfaces of
biomaterials through the biomaterial corrosion, degradation, and deposition, which will affect the integrity and performance of
biomaterials and in turn lead to subsequent biological responses. Since the interaction between biomaterials and biological systems
is dynamic and occurs gradually over time, it may lead to different observed responses between short and long term. To understand
the interaction of biomaterials with the relevant biological systems, knowledge of the principles of biology, especially in anatomy
and physiology, is required. The knowledge is important during biomaterials research and development for improving biomaterials
design and for evaluating the effects of biomaterials on biological systems.
Principles of Anatomy and Physiology

Anatomy
Anatomy is the study of the structure and organization of organisms, at both microscopic (histology) and macroscopic (gross)
levels. Anatomy is typically studied through dissections, which is often accompanied by additional staining, optical, or imaging
methods. The anatomy of an organism is understood as a hierarchical organization.
Levels of organization
Through a reductionist approach, the anatomy of an organism can be simplified to a manageable level. An organism can be divided
into levels of organization with descending complexity: from organism to organ systems, to organs, to tissues, and to cells (Fig. 1).
An organ system is a group of organs and tissues that perform similar functions; an organ is a structurally distinct group of tissues
that work together to perform a specific function, and a tissue is a group of cells. The following are the key characteristics of this
hierarchical organization:
• Each component in a level is assembled from the more basic components of a lower level. Thus, each higher level in the
hierarchy represents an increase in organizational complexity. For example, the digestive system constitutes organs such as the
stomach, liver, pancreas, small intestines, and large intestines.
• Emergent functions appear at higher levels due to cooperation of different basic components of lower levels. These functions are
not present at the lower levels. For example, digestion of food requires the cooperation of multiple organs and cannot be
performed by a single organ. The mouth mechanically breaks down food; acids and enzymes in the stomach degrade partially
digested food into chime; the liver produces bile, while the pancreas produces other enzymes to further digest food into its basic
constituents.
• Each component may communicate with and regulate the function of another component from a similar or different hierar-
chical level. For example, the nervous and endocrine system can regulate the production of enzymes by the different organs in
the digestive system.
Biomaterials: In vitro and in vivo studies of biomaterials j Anatomy and Physiology 227
Fig. 1 Levels of organization. An organism is made up of different organ systems. Each organ system consists of different organs. Each organ is
formed by different types of tissues. Each type of tissue is assembled from cells. Examples of components at each level are shown. Illustrations by
Wong Chun Xi, MBI Science Communications Unit.
Physiology
Physiology is the study of functions and processes of organisms and their components. The study includes normal and
diseased states and maintenance of homeostasis under different environmental conditions. Physiology can be studied using
physical, chemical, mechanical, and biological assays. Assays are investigative procedures for qualitative and quantitative
measurement of biological functions. These assays can be performed in vitro in laboratory vessels or in vivo on animals.
The interaction of biomaterials with biological systems can be evaluated using the mentioned assays for studying physiology.
The interaction of a scaffold with cells can be studied in vitro in cell culture plates or in vivo by implantation of the scaffold
into the animal models.
Processes of life
An organism has seven key processes of life. These physiological processes are important to keep an organism alive. A biomaterial
can be used to support or suppress some of these processes:
• Respirationda process of extracting energy out of food

• Movementdto search for food and shelter and to avoid danger
• Sensitivitydawareness of and the degree of response to changes in environmental conditions
• Growthddevelopment and repair of organism and their parts
• Reproductiondproduction of offspring
• Nutritiondacquisition of nutrients or its synthesis
• Excretiondremoval of harmful waste products
Homeostasis
To ensure survival, a biological system needs to maintain a relatively stable internal environment, despite the continuous changes in
the signals from within and outside the system. The maintenance of this dynamic equilibrium is called homeostasis. Homeostasis
requires three components for constant monitoring and adjustments of the internal environment:
1. The receptor, which senses the changes
2. The control center, which receives and processes the information sent from the receptor
3. The effector, which increases or reduces its function in response to the commands from the control center
Homeostasis is maintained through negative feedback: a response by the system to reverse the direction of change. Examples of
homeostasis are the maintenance of body temperature and blood glucose level. Biomaterials are usually used to restore but seldom
allowed to dysregulate homeostasis.
Structure to Function
Anatomy and physiology are a pair of related disciplines. They are often studied in combination to relate organism structure to func-
tion. Biomaterials interact with anatomy and impact physiology at different levels of organization. Positive effects of a biomaterial
on one component at a particular level might not necessarily translate to similar effects on another component at the same level or
other units at higher or lower levels.
Organ Systems and Organs
An organism is made up of different organ systems. Each organ system performs a specific function, and all organ systems work in an
interconnected manner to maintain the internal conditions essential to the survival of the organism. There are 11 key organ systems
in the human body (Table 1). We will use three examples of the human organ systems of particular interest to the field of bioma-
terials, the circulatory, skeletal, and integumentary systems, to explain in detail how these organ systems work.
The Circulatory System

The components of the circulatory system include the heart, blood vessels, and blood. The circulatory system delivers nutrients and
hormones throughout the body and removes waste products and excess heat generated by the metabolic activities of the body.
Biomaterials used in devices for the circulatory system often include catheters, pacemakers, heart valves, vascular grafts, and stents.
Table 1 Function of human organ systems and their components
System Functions Key organs and tissues
Integumentary system Forms a permeability barrier, regulates body temperature, • The skin (provides protection against abrasion and
reduces water loss, and helps produce vitamin D ultraviolet light)
• Hair (regulates body temperature)
• Nails (protect soft tissues from injuries)
• Sweat glands (regulates body temperature)
Skeletal system Provides structural protection and support, allows body • Bone (provides mechanical support and body structure)
movement, stores minerals and fat, and produces blood cells • Cartilage (provides firm but flexible support)
• Ligaments (prevent movements that may damage joints)
• Joints (for movement and stability of limbs)
Muscular system Produces body movements, maintains posture, and generates • Muscle (generates contractile forces)
body heat • Tendons (connect skeletal muscles to bones)
Lymphatic system Removes foreign substances from the blood and lymph, • Lymphatic vessels (drain tissue fluids back into the
combats disease, maintains tissue fluid balance, and absorbs bloodstream)
fats from the digestive tract • Lymph nodes (filtration of lymph to identify antigens)
• Thymus (maturation site of T lymphocytes)
• Spleen (filters blood)
Respiratory system Exchanges oxygen and carbon dioxide between the blood and • Nose (primary organ of smell)
air, and regulates blood pH • Larynx (sound generation)
• Pharynx (provides airflow to and from lungs)
• Trachea (provides airflow to and from lungs)
• Bronchi (provide airflow to and from lungs)
• Lungs (principal organ of respiration)
Digestive system Performs mechanical and chemical processes of digestion, • Mouth (chews/breaks down food)
absorption of nutrients, and elimination of wastes • Esophagus (channels food into the stomach)
• Stomach (breaks down protein-rich food)
• Liver (detoxifies and metabolizes chemicals and drugs)
• Pancreas (secretes enzymes to digest food)
• Gallbladder (stores and concentrates bile)
• Small intestines (absorb nutrients and minerals)
• Large intestines (absorb water)
• Mesentery (attaches intestines to the walls of the
abdomen)
• Rectum (temporary storage for feces)
• Anus (digestive tract opening)
Nervous system Detects sensations and controls intellectual functions, • Brain (provides control over the actions of the body)
movement, and physiological processes • Spinal cord (connects the peripheral nervous system to
the brain)
• Nerves (transmit nervous signals between different parts
of the body)
Table 1 Function of human organ systems and their componentsdcont'd
System Functions Key organs and tissues
Endocrine system Regulates metabolism, growth, reproduction, and many other • Hypothalamus (links the nervous system to the
functions endocrine system)
• Pituitary gland (secretes a variety of hormones)
• Pineal gland (produces melatonin)
• Thyroid gland (secretes hormones that regulate the
growth and metabolism of the body)
• Parathyroid gland (secretes hormone that regulates
calcium and phosphate levels)
• Adrenal glands (secrete hormones that regulate blood
sugar levels and blood pressure)
• Pancreas (secretes hormones that regulate blood
glucose levels)
• Ovaries (produce oocytes for reproduction and secrete
estrogen and progesterone)
• Testes (produce sperm and testosterone)
Circulatory system Transports nutrients, waste products, gases, and hormones • Heart (pumps blood for circulation)
throughout the body, mediates immune response and the • Blood vessels (transport blood throughout the body)
regulation of body temperature • Blood (carries oxygen, hormones, and waste products
and is involved in the immune response)
Urinary system Removes waste products from the blood and regulates blood • Kidneys (extract waste from blood and body fluids to
pH, ion balance, and water balance form urine)
• Ureters (carry urine from the kidney to the bladder)
• Urethra (carries urine from the bladder to outside of the
body)
Reproductive system Production of sex cells and hormones that influence sexual Male
function and behaviors • Testes (produce sperm and testosterone)
• Prostate gland (secretes prostate fluid)
• Penis (male sexual organ)
Female
• Ovaries (produce oocytes for reproduction and secrete
estrogen and progesterone)
• Uterus (nurtures fertilized ovum)
• Vagina (birth canal)
Heart and blood vessels

The heart is a muscular organ that contains four chambers. Valves at the entries and exits of the chambers maintain the correct unidi-
rectional flow of blood. Sequential contraction of the heart chambers allows the heart to receive blood and pump it out to the lungs
or to other parts of the body through closed loops of blood vessels. There are three types of blood vessels: the arteries, veins, and
capillaries. Arteries take blood away from the heart, while veins bring blood toward the heart. Arteries have comparatively thicker
walls because they experience higher blood pressure due to proximity to the heart. Veins contain valves that prevent the backflow of
blood. Arteries decrease in diameter as they branch out from the heart throughout the body and end in capillaries. Meanwhile, veins
begin from capillaries and increase in diameter as they converge toward the heart. Capillaries are tiny web of vessels of 5–10 mm in
diameter that connect arteries to veins at the other end of the heart. The thin walls of capillaries allow the exchange of gas and nutri-
ents between the blood and surrounding tissues. Blood flow and pressure are regulated by the heart rate and the contraction and
relaxation of arteries and veins. When artificial heart valves or stent are implanted into the circulatory system, they interact with the
flowing blood and the neighboring tissues that determine plaque deposition and blood flow dynamics in the local structures.
Blood
Blood is a type of connective tissue. It is composed of plasma, a liquid matrix in which blood cells are suspended. The plasma is an
aqueous solution consisting of water, blood plasma proteins and dissolved electrolytes, nutrients, gases, waste products, and other
substances, while blood cells are grouped into red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (throm-
bocytes). Red blood cells are biconcave-shaped cells with no nucleus and contain hemoglobin that helps in the transport of oxygen
and carbon dioxide. White blood cells mediate immune response to protect the body against foreign substances and further cate-
gorized into two groups based on their morphology, namely, granulocytes and agranulocytes. Granulocytes consist of neutrophils,
which phagocytose microorganisms and other substances; basophils, which promote inflammation by releasing histamine and
prevent clot formation by releasing heparin; and eosinophils, which reduce inflammation by releasing cytokines and attack certain
worm parasites. Agranulocytes consist of monocytes and lymphocytes. Monocytes leave the blood to become macrophages, whose
function is to phagocytose bacteria, dead cells, cell fragments, and other debris within tissues. Lymphocytes can be divided into
natural killer (NK) cells and B and T lymphocytes. Lymphocytes destroy microorganisms by producing antibodies and other cyto-
kines; contribute to allergic reactions, tumor control, and graft rejection; and regulate the immune system. Platelets are cell frag-
ments that take part in blood clotting.
Immune response is a reaction of the body to protect itself against substances that appear foreign, such as microorganisms, para-
sites, harmful compounds, cancer cells, and some biomaterials. Activation of the immune response may lead to hypersensitivity of
the body and rejection of a biomaterial. The immune response is mediated by certain plasma proteins in the blood and white blood
cells in the circulatory and lymphatic systems. It is regulated by cooperation of two different mechanisms: the innate and adaptive
immunities.
The innate immunity is a general resistance that is activated immediately or within hours after the body encounters certain anti-
gens, substances that activate an immune response, but the reaction to them is the same each time the body is exposed. It is activated
by the chemical properties of the antigens. Innate immune response can manifest as local or systemic inflammation and is mediated
by neutrophils, macrophages, NK cells, and complement cascade. Phagocytosis of antigens induces neutrophils and macrophages to
release chemical mediators called cytokines that regulate the inflammatory response. These cytokines may either encourage inflam-
mation by increasing vascular permeability and recruiting additional white blood cells to the site of inflammation or reduce inflam-
mation and promote tissue repair. The former cytokines are released by M1 macrophages, while the latter cytokines are released by
M2 macrophages. NK cells induce apoptosis of virus-infected and cancer cells. Complements bind to antigens and enhance their
phagocytosis by neutrophils and macrophages, promote inflammation, and assemble to form membrane pores that induce lysis
of pathogens.
The adaptive immunity is a specific resistance in which the body recognizes and memorizes antigens, and the reaction to previ-
ously encountered antigens improves each time they meet again. It is mediated by B and T lymphocytes. B lymphocytes mature in
the bone marrow and produce antibodies that bind to antigens, whereas T lymphocytes, which mature in the thymus, release cyto-
kines and induce cytotoxicity. Antigen recognition by adaptive immunity involves cell surface proteins called the major histocom-
patibility complex (MHC).
MHC receptors present processed fragments of antigens called epitopes at cell surfaces for recognition by T lymphocytes. There
are three classes of MHC receptors: MHC class I, II, and III. MHC class I receptor is expressed by all nucleated cells and platelets. It
presents epitopes to a subtype of T lymphocytes called cytotoxic T lymphocytes (CTLs). CTLs express CD8 receptor and T-cell
receptor (TCR). CD8 receptor interacts with MHC class I receptor while TCR will only bind to a specific epitope. An antigen-
presenting cell will be induced by CTL to undergo programmed cell death if the TCR could bind to an epitope presented by its
MHC class I receptor while the MHC class I receptor itself is interacting with a CD8 receptor. MHC class II receptor is normally
expressed by professional antigen-presenting cells such as macrophages, dendritic cells and B lymphocytes. It presents epitopes
to a subtype of T lymphocytes called helper T lymphocytes (Th). Th lymphocytes express CD4 receptor and TCR. CD4 receptor inter-
acts with MHC class II receptor. Naïve Th lymphocyte will undergo differentiation if its TCR could bind to an epitope presented by
an MHC class II receptor while the MHC class II receptor itself is interacting with its CD4 receptor. The naïve Th lymphocyte will
differentiate into either effector or memory Th lymphocyte that mediate immunization, or suppressor T lymphocyte that mediate
immune tolerance.
Biomaterials interaction with immune system plays a central role in any implantable materials or devices or even the extracor-
poreal devices that connect to the body via circulatory system.
Skeletal System
The skeletal system consists of bone, cartilage, and ligaments. It protects and supports the body, helps with movement, produces
blood cells, and stores important minerals. Examples of orthopedic applications of biomaterials include hydroxyapatite ceramics
for bone grafts, biodegradable polymers for bone and cartilage substitution, and scaffolds for ligament reconstruction.
Bone
Bone is a composite material made up of calcium hydroxyapatite, a mineral that provides strength and rigidity, and collagen, an
elastic protein that provides flexibility and improves fracture resistance. Since bones are hard, they form the major structural support
components of the body and protect organs such as the brain, heart, and lungs. There are two types of bones based on their struc-
tural arrangement: the compact and spongy bones. The compact bone is a dense, solid matrix that forms most of the shaft of long
bones and provides mechanical strength. On the other hand, the spongy bone, which consists of a lacy network of bone with many
small, marrow-filled spaces, provides compressional strength. Bone is a living organ that is constantly being remodeled. Bone
remodeling occurs during bone growth and regulates calcium availability. Old bone is removed by cells called osteoclasts, and
new bone is deposited by osteoblasts. Extensive work in biomaterial R&D has been conducted to stimulate bone regeneration
in vivo or prosthesis to replace lost bone function with inert biomaterials.
Cartilage and ligaments

The skeletal system also consists of connective tissues such as the cartilage and ligaments. Cartilage is a smooth, firm, resilient,
nonvascular tissue, which provides structural support and reduces friction between bones at the joints. Ligaments are fibrous tissue
that connect bones together at the joints, providing joint stability and prevent movement that might damage the joint. Their char-
acteristics are mainly determined by their extracellular matrix (ECM) composition. The matrix always contains collagens and
proteoglycans, but the types and quantities of these substances differ. Collagen is a tough, fiber-like protein that provides rigidity
and flexibility. Proteoglycans are large molecules that consist of polysaccharides attached to core proteins that attract and retain large
amounts of water. In the cartilage, the ECM consists of chondrocytes, polymeric components (type II collagen and proteoglycans),
and water. The collagen makes the cartilage tough, whereas the proteoglycans make it smooth and resilient. Thus, the cartilage is
relatively rigid but springs back to its original shape after being bent or slightly compressed, enabling it to absorb shock. On the
other hand, the ligaments contain large amounts of collagen fibers, making these structures very tough. A key focus of cartilage
and ligament tissue engineering for rehabilitation is to engineer constructs that mimic the mechanical properties and load-
bearing characteristics of the cartilage and ligaments in vivo.
Integumentary System
The integumentary system is an organ system that consists of the skin, its derivatives (sweat and sebaceous glands), nails, and hair. It
forms a physical barrier between the body and the environment; it protects the body against invasion by infectious organisms and
against ultraviolet light, reduces water loss, and provides thermal insulation. Other functions include excreting waste products
through perspiration; sensing stimuli such as touch, pressure, pain, heat, and cold; generating vitamin D under ultraviolet light;
and serving as a store for water and fat. Examples of biomaterial applications for integumentary system include skin grafts and
skin substitutes such as epidermal covers and dermal replacements. There are commercial skin grafts of different levels of sophis-
tication for implant or for drug testing in vitro. The current challenges are developing more sophisticated biomaterial support to
improve the aesthetics with micro-/nanosized features, mechanical contractility, and interaction with immune or nervous systems.
Epidermis
The human skin is composed of two major layers of tissue: the epidermis and dermis. The epidermis forms the outermost layer,
providing the initial barrier to the external environment. Cells in this layer are increasingly filled with keratin, and they eventually
die to form an outer layer of dead, hard cells that resist abrasion and also act as a permeability barrier. Another unique characteristic
of this layer is its pigmentation. Melanin, the pigment that gives the skin its color and protects it against ultraviolet rays from the sun,
is produced by melanocytes. Skin color has implications on the cosmetic value of biomaterials. Earlier generations of skin grafts
involve only the epidermis substitutes.
Dermis
Beneath the epidermis is the dermis that comprises of two layers: the papillary and reticular layers. The dermis contains connective
tissues, vessels, glands, follicles, hair roots, sensory nerve endings, and muscular tissue. Beneath the dermis is the hypodermis that is
primarily made up of adipose tissue. Substantial collagen bundles anchor the dermis to the hypodermis in a way that permits most
areas of the skin to move freely over this tissue layer. Modern skin grafts involve both dermis and epidermis layers for mimicking the
skin structure and functions.
Tissues
Tissue refers to a group of cells and their microenvironment, which perform their functions together. There are four main types of
tissues: muscle, epithelial, connective, and nerve tissues. Muscle tissue is involved in generating contractile forces, epithelial tissue
contains tightly packed cells, connective tissue contains cells enveloped by the ECM, and nerve tissue contains nerve cells that
conduct nerve impulses.
Organs, for example, the heart, are made up of different types of tissues. Cardiac muscle tissue, a subtype of muscle tissue that is
composed of aligned cardiomyocytes, mediates the contraction of the heart. The inner linings of the heart are covered by the endo-
thelium, a subtype of epithelial tissue. The endothelium forms a barrier separating the cardiac muscle tissue from the blood,
a subtype of connective tissue. Nerve tissue connects the heart to the nervous system, allowing heart rate to be regulated by the brain.
In most applications, a biomaterial will interact with biological systems at the tissue level; rarely does it interact with the organ as
a whole or only with a single cell. Tissue response is a collective response of individual cells to the environment. Guiding principles
behind response of individual cells can be applied to the tissue level as well.
Cells
Cells are the most basic component of life; they assemble and work together to form components at higher levels of organization.
Millions of cells make up an organism. Although all called cells, there are a lot of different types of cells, each have different shape,
distribution, and function. Cells respond to signals from the environment that may come in the form of ECM surrounding the cells,
soluble signals, or physical interaction with neighboring cells (Fig. 2). Cells are the functioning unit to sense and process the signals
from the environments and respond to the environmental cues. Cells are made up of many compartments that work together to
make the cell a responsive functional unit. Cells respond locally to local stimulus outside the cells, in the form of physical or chem-
ical stimulus, and convert this local signal into a cascade of intracellular signals amplified and transported to decision centers such as
Fig. 2 Tissue and cell. Tissue is composed of cells and their environment. Cells are the most basic unit of life. Cells are made up of many compart-
ments (blue): cell membrane, cytoskeleton, nucleus, mitochondria, endoplasmic reticulum, Golgi, and lysosome. It interacts with the environment
through molecules highlighted in yellow: extracellular matrix, integrins, soluble signals, receptors, and cell–cell adhesion. Components involved in
gene expression are labeled in black: DNA, RNA polymerase, RNA, ribosomes, mRNA, and polypeptide chain. Illustrations by Wong Chun Xi, MBI
Science Communications Unit.
nucleus. Decision centers would integrate all the signals from the different stimulus from different parts of the cells over a specific
period to decide a course of actions such as to move a cell, turn on a gene, and start cell differentiation or proliferation.
Structure of a Cell
The cell is like a small machine; it consists of several compartments that work together to carry out proper cell functions. Although
different cells have different shape, distribution, and function, they all have these compartments. Compartmentalization is a central
concept in biology with lipid membranes separating aqueous compositions of living organisms into smaller compartments.
Cellular compartments help to concentrate molecules on a two-dimensional space to facilitate molecular interactions, which speed
up chemical reactions.
Cell membrane
Cell membrane is a lipid bilayer that separates the inside of the cell from the outside environment. The lipid bilayer is made up of
phospholipids that consist of a hydrophilic head and hydrophobic tails, which are assembled into two-layered sheet with the hydro-
phobic tails directed toward the center of the sheet. The cell membrane forms an impermeable barrier to most molecules, allowing
only small nonpolar and lipid-soluble molecules such as oxygen, carbon dioxide, and steroids, to diffuse freely through it. Inter-
spersed throughout the cell membrane are molecules such as certain proteins, sphingolipids, and cholesterol.
The function of the cell membrane is to serve as a barrier between intracellular compartments and extracellular environment and
prevent molecules from coming in or leaving the cell freely. This allows the internal cell environment to be stabilized and not fluc-
tuate with the changes in the external environment. This stabilization is important as disruption to homeostasis in the internal envi-
ronment can lead to cell death. Biomaterials design usually considers the interaction with the cell membranes as a critical issue.
Other membrane compartments within a cell are in the form of organelles. Like the cell membrane, these internal membrane
compartments separate their contents from other parts of the cell, allowing different environments to be maintained within the cell.
Hence, substances that need to be delivered within the cell might have to cross multiple membranes to reach a particular location.
Nanomaterials or biomaterials for drug and gene delivery can penetrate the cell membranes and concentrate on the membranes
by taking advantage of the charge properties or hydrophilicity of the membrane to accelerate their intracellular delivery.
Organelles
Organelles are membrane-enclosed compartments in the cells that perform different functions.
• Nucleus
The nucleus is enveloped in a double membrane, which is contiguous to the endoplasmic reticulum (ER). On the membrane are
pores that regulate the constant flow of entry and exit of molecules into and from the nucleus. Molecules like transcription
factors translocate in and the transcribed mRNAs leave the nucleus. The nucleus contains genetic information of the cell and
proteins that maintain and transcribe the deoxyribonucleic acid (DNA).
The genetic information in the nucleus is kept in long strands of DNA molecules. The DNA is composed of four nucleotide bases,
guanine, adenine, thymine, and cytosine, short formed GATC. The bases are complementary pairs, G with C and A with T. The
bases are linked together to form a strand. The strand exists in pairs in a structure called the double helix. One strand is called the
coding strand, and the other strand is called noncoding strand.
Nuclear translocation of drug or gene delivery carriers can take advantage of the dynamic nature of the bidirectional transport of
molecules across the nuclear pore complex and importin-/exportin-mediated trafficking machinery to improve the efficiency of
the gene and drug delivery into the nucleus. Transfection of foreign genes can be much more effective during cell division when
the nuclear envelop dissolves.
• Mitochondria
The mitochondria consist of an outer and inner membrane, the outer membrane encasing the mitochondria and a curvy inner
membrane which allows for more surface area for reactions to take place. The inner membrane is where glucose is converted to
adenosine triphosphate (ATP). Mitochondria are the powerhouse of the cell where ATP, the energy currency the cell uses, is
generated. Mitochondria also play a role in regulating cell death and survival, thus a target for biomaterial applications that
involve these two processes.
• Endoplasmic reticulum
ER is organized into rough and smooth ER based on local morphology. Rough ER has attached ribosomes on its outer surface
and is involved in protein translation and folding; smooth ER plays a role in lipid metabolism. ER also acts as a calcium store,
regulating calcium levels in the cytosol. ER is the largest organelle compartment extending to every corner of a typical cell.
Besides the canonical role in protein synthesis, folding and modification, ER regulates local functions such as adhesion complex
maturation, local protein synthesis in long-term potentiation of neurons, and transient local response to stress. Recent findings
suggest that ER is a master regulator of cell functions by providing signaling highways connecting local responses to decision
centers such as the nucleus.
• Golgi apparatus
Golgi apparatus exists as many stacked vesicles in the cytoplasm. It is involved in intracellular vesicle trafficking, protein sorting,
and modification. It is important for membrane receptor recycling that innovative biomaterials R&D can explore to tune the cell
response to extracellular environmental cues.
• Lysosomes
Lysosomes are membrane organelles that have a very low pH; this environment facilitates the breakdown process that occurs
within lysosomes. pH-sensitive biomaterials have been developed to activate gene carriers to escape from organelles prior to
reaching or escape from lysosomes.
Cytosol
Cytosol consists of everything enclosed by the cell membrane, excluding the nucleus. The aqueous component in which organelles
are floating in is the cytosol. Cytosol is a relatively dense media with high viscosity such that the molecules in cytosol moves by
diffusion in short distance of a few nanometers and by active transport on cytoskeleton tracks over longer distances of micrometers.
Cytoskeleton
Cytoskeleton provides structural support to maintain the shape of the cell and serve as tracks for the directional transport of cargoes
within the cell. The cytoskeleton can be divided into three types based on the composition of their protein subunits: microfilaments,
which are made up of actins; microtubules, which are made up of tubulins; and intermediate filaments, which are made of a variety
of subunits. These subunits can reversibly polymerize into filaments or depolymerize into individual subunits, allowing the cyto-
skeleton to be dynamic and rearrange itself in response to different conditions, such as for cell migration, attachment, division, and
polarization. Polymeric gene carriers can take advantage of the transport machineries along cytoskeleton to move foreign genes into
different organelle compartments including nucleus.
Cells Interact With Extracellular Environment (Input)

Cells respond to many environmental cues to survive and function, namely, cell–ECM, cell-soluble signals, and cell–cell interac-
tions. Each signal is generated locally. For chemical signals, each ligand binding to a receptor initiates a local event and generation
of one or more secondary messengers to diffuse to the nucleus. For physical signal such as substrate stiffness, a local pinching of the
substrate by a sarcomere unit leads to the local activation of Src that transmits the amplified signal to the nucleus. The consequence
of either the physical or chemical signals interacting with the cells locally is the generation of the amplified chemical messengers to
be sent to the decision center such as nucleus to decide on whether and how the cell should respond.
Cell–ECM interaction
Cells are usually in contact with the ECM, molecules that cells secrete into the extracellular space. ECM is made up of interlocking
fibrous proteins and glycosaminoglycans, forming a mesh that provides structural and biochemical support to cells. Examples of
ECM types are fibronectin, collagen, proteoglycan, elastin, and laminin.
Structurally, ECM may affect cell function depending on its composition, stiffness, porosity, density, and alignment. Cells bind
to ECM through cell membrane receptor proteins such as integrins. Integrins are linked to the cell cytoskeleton, allowing the cells to
sense stiffness and other physical properties of the surrounding ECM, triggering different downstream pathways within the cell.
ECM is also involved in presenting biochemical cues that are crucial for cell survival. Biochemical cues usually come in the form
of soluble signals. ECM can tether the soluble signals to cell surface, increasing their effective concentration by reducing their degrees
of freedom to only two dimensional space.
It is important to understand the native ECM properties around cells and in tissues, so that biomaterial scaffold design can
recapitulate and mimic the in vivo environments. Decellularized scaffolds can support the cell growth ex vivo to
mimic in vivo conditions. Scaffolds should contain the appropriate type of ECM so that cells can bind to it. We should
also monitor the scaffold stiffness. Mammary epithelial cells cultured in stiff matrices express genes correlated to the breast
cancer phenotype, in contrast to those cultured in matrices with physiological stiffness that exhibit normal gene expression
profiles.
Cell-soluble signals interaction

Cells can communicate to other cells that are not in immediate contact with them, by releasing soluble signals into their
surrounding and bloodstream. These soluble signals may be cytokines, growth factors, hormones, neurotransmitters, salt
ions, exosomes or microvesicles containing lipids, DNA, and microRNA (miRNA). The soluble signal might either bind to recep-
tors on the cell membrane and initiate signaling or can move into the cells and interact with proteins involved in cell signaling.
Cells may receive signals released from other cells, in cases of paracrine and endocrine signaling, or those released by itself, in the
case of autocrine signaling. In autocrine signaling, cells secrete soluble signals into its surrounding; these signals can in turn bind
to receptors on the cell, acting on itself as a stimulus. An example of endocrine signaling is insulin, which is released by islet’s beta
cells in the pancreas in response to an increase in blood glucose level. Insulin is secreted into the bloodstream to signal cells in
other parts of the body such as the brain and liver to uptake glucose. Insulin does this by binding to insulin receptor, a protein
expressed on the cell membrane. Binding of insulin triggers translocation of glucose transporter, GLUT4, to the cell membrane,
enabling the cell to uptake glucose, as glucose cannot pass through cell membrane directly. Soluble signals such as growth factors
are often conjugated to biomaterials, to confer additional properties needed such as to stimulate tissue regeneration. For
example, VEGF blended in biomaterials have been used in implants to promote vascularization to facilitate wound healing
and regeneration.
Cell–cell interaction
Cell–cell interactions can be relatively stable such as those mediated by tight junction, adherens junction, desmosomes and gap
junctions, or transient such as those mediated by the cell adhesion molecules (CAMs) from the immunoglobulin and selectin fami-
lies. Tight junctions, adherens junctions, and desmosomes act as structural linkers that hold two cells together, whereas gap junc-
tions form pores that link the cytoplasm of one cell to another, allowing small molecules to diffuse through. CAMs from the
immunoglobulin and selectin families are involved in contact-dependent cell–cell signaling.
Cell–cell contact is important for maintaining structural integrity of tissues, polarization, and proliferation of the cells. Cell–cell
interaction is important for synchronizing responses in groups of cells at the tissue level, forming a communication channel
between cells that do not rely on outside signaling. Examples include calcium signal passing through gap junctions in cardiac
muscle tissue to synchronize beating of cardiomyocytes and supracellular actin cables that synchronize cell movements in
development.
Most cell lining parts of the body that contact the outside environment requires strong cell–cell interaction to form a barrier.
Examples include air tracks that contact the air breathed in; gastrointestinal track contacts the food consumed. The barrier formed
by tight cell–cell junction prevents leakage of outside molecules into the body.
For many cell types, cell–cell interaction is essential for cell survival and function. Therefore, biomaterials are designed to allow
and encourage cell–cell interaction in macroporous scaffolds or flexible nanofibers or ribbons to support cell and tissue functions
required in applications.
Signaling Cascade
All environment stimuli, whether physical or chemical, will activate the signaling cascade, a series of intracellular biochemical
reactions that allow signals to be amplified. An example is the G-protein-coupled receptor, a receptor that activates G-
proteins. Once a ligand binds to the extracellular domain of the receptor, the receptor will undergo conformational changes
and be activated, which will then lead to activation of G-proteins bound to the intracellular domain of the receptor. An
activated receptor can activate multiple G-proteins, and each of the activated G-protein can activate many downstream
secondary messengers. This results in an exponential amplification of the signal, even with only one ligand bound. The under-
standing of signaling cascade is causative. Each node of the cascade is connected to the next node downstream through
conformational changes of the downstream node via binding or modifications such as phosphorylation, palmitoylation,
or methylation.
Cells make decisions in a digital manner; a response is either triggered or not triggered. A cell makes decision based on the accu-
mulation of analog signals diffused from elsewhere; once the accumulation reaches a certain threshold at the decision center, this
triggers a response from the cell. For example, rapid changes in membrane potential in the cell body or dendrites of a neuron can
diffuse to the decision center (in this case the axon hillock) to generate an action potential. Action potential is only triggered when
the membrane potential at the decision center reaches a certain threshold, after which specific ion channels are activated that lead to
rapid and massive influx of ions to generate an action potential. This can apply to other reactions in the cell as well: only when
signals exceed a defined threshold is the response triggered. Decision is based on the accumulated signals from different locations
of the cell over a defined period.
Cell Response (Output)

Once a decision is made by the decision center such as the nucleus, cells execute or implement a decision causing a phenotype
change (such as proliferation, growth, and differentiation). One pathway does not occur in isolation from the others, and there
are overlaps and dependencies on other pathways.
Gene expression
Gene expression is the process in which protein is synthesized from genetic material. All cells in the body contain the same DNA but
differ from one another due to a different subset of genes actively expressing the corresponding subset of proteins. A gene is a specific
sequence of DNA that codes for a protein. The central dogma of molecular biology is a gene is transcribed to RNA; RNA is then read
by ribosomes, which translates the information on the RNA to proteins. Gene expression encompasses all regulation steps that
control the level of expression of a particular gene into protein. Levels of the expressed protein can be regulated at the DNA level,
RNA level, and protein level:
• Regulation at DNA level: Genetic information is stored as long strands of DNA packed in the nucleus; the level of compaction of
DNA where a particular gene is located can affect expression levels. Histone modification or methylation of base DNA can
promote or inhibit gene expression.
• Regulation at transcription level: At the transcription level, from DNA to RNA, there are many molecules that need to come
together for transcription to occur. Transcription starts when RNA polymerase is assembled at promoter site, a sequence usually
located upstream of the gene to be transcribed. Transcription factors can promote or inhibit recruitment of RNA polymerase to
the gene. Enhancer sequences are DNA sequences that if transcription factors bind to can affect transcription rate.
• Regulation at RNA level: Transcribed RNA requires processing, to mRNA, before it can leave the nucleus and be translated to
proteins. During transcription, both regions that will be translated, exons, and regions that will not be translated, introns, are
transcribed. Intron regions have to be spliced out, so the final mRNA contains only exons. Regions need to be added on to both
ends, 50 cap at the 50 end and poly-A tail at the 30 end. The 50 cap allows translation machineries to recognize the mRNA and
translate it. An mRNA can be translated multiple times, each time an A is hydrolyzed off the poly-A tail; the longer the poly-A tail,
the more times the mRNA can be translated before it is degraded. mRNA levels are also controlled by miRNA. These short strands
of noncoding RNA bind complementarily to mRNA to affect mRNA stability, readability, and integrity.
• Regulation at translation level: mRNA is translated to a polypeptide chain. The polypeptide is not always fully functional and
needs further modification. For example, insulin is translated to preinsulin polypeptide chain that is cleaved prior to being
functional. Others may require posttranslational modifications and chemical modifications to the amino acid bases, before
being fully functional.
Cell differentiation
Cell differentiation is known as a process in which cells become specialized. Pluripotency means the potential to become any cell
type and is a potential observed in stem or progenitor cells at the beginning of the differentiation process. This potential is slowly
lost as the cell differentiates and gains specialized functions. The differentiation process alters the cell dramatically, its shape, size,
and energy requirements. This process is not a linear and irreversible process. Differentiation selects a subset of genetic information
to be expressed at different stages of the differentiation process. Therefore, differentiated cells can be manipulated back to a more
primitive or stem cell-like state, by reprogramming it to express a particular set of genes.
Cell differentiation is a result of the integration of different stimulus in a spatiotemporal fashion. Cell differentiation is sensitive
to both mechanical and chemical stimulus from the environment. Improved understanding of the differentiation process can allow
us to engineer the process to achieve desired outcome. This is especially true when developing biomaterials as delivery carriers of the
stem or progenitor cells in vivo.
Cell growth and division

Cell division requires a lot of energy and resources and is a highly controlled process in the cell. There are many checkpoints in the
cell cycle: there are requirements to control whether cells can transition to the next phase. An example of what happens when the
checkpoints are bypassed is cancer. Cancer is a result of uncontrolled cell growth and division. Normally, cells rely on several
survival signals and factors to suppress the default cell-death process. In cancer, these checkpoints are bypassed, and cells grow
and divide despite no growth or dividing signals.
Majority of the cells exist in the nondividing and quiescent G0 phase. If there are environmental cues to instruct a cell to divide,
the cell enters the division cycle. Cell division is split into two phases, interphase and mitosis. Interphase is further split into four
stages, G0, G1, S, and G2. Interphase is when the cells prepare themselves for division in mitosis. The cells have to meet size require-
ments and should be able to synthesize another copy of DNA. Cells have to proceed strictly in a specific order and have to meet strict
requirements before the cell cycle can proceed. Failure to meet requirements will force the cells to exit the division cycle. This ensures
that cell division occurs only when needed and the dividing cells are healthy.
Dividing cells express unique proteins such as topoisomerase IIB, PCNA, and Ki-67 required during the division process. These
proteins are useful markers in the study of the implanted scaffolds on how the tissue is responding to the scaffold or whether cells
are migrating into the scaffold and dividing.
Cell spreading and migration

Cell spreading and migration are a few of the many processes that involve cytoskeletal rearrangement. Cell spreading is when cells in
its rounded morphology in suspension flatten out on a substrate. This is a sign of adherence to substrate, as cells need anchors on
the substrate to allow it to flatten itself out. Two ways were observed that cells can spread isotropically and anisotropically. Cells
usually spread in an anisotropic manner, where the edges extend due to actin polymerization. In isotropic spreading, edges spread
out in a circular manner and flatten rapidly. Isotropic spreading can be induced by removing serum; cells spread at a faster rate iso-
tropically. Cell migration is needed during development when cells have to move to its required location for tissue rearrangement. It
is important in wound healing and responses to injury where cells need to migrate to close up wounds or move to site of injury,
respectively.
Mechanistic Insights Into the Biomaterials Interaction With Biological Systems

To understand the mechanism of how biomaterials interact with a biological system, researchers have conducted systematic eval-
uation using in vitro and in vivo assays. In vitro assays are conducted in laboratory vessels with isolated molecules or cells, not on
the living organism itself. In vitro assays can be divided into cell-free and cell-based assays. The former evaluates the interaction of
a biomaterial with biological molecules, while the latter evaluates the interaction of a biomaterial with cultured cells or tissues. In
contrast, in vivo assays are conducted directly on living organisms to evaluate the effects of biomaterials on an organism at both
local and system levels. The results from in vivo assays are more physiologically relevant, but due to the complexity in interpreting
the results, in most cases, in vitro assays are the first assays of choice. In vivo assays will be performed to confirm the mechanistic
insights revealed by the in vitro assays.
A mechanistic study is performed to understand causation, not just correlation. Causation demonstrates the direct connection
between the input stimulation (e.g., nanosized features of a polymer substrate) and output response (e.g., cell differentiation). A
biological system exhibits short-term to long-term responses when exposed to biomaterials. Short-term responses involve early
events, which occur almost immediately, in timescales of seconds to a few minutes upon stimulation. Studying such short-term
responses should occur at local or subcellular levels in the spatioscale of a few micrometers. Such output responses are the direct
consequence of the input stimulation. In contrast, long-term responses occur after significant time has elapsed, in timescale of
hours, days, or weeks. By then, the cell phenotype changes observed are the consequence of complex feedback mechanism leading
to a steady state. Therefore, long-term responses often are studied with the aid of computational model and system biology meth-
odologies to understand what and how the decision-making centers integrate local signals and multiple signaling networks over an
extended period into a coherent understanding of the input-out causative relationship.
Further Reading
Alberts, B., Johnson, A., Lewis, J., Morgan, D., Raff, M., Roberts, K., & Walter, P. (2014). Molecular biology of the cell (6th edn.). New York: Garland Science.
Badylak, S. F. (2015). Host response to biomaterials: the impact of host response on biomaterial selection. Oxford: Academic Press.
Patton, K. T. (2015). Anatomy and physiology (9th edn.). London: Elsevier Health Sciences.
Pocock, G., Richards, C. D., & Richards, D. (2013). Human physiology (4th edn.). Oxford: Oxford University Press.
Sherwood, L. (2015). Human physiology: from cells to systems (9th edn.). Boston, MA: Cengage Learning.
Relevant Websites
https://www.khanacademy.org/science/health-and-medicine/.
http://www.nature.com/scitable/ebooks/essentials-of-cell-biology-14749010/contents.
https://www.ncbi.nlm.nih.gov/books/NBK10757/.
https://en.wikipedia.org/wiki/Human_body.
https://en.wikibooks.org/wiki/Human_Physiology.
Animal Models in Biomaterial Development
James M Anderson and Sirui Jiang, Case Western Reserve University, Cleveland, OH, United States
Introduction 237
Biomaterial and Device Perspectives in In Vivo Testing 237
Selection of Animal Models for In Vivo Tests 238
Implantation 238
Species Differences 239
Animal Models for Host Response Mechanisms 240
Further Reading 241
Introduction
Animal studies with appropriate animal models are a necessary component in the pathway to ultimately determine clinical predict-
ability in humans. The results of animal studies most commonly are utilized to determine the biocompatibility (safety) of the
biomaterial/medical device in the intended application. This process involves the design and execution of animal studies, from
the proof-of-concept stage to the pivotal preclinical study of the medical device required for regulatory submission. Preclinical
testing in animal models is an important part of the regulatory process, used to determine the safety and efficacy of devices prior
to human clinical trials. The choice of the animal model and the selection of in vitro tests should be made according to the intended
use of the respective medical device, prosthesis, or biomaterial.
Numerous factors contribute to the choice of animal experimental model. Animal models are generally divided into small
animal models (mouse, rat, and rabbits) and large-animal models (dog, goat, pig, sheep, etc.). Small animal models are usually
used for ethical, economical, and statistical considerations, whereas large-animal models are primarily used if small animal models
are not suitable for the replication of a clinical application or for proof-of-concept testing before clinical translation. Recent reviews
in the literature have discussed the advantages and limitations of small- and large-animal models and procedural and experimental
considerations vital to the implementation and evaluation of materials in these models. The use of any model for an intended clin-
ical application requires that the strengths and weaknesses of any model be discussed in the development of the experimental
strategy and plan.
Biomaterial and Device Perspectives in In Vivo Testing
Two perspectives may be considered in the in vivo assessment of tissue compatibility of biomaterials and medical devices. The
first perspective involves the utilization of in vivo tests to determine the general biocompatibility of newly developed bioma-
terials for which some knowledge of the tissue compatibility is necessary for further research and development. In this type of
situation, manufacturing and other processes necessary to the development of a final product, that is, the medical device, have
not been carried out. However, the in vivo assessment of tissue compatibility at this early stage of development can provide
additional information relating to the proposed design criteria in the production of a medical device. While it is generally rec-
ommended that the identification and quantification of extractable chemical entities of a medical device should precede bio-
logical evaluation, it is quite common to carry out preliminary in vivo assessments to determine if there may be unknown or as
yet identified chemical entities that produce adverse biological reactions. Utilized in this fashion, early in vivo assessment of the
tissue compatibility of a biomaterial may provide insight into the biocompatibility and thereby may permit further develop-
ment of the biomaterial utilized in a medical device. Obviously, problems observed at this stage of development would require
further efforts to improve the biocompatibility of the biomaterial and to identify the agents and mechanisms responsible for
the adverse reactions. As the in vivo assessment of tissue compatibility of a biomaterial or medical device is focused on the end-
use application, it must be appreciated that a biomaterial considered compatible for one application may not be compatible for
another.
The second perspective regarding the in vivo assessment of tissue compatibility of medical devices focuses on the biocom-
patibility of the final product, that is, the fabricated medical device in the condition in which it is to be implanted. Thus, issues
related to desired fabrications and interactions between biomaterials, etc., may come into play. Although medical devices in
their final form and condition are commonly implanted in carefully selected animal models to determine function and biocom-
patibility, it may be inappropriate to carry out all of the recommended tests necessary for regulatory approval on the final
device. In these situations, some tests may initially be carried out on biomaterial components of devices that have been
prepared under manufacturing and sterilization conditions and other processes utilized in the development of the final
product.

238 Biomaterials: In vitro and in vivo studies of biomaterials j Animal Models in Biomaterial Development
Selection of Animal Models for In Vivo Tests
Animal models are used to predict the clinical behavior, safety, and biocompatibility of medical devices in humans (Table 1). The
selection of animal models for the in vivo assessment of tissue compatibility must consider the advantages and disadvantages of the
animal model for human clinical application. Several examples follow, which exemplify the advantages and disadvantages of
animal models in predicting clinical behavior in humans.
A single test animal species may not assess all pertinent clinically important applications. For example, as described earlier, sheep
are commonly used for the evaluation of heart valves. This is based on size considerations and also the propensity to calcify tissue
components of bioprosthetic heart valves and thereby be a sensitive model for this complication. Thus, the choice of this animal
model for bioprosthetic heart valve evaluation is made on the basis of possible accelerated calcification, the major clinical problem,
assessed in rapidly growing animals, which has its clinical correlation in young and adolescent humans. Nevertheless, normal sheep
may not provide a sensitive assessment of the propensity of a valve to thrombosis, which may be potentiated by the reduced flow
seen in abnormal subjects, but diminished by the specific coagulation profile of sheep.
The in vivo assessment of tissue responses to vascular graft materials is an example in which animal models present a particularly
misleading picture of what generally occurs in humans. Virtually, all animal models, including nonhuman primates, heal rapidly
and completely with an endothelial blood-contacting surface. Humans, on the other hand, do not show extensive endothelializa-
tion of vascular graft materials, and the resultant pseudointima from the healing response in humans has potential thrombogenic-
ity. Consequently, despite favorable results in animals, small-diameter vascular grafts (< 4 mm in internal diameter) usually yield
early thrombosis in humans, the major mechanism of failure that is secondary to the lack of endothelialization in the luminal
surface healing response.
Originally, the porcine coronary artery model was considered the model of choice for the evaluation of arterial stents. More
recently, the rabbit iliac artery model for the evaluation of drug-eluting stents has been considered to be more realistic, as endothe-
lialization is slower in the rabbit model than in the porcine model and inflammation is not as extensive in the rabbit. Thus, endo-
thelialization, healing, and inflammation in the rabbit iliac artery model may be closer to these responses in humans than the
porcine coronary artery model.
Table 2 is a short, incomplete list of animal tissue/organ modifications that have been used in biomaterial development. This is
considered to be incomplete but does offer a perspective on the variety of animals that have provided, in some cases, appropriate
and adequate study responses.
Implantation
Implantation tests assess the local pathological effects on the structure and function of living tissue induced by a sample of a material
or final product at the site where it is surgically implanted or placed into an implant site or tissue appropriate to the intended appli-
cation of the biomaterial or medical device. In some cases, the anatomic site of implantation used for biocompatibility evaluation is
not the same as the site of ultimate use, but has representative mechanisms and consequences of tissue–biomaterial interaction
Table 1 Animal models for the in vivo assessment of medical devices
Device classification Animal
Cardiovascular
Heart valves Sheep
Vascular grafts Dog, pig
Stents Pig, dog, rabbit
Ventricular assist devices Calf
Artificial hearts Calf
Ex vivo shunts Baboon, dog
Orthopedic/bone
Bone regeneration/substitutes Rabbit, dog, pig, mouse, rat
Total jointsdhips, knees Dog, goat, nonhuman primate
Vertebral implants Sheep, goat, baboon
Craniofacial implants Rabbit, pig, dog, nonhuman primate
Cartilage Rabbit, dog
Tendon and ligament substitutes Dog, sheep
Neurological
Peripheral nerve regeneration Rat, cat, nonhuman primate
Electric stimulation Rat, cat, nonhuman primate
Ophthalmological
Contact lens Rabbit
Intraocular lens Rabbit, monkey
Biomaterials: In vitro and in vivo studies of biomaterials j Animal Models in Biomaterial Development 239
Table 2 Animal tissue/organ modifications in biomaterial development
Species Tissue modification Study response
Canine Female/male blood cross transfusion Endothelium development on synthetic vascular grafts
Mice Muscle crush, ischemia, laceration, or toxin injection Muscle nerve and vascular regeneration
Rat
Mice
Rabbit Critical defect in the bone Bone regeneration and remodeling
Canine
Others
(e.g., subcutaneous implantation in rodents of bioprosthetic heart valve materials to study calcification that occurs as a major clin-
ical limitation in humans). The most basic evaluation of the local pathological effects is carried out at both the gross level and the
microscopic level. Histological (microscopic) evaluation is used to characterize various biological response parameters. To address
specific questions, more sophisticated studies may need to be done. Examples include immunohistochemical staining of histolog-
ical sections to determine the types of cells present and studies of collagen formation and remodeling of the fibrous capsule. For
short-term implantation evaluation out to 12 weeks, mice, rats, guinea pigs, or rabbits are the most common animals utilized in
these studies. For longer-term testing in the subcutaneous tissue, muscle, or bone, animals such as dogs, sheep, goats, pigs, and other
animals with relatively long life expectancy are suitable. If a complete medical device is to be evaluated, larger species may be
utilized so that human-sized devices may be used in the site of intended application. For example, substitute heart valves are usually
tested as heart valve replacements in sheep, whereas calves are usually the animal of choice for ventricular assist devices and total
artificial hearts.
In all aspects of biocompatibility testing, it is important to recognize that the effects of the material on the surrounding tissues
are generally superimposed on the events occurring during physiological wound repair induced by the surgery of implantation. This
is particularly important in shorter-term experiments.
The use of canines in determining the blood compatibility of candidate materials in cardiovascular applications has been shown
to be affected by the activity of the respective coagulation system in these animals. Early studies on the blood compatibility of
vascular grafts reveal that some canines have a high level of coagulation activity, whereas other canines have a low activation poten-
tial. This broad range of blood coagulation activity in canines initially led to problems in the appropriate determination of blood
compatibility of vascular graft materials. An issue such as this requires prior identification and characterization of the tissue activa-
tion potential of the respective animals utilized in any choice of animal model. Currently, the Food and Drug Administration (FDA)
strongly suggests that the nonanticoagulated venous implants (NAVI) usually in the shape of a catheter be utilized in canines for
periods up to 4 h and then gross evaluation of the amount of apparent thrombus on the surface be performed. Information derived
from the NAVI model may be compromised by variation in the implant position, the implant technique, the extent of device–vessel
wall contact and the time/incubation, the explant technique, the material/material surface, the nonthromboadherent materials that
may be labeled nonthrombogenic, recipient/subject thrombotic potential, the statistical power, and the expertise of the evaluator
utilizing this model.
These caveats, although suggested for the NAVI model, hold for the choice of any model system utilized in the early biocom-
patibility evaluation of biomaterial and the later biocompatibility evaluation of medical devices containing component
biomaterials.
Species Differences
Species differences may play a significant role in the early determination of biomaterial biocompatibility and especially in the latter
biological response evaluation with risk assessment of medical devices. While rats, mice, and rabbits are most commonly used for
the early in vivo assessment of biomaterial biocompatibility, the in vivo assessment of tissue compatibility of the medical device
usually requires larger animals. The limitations and the strengths and weaknesses of the choice of any animal model for biological
response (biocompatibility) assessment must be considered in the selection of an appropriate animal model for medical device
evaluation.
In addition to the biological response evaluation of a medical device, the appropriate choice of an animal model may provide
new insight into the biological interactions that may be discovered.
For example, mini pigs have been most commonly used to evaluate the tissue response to the implantation of coronary stents.
However, recent studies have demonstrated that the evaluation of drug-eluting stents in the atherosclerotic rabbit arteries is more
predictive of neointimal progression and healing than normal rabbit iliac or porcine coronary arteries when compared with these
responses in atherosclerotic human coronary arteries. While endothelial regrowth is significantly greater in the atherosclerotic rabbit
model and occurs more rapidly than in humans, the time frame for these responses in the rabbit is much closer to that in humans
than in mini pigs. These findings demonstrate two significant principles in the selection of the appropriate animal model. First, the
healing response to coronary arteries in mini pigs is much faster than in rabbits that are much faster than in humans. Animal models
240 Biomaterials: In vitro and in vivo studies of biomaterials j Animal Models in Biomaterial Development
may not adequately and appropriately predict the time-dependent nature of biological responses in humans. Secondly, the utiliza-
tion of a diseased model may provide more significant information regarding the biological response assessment than when non-
diseased animal models are utilized. However, rarely are diseased animal models used in biocompatibility, that is, biological
response evaluation with risk assessment studies.
Another example where the appropriate choice of animal models has revealed new information regarding the human response is
studies from the early 1960s with the identification of blood-borne progenitor cells, presumably stem cells, in canines provided for
the endothelialization of vascular grafts, and the endothelialization generally observed in animal studies with grafts and canines is
not due to ingrowth either through the vascular graft or from the anastomosis of the vascular graft with the artery. Considering the
early time frame, 1960s, these blood studies with canines were novel and innovative at that time. These canine studies utilize the
identification of chromosomal markers in male recipients of vascular grafts after bone marrow radiation combined with exchange
transfusions of female blood at the time of implantation of the vascular graft. The developed endothelial surfaces of these vascular
grafts receiving female blood were identified to originate from the canine female blood.
These cross transfusion studies in canines and the identification of the source of the endothelial cells may explain the lack of
endothelialization in humans where blood-circulating progenitor cells, that is, stem cells, in humans are at a significantly lower
concentration than that found in the various animal studies with vascular grafts that, in addition to canines, include rabbits,
pigs, sheep, and significantly primates such as chimpanzees, baboons, and monkeys.
These two examples clearly demonstrate that the appropriate choice of animal model may provide insight into biological
responses and findings from such studies must be incorporated into the predictability of the animal model for human clinical appli-
cation of the medical device.
Infection is one of the most common complications associated with medical devices in clinical application. Some studies have
been developed to address this common complication of infection and are considered to be significantly important in the devel-
opment of wound dressings and tissue engineering. When selecting or designing an effective animal model, it is important to
consider the host animal species and strain, the host animal effect, the pathogen species and strain, the inoculum concentration
and vehicle, and many other factors specific to the disease state of interest. It is critical to fully explore previous animal models avail-
able in the literature and often necessary to conduct pilot studies to ensure an infection that has been created is self-sustaining but
does not overwhelm the host. In this regard, specific attention to detail is required in the early development of the experimental plan
and strategy for evaluation.
Animal Models for Host Response Mechanisms
Table 3 provides an incomplete list of transgenic models in biomaterial development where the focus or target of the study has been
the identification of mechanisms of interaction in the early inflammatory responses leading to final end-stage healing with fibrous
capsule formation. These “knockout” animal models have been used to identify the participation of the various types of inflamma-
tory cells that may contribute to the ultimate end-stage healing or the cytokines and other humoral factors that may play a significant
role in the cellular and humoral responses leading to end-stage healing. Currently, this is an active area that is attempting to identify
those important factors that may control or modulate the focal foreign-body reaction or the development of the final fibrous
capsule that surrounds or encapsulates the implant. As such, the identification of these factors offers a target for intervention
and inhibition of factors that may affect macrophage activation and adhesion to biomaterial surfaces. Macrophage fusion forms
foreign-body giant cells that are adherent to the surface of the biomaterials or the inhibition of fibrous capsule formation. These
are important considerations in the subsequent development of biosensors where the focal foreign-body reaction and the fibrous
capsule have been inhibited in the development of appropriate sensors for clinical application.
Table 3 Transgenic models in biomaterial development
Species Genetic modification Biological response
Mice DAP12 deficient Inhibits macrophage fusion (FBGC/osteoclast formation)

Mice Syk deficient No macrophage fusion (FBGC formation)
Mice Monocyte chemoattractant protein-1 (MCP-1) Reduced accumulation and fusion reduced MMP-9
Mice NIrp3/ inflammasome Nb IL-1b secretion, reduced scaffold cell, infiltration and phenotypes
Mice Bat 3/ Deficient in cytotoxic T lymphocytes and IL12, induction, reduced IL6 and G-CSF
CD8 (þ) dendritic cell knockout
Mice TGF-b receptor II knockout Photoactivation of latent transforming growth factor-b1 on dental stem cell
differentiation
Mice SCID Severe combined immunodeficiency
Mice muMT B-cell deficient, decreased fibrosis
Mice Reg2/IL2ry T- and B-cell deficient, no fibrosis, macrophage dysfunction
Mice a-MF Macrophage depletion or inhibition, no fibrosis
Mice a-CXCL13 Decreased B cells, decreased fibrosis
Biomaterials: In vitro and in vivo studies of biomaterials j Animal Models in Biomaterial Development 241
Knockout animals provide insight into molecular mechanisms and potential targets for the intervention of selected host
responses. However, it must be understood that knockout and transgenically modified animal models may have other molecular
and cellular host responses that are modified but not identified in the host response assessment. Therefore, caution is recommended
in the use of these systems, and the experimental study should be as comprehensive as possible to identify all possible host
responses. Particular attention should be given to the biocompatibility of bioactive biomaterials and tissue-engineered systems.
Further Reading
An, Y. H., & Friedman, R. J. (1999). Animal models in orthopedic research. Boca Raton, FL: CRC Press.
Anderson, J. M., & Schoen, F. J. (2013). In vivo assessment of tissue compatibility. In B. D. Ratner, A. S. Hoffman, F. J. Schoen, & J. E. Lemons (Eds.), Biomaterials science, an
introduction to materials in medicine (3rd edn, pp. 609–617). Oxford, England: Elsevier Academic Press.
Boutrand, J.-P. (Ed.). (2012). Biocompatibility and performance of medical devices. Oxford: Woodhead Publishing Limited.
Nakazawa, G., Nakano, M., Otsuka, F., Wilcox, J. N., Melder, R., et al. (2011). Evaluation of polymer-based comparator drug-eluting stents using a rabbit model of iliac artery
atherosclerosis. Circulation. Cardiovascular Interventions, 4, 38–46.
Shah, S. R., Young, S., Goldman, J. L., Jansen, J. A., Wong, M. E., & Mikos, A. G. (2016). A composite critical-size rabbit mandibular defect for evaluation of craniofacial tissue
regeneration. Nature Protocols, 11(10), 1989–2009.
Tatara, A. M., Shah, S. R., Livingston, C. E., & Mikos, A. G. (2015). Infected animal models for tissue engineering. Methods, 84, 17–24.
Veiseh, O., Doloff, J. C., Ma, M., Vegas, A. J., Tam, H. H., et al. (2015). Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-
human primates. Nature Materials, 14(6), 643–651.
Wolf, M. F., & Anderson, J. M. (2012). Practical approach to blood compatibility assessments: general consideration and standards. In J.-P. Boutrand (Ed.), Biocompatibility and
performance of medical devices (pp. 159–200). Oxford: Woodhead Publishing Limited.
Yamamoto, S., Urano, K., Koizumi, H., Wakana, S., Hioki, K., et al. (1998). Validation of transgenic mice carrying the human prototype c-Ha-ras gene as a bioassay model for rapid
carcinogenicity testing. Environmental Health Perspectives, 106(Suppl. 1), 57–69.
Yang, Y., Jao, B., McNally, A. K., & Anderson, J. M. (2014). In vivo quantitative and qualitative assessment of foreign body giant cell formation on biomaterials in mice deficient in
natural killer lymphocyte subsets, mast cells, or the interleukin-4 receptor f and in severe combined immunodeficient mice. Journal of Biomedical Materials Research. Part A,
102A, 2017–2023.
Blood–Biomaterial Interactions
Nicholas P Rhodes, University of Liverpool, Liverpool, United Kingdom
Introduction 242
Biology of Blood 243
In Vitro Observations 243
Plasma Proteins 243
Coagulation and Contact Phase Activation 244
Platelets 245
Leukocytes 246
Complement Activation 246
In Vivo Observations 246
Rheological Factors 247
Surface Topography 247
Further Reading 248
Glossary
Biocompatibility The ability of a biomaterial to perform its desired function without eliciting undesirable local or systemic
effects but generating the most appropriate response in that specific application.
Complement system activation The activation of a protein cascade representing the noncellular immune system that
ultimately results in the formation of a cell destruction complex.
Contact-phase activation The surface-mediated autoactivation of factor XII (or Hageman factor) resulting in the initiation of
the coagulation cascade which ultimately results in blood plasma clotting.
Platelet activation Formation of an active form of platelets resulting from the attachment of surface-phase fibrinogen and
collagen to glycoprotein receptors GPIIb/IIIa and Ib respectively, resulting in platelet spreading, the secretion of platelet
granules and expression of procoagulant lipid on the plasma membrane of the platelets.
Introduction
Researchers have, for many decades, expected that the performance of future blood-contacting biomaterials would match that of
nonthrombogenic, natural endothelium, that is, with no blood coagulation and without blood cell adhesion or activation. Despite
the wealth of knowledge accumulated regarding the interfacial phenomena of surfaces with protein mixtures and cells, it has not
been possible to design such a surface, and the interactions of blood elements with biomaterials placed intravascularly can cause
significant morbidity. Coronary arteries, for example, are routinely bypassed using saphenous vein autografts, as synthetic alterna-
tives cannot be used.
The failure of the medical engineering community over more than half a century to meet this challenge may, in part, be due to
the lack of understanding that the phenomena observed in vitro, such as coagulation cascade protein activation or platelet adhesion,
may be of relative unimportance in vivo at particular anatomical locations within the circulation and the fact that the response of
vascular endothelium to intravascular biomaterial implantation is rarely considered.
Described in this article are the in vitro phenomena usually considered when describing blood–biomaterial interactions and
a selection of in vivo reactions that are often observed after intravascular placement of blood-contacting devices. Although not
an exhaustive list, the range of possible effects due to poor blood compatibility of an intravascular device can be listed as:
• Thrombosis
• Embolism
• Thrombocytopenia and leukopenia
• Immune and allergenic reactions
• Infection
• Inflammation
• Fibrosis
• Material degradation
• Tumorigenesis
• Mutagenicity

Biomaterials: In Vitro and in Vivo Studies of Biomaterials j Blood–Biomaterial Interactions 243
Thus, coagulation and thrombosis continue to be problematic in certain blood-contact applications, as no generalized theory
currently exists that relates physical or chemical surface features to in vivo performance of a biomaterial. Introduction of a bioma-
terial into blood triggers a complex cascade of interrelated reactions that may, or may not, relate to the long-term performance of
that biomaterial. The picture is made more complex by the recent discovery of surface interactions with biological molecules not
previously identified, and the greater complexity of coagulation cascade protein cleavage than previously hypothesized.
Biology of Blood
Blood is composed of red blood cells (RBCs), also known as erythrocytes, white blood cells (or leukocytes) and platelets (originally
known as thrombocytes), proteins, ions, lipids and carbohydrates in an aqueous medium (Tables 1 and 2). RBCs are both phys-
ically large and great in number, comprising almost half of the volume of blood. Their role in blood–biomaterial interactions is
largely limited to transport phenomena in flowing blood. Platelets are physically small, outnumbered by RBCs by a factor of
20:1 and so represent a small percentage of blood volume, but play a central role in the control and propagation of blood reactions,
interacting with the coagulation system proteins. Leukocytes are similar in size to RBCs but represent only 0.1% of the total blood
cell number. These interact with the host’s immune and inflammatory systems and play an important role in reactions with the
endothelium.
There are several hundred different plasma proteins within blood, but albumin, fibrinogen and immunoglobulins represent
98% of the total protein mass. There are a number of important protein systems: the coagulation cascade, complement cascade,
protease inhibitors and fibrinolytic system being predominant in potentiating the reactions of blood with biomaterials.
In Vitro Observations
Developers of intravascular biomaterials must initially test the biocompatibility of their designs rigorously in vitro. So while
phenomena that can be observed in vitro may not be clinically relevant, material scientists have a requirement to perform a basic
set of tests that may point to aspects of a biomaterial’s performance in vivo.
It clearly follows that the surface chemistry, energetics and morphology of a biomaterial will have some bearing on its long-term
clinical performance, but only when the significance of all the component parts is fully understood will a quantum leap in device
performance be possible.
Plasma Proteins
All cellular interactions with implanted biomaterials are controlled by a layer of proteins formed after the initial contact of the mate-
rial with a biological fluid. When a biomaterial is placed into blood, dissolved plasma proteins adsorb to its surface within milli-
seconds, following a small quantity of water and inorganic ion adsorption. This is largely due to the polar nature of amino acids, the
relative lack of their solubility and the nonhydrated nature of the majority of implantable biomaterials. Three-dimensional rear-
rangement of the plasma proteins allow the hydrophobic domains to retreat towards the material surface, hydrophilic domains
facing the aqueous milieu.
Typical blood-contacting polymers have a relatively narrow range of surface energies, that is they are moderately hydrophobic.
Placing materials that are very much more hydrophilic, such as hydrogels, or extremely hydrophobic, can have a dramatic influence
on the course of protein adsorption and subsequent cellular events. Researchers have over the years attempted to reach conclusions
about the best, or an acceptable range of surface energy a biomaterial should possess, but unfortunately, their conclusions vary
massively, and in some cases directly contradict each other. That is, some studies have concluded that long-term biocompatibility
improves with increasing surface energy, while other studies conclude the opposite. Hydrophobic materials, however, tend to
absorb more protein molecules than a surface which is correspondingly hydrophilic.
Table 1 Cellular composition of blood
Cell type Approx. diameter (mm) Approx. concentration in blood (number per mL)
Red blood cell 7.5 5000 106

Platelet 3 250 106
Leukocyte 6–10 7.5 106
Lymphocyte 7 2 106
Monocyte 9 0.5 106
Neutrophil 8 5 106
Basophil 8 0.2 106
Eosinophil 8 0.1 106
244 Biomaterials: In Vitro and in Vivo Studies of Biomaterials j Blood–Biomaterial Interactions
Table 2 Noncellular composition of blood
Constituent Approx. concentration in plasma (g per L)
Proteins 720
Albumin 56% of total protein
Immunoglobulins 38% of total protein
Fibrinogen 4% of total protein
Ions 98
Lipids 83
Carbohydrate 17
The composition of the protein layer is initially dependant on the concentration of proteins within blood, that is albumin,
fibrinogen and immunoglobulins predominate. Where a surface has higher affinity for proteins that are less abundant, including
those involved in the coagulation or complement cascades (e.g., C3 or factor XII), the concentration of proteins in that initial layer
is gradually reduced in favor of those higher-affinity proteins due to the random arrivals and departure of individual molecules,
a process known as the ‘Vroman Effect’. This change in the identity of proteins adsorbed to the biomaterial surface results in selected
proteins to become susceptible to cleavage and others that are able to form bonds with cell surface receptors, thereby causing
cellular activation.
Fibrinogen, when adsorbed in the correct conformation, will cause the adhesion of platelets, as described in detail below, and
subsequently lead to their activation, an interaction that is not generally observed, at least initially, on surfaces that preferentially
adsorb albumin.
Thus, the actual concentrations of specific proteins adsorbed on a biomaterial surface is of less importance than the precise
conformational rearrangements they subsequently adopt, which develops dynamically over time. The specificity of the interactions
required for cleavage and receptor activation, as described above, are difficult to predict, making the design of surfaces that have
good blood compatibility challenging to achieve.
Coagulation and Contact Phase Activation

Within the closed environment of a blood vessel in which a biomaterial has been placed, coagulation is initiated when factor XII
(fXII) is cleaved to form fXIIa, a process known as contact phase activation (Fig. 1) of the intrinsic coagulation cascade. This reaction
occurs when fXII, a serine protease in an inactive form, adopts a conformation that renders it susceptible to cleavage. The three prin-
cipal routes of activation are autoactivation (i.e., activation by other molecules of fXIIa), activation of fXII by kallikrein that has been
formed by the cleavage of prekallikrein by other fXIIa molecules, both of which are surface-mediated reactions the rate of which are
dependent on the procoagulant nature of that surface, and by activated platelets, which could be either a surface or cell-mediated
process. Other physiological activators of fXII include platelet-derived microparticles, acellular RNA molecules and collagen.
The precise mechanisms of fXII cleavage have been intensely debated over the past decades. Most seminal literature concluded
that negatively charged surfaces are most liable to activate fXII although recent studies have now shown this to not be the case. In
fact, these studies have shown that both highly hydrophobic and highly hydrophilic surfaces are potent activators.
Many textbooks indicate that fXII can be cleaved to form two separate activated forms, a small b-fXIIa cleavage product, and
a larger activated fragment denoted a-fXIIa, both able to cleave factor XI. Current research, however, has shown that there at least
6, and possibly an even greater number of cleavage points on fXII, depending on the activator, and resulting in the production of
multiple protein fragments of fXII due to autoactivation, some which initiate plasma coagulation, and some that do not.
The major confusion in defining how susceptible a biomaterial is to initiating plasma clotting that has persisted for more than
two decades has been due to the use of a popular method of quantifying contact activation. Some fragments of fXII that do not
coagulate plasma have been found to activate a widely used chromogenic substrate assay, utilizing S-2302.
Finally, activated fXII fragments also appear to undergo a form of autoinhibition and suppression of autoactivation, although
the mechanisms of this remain unclear.
The main feature of the coagulation pathway is the cascading effect of factors, whereby small concentrations of initiating stim-
ulus generate large concentrations of thrombin through a series of reactions of ever increasing magnitude. Each factor circulates
within blood in an inactive state but is then cleaved by its activator (the previous factor in the pathway), conveying proteolytic
activity on it. Each active factor is a serine protease, whose active site is composed of the same amino acid sequence: Gly-Asp-
Ser-Gly-Gly-Pro which cleaves an Arg-X bond within the inactive factor. Despite the similarity of the active sites, the factors are
highly specific, the three-dimensional architecture and chemistry of the molecules providing steric and intramolecular forces which
are important in coagulation factor recognition.
The cascade is controlled by the anticoagulant and fibrinolytic pathways, independent mechanisms which prevent excess gener-
ation of fibrin. There are also a variety of protein inhibitors, the most important being Antithrombin III, C1-esterase inhibitor, a2-
macroglobulin and a1-antiproteinase inhibitor. These react with active factors and inhibit them. The activity of antithrombin III is
enhanced by its interaction with heparin.
Fig. 1 Intrinsic coagulation pathway.
Not shown in Fig. 1 is the extrinsic pathway, which has physiological relevance in the prevention of hemorrhage. Both pathways
activate factor X and have a common terminal pathway, but with different initiating steps.
Platelets
Platelets are small, anucleate cells that undergo a variety of interactions which, physiologically, are central to hemostasis and an
element of the inflammatory response, broadly described as: adhesion, aggregation, activation and microparticle shedding. The
ability of a biomaterial to influence the magnitude of any of these interactions is critically dependant on the rate of collision of
the platelets with the surface, as discussed in “Rheological Factors” section. However, the following interactions have always served
as a central part of any blood compatibility assessment of a biomaterial.
Platelets become adhered to biomaterials predominantly via the reaction of adsorbed fibrinogen with the aIIbb3 integrin
receptor, also known as GPIIb/IIIa. Platelets also possess surface receptor affinity for many other adhesive proteins, including
collagen, fibronectin, vitronectin and von Willebrand’s factor (vWf). In its natural conformation, fibrinogen does not react
with this receptor, but adsorption to a surface can expose a binding site for resting aIIbb3 on platelets. Fibrinogen has a total
of 4 potential platelet-binding sites, two 12 amino acid sites (HHLGGAKQAGDV), the most commonly-observed binding
sites, and two RGD sequences. Due to their small physical size, the adhesion of platelets onto a biomaterial rarely presents
a problem in its own right, but subsequent reactions can initiate thrombosis, embolism, inflammation and immune
responses.
Platelet activation occurs when the level of stimulation of aIIbb3 is great enough and is characterized by the influx of extracellular
calcium into the cytoplasm and release of the contents of alpha, dense and lysosomal granules via the open canalicular system to the
extracellular medium. Alpha granules contain coagulation factors, chemotactic and growth factors which allow interaction with
inflammatory cells and proteins. Actin filaments are mobilized within the platelet cytoskeleton causing spreading and expression
of granule membrane proteins such as P-selectin (CD62P) and GP-53 (CD63) on the platelet surface, potential ligands for the adhe-
sion of leukocytes. Prior to the full activation of platelets, however, a procoagulant stage is expressed when the membrane phos-
pholipids convert to phosphatidylserine, which supports the complexation of coagulation factors on the surface, supporting the
propagation of the clotting cascade. Strong activation of the coagulation cascade, even in flowing blood, can result in occlusion
of the vessel.
Platelet aggregation occurs when two or more platelets are joined together via fibrinogen or vWf. This type of reaction is observed
in conditions of high shear stress when a platelet receptor GPIb-binding domain is exposed on vWf. The vWf-GPIb interaction
causes a stimulation of the aIIbb3 receptor via internal signaling which allows it’s interaction with soluble and nonconformationally
disturbed fibrinogen, and vWf. Large aggregates are potentially lethal by forming emboli within the narrow capillary structures of
major organs and the brain.
Platelets also produce microparticles, small vesicles of procoagulant lipid membrane which also express some platelet receptors,
including aIIbb3. These have a volume many orders of magnitude smaller than a platelet, but have the potential for propagating
reactions in the coagulation cascade, inflammatory and immune responses, in much the same way that do platelets. The release
of microparticles has generally been associated with the activation of aIIbb3 by fibrinogen, or stimulation of the platelet by strong
agonists. However, shear forces alone can also be responsible for the shedding of microparticles which express procoagulant activity.
The significance of microparticles is that their behavior in flowing blood is different from that of platelets. Evidence suggests that the
cyclo-oxygenase pathway is not utilized in the shedding of microparticles, but rather the phosphorylation of protein kinase C. Treat-
ments such as aspirin therapy do not, therefore, affect the production of microparticles, nor is it a calcium-dependant mechanism.
Leukocytes
The adhesion of white blood cells (leukocytes) onto an implanted biomaterial is normally considered to be controlled by the gener-
ation of activated fragments of complement protein at the biomaterial surface. Many in the field of blood compatibility do not
consider the interaction of white blood cells with biomaterials of any great importance. This view has largely come about due to
the observations of profound platelet activity after the contact of a biomaterial with blood in vitro whilst at the same time failing
to observe any dramatic leukocyte activity. However, when an implant is deployed in vivo it is not possible for there to be solely
blood contact without its interaction with blood-derived leukocytes in the surrounding tissue. Activation of resident macrophages
and blood leukocytes recruited to the implantation site will ultimately lead to inflammation and the generation of a thrombosis via
activation of the endothelium. Activation of leukocytes and complement in blood will often result in the same cycle of events occur-
ring. Release of species such as cathepsin G from granulocytes directly activate platelets, whilst elastase secretion results in fibrino-
lytic activity. A thrombosis that is observed in vivo is generally attributed to the action of the coagulation cascade and platelets,
whereas there is mounting evidence that the initial impetus for the reaction is often initiated outside of the circulation, a result
of inflammation in the tissue surrounding the blood vessel.
Complement Activation
The system of complement proteins represents the humoral arm of the innate immune system. In a process similar to coagulation,
a number of factors interact without cellular involvement in such a manner as to generate a multimeric complex called MAC
(membrane attack complex), comprising C5b, C6, C7, C8 and C9. This structure resembles a tube and is naturally energetically
able to reside within the lipid structure of a cell membrane. This neatly causes egress of the cell cytoplasm contents and results
in the death of the cell. Thus pathological activation of the complement cascade is not a desirable outcome of placing a biomaterial
into blood.
Activation of complement proteins on biomaterials results in the adhesion and activation of leukocytes, and the possible
involvement of an immune response. The complement system is split into alternate and classical pathways, but the involvement
of the alternate pathway is more important when considering the reaction of blood with biomaterials.
The alternate pathway relies on the continuous hydrolysis of C3. The fragment C3b thus formed will adsorb to a nearby surface.
The conformational rearrangements which take place if the surface is complement protein activating allow the attachment of factor
B and the initiation of the pathway. The conformation that C3b adopts when adsorbed on a nonactivating surface allow the attach-
ment of factor H, leading to pathway inactivation.
The presence of C3b on the surface of a biomaterial is thought to be the main focal point for the adhesion of neutrophils, via the
Mac-1 receptor.
A number of strategies have been proposed to limit complement activation on blood-contacting biomaterials. The development
of heparin coatings has been utilized extensively, which aim to capture factor H and so inactivate any pathway activation. However,
heparin has been shown to have some undesirable consequences following its interaction with a number of plasma proteins, and
ultimately, activated complement fragments are nevertheless produced by such surfaces. Creating nanostructures that sterically
inhibit the formation of activated complement complexes on the surface have shown some success. In one example, reducing
the diameter of surface pores from 200 to 20 nm successfully reduced complement activation, despite the surface area of the bioma-
terial being greater in the small-pore version. Each surface area both inside and around the pores was simply too small for the molec-
ular assembly of the C3 convertase.
In Vivo Observations
Biomaterials and vascular devices placed in the circulation that fail overwhelmingly suffer from thrombosis or embolism,
both events resulting from the activation of the coagulation cascade and involving the activation of platelets. There are
a number of obvious differences between in vitro and in vivo testing of a biomaterial, for example, fresh blood continuously
arrives at the site of blood-material contact during in vivo contact, resulting in the dilution of activated factors or cells in
their unactivated form. In addition, the blood-material interaction is combined with a response from endothelial cells
and other extrinsic factors.
The usual definition of biocompatibility refers to the ability of a biomaterial to successfully fulfill its function in a particular
application at the anatomical site relevant to that device. For large-diameter vascular grafts, Dacron has successfully been utilized
as a vascular graft for many years, despite experiments in vitro demonstrating that Dacron has rather unremarkable biocompati-
bility. However, the same material cannot be used for the replacement of a coronary artery due to rapid occlusion observed in exper-
imental animals.
Thus, important factors that must be considered when designing a material for use in a blood-contacting application in vivo are
protein adsorption leading to coagulation cascade activation, platelet adhesion, activation and aggregation and activation of the
endothelium, but taking into account the dynamics of blood flow and the rate of reactant turnover. Studies have also shown
that thrombosis can be initiated without contact activation of the coagulation cascade. In addition to these, the importance of
a number of additional factors in thrombosis has recently emerged.
Polyphosphates having both short and long-chain lengths (100–1000 phosphates) are secreted from both platelet dense gran-
ules and microorganisms, respectively. Microbial polyphosphates, having a large charge density are potent autoactivators of fXII,
leading to rapid contact-activation. Cell-free nucleic acids similarly activate fXII and are secreted from microorganisms and
apoptotic or necrotic host cells. Activated neutrophils release fibers comprising a structure knows as Neutrophil extracellular traps,
intended to entrap microbes but which are highly prothrombotic due to the presence of DNA-histones that simultaneously activate
fXII and platelets, and tissue factor that is a potent extrinsic activator of the coagulation cascade.
Any placement of a vascular biomaterial within the circulation necessarily results in a degree of damage to the endothelium.
Such damaged endothelial cells release tissue factor leading to the activation of the extrinsic coagulation cascade. Inflammation
within the tissue surrounding the site of implantation results in the expression of intercellular adhesion molecule 1 (ICAM-1)
and vascular cell adhesion molecule 1 (VCAM-1) on the endothelial cells following the expression of TNFa and IL-6 within the
soft tissue outside the circulation. Upregulation of these adhesion molecules results in the extraction of leukocytes through a process
known as rolling and diapedesis, which perpetuates the inflammatory reaction within the external tissue. Activation of the endo-
thelium also causes reduced secretion of prostacyclin, leading to reduced resistance to thrombosis.
Rheological Factors
The type of reaction observed when a biomaterial is placed in blood is dependent on the level and type of blood flow that is
experienced. Blood is a non-Newtonian fluid and under steady flow conditions will result in a greater concentration of red blood
cells within the central portion of the flow profile. This has the effect of pushing platelets to the outer wall of the vessel, making
contact with a biomaterial more likely. In steady state flow within a tube (the blood’s natural environment), platelet adhesion
onto a surface will be reaction rate limited, that is the number of platelets adhering will be proportional to the affinity of the cells
to the surface.
At physiological wall shear rates the effect of the flow will not in itself be a source of cell activation. The distribution of velocities
within the vessel has the effect of unfolding vWf, which allows platelets to aggregate via the interaction of vWf with platelet receptor
GPIb/IX. At very high wall shear rates the shedding of platelet-derived microparticles is observed. When the flow is disturbed (turbu-
lent), however, the degree of platelet activation, aggregation and microparticle shedding is dramatically increased.
The presence of high blood flow rates has the effect of diluting activated cells and proteins, preventing thrombosis. However,
these conditions increase the probability of embolism occurring, where small fragments of clots that begin to form, especially in
combination with highly prothrombotic platelet-derived microparticles, break away and become coagulated at distant sites in
low flow environments. This is particularly dangerous within the capillary beds of organs such as the lungs or brain.
Surface Topography
Generally, surface features on biomaterials give rise to increased material surface area, resulting in protein and cellular reactions that
are increased relative to a smooth surface of the same biomaterial. Roughness on the surface of a static device within flowing blood
is likely to capture cells that would otherwise flow downstream away from the site of activation. Thus, in most cases, surface features
are not helpful in improving the biocompatibility of blood-contacting devices.
However, there are some notable exceptions. First of all, surfaces that are mobile, in which, for example, long side chains of poly-
ethylene glycol, constantly move in relation to one another and so provide an unstable foundation for the complexation of pro-
coagulant peptides, thus preventing coagulation and possibly complement activation occurring.
Nanotopographical structures could be used to more precisely control the adsorption of specific proteins, in defined orientations
and conformations. However, such investigations are at an early stage of research, but may in future provide better control of long-
term performance of blood-contacting biomaterials.
Further Reading
Baier, R. E., & Kurusz, M. (2012). Understanding blood/material interactions: Contributions from the Columbia University biomaterials seminar. ASAIO Journal, 58, 450–454.
Bauer, J., Xu, L.-C., Vogler, E. A., & Siedlecki, C. A. (2017). Surface dependent contact activation of factor XII and blood plasma coagulation induced by mixed thiol surfaces.
Biointerphases, 12, 02D410-1-10.
Ekdahl, K. N., Lambris, J. D., Elwing, H., Ricklin, D., Nilsson, P. H., et al. (2011). Innate immunity activation on biomaterial surfaces: A mechanistic model and coping strategies.
Advanced Drug Delivery Reviews, 63, 1042–1050.
Golas, A., Yeh, C.-H. J., Siedlecki, C. A., & Vogler, E. A. (2011). Amidolytic, procoagulant, and activation-suppressing proteins produced by contact activation of blood factor XII in
buffer solution. Biomaterials, 32, 9747–9757.
Kalathottukaren, M. T., & Kizhakkedathu, J. N. (2018). Mechanisms of blood coagulation in response to biomaterials: Extrinsic factors. In C. A. Siedlecki (Ed.), Hemocompatibility of
biomaterials for clinical applications (pp. 29–49). Elsevier.
Xu, L.-C., Bauer, J., & Siedlecki, C. A. (2014). Proteins, platelets, and blood coagulation at biomaterial interfaces. Colloids and Surfaces. B, Biointerfaces, 124, 49–68.
Interaction Between Mesenchymal Stem Cells and Immune Cells in Tissue
Engineering
Rong Huang, Yinghong Zhou, and Yin Xiao, Queensland University of Technology, Brisbane, QLD, Australia
T Cell 251
B Cell 252
Macrophage 252
Natural Killer (NK) Cell, DC Cell and Neutrophil 253
References 254
Glossary
Antigen-presenting cells Cells which displays antigen complexed with major histocompatibility complexes on their surfaces
and play an important role in adaptive immune response.
Cognate immunity Also known as the acquired immunity or adaptive immunity, which creates immunological memory after
an initial response to a specific pathogen, and leads to an enhanced response to subsequent encounters with that pathogen.
Histocompatibility Compatibility between the tissues of different individuals, so that one accepts a graft from the other
without giving an immune reaction.
Major histocompatibility complex A set of cell surface proteins essential for the acquired immune system to recognize foreign
molecules in vertebrates, which in turn determines histocompatibility.
Non-immunogenic Not capable of inducing an immune response; not antigenic.
Paracrine action Relating to or denoting a hormone which has effect only in the vicinity of the gland secreting it.
Abbreviations
ALP Alkaline phosphatase
APCs Antigen-presenting cells
C3 Complement component-3
cAMP Cyclic adenosine monophosphate
CCL C–C motif chemokine ligand
CD Cluster of differentiation
CD45RO Cluster of differentiation 45 isoform
DAMPs Damage-associated molecular patterns
ELISA Enzyme-linked immunosorbent assay
EP Prostaglandin E receptor
FGF Fibroblast growth factors
GM-CSF Granulocyte/macrophage colony-stimulating factor
GvHD Graft versus host disease
HLA-DR Human leukocyte antigens
HLA-G5 Human leucocyte antigen-G5
IDO Indoleamine 2,3-dioxygenase
IFN-g Interferon gamma
Ig Immunoglobulin
IL Interleukin
LPS Lipopolysaccharide
M1 Classically activated macrophages
M2 Alternatively activated macrophages
M-CSF Macrophage colony-stimulating factor
MHC Major histocompatibility complex
MMP Matrix metallopeptidase

250 Biomaterials: In vitro and in vivo studies of biomaterials j Interaction Between MSCs and Immune Cells
MSCs Mesenchymal stem cells

MyD88 Myeloid differentiation factor 88
NK cells Natural killer cells
PAMPs Pathogen-associated molecular pattern molecules
PBMCs Peripheral blood mononuclear cells
PD-1 Programmed death 1
PGE2 Prostaglandin E2
RT-PCR Real-time reverse transcription-polymerase chain reaction
TC cells/CTLs Cytotoxic T cells
TCR T cell receptor
Teff cells Effector T cells
TGF-b Transforming growth factor-beta
Th cells T helper cells
TLR Toll-like receptor
TNF-a Tumor necrosis factor-alpha
Tol DC Tolerogenic DC
TSG Tumor necrosis factor-inducible gene
The definition of tissue engineering evolved from biomaterials covers interdisciplinary fields involving basic principles of life science
and engineering to replace or improve tissue structure and function (Langer and Vacanti, 1993). Currently, the treatment for cardio-
vascular failure (Surder et al., 2013; Yerebakan et al., 2011), liver diseases (Kharaziha et al., 2009), degeneration of the spinal cord
(Frolov and Bryukhovetskiy, 2012; Cashman et al., 2008), and the use of substitutes to repair damaged tissues, such as cartilage
(Wakitani et al., 2011) and bone (Nagata et al., 2012) have offered insights to the tissue engineering and regenerative medicine.
Although tissue function can be restored to a certain level using conventional modalities, some drawbacks such as limited donor
tissue, implant rejection, and procedure-related complications remain major challenges for the first-line clinicians (Herford and
Dean, 2011; Paul et al., 2009). One of the highlights of tissue engineering is to repair and regenerate without donor site morbidity
which is different from the traditional surgery that needs to sacrifice tissues from the donor site (Dimitriou et al., 2011). Extensive
studies have confirmed the feasibility of generating bone to repair large defects (Viateau et al., 2007; Liu et al., 2008; Cao et al.,
2012), and the outcomes of other ongoing clinical trials are quite promising (Ardjomandi et al., 2015; Yamada et al., 2008).
To date, the use of autografts serves as a gold standard for tissue repair and regeneration. Combined with the cell-based therapy
with autologous cells that can be manipulated to repair or replace damaged tissues, the field of tissue engineering is being explored
with less ethical issues (Dimitriou et al., 2011). Recent advances in regenerative medicine, in particular the stem cell biology have
enabled the development of stem cell-based therapies which are histocompatible and non-immunogenic. For example, bone regen-
eration has shown that more reliable and effective results have been found in the management of bone defects using stem cell
delivery compared to the traditional methods (Lo et al., 2012; Dupont et al., 2010). Mesenchymal stem cells (MSCs) is one of
the useful cell types which can be easily isolated from patients and transplanted back with less ethical issues. The generation of suffi-
cient MSCs for transplantation can be simply achieved by in vitro expansion, suggesting the possibility of their broad application in
cell-based therapy (Kuroda, 2016; Chanda et al., 2010). The anti-inflammatory and immunosuppressive properties (MacFarlane
et al., 2013; Kuo et al., 2012), migratory ability to defect sites (Mokbel et al., 2011; Sordi, 2009) and capability to modulate the
microenvironment by paracrine actions (Meyerrose et al., 2010) make them a promising candidate for tissue engineering.
MSCs are fibroblast-like plastic cells with self-renewal and pluripotency. After their initial discovery in bone marrow, MSCs have
been identified from a wide range of tissues including dental pulp, umbilical cord, adipose tissue, skin, periosteum, muscle, skel-
eton, liver, spleen, brain, tendon, and synovial. They are positive for expression of stem cell surface markers such as cluster of differ-
entiation 73 (CD73), cluster of differentiation 90 (CD90) and cluster of differentiation 105 (CD105) and negative for expression of
the hematopoietic cell surface markers such as cluster of differentiation 11 (CD11), cluster of differentiation 34 (CD34), cluster of
differentiation 45 (CD45), cluster of differentiation 79a (CD79a) and major histocompatibility complex (MHC) class II cell surface
receptor encoded by human leukocyte antigens (HLA-DR). They own the ability to differentiate into osteoblasts, chondrocytes,
adipocytes and many other cell types (Kolf et al., 2007; Pittenger et al., 1999). MSCs have been well characterized with their effects
on immune disorders, including graft versus host disease (GvHD), type I diabetes, rheumatoid arthritis, and inflammatory bowel
diseases (Hinsenkamp et al., 2012; Polchert et al., 2008; Urban et al., 2008; Hayashi et al., 2008). It is reported that MSCs secret pro-
inflammatory cytokines such as interleukin (IL)-1, interleukin (IL)-6, interleukin (IL)-8 and tumor necrosis factor-alpha (TNF-a),
which are involved in inflammation-related diseases (Kim et al., 2005). Meanwhile, MSCs are able to inhibit T cell and NK cell
activity and play an immunosuppressive role in dendritic cell differentiation and maturation (Rasmusson et al., 2003; Zhang
et al., 2004). Most importantly, MSCs are non-targeted by MHC-mismatched immune cells, establishing an environment free
from cellular attack.
Biomaterials: In vitro and in vivo studies of biomaterials j Interaction Between MSCs and Immune Cells 251
The mechanisms of MSC immunoregulation are based on two paradigms. First of all, MSCs can sense the microenvironment
change and act accordingly. Secondly, they become polarized toward different phenotypes depending on the toll-like receptor
(TLR) signals received (Auletta et al., 2012; Waterman et al., 2010). MSCs can regulate the survival and activation of immune cells
on one hand; on the other hand, they can inhibit inflammation and promote tissue repair (Fig. 1). When tissue injury occurs, MSCs
migrate to the injury site where they are directly activated through TLR stimulation including pathogen-associated molecular pattern
molecules (PAMPs) or damage-associated molecular patterns (DAMPs) (Prockop and Youn Oh, 2012). MSCs are indirectly acti-
vated by several pro-inflammatory cytokines such as TNF-a and interferon gamma (IFN-g) which bond to MSC receptors. The acti-
vation of complement component-3 (C3) is triggered to target MSCs for complement lysis in normal circumstances. However,
MSCs express a complement regulatory protein cluster of differentiation 59 (CD59) and release complement factor H to protect
them from complement lysis. In response to the pathogen, MSCs either help clear pathogen or modulate immune responses
depending on the stimuli received (Fig. 1). MSCs may promote pathogen clearance through secretion of IL-6 and IL-8, evoke
the polarization of the pro-inflammatory phenotype of macrophages, increase bacterial clearance through the enhanced survival
and function of neutrophils and induce the apoptosis of effector T (Teff) cells. In addition, MSCs are reported to regulate immune
responses through the secretion of immunosuppressive soluble factors and evoke the polarization of the anti-inflammatory pheno-
type of macrophages (M2), tolerogenic DC (Tol DC) and Tregs (English, 2013).
The following article will introduce the interaction between MSCs and immune cells to further understand the immunoregula-
tory properties of MSCs in tissue engineering.
T Cell
Lymphocytes originate in the bone marrow and migrate to other parts of the lymphatic system such as the thymus, lymph nodes
and spleen. In our immune system, T cells are the primary lymphocyte effectors and their functional properties are central to antigen
specificity and memory associated with cognate immunity. T cells consist of T helper cells (Th cells), cytotoxic T cells (TC cells or
CTLs), memory T cells, Tregs and a few other categories. T helper cells are also called cluster of differentiation 4 þ (CD4 þ) T cells
because they express the CD4 glycoprotein on the cell surfaces. Antigenic peptides bound to self MHC class II molecules which are
expressed on the surface of antigen-presenting cells (APCs) induce the activation of T helper cells. Once activated, T helper cells
divide rapidly and secrete cytokines that assist other immune cells in immunologic processes, including the differentiation of B cells
into plasma cells and memory B cells, as well as the activation of macrophages and cytotoxic T cells (Gutcher and Becher, 2007).
Cytotoxic T cells are known as CD8 þ T cells because they express the CD8 glycoprotein on the cell surfaces. They are responsible for
destroying virus-infected cells and tumor-related cells, and are also implicated in transplant rejection. Cytotoxic T cells bind to
antigen associated with MHC class I molecules and secret cytokines for cell lysis. Memory T cells are a subset of antigen-specific
T cells that persist long-term after an infection has resolved. They can be either CD4 þ or CD8 þ T cells and typically express cluster
of differentiation 45 isoform (CD45RO) on the cell surfaces (Willinger et al., 2005; Akbar et al., 1988). Tregs are formerly known as
suppressor T cells which are important for the maintenance of immunological tolerance. Tregs come in many forms and CD4 þ,
CD25 þ, and FoxP3 þ are three major classes of Tregs. They play an important role in T cell-mediated immunity and suppression
of auto-reactive T cells (Abbas et al., 2013; Singh et al., 2013).
MSCs are reported to suppress T cell proliferation induced by alloantigens, nonspecific mitogens and anti-CD3 and anti-CD28
antibodies in vitro (Di Nicola et al., 2002). MSCs have a similar effect on naive T cells, memory T cells, as well as CD4 þ and CD8 þ
T cells, which do not require major MHC restriction. MSCs can selectively suppress T lymphocytes by a cell-to-cell contact mech-
anism via the inhibition of programmed death 1 (PD-1) protein and its ligands PD-L1 (also known as B7 homolog 1 (B7-H1))
and PD-L2 (Freeman et al., 2000). PD-1 is a cell surface membrane protein of the immunoglobulin superfamily and negatively
regulates T cell receptor (TCR) signals. PD-1 also inhibits T cell proliferation and IFN-g secretion via the inhibition of IL-2 secre-
tion (Carter et al., 2002). Additionally, several reports suggest that cell-to-cell contact is not a compulsory requirement for MSCs to
suppress the immune response of T cells (Groh et al., 2005). Cell inhibition is also due to cytokines secreted by MSCs such as IFN-
g, IL-1b and growth factors such as transforming growth factor (TGF)-b1 and hepatocyte growth factor (HGF). The immunomod-
ulatory activity of MSCs is through indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). These growth factors and
cytokines bind to their receptors on cell surfaces and trigger enzyme cascade of T cells (Glennie et al., 2005). The secretion of
human leucocyte antigen-G5 (HLA-G5) by MSCs is also regarded essential for their effects of T cell suppression, shift of the
Fig. 1 Illustration of MSC immunoregulation. When tissue infection or injury occurs, MSCs migrate to the injury site where they are directly acti-
vated through TLR stimulation including pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular patterns
(DAMPs). MSCs also express other receptors such as Fas, TNFR and IFNGR depending on the signals received. MSCs are able to regulate the path-
ogen clearance, inhibit inflammation and promote tissue repair.
activated T cell response to a T helper type 2 (Th2) cytokine profile and induction of CD4 þ, CD25 þ, and FoxP3 þ regulatory
T cells (Selmani et al., 2008).
MSCs also modulate immune responses through the induction of regulatory T cells. Regulatory T cells actively suppress activa-
tion of the immune system and prevent pathological self-reactivity in autoimmune diseases. MSCs are thought to induce the forma-
tion of CD8 þ regulatory T cells that are responsible for the inhibition of allogeneic lymphocyte proliferation. An increasing number
of CD4 þ CD25 þ T cells, displaying a regulatory phenotype such as FoxP3 þ cells, have been demonstrated in mitogen stimulated
peripheral blood mononuclear cell (PBMC) cultures with MSCs. On the contrary, depletion of CD4 þ CD25 þ regulatory T cells
have no effect on the inhibition of T cell proliferation by MSCs (Aggarwal and Pittenger, 2005). The molecular mechanism by which
regulatory T cells exert their regulatory activity has not been identified. The in vitro experiments have given mixed results regarding
the requirement of cell-to-cell contact for effector cells to be suppressed. The immunosuppressive cytokines TGF-b and IL-10 have
also been implicated in regulatory T cell function (Agarwal et al., 2014). Recent studies have shown that the MSC-induced formation
of regulatory T cells is driven by MSC-derived TGF-b1 and by CeC motif chemokine ligand 18 (CCL18) which is produced by
monocytes upon the interaction with MSCs (Melief et al., 2013). Further understanding of the interplay between MSCs and
T cells is helpful in the development of promising approaches to improve cell-based regenerative medicine.
B Cell
In mammals, immature B cells are derived from the bone marrow and distinguished from other lymphocytes by the presence of a B
cell receptor (BCR) on the cell surfaces. This specialized receptor protein allows B cells to bind to the specific antigens. B cells have
been considered mainly as positive regulators of immune responses and central contributors to the pathogenesis of immune-related
diseases because they are able to produce antibodies such as immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglob-
ulin M (IgM). B cells are characterized by the capacity of antigen presentation, the production of multiple cytokines, and a suppres-
sive capacity by secretion of IL-10 (Mauri and Bosma, 2012).
MSCs may also regulate the immune response through interaction with B cells. Co-culture of MSCs and B cells with stimuli can
inhibit the proliferation of B cells and immunoglobulin expression. In addition, MSCs can suppress immunoglobulin production as
a result of MSC-derived CeC motif chemokine ligand 2 (CCL2) and CeC motif chemokine ligand 7 (CCL7) which have influence
on the chemotactic property of B cells. Inhibition of CCL2 in MSC impaired their suppressive capacity for B cells (Lee et al., 2017).
Inhibition of matrix metallopeptidase (MMP) enzymatic activity may abolish the suppressive effect of MSCs because MMP-
processed CCL2 can suppress the signal transducer and activator of transcription3 (STAT3) activation in plasma cells (Rafei
et al., 2008). However, depending on the level of stimulation, the secretion of IgG by induced B cells can be stimulated or inhibited
after the addition of MSCs, which leads to different results (Corcione et al., 2006). Interestingly, MSCs possess the capacity to inhibit
the proliferation of activated B cells in the presence of pro-inflammatory cytokines such as IFN-g. MSCs negatively regulate antibody
production depending on the activation state of the B cells and the dose of MSCs themselves. Although the expression of migration
related chemokines for B cell is inhibited by MSCs, MSCs have no influence on the expression of co-stimulatory molecules and cyto-
kines (Krampera et al., 2006; Bernardo et al., 2009). Some in vivo and in vitro investigations have also illustrated that MSCs can
modulate the expression of B cells and protect the bone healing process in the defect sites (Corcione et al., 2006).
Macrophage
Macrophages are a type of circulating monocytes derived from hematopoietic progenitors or myeloblasts that engulf microbes and
antigens in a process called phagocytosis. Macrophages perform an important role in tissue homeostasis and regeneration by
acting as scavengers for potential pathogens. They participate in non-specific innate immunity and also initiate specific adaptive
immunity by recruiting other immune cells (Ovchinnikov, 2008). In response to infection or injury, macrophages migrate to the
local sites and contribute to different inflammation processes (Varin and Gordon, 2009; Lichtnekert et al., 2013). The differenti-
ation, activation, and polarization of macrophages depend on specific cytokines such as granulocyte/macrophage colony-
stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and pro-inflammatory cytokines. Based on distinct
functional properties, surface markers and inducers, macrophage phenotypes have been characterized broadly into M1 (classically
activated) and M2 (alternatively activated), mirroring the Th1 and Th2 nomenclature described for T helper cells (Mills et al.,
2000).
Cytokines such as IFN-g and bacterial endotoxins such as lipopolysaccharide (LPS) can activate macrophages. Activated macro-
phages have increased metabolism, enhanced levels of lysosomal proteins and a greater potential to phagocytosis. They also
undergo many changes which allow them to engulf invading bacteria and infected cellular debris. Proteases, neutrophil chemotactic
factors, superoxide, cytokines such as TNF-a, IL-1 and IL-8, eicosanoids and some growth factors are released which result in tissue
destruction. Another important role of macrophages in the immune system is antigen presentation. Macrophages are APCs that
present antigens to effector T lymphocytes. Macrophages degrade protein exogenous antigens into peptides and attach them to
MHC-II molecules. The MHC-II peptide epitopes are then placed on the surfaces of the macrophages where they can be recognized
by complementary shaped TCRs and CD4 molecules on the surfaces of T cells to initiate the antigen presenting process (Hume,
2008).
Increasing evidence demonstrates that MSC-mediated regulation of macrophages is critical for inflammatory response and tissue
repair. MSCs interfere with the acquisition of M1 phenotype, and promote M2 polarization. By increasing IL-10 and decreasing IL-2
and TNF-a production, human MSCs can promote the generation of alternatively activated macrophages, characterized by a high
expression of cluster of differentiation 206 (CD206) and a typical cytokine profile of M2 macrophages (Kim and Hematti, 2009).
When inflammatory stimuli occur, bacterial toxins such as LPS act on the toll-like receptor 4 (TLR4) on the cell surface of MSCs and
macrophages. MSCs then receive a second signal from macrophage-derived TNF-a. These events initiate a signaling cascade to acti-
vate myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B (NF-kB), upregulate cycloxygenase 2 (Cox2) expression
and further increase the synthesis of PGE2. Thereafter, PGE2 secreted by MSCs binds to its receptors EP2 (prostaglandin E receptor
2) and EP4 (prostaglandin E receptor 4) on the surfaces of macrophages. Activation of EP2 and EP4 increases intracellular cAMP
levels leading to direct suppression of multiple pro-inflammatory cytokines and promotion of anti-inflammatory cytokine IL-10
(Tyndall and Pistoia, 2009).
Bone macrophages are a discrete population of resident macrophages involved in bone homeostasis. They are found to be inter-
calated throughout murine and human osteal tissues and distributed among other bone lining cells within both endosteum and
periosteum. Researchers have demonstrated that bone macrophages are required for efficient osteoblast mineralization in response
to the physiological remodeling stimulus. Removing them from calvarial cultures can down-regulate osteocalcin expression and
inhibit osteoblast mineralization in vitro. Depletion of bone macrophages in vivo can cause complete loss of osteoblast bone-
forming surface at this modeling site. The previous study has also shown that it is bone macrophages and not osteoblasts that
respond to pathophysiological concentrations of LPS (Chang et al., 2008). Our study has shown that MSCs regulate the activation
of macrophage during osteogenesis (Zhou et al., 2017). These observations implicate that macrophages are closely associated with
MSCs in bone tissues. In terms of acquiring anti-inflammatory properties, expanding lifespan, and increasing motility with proper
engraftment of MSCs, the interaction could be explored in therapeutic applications.
Natural Killer (NK) Cell, DC Cell and Neutrophil
NK cells are first identified as large granular lymphocytes with natural cytotoxicity against tumor cells and are later recognized as
a separate lymphocyte lineage with cytokine-producing effector functions. Activating NK cell receptors detect the presence of ligands
such as the stress-induced self-ligands, infectious nonself-ligands and TLR ligands. The in vitro exposure to TLR ligands promotes
IFN-g production and enhances cytotoxicity of NK cells. However, this process is more efficient when accessory cells are present in
the cultural environment of NK cells, suggesting that the TLR on cell surfaces of NK cells may play an indirect role in vivo (Vivier
et al., 2008). MSCs have been shown to differentially affect the cytotoxicity of NK cells. MSCs inhibit IL-2-induced NK cell prolif-
eration by secretion of the soluble factors such as TGF-b, HLA-G and PGE2 as well as direct cell-to-cell contact. MSCs also prevent
effector functions of NK cells, such as cytotoxicity against virus-infected cells, by the inhibition of IFN-g secretion. This inhibitory
effect is primarily mediated by IDO and PGE2 to reduce the expression of the activating NK cell receptors. Although MSCs do not
directly inhibit the functions of NK cells, reduced cytolytic potential has been demonstrated, which is more pronounced against
HLA class I-positive than HLA class I-negative cells (Sotiropoulou et al., 2006; Shi et al., 2011).
DCs are APCs of the mammalian immune system. DCs play a role in the initiation, maintenance and regulation of immune
responses by promoting antigen specific T cell activation and inducing cells of the innate immune system. DCs act through antigen
presentation and activation of T cells. It is also suggested that a different class of DCs exists with the function of initiating and main-
taining immune tolerance. The third category of DCs, known as follicular DCs, appears to maintain immune memory in tandem
with B cells (Wu et al., 2012). In contrast to their effects on effector immune cells, MSCs are able to down-regulate the expression of
the co-stimulatory molecules such as cluster of differentiation 40 (CD40), cluster of differentiation 80 (CD80) and cluster of differ-
entiation 86 (CD86) on the surfaces of DCs and negatively affect DC differentiation in vitro. In fact, MSCs prevent DC maturation
through PGE2 secretion. Recent studies show that MSCs inhibit DC maturation and function through the secretion of tumor
necrosis factor-inducible gene-6 (TSG-6) (Liu et al., 2014). DCs cultured in the presence of MSCs release lower amounts of IL-
12 and TNF-a, but higher levels of IL-1b and IL-10, and further express low levels of MHC-II surface antigens (Banchereau and Stein-
man, 1998).
Neutrophils are derived from stem cells in the bone marrow which can be subdivided into segmented neutrophils and banded
neutrophils. They form part of the polymorphonuclear cell family which are short-lived and highly motile. In response to the
inflammatory stimuli, neutrophils migrate from the circulating blood to the infected tissues, where they efficiently engulf and inac-
tivate bacteria. Neutrophils also degranulate and secret antimicrobial factors into the extracellular medium (Witko-Sarsat et al.,
2000). MSCs significantly inhibit apoptosis of resting and IL-8 activated neutrophils. As shown by transwell experiments, the
anti-apoptotic property of MSCs does not require cell-to-cell contact. IL-6 expression is detected in MSCs by real-time reverse
transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Antibody neutralization
experiments have demonstrated that IL-6 is responsible for neutrophil protection from apoptosis and it is signaled by activating
STAT3 transcription factor. Researchers have also found that recombinant IL-6 can protect neutrophils from apoptosis in a dose-
dependent manner. However, MSCs perform no effect on neutrophil phagocytosis, expression of adhesion molecules and chemo-
taxis (Raffaghello et al., 2008; Fig. 2).
In order to bypass the ethical controversy surrounding embryo destruction, stem cell obtained from differentiated somatic cells is
an alternative way for tissue engineering. MSCs are ideal transplanted cells not only because of their own self-renewal capacity and
Fig. 2 Illustration of the effect of MSCs on immune cells. Immune cells including T cells, B cells, neutrophils, NK cells, dendritic cells, monocyte
and macrophages are presented in the frame on the left-hand side, and the main cytokines secreted by MSCs to regulate immune responses are pre-
sented on the right-hand side. MSCs inhibit T cell and B cell proliferation, induce neutrophil apoptosis, inhibit dendritic cellular maturation and limit
division of NK cells. At the same time, MSCs induce differentiation of Treg cells. The green arrows indicate the cytokines produced by MSCs. The
black line indicates the positive effect and the blunt-end line indicates the negative effect of MSCs.
pluripotency, but they are also a powerful tool in the regulation of immune cells for assisting transplantation (Fig. 2). For example,
the transplantation of allogeneic skin grafts has been associated with a potent inflammatory immune response leading to the rapid
elimination of donor cells and rejection of the graft (Benichou et al., 2011). When MSCs are applied in the surgery, MSCs can
suppress lymphocyte proliferation and prolong skin graft survival (Bartholomew et al., 2002). Researchers have also found out
that autologous bone grafts with MSCs and fibroblast growth factors-2 (FGF-2) can accelerate bone union in large bone defects
(Murakami et al., 2016). To sum up, research on stem cell biology has demonstrated exciting opportunities and great potentials
for cell-based tissue regeneration. Combined with new delivery strategies, desired properties for tissue grafts can be created along
with less ethical concerns. As the field of tissue engineering continuous to evolve, more ideal cell sources will be developed for
promising therapeutic applications.
References
Abbas, A. K., Benoist, C., Bluestone, J. A., Campbell, D. J., Ghosh, S., et al. (2013). Regulatory T cells: Recommendations to simplify the nomenclature. Nature Immunology, 14,
307–308.
Agarwal, A., Singh, M., Chatterjee, B. P., Chauhan, A., & Chakraborti, A. (2014). Interplay of T helper 17 cells with CD4(þ)CD25(high) FOXP3(þ) Tregs in regulation of allergic
asthma in pediatric patients. International Journal of Pediatrics, 2014, 636238.
Aggarwal, S., & Pittenger, M. F. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 105, 1815–1822.
Akbar, A. N., Terry, L., Timms, A., Beverley, P. C., & Janossy, G. (1988). Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. Journal of Immunology, 140,
2171–2178.
Ardjomandi, N., Duttenhoefer, F., Xavier, S., Oshima, T., Kuenz, A., & Sauerbier, S. (2015). In vivo comparison of hard tissue regeneration with ovine mesenchymal stem cells
processed with either the FICOLL method or the BMAC method. Journal of Cranio-Maxillo-Facial Surgery, 43, 1177–1183.
Auletta, J. J., Deans, R. J., & Bartholomew, A. M. (2012). Emerging roles for multipotent, bone marrow-derived stromal cells in host defense. Blood, 119, 1801–1809.
Banchereau, J., & Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature, 392, 245–252.
Bartholomew, A., Sturgeon, C., Siatskas, M., Ferrer, K., McIntosh, K., et al. (2002). Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival
in vivo. Experimental Hematology, 30, 42–48.
Benichou, G., Yamada, Y., Yun, S. H., Lin, C., Fray, M., & Tocco, G. (2011). Immune recognition and rejection of allogeneic skin grafts. Immunotherapy, 3, 757–770.
Bernardo, M. E., Locatelli, F., & Fibbe, W. E. (2009). Mesenchymal stromal cells. Annals of the New York Academy of Sciences, 1176, 101–117.
Cao, L., Liu, X., Liu, S., Jiang, Y., Zhang, X., et al. (2012). Experimental repair of segmental bone defects in rabbits by angiopoietin-1 gene transfected MSCs seeded on porous
beta-TCP scaffolds. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 100, 1229–1236.
Carter, L., Fouser, L. A., Jussif, J., Fitz, L., Deng, B., et al. (2002). PD-1:PD-L inhibitory pathway affects both CD4(þ) and CD8(þ) T cells and is overcome by IL-2. European
Journal of Immunology, 32, 634–643.
Cashman, N., Tan, L. Y., Krieger, C., Madler, B., Mackay, A., et al. (2008). Pilot study of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells in
amyotrophic lateral sclerosis (ALS). Muscle & Nerve, 37, 620–625.
Chanda, D., Kumar, S., & Ponnazhagan, S. (2010). Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in diseases of the skeleton. Journal of Cellular
Biochemistry, 111, 249–257.
Chang, M. K., Raggatt, L. J., Alexander, K. A., Kuliwaba, J. S., Fazzalari, N. L., et al. (2008). Osteal tissue macrophages are intercalated throughout human and mouse bone lining
tissues and regulate osteoblast function in vitro and in vivo. Journal of Immunology, 181, 1232–1244.
Corcione, A., Benvenuto, F., Ferretti, E., Giunti, D., Cappiello, V., et al. (2006). Human mesenchymal stem cells modulate B-cell functions. Blood, 107, 367–372.
Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P. D., et al. (2002). Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or
nonspecific mitogenic stimuli. Blood, 99, 3838–3843.
Dimitriou, R., Jones, E., McGonagle, D., & Giannoudis, P. V. (2011). Bone regeneration: Current concepts and future directions. BMC Medicine, 9, 66.
Dupont, K. M., Sharma, K., Stevens, H. Y., Boerckel, J. D., Garcia, A. J., & Guldberg, R. E. (2010). Human stem cell delivery for treatment of large segmental bone defects.
Proceedings of the National Academy of Sciences of the United States of America, 107, 3305–3310.
English, K. (2013). Mechanisms of mesenchymal stromal cell immunomodulation. Immunology and Cell Biology, 91, 19–26.
Freeman, G. J., Long, A. J., Iwai, Y., Bourque, K., Chernova, T., et al. (2000). Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative
regulation of lymphocyte activation. Journal of Experimental Medicine, 192, 1027–1034.
Frolov, A. A., & Bryukhovetskiy, A. S. (2012). Effects of hematopoietic autologous stem cell transplantation to the chronically injured human spinal cord evaluated by motor and
somatosensory evoked potentials methods. Cell Transplantation, 21(Suppl. 1), S49–55.
Glennie, S., Soeiro, I., Dyson, P. J., Lam, E. W., & Dazzi, F. (2005). Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood, 105,
2821–2827.
Groh, M. E., Maitra, B., Szekely, E., & Koc, O. N. (2005). Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells. Experimental
Hematology, 33, 928–934.
Gutcher, I., & Becher, B. (2007). APC-derived cytokines and T cell polarization in autoimmune inflammation. Journal of Clinical Investigation, 117, 1119–1127.
Hayashi, Y., Tsuji, S., Tsujii, M., Nishida, T., Ishii, S., et al. (2008). Topical implantation of mesenchymal stem cells has beneficial effects on healing of experimental colitis in rats.
Journal of Pharmacology and Experimental Therapeutics, 326, 523–531.
Herford, A. S., & Dean, J. S. (2011). Complications in bone grafting. Oral and Maxillofacial Surgery Clinics of North America, 23, 433–442.
Hinsenkamp, M., Muylle, L., Eastlund, T., Fehily, D., Noel, L., & Strong, D. M. (2012). Adverse reactions and events related to musculoskeletal allografts: Reviewed by the World
Health Organisation Project NOTIFY. International Orthopaedics, 36, 633–641.
Hume, D. A. (2008). Macrophages as APC and the dendritic cell myth. Journal of Immunology, 181, 5829–5835.
Kharaziha, P., Hellstrom, P. M., Noorinayer, B., Farzaneh, F., Aghajani, K., et al. (2009). Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem
cell injection: A phase I–II clinical trial. European Journal of Gastroenterology & Hepatology, 21, 1199–1205.
Kim, J., & Hematti, P. (2009). Mesenchymal stem cell-educated macrophages: A novel type of alternatively activated macrophages. Experimental Hematology, 37,
1445–1453.
Kim, D. H., Yoo, K. H., Choi, K. S., Choi, J., Choi, S. Y., et al. (2005). Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived
mesenchymal stem cell. Cytokine, 31, 119–126.
Kolf, C. M., Cho, E., & Tuan, R. S. (2007). Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: Regulation of niche, self-renewal and differentiation. Arthritis
Research & Therapy, 9, 204.
Krampera, M., Cosmi, L., Angeli, R., Pasini, A., Liotta, F., et al. (2006). Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem
cells. Stem Cells, 24, 386–398.
Kuo, Y. R., Chen, C. C., Goto, S., Huang, Y. T., Wang, C. T., et al. (2012). Immunomodulatory effects of bone marrow-derived mesenchymal stem cells in a swine hemi-facial
allotransplantation model. PLoS One, 7, e35459.
Kuroda, S. (2016). Current opinion of bone marrow stromal cell transplantation for ischemic stroke. Neurologia Medico-Chirurgica (Tokyo), 56, 293–301.
Langer, R., & Vacanti, J. P. (1993). Tissue engineering. Science, 260, 920–926.
Lee, H. K., Kim, H. S., Kim, J. S., Kim, Y. G., Park, K. H., et al. (2017). CCL2 deficient mesenchymal stem cells fail to establish long-lasting contact with T cells and no longer
ameliorate lupus symptoms. Scientific Reports, 7, 41258.
Lichtnekert, J., Kawakami, T., Parks, W. C., & Duffield, J. S. (2013). Changes in macrophage phenotype as the immune response evolves. Current Opinion in Pharmacology, 13,
555–564.
Liu, G., Zhao, L., Zhang, W., Cui, L., Liu, W., & Cao, Y. (2008). Repair of goat tibial defects with bone marrow stromal cells and beta-tricalcium phosphate. Journal of Materials
Science. Materials in Medicine, 19, 2367–2376.
Liu, Y., Yin, Z., Zhang, R., Yan, K., Chen, L., et al. (2014). MSCs inhibit bone marrow-derived DC maturation and function through the release of TSG-6. Biochemical and Biophysical
Research Communications, 450, 1409–1415.
Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., et al. (2012). Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested
from lipoaspirate. Journal of Visualized Experiments, 68, e4221.
MacFarlane, R. J., Graham, S. M., Davies, P. S., Korres, N., Tsouchnica, H., et al. (2013). Anti-inflammatory role and immunomodulation of mesenchymal stem cells in systemic
joint diseases: Potential for treatment. Expert Opinion on Therapeutic Targets, 17, 243–254.
Mauri, C., & Bosma, A. (2012). Immune regulatory function of B cells. Annual Review of Immunology, 30, 221–241.
Melief, S. M., Schrama, E., Brugman, M. H., Tiemessen, M. M., Hoogduijn, M. J., et al. (2013). Multipotent stromal cells induce human regulatory T cells through a novel pathway
involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells, 31, 1980–1991.
Meyerrose, T., Olson, S., Pontow, S., Kalomoiris, S., Jung, Y., et al. (2010). Mesenchymal stem cells for the sustained in vivo delivery of bioactive factors. Advanced Drug Delivery
Reviews, 62, 1167–1174.
Mills, C. D., Kincaid, K., Alt, J. M., Heilman, M. J., & Hill, A. M. (2000). M-1/M-2 macrophages and the Th1/Th2 paradigm. Journal of Immunology, 164, 6166–6173.
Mokbel, A. N., El Tookhy, O. S., Shamaa, A. A., Rashed, L. A., Sabry, D., & El Sayed, A. M. (2011). Homing and reparative effect of intra-articular injection of autologus
mesenchymal stem cells in osteoarthritic animal model. BMC Musculoskeletal Disorders, 12, 259.
Murakami, H., Nakasa, T., Ishikawa, M., Adachi, N., & Ochi, M. (2016). Autologous bone grafts with MSCs or FGF-2 accelerate bone union in large bone defects. Journal of
Orthopaedic Surgery and Research, 11, 105.
Nagata, M., Hoshina, H., Li, M., Arasawa, M., Uematsu, K., et al. (2012). A clinical study of alveolar bone tissue engineering with cultured autogenous periosteal cells: Coordinated
activation of bone formation and resorption. Bone, 50, 1123–1129.
Ovchinnikov, D. A. (2008). Macrophages in the embryo and beyond: Much more than just giant phagocytes. Genesis, 46, 447–462.
Paul, J., Sagstetter, A., Kriner, M., Imhoff, A. B., Spang, J., & Hinterwimmer, S. (2009). Donor-site morbidity after osteochondral autologous transplantation for lesions of the talus.
Journal of Bone and Joint Surgery. American Volume, 91, 1683–1688.
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., et al. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143–147.
Polchert, D., Sobinsky, J., Douglas, G., Kidd, M., Moadsiri, A., et al. (2008). IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host
disease. European Journal of Immunology, 38, 1745–1755.
Prockop, D. J., & Youn Oh, J. (2012). Mesenchymal stem/stromal cells (MSCs): Role as guardians of inflammation. Molecular Therapy, 20, 14–20.
Rafei, M., Hsieh, J., Fortier, S., Li, M., Yuan, S., et al. (2008). Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation
and PAX5 induction. Blood, 112, 4991–4998.
Raffaghello, L., Bianchi, G., Bertolotto, M., Montecucco, F., Busca, A., et al. (2008). Human mesenchymal stem cells inhibit neutrophil apoptosis: A model for neutrophil preservation
in the bone marrow niche. Stem Cells, 26, 151–162.
Rasmusson, I., Ringden, O., Sundberg, B., & Le Blanc, K. (2003). Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes
or natural killer cells. Transplantation, 76, 1208–1213.
Selmani, Z., Naji, A., Zidi, I., Favier, B., Gaiffe, E., et al. (2008). Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and
natural killer function and to induce CD4 þ CD25highFOXP3 þ regulatory T cells. Stem Cells, 26, 212–222.
Shi, M., Liu, Z. W., & Wang, F. S. (2011). Immunomodulatory properties and therapeutic application of mesenchymal stem cells. Clinical and Experimental Immunology, 164, 1–8.
Singh, B., Schwartz, J. A., Sandrock, C., Bellemore, S. M., & Nikoopour, E. (2013). Modulation of autoimmune diseases by interleukin (IL)-17 producing regulatory T helper (Th17)
cells. Indian Journal of Medical Research, 138, 591–594.
Sordi, V. (2009). Mesenchymal stem cell homing capacity. Transplantation, 87, S42–5.
Sotiropoulou, P. A., Perez, S. A., Gritzapis, A. D., Baxevanis, C. N., & Papamichail, M. (2006). Interactions between human mesenchymal stem cells and natural killer cells. Stem
Cells, 24, 74–85.
Surder, D., Manka, R., Lo Cicero, V., Moccetti, T., Rufibach, K., et al. (2013). Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial
infarction: Effects on global left ventricular function. Circulation, 127, 1968–1979.
Tyndall, A., & Pistoia, V. (2009). Mesenchymal stem cells combat sepsis. Nature Medicine, 15, 18–20.
Urban, V. S., Kiss, J., Kovacs, J., Gocza, E., Vas, V., et al. (2008). Mesenchymal stem cells cooperate with bone marrow cells in therapy of diabetes. Stem Cells, 26, 244–253.
Varin, A., & Gordon, S. (2009). Alternative activation of macrophages: Immune function and cellular biology. Immunobiology, 214, 630–641.
Viateau, V., Guillemin, G., Bousson, V., Oudina, K., Hannouche, D., et al. (2007). Long-bone critical-size defects treated with tissue-engineered grafts: A study on sheep. Journal of
Orthopaedic Research, 25, 741–749.
Vivier, E., Tomasello, E., Baratin, M., Walzer, T., & Ugolini, S. (2008). Functions of natural killer cells. Nature Immunology, 9, 503–510.
Wakitani, S., Okabe, T., Horibe, S., Mitsuoka, T., Saito, M., et al. (2011). Safety of autologous bone marrow-derived mesenchymal stem cell transplantation for cartilage repair in 41
patients with 45 joints followed for up to 11 years and 5 months. Journal of Tissue Engineering and Regenerative Medicine, 5, 146–150.
Waterman, R. S., Tomchuck, S. L., Henkle, S. L., & Betancourt, A. M. (2010). A new mesenchymal stem cell (MSC) paradigm: Polarization into a pro-inflammatory MSC1 or an
immunosuppressive MSC2 phenotype. PLoS One, 5, e10088.
Willinger, T., Freeman, T., Hasegawa, H., McMichael, A. J., & Callan, M. F. (2005). Molecular signatures distinguish human central memory from effector memory CD8 T cell
subsets. Journal of Immunology, 175, 5895–5903.
Witko-Sarsat, V., Rieu, P., Descamps-Latscha, B., Lesavre, P., & Halbwachs-Mecarelli, L. (2000). Neutrophils: Molecules, functions and pathophysiological aspects. Laboratory
Investigation, 80, 617–653.
Wu, W., Shan, J., Li, Y., Luo, L., Sun, G., et al. (2012). Adoptive transfusion of tolerance dendritic cells prolongs the survival of cardiac allograft: A systematic review of 44 basic
studies in mice. Journal of Evidence-Based Medicine, 5, 139–153.
Yamada, Y., Nakamura, S., Ito, K., Kohgo, T., Hibi, H., et al. (2008). Injectable tissue-engineered bone using autogenous bone marrow-derived stromal cells for maxillary sinus
augmentation: Clinical application report from a 2–6-year follow-up. Tissue Engineering. Part A, 14, 1699–1707.
Yerebakan, C., Kaminski, A., Westphal, B., Donndorf, P., Glass, A., et al. (2011). Impact of preoperative left ventricular function and time from infarction on the long-term benefits
after intramyocardial CD133(þ) bone marrow stem cell transplant. Journal of Thoracic and Cardiovascular Surgery, 142, 1530-9.e3.
Zhang, W., Ge, W., Li, C., You, S., Liao, L., et al. (2004). Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells.
Stem Cells and Development, 13, 263–271.
Zhou, Y., Huang, R., Fan, W., Prasadam, I., Crawford, R., & Xiao, Y. (2017). Mesenchymal stromal cells regulate the cell mobility and the immune response during osteogenesis
through secretion of vascular endothelial growth factor A. Journal of Tissue Engineering and Regenerative Medicine.
Relevant Websites
http://www.bloodjournal.org/?sso-checked¼true.
https://www.jci.org/.
http://www.jimmunol.org/.
http://www.liebertpub.com/overview/stem-cells-and-development/125/.
https://www.nature.com/ni/.
http://www.nature.com/nature/index.html.
http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291932-7005.
http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1526-968X.
http://www.sciencemag.org/journals.
http://stemcellsjournals.onlinelibrary.wiley.com/hub/journal/10.1002/(ISSN)1549-4918/.
Osseointegration of Permanent and Temporary Orthopedic Implants
JS Hayes, NUI Galway, Galway, Ireland
RG Richards, AO Research Institute, Davos, Switzerland
Historical Review of Internal Fixation 257

Plate Design 257
Metals Used in Orthopedics 258
The Cause of Bone Loss under Plates – Stressing Shielding or Temporary Porosis? 260
Implants – Remove or Not to Remove? 260
Reasoning for Elective Implant Removal 261
Reasoning against Elective Implant Removal 262
Biological Performance – Empirically Determining the Cell/Tissue–Implant Interaction 262
Surface Chemistry 264
Surface Topography 264
Substrate Stiffness 265
Porosity/Pore Size 265
Bioresorbable Implants – Temporary Implants to Provide a Permanent Solution? 266
Treatment of Nonunions – A Brief Perspective into Scaffolds for Tissue Engineering 266
Summary 267
References 267
Glossary
Anodization Electrolytic passivation process to increase the thickness of the natural oxide layer.
Biocompatible Perform with an appropriate host response in a specific application.
Ductile Plastically deform without fracture.
Electropolishing Electrochemical process that removes material from a metal implant.
Microtopography Micron scale variations in the height and roughness of an implant surface.
Osseointegration Direct structural and functional connection between bone and the surface of a load-bearing implant.
Paste polishing Mechanically abrasive process to remove material from a metal implant.
Stiff Resist deformation by an applied force.
Strong Withstand applied stresses without failure.
Historical Review of Internal Fixation

Plate Design
Despite the medical management of fractures being described for thousands of years, it is only in the last hundred years or so that
the metal internal fixators that we rely on today were introduced. Since the first introduction of the metal plate by Lane in 1895,
there have been several improvements on design, mechanical properties, and material choice that have allowed for great advances
in fracture fixation. The initial plate developed by Lane was flat and because of issues with corrosion, subsequent improvements
ensued. In the early 1900s, Lambotte introduced a curved plate in an effort to better fit the natural curvature of bone, as well as
a threaded screw which eventually gave rise to the current threaded cortical screws found in clinics today. Shortly afterward
in 1912, Sherman was credited with the development of self-tapping screws. While material and design advancements did
demonstrate improved corrosion resistance over the Lane plate they too were ultimately abandoned due to inadequate strength
and fixation instability.
An important development in internal fracture fixation came in 1949 with the development of Danis’ ‘coapteur’ or compression
plate (Figure 1). This design limited interfragmentary movement while improving the stability of the fixation. This new approach
of absolute stability resulted in what Danis described as soudure autogène, a process we now recognize as primary bone healing
(healing without the presence of fracture callus). While Danis’ compression plate was introduced less than a century ago, it is
this design that influenced all subsequent plate designs. In fact, Danis’ description of diaphyseal fracture healing in the absence
of callus formation spurred the interest of a young Swiss surgeon by the name of Maurice E Müller, who subsequently met with
Robert Danis to discuss his observations in 1950. This meeting resulted in the establishment of the Arbeitsgemeinschaft für

258 Biomaterials: In Vitro and in Vivo Studies of Biomaterials j Osseointegration of Permanent and Temporary Orthopedic
Figure 1 The evolution of internal fixation plate design. Lambotte’s plate (a) was introduced subsequent to Lane’s plate due to issues of corrosion.
Insufficient strength and problems with stability eventually led to the naissance of Danis’ ‘coapteur’ plate (b). The latter allowed for interfragmentary
compression, the result of which is primary bone healing (absence of callus formation). (c) It was this design that revolutionized all subsequent
designs including the dynamic compression plate and later the limited contact dynamic compression plate which arose due to perceived issues
relating to bone necrosis (see Section The Cause of Bone Loss under Plates – Stressing Shieding or Temporary Porosis? for discussion). (d) The
point contact fixator (PC-fix) is another example of subsequent plate designs that focused on preserving the periosteal blood supply underneath
the plate for appropriate healing to occur. This type of design limits the plates ‘footprint’ along the bone. (e) The development of the locking
compression plate came from the continuous requests of surgeons for a synergistic system of a threaded and a conventional screw hole allowing
for the greatest possible flexibility for the treatment of fractures. Images (a) and (b) reproduced with kind permission of the Journal of Orthopaedic
Science (Uhthoff, H.K., Poitras, P., Backman, D.S., 2006. Internal plate fixation of fractures: short history and; recent developments. J. Orthop. Sci.
11, 118–126). Images ((c)–(e)) reproduced with kind permission of the AO Foundation from AO principles of fracture management, 2nd Edition.
Osteosynthesefragen/Association for the study of Internal Fixation (AO/ASIF) by Müller and others in 1958 and the subsequent
Laboratory for Experimental Surgery in Davos, Switzerland which is responsible for much of what is known today relating to
fracture healing and fixation.
The AO contributed to the development of the dynamic compression plate (DCP) in 1969 (Figure 1). However, observations of
interrupted cortical blood perfusion leading to cortical necrosis (Akeson et al., 1976; Perren et al., 1988) led to the development of
the limited contact dynamic compression plate (LC-DCP) by Perren in 1990. With plating, most of the iatrogenic disruption to
bone has been traced to the regions of direct bone–implant contact and is believed to result directly from periosteal blood supply
disruption (Perren et al., 1988). This finding was the main motivation behind the large investment in the development of new
generation internal fixation systems that effectively reduce the extent of contact of the plate with bone consequently avoiding
disrupted periosteal vascularity. Here the AO again pioneered much of the developments within this area firstly with the
development of point contact fixator or PC-fix system (Figure 1), which allowed fixation by shear forces between the screw and
bone rather than frictional forces generated between the bone and plate as with conventional plating methods (Tepic and Perren,
1995). Subsequent designs include the less invasive stabilization system (LISS) which was developed for application specifically
within metaphyseal and epiphyseal regions as the PC-fix was unsuitable in these areas. The locking compression plate (LCP;
Figure 1) was developed in response to increasing requests from orthopedic surgeons for the availability of a system that could offer
a synergy between a threaded and a conventional screw hole allowing for the greatest possible flexibility for the treatment of
fractures (Frigg, 2003). The operating surgeon thus has the scope of deciding which variation of the system would be applicable
for treatment depending on type of fracture and quality of the bone since the system can be applied in either a conventional
technique (compression), bridging method (internal fixator), or combination of both (Wagner, 2003).
Metals Used in Orthopedics

Desirable properties such as high stiffness, strength, biological toleration, and reliable function have meant that metal implants
have gained unrivaled success in fracture fixation for many years. Before the advent of metals in orthopedics fracture management
Biomaterials: In Vitro and in Vivo Studies of Biomaterials j Osseointegration of Permanent and Temporary Orthopedic 259
Table 1 Despite the excellent clinical success of stainless steel and titanium in fracture fixation, there are several key properties which differ between
materials. As noted in Figure 2 stainless steel has a smooth mirrorlike finish compared to the microrough finish of titanium. In addition,
differences in density, oxide chemistry, thickness, and regeneration time as well as modulus of elasticity all influence the materials clinical
performance (Section Metals Used in Orthopaedics)
Young’s Main Thickness of Innate oxide

Density modulus components natural oxide regeneration
Material (g cm3) (GPa) Surface of oxide layer later time (min) Annealed Cold-worked Cold-drawn
Stainless 7.9 186 Smooth O, C, Cr, Fe, 2 nm 35 UTS ¼ 490– UTS ¼ 860– UTS ¼ 1350–
steel 0.1 ms Mo, Si 690 MPa 1100 MPa 1600 MPa
Cerclage wire Bone screws, Schanz screws,
plates, IM nails Krischner
wire
Titanium 4.5 110 Microrough Ti, O, C 6 nm 8 UTS ¼ 200– UTS ¼ 680 –
1 mm 550 MPa
CMF plates Bone screws,
plates, IM nails
Content adapted from Hayes, J.S., Richards, R.G., 2010b. The use of titanium and stainless steel in fracture fixation. Expert Rev. Med. Devices 7 (6), 843–853.
mainly involved immobilization in plaster or by traction but this approach greatly inhibited function during healing. The
introduction of stainless steel and titanium in the early twentieth century would see internal fixation transformed. The reader
is directed to the review paper by Hayes and Richards (2010b) for a more comprehensive insight to the properties and clinical
applications of stainless steel and titanium that will be summarized here.
Despite their similar clinical success, stainless steel and titanium have many different properties that are important for fracture
fixation. Ideally, an implant for internal fixation applications should be ductile, strong, and stiff, perform well under different
mechanical demands and importantly be biocompatible. Both stainless steel and titanium fulfill these requirements to different
degrees. Stainless steel has a density of 7.9 g cm3 which for small implants is not generally considered to be problematic, however,
it is worth noting that it is more than twice as dense as titanium implants of the same design (Disegi and Eschbach, 2000). The
modulus of elasticity of steel is also higher than titanium (186 GPa compared to 110 GPa). In terms of transferring load to the
adjacent bone (approximately 20 GPa) this means stainless steel is less effective than titanium which may be significant in terms
of stress shielding. In contrast, stainless steel is more ductile than titanium which permits more deformation of the implant for
intraoperative contouring by the surgeon to compensate for anatomical requirements. To counter this limitation of titanium
anatomically designed implants are produced to restrict the need for contouring. Three main tensile versions of stainless steel
are fabricated for clinics namely (listed from lowest to highest strength), annealed (i.e., cerclage wire, reconstruction plates),
cold-worked (i.e., bone screws, bone plates, intramedullary nails), and cold-drawn (i.e., Schanz screws, Kirschner wire). These
conditions vary in strength allowing for use in several different implant applications depending on strength and ductility
requirements. For instance, annealed implants are generally the ‘softer’ version of the material and thus allow for deformation
and are normally used in low stress loading applications whereas cold-worked materials represent the increased strength version
of the material where ductility and high strength are favored. Titanium is fabricated for clinics in two mechanical forms, annealed
(i.e., craniofacial plates, mini-fragment plates) and cold-worked (i.e., bone plates, bone screws, intramedullary nails) (Table 1).
In terms of their biological reaction, stainless steel and titanium differ in two major areas. Firstly, stainless steel is fabricated for
clinical applications with a smooth electropolished surface (an approximate microroughness of 0.1 mm) whereas titanium and its
alloys are produced with a microrough surface of approximately 1 mm (Figure 2). As outlined in Section Surface Topography,
the surface microtopography can have a profound biological effect on tissue integration. Another important difference of these
materials is the chemical makeup of their surface oxides. This is especially important as it is the presence of an oxide film that is
responsible for the implants’ increased corrosion resistance and biocompatibility. The reader is directed to the review by Hayes
et al., (2010b) for more detail. Briefly, the passive film of stainless steel consists mainly of iron, nickel, and chromium. The
chromium in stainless steel reacts with oxygen to form the 2 to 3-nm-thick characteristic passive film that provides increased
corrosion resistance for these devices. In contrast, titanium oxide layer mainly consists of titanium, oxygen, and carbon which
produces an innate oxide layer of approximately 5–6 nm. However, to improve clinical performance, the commercial technique
of anodizing is normally employed for both stainless steel and titanium devices which can result in oxide layers of approximately
200 nm.
As mentioned, it is this delicate oxide layer that is responsible for maintenance of biocompatibility by preventing the release of
toxic ions from the underlying highly reactive bulk material. If abraded both stainless steel and titanium maintain the ability of
regenerating the layer. Stainless steel devices tested in saline solution take approximately 35 min to regenerate compared with
approximately 8 min for titanium and titanium alloys (Hanawa, 2004). This can have consequences for stainless steel compatibility
given that the ions within the bulk material are more highly reactive than titanium and its oxide layer regenerates at a slower rate,
thereby potentially permitting increased release of toxic ions once abraded.
Figure 2 Examples of the surface of electropolished stainless steel (a) and commercially pure titanium (b) used for orthopedic devices. Note the
extremely smooth surface of stainless steel compared to the 3D microrough surface of titanium. Despite unrivaled clinical success in orthopedics,
both materials differ in several key areas (Table 1). The difference in surface appearance is translated to differential tissue responses at the implant
interface in vivo. Generally, stainless steel supports a fibro–osseous interface (soft and hard tissue interface) while titanium supports direct
osseointegration.
The Cause of Bone Loss under Plates – Stressing Shielding or Temporary Porosis?
The occurrence of temporary osteoporosis adjacent to the implant contact area has long been observed and recognized. Specifically,
there appears to be an enlarging of the intramedullary cavity coupled with cortical bone loss via endosteal resorption (Akeson et al.,
1976). During the nineteenth century, Julius Wolff developed the concept that bone will adapt to the load under which it is placed.
According to Wolff’s law if the load applied to bone is decreased then its density and structure will diminish (and vice versa).
Applying this theory to osteosynthesis initially appeared logical: the implant would inadvertently transfer load away from bone
to the device, thereby, protecting or shielding the bone from load therefore adapting its structure accordingly. Hence, experimental
evidence to support the theory of stress shielding, also known as stress protection has been published for over 40 years.
However, the term ‘stress protection’ in terms of bone plate fixation has become debated since the theory of disturbed vascularity
has come to light. Arguing against stress protection, Perren and colleagues (1988) suggest that early osteoporosis observed within
the first 6 months of fixation was due to cortical necrosis, secondary to excessive plate–bone contact interfering with cortical
perfusion. Their hypothesis was that the initial increase in bone porosity was a result of the host attempting to eliminate necrotic
bone and ultimately remodel the site. Studies demonstrating the ability of fixation devices to hamper perfusion have been available
for over 50 years (Rhinelander, 1965; Gunst et al., 1979). These findings were later supported in a study where railed and contact
plates were used in a dog model (Uhthoff et al., 1994). As a result of Perren and colleagues’ findings, significant developments were
made to plate design with subsequent designs incorporating a limited contact approach as demonstrated by PC-fix, the LC-DCP,
and LISS systems (Figure 3).
Based on Perren’s theory it would seem that porosis is transient and is reversed upon completion of remodeling of the necrotic
area. As a major critic of the theory of temporary porosis Uhthoff and colleagues (1994) addressed this issue and found no evidence
to support the notion (Uhthoff et al., 1994). They did, nevertheless, show in other studies that the immobilization of canine limbs
with plaster casts led to cortical thinning and widening of the medullary canal (Uhthoff and Jaworski, 1978; Jaworski et al., 1980).
Uhthoff also maintains that if the initial hypothesis put forth by Perren and colleagues was true then the increased porosity would
be found at the area of necrosis. Recently, Tepic (2008) showed that for PC-fix plates the area of porosity was increased in the
vicinity of necrotic tissue. However, it has also been demonstrated that bone porosity is increased more in the endosteal part
of the cortex rather than the periosteal part where necrosis is most evident (Akeson et al., 1976; Uhthoff et al., 1994). Despite
the ambiguity surrounding this issue, these observations and clinical outcomes have resulted in the routine removal of many of
these internal fracture repair devices.
Implants – Remove or Not to Remove?
The ubiquitous use and success of biomaterials in medical applications is undeniable. Nevertheless, when referred to in the context
of medical applications it is generally accepted as referring to long-term implantation. Almost all hardware removal procedures in
adults are a result of a complication. In contrast, for pediatric patients, the accepted protocol was removal of the device once its
purpose had been spent (Schmalzried et al., 1991). In this section we examine the main clinical considerations for and against
elective implant removal.
Figure 3 Decreased cortical perfusion is evident with increased bone–plate contact with contact plates (left) compared to railed plates (right) as
demonstrated with intravital disulfine administration. Two main arguments exist to debate the bone changes noted beneath plates (Section The Cause
of Bone Loss under Plates – Stressing Shieding or Temporary Porosis?). Perren and colleagues suggest that early porosis is due to cortical necrosis,
secondary to excessive plate–bone contact interfering with cortical perfusion. Others advocate the theory of ‘stress protection’ which outlines that
load is transferred away from bone as it is shielded by the plate and as a result undergoes a weakening in the absence of appropriate mechanical
stimulus. Images from Uhthoff, H.K., Poitras, P., Backman, D.S. 2006. Internal plate fixation of fractures: short history and recent developments.
J. Orthop. Sci. 11, 118–126.
Reasoning for Elective Implant Removal

Many complications surround the use of metal fixation devices but are normally alleviated upon extraction and are therefore used as
grounds for electively removing devices. Common ailments experienced include pain upon implantation, irritation of adjacent soft
tissues, and acute aseptic inflammation. The formation of restrictive adhesions and subsequent limited motion is one of the most
challenging areas of hand surgery. Specifically the tendon sheath if irritated produces a severe inflammatory response (Khan et al.,
2000). Here, stainless steel devices are favored (Sinicropi et al., 2005) although recent developments in the modification of titanium
alloy plates have proved successful (Hayes et al., 2012a). Moreover, the device may encroach into the joint space resulting in pain,
loss of motion, and fracture instability. In severe cases an implant may migrate. Devices implanted originally in the humerus,
mandible, and shoulder have been reported to migrate to the mediastinum, spinal cord, and jugular foramen, respectively (Seipel
et al., 2001).
Infection is practically impossible to treat unless the cause, most often the implant, is removed. This complication can arise at
any time and is not restricted to the early events that occur upon implantation. Highland and LaMont (1985) report the occurrence
of deep late infection from 7 to 24 months after implantation. In a case study cited by Peterson (2005), an eighteen-year-old male
developed osteomyelitis some 50 years after initial treatment. It is possible that this was a result of a recent hematogenous infection
rather than the organism remaining quiescent for those years, nevertheless, the persistence of bacteria intracellularly has been
reported (Vaudaux and Lew, 2006; Broekhuizen et al., 2008). Orthopedic device-related infections are notoriously difficult to treat
and several studies have reported the varying effect of antibiotics in treating pyogenic (pus producing) infections (Lew and
Waldvogel, 2004). In many instances antibiotics are given prophylactically, however, the cost of continued treatment for a patient
with an infected device can be staggering.
Some patients demonstrate hypersensitivity to metal devices. However, it is not believed that metal hypersensitivity contributes
to endoprosthesis loosening (Milavec-Puretic et al., 1998). A retrospective study reported elevated levels of metal ion (titanium,
vanadium, and aluminum) concentrations in the serum (35% of patients (70–90 ppb while 20 ppb was considered ‘normal’)
and hair (24% of patients (20–30 mg g1) compared to 3 and 19 mg g1) considered as ‘normal’ for titanium and aluminum,
respectively) of patients with titanium alloy fixation devices (Kasai et al., 2003). Nevertheless, stainless steel devices tend to
demonstrate more problems than titanium counterparts due to the presence of chromium, cobalt, and nickel. Current stainless steel
implants comprise approximately 13–16 wt% nickel despite this being the most common skin-contact allergen. Approximately
1–2% of patients present with allergic reactions to Ni-containing stainless steel devices after internal fixation. Furthermore, nickel,
chromium, and cobalt have been shown to produce reactive oxygen species (Valko et al., 2006) and are thought to contribute to the
development and potential progression of neurodegenerative disorders (Olivieri et al., 2002). Directives are in place to define
acceptable concentrations of metal debris within patients (Morgan and Shaller, 1999); however, in reality these are often exceeded
(Schaffer et al., 1999; Pilger et al., 2002; Lhotka et al., 2005; Witzleb et al., 2006). Moreover, the concentration of metal ions for
adverse responses to occur varies considerably. For instance, Hallab et al. (2008) found that metals such as cobalt and vanadium
were toxic at concentrations as low as 0.5 mM, while other metals (aluminum, chromium, iron, molybdenum, and nickel) were
toxic at much higher concentrations (>10 mM). While there is still much to learn regarding the tolerated concentrations of metal
debris/ion release, evidence does exist to suggest that exposure can be influential in a variety of biological systems. Kanojia and
colleagues (1998) has reported increased levels of cobalt and chromium in the cord blood of pregnant women. These patients
all had metal fixation devices and had become pregnant subsequent to implantation. This is of particular concern given that these
metal ions are known inducers of developmental toxicity (Domingo, 1994; Elbetieha and Al-Hamood, 1997; Keegan et al., 2007).
The negative biological impacts of metal debris and ions have also been observed in cardiovascular, respiratory, urinary, and
reproductive systems (Elbetieha and Al-Hamood, 1997; Oliveira et al., 2006; Keegan et al., 2007). Regardless, stainless steel
maintains one of the leading materials used and is generally considered biocompatible.
In pediatric patients the risk of premature physeal closure is increased when an implant transverses a physis. Thus removal of the
device is strongly advocated in the case of patients who have significant growth potential unless the growth cessation is desired
(Peterson, 2005). Unfortunately, the occurrence of premature unwanted physeal closure due to the presence of a fixation device
is high. For instance, Chen and colleagues (2002) observed a 41% occurrence of premature closure of physis while Bagatur and
Zorer (2002) report its presence in 65% of the patients included in their retrospective study.
Other factors such as return to sports, carcinogenicity and peri-implant fracture are also concerns to be taken into account. In
terms of carcinogenicity and peri-implant fracture, the literature indicates that their association with metal implants appears to
be negligible and thus do not warrant routine removal. Nevertheless, some of those reported hold reason for revision. For instance,
Keel and colleagues (2001) reported that sarcomas related to fixation devices tend to be high grade, to behave aggressively and to
metastasize frequently. In the 12 cases they reviewed, 10 were found in bone (the other 2 were found in adjacent soft tissues) and
follow up was permitted in only 8. Of these, 7 died between 2 and 30 months after diagnosis. In the case of patients wishing to
return to sports, there seems to be a lack of consensus here too. Some believe that devices that restrict joint or muscle action should
be removed prior to the patient returning to sport (Labosky et al., 1990). In contrast, 87% of professional rugby players returned to
full training with retained fixation devices (Evans and Evans, 1997).
Reasoning against Elective Implant Removal

Many surgeons, engineers, and researchers alike fail to find sufficient relevant clinical or experimental evidence to convince them to
adopt an attitude that routine elective hardware removal is the best option in the end treatment of fixation. Although few reasons
exist for this failure those that do are just as valid and compelling. From a purely economic view point elective removal can be costly
and time consuming. Hospitalization, anesthesia, imaging, antibiotics therapy, and operating equipment are all points that should
be considered. Cost to patient earning is rarely considered but is equally valid.
Although the carcinogenicity risk associated with retained devices in animal models has been well documented there appear to
be few cases of implant-associated tumors in humans. Thus, advocates of implant retention suggest that this risk is impossible to
quantify accurately and does not warrant the application of routine removal based on its occurrence. A retrospective study of more
than 116 000 patients reported no correlation between implant retention and increased cancer risk when compared to the general
population (Signorello et al., 2001; Fryzek et al., 2002). They did, however, report an increase in prostate cancer and melanoma
(standardized incidence ratio of 1.16 and 1.86, respectively). Of the patients that had implants retained no increase was noted
for bone or other connective tissue-related cancers (Signorello et al., 2001). A later study also found that patients with knee pros-
theses did not present with an increase risk of cancer compared to the general population (Fryzek et al., 2002). Again, elevated risk
was observed for prostate cancer (standardized incidence ratio of 1.20) and may warrant due attention. A risk in bone cancer was
detected in this study (standardized incidence ratio 6.00), however, the authors believe these incidents (3 of the total
120 000 þ patients studied) to be unrelated to the implant itself (Fryzek et al., 2002).
It is maintained that conclusions regarding the carcinogenic potential of implants in patients are particularly difficult to make
since variables such as the quantity of wear debris produced and patient age may be possible confounding factors (Heath et al.,
1971). The question remains whether malignancy is correlated to the presence of long-term implants or if it is in fact caused by
them. Katzer and colleagues (2003) reported that neither chromium cobalt molybdenum nor titanium aluminum wear particles
produced toxic or mutagenic effects, whereas Doran and co-workers (1998) document significant increases in cell transformation
for soluble forms of the same materials and appeared to relate directly to toxicity. Furthermore, macrophages have been previously
shown in vitro to phagocytose laboratory produced debris from steel and titanium and a titanium alloy in a dose dependent manner
(Wilson, 1999). Steel particles are reported to be approximately 0.5 mm in size (ap Gwynn and Wilson, 2001), and thus, are easily
dispersed throughout the body and have been noted in organs remote from the implantation site (Case et al., 1994). In contrast,
particles generated from titanium are approximately 10 mm in size (ap Gwynn and Wilson, 2001) and have been reported to be
found in tissue adjacent to the implanted site giving the harmless discoloration of the tissue. Nevertheless, the correlation to cancer
remains unfounded (Signorello et al., 2001; Fryzek et al., 2002).
Complications associated with the surgical aspect of device retrieval are a principal basis for hardware retention for many
surgeons. Inherent risks such as iatrogenic impairment and complications with anesthesia are considerations for any surgical
procedure. In addition, the actual procedure of implant retrieval can add to the already serious preexisting concerns.
For instance, refracture, nerve damage, excessive blood loss, infection, and difficulty in removing the device e.g., stripping of
the screw head or even breakage at the screw neck are all potential obstacles that must be reviewed carefully for one to carry out
a successful retrieval. There have been steps in recent years to address this issue (Pearce et al., 2008; Hayes et al., 2009; Hayes
et al., 2010a) which is of particular benefit for pediatric fixation.
Biological Performance – Empirically Determining the Cell/Tissue–Implant Interaction
There are several definitions available to define a biomaterial in terms of tissue regeneration. One such definition by the National
Institute of Health defines a biomaterial as “Any substance (other than a drug) or combination of substances, synthetic or natural in
origin, which can be used for any period of time, as a whole or as a part of a system which treats, augments, or replaces any tissue,
organ, or function of the body.” This encompasses several variations of biomaterial for several different clinical applications.
Regardless of application, one commonality is that the surface properties play a determining role in the tissue response upon
implantation. Ultimately, therefore, it is this delicate interaction between material and host tissue that has resulted in a billion
dollar industry emerging to empirically design implants in an effort to regulate the ensuing biological response.
In terms of a substrate dependent response, it is all down to what a cell ‘sees’ once it comes into contact with a material, be it gel,
polymer, demineralized matrix, or specifically in this case, metal. The primary interaction upon implantation of a metal device is
the formation of a water biolayer (Figure 4). This process occurs within nanoseconds of the device impacting the surrounding
biological niche and is followed instantaneously by contact with blood. As the implantation of a device is essentially recognized
as an injury response, blood coagulates at the implant interface forming a hematoma. This hypoxic environment, similar to fracture
healing itself, provides a protein and cell rich milieu at the surface of the device resulting in an inflammatory response eventually
leading to the recruitment of repair cells. A more in-depth explanation can be found in several additional resources (Hayes et al.,
2010a, 2012).
In terms of tissue repair at the implant interface, there are several key factors that knowingly elicit specific tissue responses. Here
we will deal briefly with topography, chemistry, stiffness, and porosity/pore size. It is also important to note that while all factors
individually will bring about a specific cell response, in reality it is a combination of several surface properties that elicit the overall
biological response.
Figure 4 Summary of the cell surface interaction. (a) A standard microrough titanium surface. (b) The same titanium surface with a cell to
demonstrate what a cell ‘sees’ in terms of its underlying surface. As outlined in Section Surface Topography, the cell will be sensitive to substrate
microdiscontinuities which exert effect via the cytoskeleton resulting in differential transduction of signals to the nucleus to determine genotypic
and phenotypic responses. (c) Depicts the initial interaction of a device when implanted. When implanted the device comes into instant contact
with biofluid i.e., plasma, blood, water, etc. The primary interaction upon implantation is the formation of a water biolayer which is followed
instantaneously by contact with blood. As the implantation of a device is essentially recognized as an injury response, blood coagulates at the
implant interface forming a hematoma. This hypoxic environment, similar to fracture healing itself, provides a protein and cell rich milieu at the
surface of the device resulting in an inflammatory response eventually leading to the recruitment of repair cells. (d) Owing to lack of anchorage
a smooth surface does not permit fibrin to remain attached during wound healing contraction. In contrast, the microrough implant provides
increased anchorage for fibrin via its 3D morphology thereby permitting improved resistance to the forces generated during contraction. This allows
for continuous cell migration to the implant interface and consequently, direct osseointegration. Content of D adapted from Hayes, J.S., Richards,
R.G., 2010a. Surfaces to control tissue adhesion for osteosynthesis with metal implants: in vitro and in vivo studies to bring solutions to the patient.
Expert Rev. Med. Devices 7 (1), 131–142.
Surface Chemistry
Focusing on metal implants, the biocompatibility of these devices relies primarily on their oxide layer. This layer forms innately,
measures a few nanometers, can spontaneously regenerate given time, and determines the primary protein interaction upon
implantation. Ultimately, the responses elicited by the oxide layer mean that cells never truly interact with a ‘bare’ metal
surface and consequently, they are the principle players responsible for the accepted biocompatibility of metal implants. To
empirically determine a cell–implant interaction chemical (i.e., calcium phosphate coatings) and/or biological modifications
(i.e., peptide-based strategies) can be made to the surface chemistry of an implant via biological or chemical processes. The majority
of biological modifications involve the immobilization of extracellular proteins or peptide sequences to improve cell attachment
and subsequent tissue response. A more comprehensive review is covered in Hayes et al. (2012). Briefly, coating implant surfaces
with the Arg-Gly-Asp (RGD) sequence is currently the most common peptide-based strategy. The RGD sequence has been identified
as a cell attachment motif present on several plasma and extracellular matrix (ECM) proteins, including collagen type I, fibronectin
and vitronectin, among others. In turn these proteins interact with integrins, cell membrane proteins that bind a myriad of cell
surface and extracellular matrix-associated ligands. In vitro efficacy of peptide immobilization strategies have long been recognized
with several studies demonstrating improved attachment and increased osteospecific gene expression in vitro (Zreiqat et al., 2003;
Rivera-Chacon et al., 2013; Mendes et al., 2013). This effect is further advocated in vivo where peptide immobilization was shown to
improve bone healing and direct bone-implant contact while also expediting remodeling around the implant (Rammelt et al.,
2004).
In terms of chemical modification, the incorporation of calcium phosphates has dominated (Schlegel et al., 2009; O’Hare et al.,
2010; Costa et al., 2013; Sun et al., 2013). This approach of incorporating hydroxyapatite to improve osteoconductivity has
demonstrated efficacy for improving osseointegration. A major drawback of this approach is that during long-term implantation
the surface coating can delaminate from the underlying implant thereby compromising stability. Some efforts have been made
to address this issue. For instance, chemical modifications of the surface to incorporate the hydroxyapatite into the oxide layer
of the implant have been made (Schlegel et al., 2009; O’Hare et al., 2010). In essence, the hydroxyapatite alters the chemical
composition of the surface thereby eliminating issues of delamination associated with conventional coating technologies.
Surface Topography
When referring to surface topography in the context of metal implants both nano- and microtopography are key. In fact, this
interplay is so delicate that quantitative and qualitative responses vary from cell type to cell type for any one particular micro-
or nanofeature. Mesenchymal stem cells appear more reactive to nanofeatures than microtopography and fibroblast growth is
hindered on a titanium alloy while primary osteoblasts are not. In our own experience we have shown that surface topography
influences fibroblast growth, spreading and cytoskeletal organization (Meredith, 2006; Meredith et al., 2007). Furthermore, liquid
filled capsule formation (specifically important for free gliding of tendons over hand implants) can be directly controlled via surface
microtopography manipulation (Hayes et al., 2012). For bone, we and others have demonstrated in vitro that microrough surfaces
increase ‘osteospecific’ factors, local factor production and upregulate key factors in osteoblast differentiation (Olivares-Navarrete
et al., 2011; Gittens et al., 2011; Hayes et al., 2010; Boyan et al., 2003) and consequently, bony integration (osseointegration) of
a device can be directly influenced by the microtopography of a material (Pearce et al., 2008; Hayes et al., 2009; Hayes et al., 2010;
Davies et al., 2013). Several of the aforementioned studies demonstrate that the same material can elicit differing tissue responses
depending on whether it is smooth (approximately 0.2 mm) or microrough (approximately 1 mm). This is poignant in terms of
implant removal. Titanium is generally favored for internal fracture fixation but its microrough surface is known to innately induce
bone on-growth. It is this affinity to bone that makes titanium and its alloys particularly difficult to remove. In a series of preclinical
studies using bone screws, compression plates, and intramedullary nails we have shown that surface polishing (removal of surface
microroughness via mechanical (paste) or electropolishing) can significantly reduce the mechanical force required for implant
removal compared to clinical microrough counterparts (Pearce et al., 2008; Hayes et al., 2009; Hayes et al., 2010, 2012). In clinical
terms, this approach has seen great efficacy in hand surgery (Hayes et al., 2012) with commercial adaptation under way for other
sites (Figure 5). Again the reader is directed to more in-depth focused reviews (Schwartz et al., 2005; Hayes and Richards, 2010,
Hayes et al., 2012).
More recently, nanotopography has been implicated as a means of controlling an osteogenic response. One such study
investigated 15, 55, and 100 nm titanium nanopillars in terms of adhesion, spreading, cytoskeletal organization, and differentiation
of mesenchymal stem cells (Sjöström et al., 2009). Sjöström and colleagues claim that the 15 nm pillars proved the most
reproducible for osteogenesis specifically in terms of nodule formation after 21 days in vitro. While several other studies have
emerged in recent years advocating the role of nanotopography for eliciting an osteogenic response in vitro, data supporting this
role in vivo are yet to prove conclusive. A possible explanation for this is that the nanotopographical dependent adhesive,
cytoskeletal and phenotypic changes observed in vitro may be masked in vivo by the protein layer that adsorbs onto the surface
upon implantation (Hayes and Richards, 2010).
It is more likely that the benefit of cell differentiation dependent on nanotopography will be found within the remit of tissue
engineering. Recently, for example, McMurray and colleagues (2011) have demonstrated that ordered nanotopographical surface
features were effective in maintaining mesenchymal stem cell (MSC) phenotype for up to 8 weeks in culture. Interestingly, the
authors showed that subsequent to prolonged culture on the ordered surfaces, cells could be removed and replated on culture
Figure 5 Bone has a high affinity for titanium and its alloys. (a) Here we show a titanium bone plate and screws that has been implanted on
a sheep tibia. Note how the bone has overgrown the device. This excessive bony overgrowth is a significant clinical problem for device removal
especially in pediatric patients were device removal is favored. The reason for this direct and excessive osseointegration is the microrough surface
of clinical titanium implants (inset (b)). As shown in (b), which is an image of a standard microrough titanium alloy intramedullary nail implanted in
sheep tibia for 12 months, this results in direct and strong bone contact. (c) By reducing the surface topography via mechanical polishing (inset (c))
we have shown that this excessive bone overgrowth can be markedly reduced causing a significant reduction in the force required to remove the
implant which is partly a consequence to the fibro–osseous interface produced by the smoothened surface. This effect has been noted for screws,
bone plates, and intramedullary nails.
plastic and retained multipotency until the appropriate media supplementation. Furthermore, the authors cultured MSCs on the
ordered surfaces in the presence of osteogenic supplemented media and observed minimal differentiation, thus supporting the
idea that specifically ordered surfaces could maintain cell phenotype. It is an important finding in the context of cell propagation
for regenerative medicine where appropriate cell number and the cost associated with media additives can be crippling.
Substrate Stiffness
Substrate stiffness elicits its effect on cell response via the delicate interplay of cell adhesion, force-sensing, and signal transduction.
The reader is referred to Dalby (2005) and Hayes et al. (2012) for a more comprehensive explanation. In brief, integrins bind
extracellular matrix ligands and link to the cytoskeleton. Signaling proteins are then differentially regulated to ultimately influence
cell adhesion. One analogy to help is to visualize a spider’s web. The points attaching the web to its surroundings would be the
cell adhesion proteins, namely integrins. The force transmitted through the individual silk fibers relates to the transduction of
mechanical signals through the cytoskeleton, specifically the actin filaments. Similar to a spider sensing these disruptions within
the web, the forces exerted on the cell are ultimately relayed to the nucleus which signals the appropriate phenotypic response.
Several studies eloquently demonstrated how manipulation of substrate stiffness, or elasticity, can be used to control cell
differentiation. One such study by Engler and colleagues (2006) reveal that neurogenic (nerve), myogenic (muscle), and osteogenic
(bone) lineage specification pertaining to cell morphology, RNA profiles, cytoskeletal markers, and transcription factors are
dependent on matrix stiffness. In a more sophisticated recent model, Young and Engler (2011) use time dependent increases in
substrate stiffness to mimic the developmental stiffening of mesoderm into adult myocardium (heart muscle). In brief, they
show that cells cultured on dynamic substrates exhibit a threefold increase in mature cardiac specific markers and up to 60%
more maturing muscle fibers than cells grown on compliant but static polyacrylamide hydrogels.
Porosity/Pore Size
While not directly implicated in traditional metal implants, surface porosity (percentage of void space in a solid) and pore size have
been implicated in regulating the cell–surface interaction. It is worth briefly covering here as many second generation metal
implants now consist of coatings such as hydroxyapatite that include porous surfaces. Furthermore, advancements in biomaterial
design have seen the introduction of porous metal devices. Recent studies suggest comparable healing of segmental defects to
hydroxyapatite scaffolds after 24 weeks implantation in rabbit (Jung et al., 2013; Zhang et al., 2013). This is particularly promising
given that titanium porous implants have superior biomechanical properties compared to hydroxyapatite scaffolds. More recent
developments have also seen successful incorporation of bioactive factors to further enhance osseointegration (Shim et al.,
2013). Porous surfaces provide an interconnected network that distributes oxygen and nutrients as well as eliminating
cytotoxic waste material. Currently, definitive pore sizes for the regeneration of different tissues are not reported. Consequently,
porosity and pore sizes of varying degrees are published for tissues such as bone, cartilage, and muscle. Nevertheless, several key
guidelines have emerged and are highly dependent on the tissue under study. For instance, limited nutrient exchange and cell
migration is achieved if pores are too small. On the contrary, if pores are too large this results in a significant decrease in surface
area thereby limiting cell adhesion and increasing porosity affects the load-bearing capacity of the material (Khoda et al., 2011).
Importantly, the selection of the biomaterial with certain pore size depends on the location to where it is going to be transplanted.
For example, a minimum pore size of 100 mm was initially suggested to be required to generate mineralized bone, however, pores
above 300 mm result in vascularized tissue formation (Karageorgiou and Kaplan, 2005). The latter is imperative for appropriate
bone formation. Even within a tissue such as bone, location will determine the porosity required (Cortical bone has porosity of
only 5–10% whereas trabecular bone ranges between 78% and 92%). Finally, the biomaterial itself may influence effect of
porosity/pore size relating to tissue response (Karageorgiou and Kaplan, 2005).
Bioresorbable Implants – Temporary Implants to Provide a Permanent Solution?
Metal remains the material of choice in loaded regions. The main reason for this monopoly is simply due to the fact that no
alternative material has yet been developed to rival the strength and established clinical success of metal fixators. One promising
material to emerge for use in long bone fracture fixation is the nondegradable polymer poly-(ether–ether)-ketone, or PEEK. Several
characteristics of PEEK have proven desirable for osteosynthesis namely a similar Young’s modulus to bone (thereby limiting the
potential occurrence of stress protection) and its radiolucency (allowing real time imaging of fracture sites without the occurrence of
artifacts commonly associated with the presence of metal implants). A major clinical limitation of its application, however, is the
fact that PEEK is not bioactive. In essence, therefore, it does not interact with bone to produce an integrated surface which is
necessary for stability in loaded regions. As the market leader of PEEK for medical device applications, Invibio (2013) have explored
several approaches of surface modification for ‘biologically activating’ the surface of PEEK to improve osseointegration including
the incorporation of osteoconductive hydroxyapatite. More recently, it has also been shown that a PEEK biomimetic surface can
be achieved using plasma treatment, a method of chemically altering the outermost surface layers. This method successfully
incorporates biomolecules that have been shown to enhance biological functionality (Hayes et al., 2013).
In terms of temporary devices, in that the device is implanted with no intention of removal and in the hope that it will resorb
subsequent to its function being fulfilled, success is mainly limited to nonloaded regions such as craniomaxillofacial bones. Again,
this is a direct result of the current lack of bioresorbable materials that have the strength to withstand high recurring loads. Some
recent studies have shown promise in preclinical in vivo models but have noted that full-weight bearing may not be indicated
initially to allow healing to occur (Neumann et al., 2013). Nevertheless, experimental data within craniomaxillofacial regions
have been promising (Turvey et al., 2011) and is advocated for pediatric fractures particularly (Ahmed et al., 2013; Degala et al.,
2013). However, there remains some concern relating to inadequate fracture stability and implant breakage (Singh et al., 2013).
The most popular polymers used in these applications are variations of polylactic and polyglycolic acid given that their products
are considered innocuous although inflammation and osteolysis have been reported (Simon et al., 1997; Turvey et al., 2011).
The developmental leap to long bone fracture fixation remains a major advance that has yet to be achieved and as such metal
remains the material of choice for the foreseeable future, with the exception of nondegradable polymer composites such as
biologically modified PEEK.
Treatment of Nonunions – A Brief Perspective into Scaffolds for Tissue Engineering
While the evolution of fracture fixation devices has occurred for over a century it is only since the 1970s that cells were combined
with scaffolds in an effort to ‘engineer’ tissue. Since then the field of tissue engineering, or regenerative medicine as it is more
commonly referred to nowadays, has expanded at a staggering rate. In terms of research alone, this has been represented by
a mere 12 manuscripts being published in 1990, increasing to over 400 in 2000 and more recently over 11 000 in 2013 alone.
This trend is also reflected in the billion dollar investments within this field and the equal revenue generated.
In keeping with bone, approximately 5–13% of fractures result in delayed/nonunion which results in the need for secondary
intervention such as bone grafting. Unfortunately, current reconstructive strategies have yet to produce a satisfactory level of
clinical predictability as well as encountering several other limitations such as immune rejection and severe tissue donor shortages.
Therefore, research focus has shifted largely to degradable and nondegradable synthetic and natural matrices as a cell therapy
approach for addressing this issue. As will be noted in previous sections, scaffolds for bone tissue engineering can be crudely divided
into degradable (temporary) or nondegradable (permanent) materials. Materials include ceramics such as hydroxyapatite and
or tricalcium phosphate (in various forms and chemical ratios), natural (collagen, platelet rich plasma) or synthetic polymers
(polylactic acid, polyglycolic acid) and more recently composite or hybrid materials. Several studies exist that have used
combinations of the approaches listed above to produce bone (Chen et al., 2010). A limitation of this strategy however, is use
of osteoconductive materials (a material that will support cell attachment but will not drive differentiation). The inclusion of
bone ‘inducing’ growth factors such as bone morphogenetic protein (BMP)-2 and BMP-7 has provided an osteoinductive (ability
to induced osteoblast differentiation/function) dimension to tissue engineering.
While limited success has been achieved the new generation of bone tissue constructs tend to favor multiphase materials that
improve overall mechanical strength and allow different rates of degradation and thus differing rates of growth factor release
to improve tissue integration. Again, the data to emerge from these studies have attracted interest, nonetheless, experimental
inconsistencies across laboratories have meant the ‘correct’ combination of factors, their concentration, time of release, and carrier
type are all widely debated (Chen et al., 2010). Moreover, preclinical data show promise for cell free (Lee et al., 2011; Lyons et al.,
2010) and cell seeded scaffolds (Janicki et al., 2010) for de novo bone formation, the major clinical limitation for widespread
application of these therapeutic strategies is lack of vascularization. Bone cannot survive without the presence of local vasculature.
Therefore, the recently revised approach for bone tissue engineering incorporates the need for angiogenesis, the formation of blood
vessels. It is fair to state that research in this area is relatively exploratory with some noteworthy data emerging (Wernike et al., 2010;
Amini et al., 2012; Nguyen et al., 2012). Nevertheless, reproduction at a clinical scale is yet to be achieved and remains the major
restraint for routine clinical application of engineered bone constructs (Nguyen et al., 2012, for review).
Summary
Many noteworthy developments have been made for improving and controlling tissue integration upon implantation of a device.
Despite advances, the widespread clinical application and success of these developments are relatively limited. Currently metal still
remains the material of choice for fracture fixation given that its desirable mechanical properties are yet to be equaled by its
alternatives. Nonetheless, more eloquent surface modifications have seen a new wave of materials such as PEEK becoming
more serious contenders. There remains much debate regarding the reasoning behind elective removal of devices. However,
microtopographical polishing of titanium and its alloys have proved to ease the intraoperative issues of excessive bony
on-growth thus allowing the debate to fundamentally focus on secondary health issues. Bioresorbable implants have proven
effective for low stress loaded regions such as craniomaxilliofacial; however, their adaption for long bone fracture remains
exploratory. In the field of bone tissue engineering, the overwhelming differences in experimental ideals have meant that progress
to the clinics has been slow. It is predicated that this will remain to be the case until more definitive requirement guidelines that
incorporate the need for vascularization are agreed.
References
Ahmed, W., Ali Bukhari, S. G., Janjua, O. S., et al. (2013). Bioresorbable versus titanium plates for mandibular fractures. J. Coll. Physicians Surg. Pak., 23(7), 480–483.
Akeson, W. H., Woo, S. L., Rutherford, L., et al. (1976). The effects of rigidity of internal fixation plates on long bone remodeling. A biomechanical and quantitative histological study.
Acta Orthop. Scand., 47, 241–249.
Amini, A. R., Laurencin, C. T., & Nukavarapu, S. P. (2012). Differential analysis of peripheral blood- and bone marrow-derived endothelial progenitor cells for enhanced
vascularization in bone tissue engineering. J. Orthop. Res., 30(9), 1507–1515.
ap Gwynn, I., & Wilson, C. (2001). Characterizing fretting particles by analysis of SEM images. Eur. Cell. Mater., 1, 1–11.
Bagatur, A. E., & Zorer, G. (2002). Complications associated with surgically treated hip fractures in children. J. Pediatr. Orthop. B, 11, 219–228.
Boyan, B. D., Lossdorfer, S., Wang, L., et al. (2003). Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies. Eur. Cell.
Mater., 6, 22–27.
Broekhuizen, C. A., de Boer, L., Schipper, K., et al. (2008). Staphylococcus epidermidis is cleared from biomaterial implants but persists in peri-implant tissue in mice despite
rifampicin/vancomycin treatment. J. Biomed. Mater. Res. A, 85(2), 498–505.
Case, C. P., Langkamer, V. G., James, C., et al. (1994). Widespread dissemination of metal debris from implants. J. Bone Joint Surg. Br., 76(5), 701–712.
Chen, C. E., Ko, J. Y., & Wang, C. J. (2002). Premature closure of the physeal plate after treatment of a slipped capital femoral epiphysis. Chang Gung Med. J., 25, 811–818.
Chen, F. M., Zhang, M., & Wu, Z. F. (2010). Toward delivery of multiple growth factors in tissue engineering. Biomaterials, 31(24), 6279–6308.
Costa, D. O., Prowse, P. D., Chrones, T., et al. (2013). The differential regulation of osteoblast and osteoclast activity by surface topography of hydroxyapatite coatings. Biomaterials,
34(30), 7215–7226.
Dalby, M. J. (2005). Topographically induced direct cell mechanotransduction. Med. Eng. Phys., 27(9), 730–742.
Davies, J. E., Ajami, E., Moineddin, R., et al. (2013). The roles of different scale ranges of surface implant topography on the stability of the bone/implant interface. Biomaterials,
34(14), 3535–3546.
Degala, S., Shetty, S., & Ramya, S. (2013). Fixation of zygomatic and mandibular fractures with biodegradable plates. Ann. Maxillofac. Surg., 3(1), 25–30.
Disegi, J., & Eschbach, L. (2000). Stainless steel in bone surgery. Injury, Int. J. Care Injured, 31, SD2–6.
Domingo, J. (1994). Metal-induced developmental toxicity in mammals: a review. J. Toxicol. Environ. Health, 42, 123–141.
Doran, A., Law, F. C., Allen, M. J., & Rushton, N. (1998). Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants. Biomaterials,
19, 751–759.
Elbetieha, A., & Al-Hamood, M. H. (1997). Long-term exposure of male and female mice to; trivalent and hexavalent chromium compounds: effect on fertility. Toxicology, 116,
39–47.
Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (August 25, 2006). Matrix elasticity directs stem cell lineage specification. Cell, 126(4), 677–689.
Evans, N. A., & Evans, R. O. (1997). Playing with metal: fracture implants and contact sport. Br. J. Sports Med., 31, 319–321.
Frigg, R. (2003). Development of the LCP. Injury, 34, 31–42.
Fryzek, J. P., Ye, W., Signorello, L. B., et al. (2002). Incidence of cancer among patients with knee implants in Sweden, 1980-1994. Cancer, 94(11), 3057–3062.
Gittens, R. A., McLachlan, T., Olivares-Navarrete, R., et al. (2011). The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation
and differentiation. Biomaterials, 32(13), 3395–3403.
Gunst, M. A., Suter, C., & Rahn, B. A. (1979). Bone perfusion after plate osteosynthesis: a study of the intact rabbit tibia with disulfin blue vital staining. Helv. Chir. Acta, 46,
171–175.
Hallab, N. J., Caicedo, M., Finnegan, A., & Jacobs, J. J. (2008). Th1 type lymphocyte reactivity to metals in patients with total hip athroplasty. J. Orthop. Surg. Res., 3, 6–16.
Hanawa, T. (2004). Metal ion release from metal implants. Mater. Sci. Eng., 24, 745–752.
Hayes, J. S., & Richards, R. G. (2010a). Surfaces to control tissue adhesion for osteosynthesis with metal implants: in vitro and in vivo studies to bring solutions to the patient. Expert
Rev. Med. Devices, 7(1), 131–142.
Hayes, J. S., & Richards, R. G. (2010b). The use of titanium and stainless steel in fracture fixation. Expert Rev. Med. Devices, 7(6), 843–853.
Hayes, J. S., Vos, D. I., Hahn, J., et al. (2009). An in vivo evaluation of surface polishing of TAN intermedullary nails for ease of removal. Eur. Cell. Mater., 18, 15–26.
Hayes, J. S., Seidenglanz, U., Pearce, A. I., et al. (2010a). Surface polishing positively influences ease of plate and screw removal. Eur. Cell. Mater., 26(19), 117–126.
Hayes, J. S., Khan, I. M., Archer, C. W., & Richards, R. G. (2010b). The role of surface microtopography in the modulation of osteoblast differentiation. Eur. Cell. Mater., 20,
98–108.
Hayes, J. S., Welton, J. L., Wieling, R., & Richards, R. G. (2012a). In vivo evaluation of defined polished titanium surfaces to prevent soft tissue adhesion. J. Biomed. Mater. Res. B
Appl. Biomater., 100(3), 611–617.
Hayes, J. S., Czekanska, E. M., & Richards, R. G. (2012b). The cell-surface interaction. Adv. Biochem. Eng. Biotechnol., 126, 1–31.
Hayes, J. S., Devine, D. M., & Murphy, M. (2013). Plasma Biofunctionalisation of poly-(ether-ether)-ketone for improved human mesenchymal stem cell functionality. In Proceedings
of the Orthopaedic Research Society Annual Meeting 2013. Paper 0145.
Heath, J. C., Freeman, M. A., & Swanson, S. A. (1971). Carcinogenic properties of wear particles from prostheses made in cobalt-chromium alloy. Lancet, 1, 564–566.
Highland, T. R., & LaMont, R. L. (1985). Deep, late infections associated with internal fixation in children. J. Pediatr. Orthop., 5, 59–64.
Invibio Website, 2013. http://www.invibio.com/biocompatible-polymers/biocompatible-polymers.php (accessed August 2013).
Janicki, P., Kasten, P., Kleinschmidt, K., et al. (2010). Chondrogenic pre-induction of human mesenchymal stem cells on beta-TCP: enhanced bone quality by endochondral
heterotopic bone formation. Acta Biomater., 6(8), 3292–3301.
Jaworski, Z. F. G., Liskova-Kiar, M., & Uhthoff, H. K. (1980). Regional disuse osteoporosis and factors influencing its reversal. In H. K. Uhthoff (Ed.), Current Concepts of Internal
Fixation of Fractures (pp. 17–26). Berlin: Springer.
Jung, H. D., Yook, S. W., Han, C. M., et al. (2013). Highly aligned porous Ti scaffold coated with bone morphogenetic protein-loaded silica/chitosan hybrid for enhanced bone
regeneration. J. Biomed. Mater. Res. B Appl. Biomater. PMID: 24259198 [Epub ahead of print].
Kanojia, R. K., Junaid, M., & Murthy, R. C. (1998). Embryo and fetotoxicity of hexavalent chromium: a long-term study. Toxicol. Lett., 95(3), 165–172.
Karageorgiou, V., & Kaplan, D. (2005). Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials, 26(27), 5474–5491.
Kasai, Y., Iida, R., & Uchida, A. (2003). Metal concentrations in the serum and hair of patients with titanium alloy spinal implants. Spine, 15, 1320–1326.
Katzer, A., Hockertz, S., Buchhorn, G. H., & Loehr, J. F. (2003). In vitro toxicity and mutagenicity of CoCrMo and Ti6Al wear particles. Toxicology, 190, 145–154.
Keegan, G. M., Learmonth, I. D., & Case, C. P. (2007). Orthopaedic metals and their potential toxicity in the arthroplasty patient. J. Bone J. Surg., 89(5), 567–573.
Keel, S. B., Jaffe, K. A., Petur, N., et al. (2001). Orthopaedic implant related sarcoma: a study of 12 cases. Mod. Pathol., 14, 969–977.
Khan, U., Kakar, S., Akali, A., et al. (2000). Modulation of the formation of adhesions during healing of injured tendons. J. Bone Joint Surg. Br., 82, 1054–1058.
Khoda, A. K., Ozbolat, I. T., & Koc, B. (2011). Engineered tissue scaffolds with variational porous architecture. J. Biomech. Eng., 133(1), 011001.
Labosky, D. A., Cermak, M. B., & Waggy, C. A. (1990). Forearm fracture plates: to remove or not remove. J. Hand Surg., 15A(2), 294–300.
Lee, G. S., Park, J. H., Shin, U. S., & Kim, H. W. (2011). Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering.
Acta Biomater., 7(8), 3178–3186.
Lew, D. P., & Waldvogel, F. A. (2004). Osteomyelitis. Lancet, 364, 369–379.
Lhotka, C., Szekeres, T., Steffan, I., Zhuber, K., & Zweymüller, K. (2005). Four-year study of cobalt and chromium blood levels in patients managed with two different metal-on-
metal total hip replacements. J. Orthop. Res., 21(2), 189–195.
Lyons, F. G., Al-Munajjed, A. A., Kieran, S. M., et al. (2010). The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered
constructs. Biomaterials, 31(35), 9232–9243.
McMurray, R. J., Gadegaard, N., Tsimbouri, P. M., et al. (2011). Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat.
Mater., 10(8), 637–644.
Mendes, L. S., Saska, S., Martines, M. A., et al. (2013). Nanostructured materials based on mesoporous silica and mesoporous silica/apatite as osteogenic growth peptide carriers.
Mater. Sci. Eng. C Mater. Biol., 33(7), 4427–4434.
Meredith, D. O. (2006). Biocompatibility of Orthopaedic Metal Implants: The Influence of Surface Chemistry and Topography. Davos, CH: Thesis from AO Research Institute. Chapter
2. Metals & Surfaces, pp. 19–41.
Meredith, D. O., Riehle, M. O., Curtis, A. S., et al. (2007). Microtopography of metal surfaces influence fibroblast growth by modifying cell shape, cytoskeleton & adhesion. J.Orthop.
Res., 25(11), 1523–1533.
Milavec-Puretic, V., Orlic, D., & Marusic, A. (1998). Sensitivity to metals in 40 patients with failed hip endoprosthesis. Arch. Orthop. Trauma Surg., 117(6–7), 383–386.
Morgan, M. S., & Schaller, K. H. (1999). An analysis of criteria for biological limit values developed in Germany and in the United States. Int. Arch. Occup. Environ. Health, 72(4),
195–204.
Neumann, H., Schulz, A. P., Gille, J., et al. (2013). Refixation of osteochondral fractures by ultrasound-activated, resorbable pins: an ovine in vivo study. Bone Joint Res., 2(2),
26–32.
Nguyen, L. H., Annabi, N., Nikkhah, M., et al. (2012). Vascularized bone tissue engineering: approaches for potential improvement. Tissue Eng. Part B Rev., 18(5), 363–382.
O’Hare, P., Meenan, B. J., Burke, G. A., et al. (2010). Biological responses to hydroxyapatite surfaces deposited via a co-incident microblasting technique. Biomaterials, 31(3),
515–522.
Olivares-Navarrete, R., Hyzy, S. L., Park, J. H., et al. (2011). Mediation of osteogenic differentiation of human mesenchymal stem cells on titanium surfaces by a Wnt-integrin
feedback loop. Biomaterials, 32(27), 6399–6411.
Oliveira, H., Santos, T. M., Ramalho-Santos, J., et al. (2006). Histopathological effects of hexavalent chromium in mouse kidney. Bull. Environ. Contam. Toxicol., 76, 977–983.
Olivieri, G., Novakovic, M., Savaskan, E., et al. (2002). The effects of b-estradiol on SHSY5Y neuroblastoma cells during heavy metal induced oxidative stress, neurotoxicity and
betaamyloid secretion. Neuroscience, 113, 849–855.
Pearce, A. I., Pearce, S. G., Schwieger, K., et al. (2008). Effect of surface topography on removal of cortical bone screws in a novel sheep model. J. Orthop. Res., 26(10),
1377–1383.
Perren, S. M., Cordey, J., Rahn, B. A., et al. (1988). Early temporary porosis of bone induced by internal fixation implants: a reaction to necrosis, not to stress protection? Clin.
Orthop., 232, 139–151.
Peterson, H. A. (2005). Metallic implant removal in children. J. Pediatr. Orthop., 25, 107–115.
Pilger, A., Schaffer, A., Rüdiger, H. W., & Osterode, W. (2002). Urinary 8-hydroxydeoxyguanosine and sister chromatid exchanges in patients with total hip replacements. J. Toxicol.
Environ. Health A, 65(9), 655–664.
Rammelt, S., Schulze, E., Witt, M., et al. (2004). Collagen type I increases bone remodelling around hydroxyapatite implants in the rat tibia. Cells Tissues Organs, 178(3), 146–157.
Rhinelander, F. W. (1965). Some aspects of the microcirculation of healing bone. Clin. Orthop., 40, 12.
Rivera-Chacon, D. M., Alvarado-Velez, M., Acevedo-Morantes, C. Y., et al. (2013). Fibronectin and vitronectin promote human fetal osteoblast cell attachment and proliferation on
nanoporous titanium surfaces. J. Biomed. Nanotechnol, 9(6), 1092–1097.
Schaffer, A. W., Pilger, A., Engelhardt, C., Zweymueller, K., & Ruediger, H. W. (1999). Increased blood cobalt and chromium after total hip replacement. J. Toxicol. Clin.Toxicol.,
37(7), 839–844.
Schlegel, P., Hayes, J. S., Frauchiger, V. M., et al. (2009). An in vivo evaluation of the biocompatibility of anodic plasma chemical (APC) treatment of titanium with calcium
phosphate. J. Biomed. Mater. Res. B Appl. Biomater., 90(1), 26–34.
Schmalzried, T. P., Grogan, T. J., Neumeier, R. N., & Dorey, F. J. (1991). Metal removal in a pediatric population: benign procedure or a necessary evil? J. Pediatr. Orthop, 11(1),
72–76.
Schwartz, Z., Nasazky, E., & Boyan, B. D. (2005). Surface microtopography regulates osteointegration: the role of implant surface microtopography in osteointegration. Alpha
Omegan, 98(2), 9–19.
Seipel, R. C., Schmeling, G. J., & Daley, R. (2001). Migration of a K-wire from the distal radius to the heart. Am. J. Orthop., 30, 147–151.
Shim, I. K., Chung, H. J., Jung, M. R., et al. (2013). Biofunctional porous anodized titanium implants for enhanced bone regeneration. J. Biomed. Mater. Res. A. PMID: 24265190
[Epub ahead of print].
Signorello, L. B., Ye, W., Fryzek, J. P., et al. (2001). Nationwide study of cancer risk among hip replacement patients in Sweden. J. Natl. Cancer Inst., 93, 1405–1410.
Simon, J. A., Ricci, J. L., & Di Cesare, P. E. (1997). Bioresorbable fracture fixation in orthopedics: a comprehensive review. Part II. Clinical studies. Am. J. Orthop. (Belle Mead NJ),
26(11), 754–762.
Singh, V., Kshirsagar, R., Halli, R., et al. (2013). Evaluation of bioresorbable plates in condylar fracture fixation: a case series. Int. J. Oral Maxillofac. Surg. S0901-5027(13)
00286–5.
Sinicropi, S. M., Su, B. W., Raia, F. J., et al. (2005). The effects of implant composition on extensor tenosynovitis in a canine distal radius fracture model. J. Hand Surg. Am., 30,
300–307.
Sjöström, T., Dalby, M. J., Hart, A., et al. (2009). Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells. Acta Biomater.,
5(5), 1433–1441.
Sun, Y., Deng, Y., Ye, Z., et al. (2013). Peptide decorated nano-hydroxyapatite with enhanced bioactivity and osteogenic differentiation via polydopamine coating. Colloids Surf. B
Biointerfaces, 111C, 107–116.
Tepic, S., 2008. Locking Screw Implants in Internal Fixation. Presentation: 15.06.2008 at European cells and Materials IX congress: Muscloskeletal trauma: 50 years of AO
research.
Tepic, S., & Perren, S. M. (1995). The biomechanics of the PC-Fix internal fixator. Injury, 26, 5–10.
Turvey, T. A., Proffit, W. P., & Phillips, C. (2011). Biodegradable fixation for craniomaxillofacial surgery: a 10-year experience involving 761 operations and 745 patients. Int. J. Oral
Maxillofac. Surg., 40(3), 244–249.
Uhthoff, H. K., & Jaworski, Z. F. (1978). Bone loss in response to long-term immobilisation. J. Bone Joint Surg, 60, 420–429.
Uhthoff, H. K., Boisvert, D., & Finnegan, M. (1994). Cortical porosis under plates. Reaction to unloading or to necrosis? J. Bone Joint Surg Am., 76, 1507–1512.
Uhthoff, H. K., Poitras, P., & Backman, D. S. (2006). Internal plate fixation of fractures: short history and; recent developments. J. Orthop. Sci., 11, 118–126.
Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., & Mazur, M. (2006). Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact, 160, 1–40.
Vaudaux, P., & Lew, D. P. (2006). Tolerence of staphylococci to bactericidal antibiotics. Injury, 37, 15–19.
Wagner, M. (2003). General principles for the clinical use of the LCP. Injury, 34, 31–42.
Wernike, E., Montjovent, M. O., Liu, Y., et al. (2010). VEGF incorporated into calcium phosphate ceramics promotes vascularisation and bone formation in vivo. Eur. Cell. Mater.,
22(19), 30–40.
Wilson, C. M. G. (1999). In vitro Biocompatibility Evaluation and Morphological Description of Fretting Wear Debris from Orthopaedic Implant Materials. PhD thesis: Institute of
biological sciences. Aberystwyth, UK: University of Wales.
Witzleb, W. C., Ziegler, J., Krummenauer, F., et al. (2006). Exposure to chromium, cobalt and molybdenum from metal-on-metal total hip replacement and hip resurfacing
arthroplasty. Acta Orthop., 77(5), 697–705.
Young, J. L., & Engler, A. J. (2011). Hydrogels with time-dependent material properties enhance cardiomyocyte differentiation in vitro. Biomaterials, 32(4), 1002–1009.
Zhang, M., Wang, G. L., Zhang, H. F., et al. (2013). Repair of segmental long bone defect in a rabbit radius nonunion model: comparison of cylindrical porous titanium and
hydroxyapatite scaffolds. Artif. Organs. PMID: 24372398 [Epub ahead of print].
Zreiqat, H., Akin, F. A., Howlett, C. R., et al. (2003). Differentiation of human bone-derived cells grown on GRGDSP-peptide bound titanium surfaces. J. Biomed. Mater. Res. A, 64(1),
105–111.
Tissue Response to Biomaterials
Jiao Jiao Li, University of Sydney, Sydney, NSW, Australia; Kolling Institute, Northern Sydney Local Health District, St Leonards,
NSW, Australia; and Sydney Medical School Northern, University of Sydney, St Leonards, NSW, Australia
Hala Zreiqat, University of Sydney, Sydney, NSW, Australia

Sequence of Tissue Responses to Biomaterials 271
Initial Response to Injury 271
Provisional matrix formation 271
Inflammation 272
Acute inflammation 272
Chronic inflammation 272
Granulation tissue formation 273
Foreign Body Reaction 273
Fibrous Capsule Formation 273
Influence of Biomaterial Properties on Directing Tissue Responses 273
Bioinert, Bioactive, and Biodegradable Materials 274
Metals 274
Polymers 274
Ceramics 275
Summary 277
Further Reading 277
Glossary
Apoptosis Process of programmed cell death.
Autograft A tissue graft transplanted from one part of the body to another in the same person.
Cytokines Small proteins secreted by cells with specific effects on the interactions and communications between cells.
Extracellular matrix A collection of extracellular molecules secreted by cells that provide structural and biochemical support to
the surrounding cells.
Exudation Escape of fluid, proteins, and blood cells from the vascular system into tissues in response to injury.
Growth factors Proteins that can stimulate or regulate many aspects of cell function, such as survival, proliferation, migration,
and differentiation.
Neovascularization Formation of new blood vessels or capillaries through the proliferation, maturation, and organization of
endothelial cells.
Osseointegration Direct structural and functional connection between living bone and the surface of a load-bearing artificial
implant.
Phagocytosis Process by which living cells ingest or engulf other cells, particles, or material fragments.
Scaffold Biomaterial(s) engineered into a three-dimensional construct to support or induce cellular activities necessary for
tissue repair or regeneration.
Stress shielding Bone resorption and reduction in bone density due to the removal of typical levels of stress from the bone by
an orthopedic implant.
Tissue Response to Biomaterials
Biomaterials are natural or synthetic materials that are intended for use in a biological environment, often in the form of implants or
medical devices. They are the central element of biomedical engineering solutions to replace, restore, or improve the function of
diseased or damaged tissues or organs. Bioactive biomaterials can even be used to induce natural tissue healing. All biomaterials
placed inside the body will elicit tissue responses, the nature and extent of which depends on the biocompatibility and bioactivity
of the biomaterial. This article will introduce the sequence of tissue responses to biomaterials, including injury, provisional matrix
formation, acute inflammation, chronic inflammation, granulation tissue formation, foreign body reaction, and fibrous capsule
development. This will be followed by a summary of characteristics of the common classes of biomaterials for use in medical

Biomaterials: In vitro and in vivo studies of biomaterials j Tissue Response to Biomaterials 271
applications, including metals, polymers, and ceramics, and the influence of these characteristics on directing the tissue response to
different classes of biomaterials.
Sequence of Tissue Responses to Biomaterials
The implantation of a biomaterial into the body triggers a cascade of tissue responses involving inflammatory and wound healing
responses to the injury, as well as the body’s responses to the foreign material (Fig. 1). The inflammatory and wound healing
responses to injury are common across different classes of biomaterials, but the nature and extent of the body’s responses to foreign
material are determined by the properties of the biomaterial, which include but are not limited to its composition, degradation rate,
morphology, shape and size, porosity, roughness, and surface chemistry.
Initial Response to Injury

Immediately following the injury elicited by biomaterials implantation, changes in blood flow occur that cause the exudation of
fluid, proteins, and cells from the vascular system into the injured tissue. The response to injury in vascularized connective tissue
is similar across different tissues and organs. This response triggers the cellular events that later occur as part of the inflammatory
response.
Provisional matrix formation

The initial blood–biomaterial interactions result in blood protein deposition on the biomaterial surface, and are the first step in
provisional matrix formation. The provisional matrix, or blood clot, forms within minutes to hours of biomaterial implantation
and consists of several components, including fibrin, inflammatory products released by the complement system, activated blood
platelets, inflammatory cells, and endothelial cells. Fibrin is the blood-coagulating protein produced from fibrinogen by activation
of the coagulation and thrombosis systems. It is the key component of the provisional matrix and has an important role in facil-
itating neovascularization to support the wound healing process. Platelets are activated during the formation of the fibrin network,
and contribute to fibroblast recruitment by releasing a number of growth factors such as platelet-derived growth factor and
Fig. 1 Sequence of tissue responses following biomaterials implantation.

272 Biomaterials: In vitro and in vivo studies of biomaterials j Tissue Response to Biomaterials
transforming growth factor b. The fibrin network is also decorated with adhesive proteins such as fibronectin and thrombospondin,
providing substrates for cell adhesion and migration. The provisional matrix hence acts as a sustained release system, supplying
a rich mixture of substances that initiate tissue repair, recruit inflammatory cells and fibroblasts, and facilitate ongoing wound heal-
ing processes.
Inflammation
Inflammation is defined as the reaction of vascularized living tissue to local injury. It serves to contain, neutralize, dilute, or isolate
the agent or process causing the injury. In the context of biomaterials implantation, inflammation triggers a series of events that may
heal and reconstitute the implant site through the formation of fibroblastic scar tissue, or if possible, regeneration of the original
tissue. The intensity and duration of the inflammatory response depends on the severity or invasiveness of the implantation proce-
dure, and the biocompatibility of the implant. The inflammatory response comprises an initial acute phase and a subsequent
chronic phase, which are followed by granulation tissue formation (Fig. 2).
Acute inflammation
Acute inflammation is usually of short duration, lasting from minutes to days depending on the severity of the injury. It is marked
by the release of fluid and blood plasma proteins, and the arrival of leukocytes which initially comprise neutrophils and later macro-
phages. Excess blood flow into the site of injury due to vessel dilation results in the influx of numerous blood and tissue proteins
such as cytokines and growth factors. These attract the infiltration and accumulation of leukocytes from the blood vessels into the
injury site, which constitute the most important feature of the inflammatory reaction. Neutrophils are the primary cell population
during the first few days following injury. They arrive in large numbers and are primarily involved in the early prevention of infec-
tion, by phagocytosing microorganisms and foreign materials, and cleaning up injury-related debris. Neutrophils are relatively short
lived and typically disintegrate and disappear after 24–48 h. In the following days to weeks, the neutrophils are replaced by mono-
cytes which differentiate into macrophages. These cells are very long lived and can persist for up to months. The macrophages
phagocytose microorganisms, while also cleaning up the remains of dead tissue cells and neutrophils. Acute inflammation usually
resolves within a week, a prolonged course of which is probably indicative of infection.
Chronic inflammation
Chronic inflammation is caused by persistent inflammatory stimuli, such as the continuous presence of a biomaterial implant.
Chronic inflammation can persist for weeks to months or even years, and the extent of the response depends on the degree of injury.
It is generally characterized by the presence of monocytes, macrophages, and lymphocytes. This is accompanied by the proliferation
of blood vessels, which is essential for supplying the necessary nutrients to support wound healing, and proliferation of connective
tissue to reconstruct the injury site. Of the cell types present in chronic inflammation, macrophages are the most important as they
secrete a large collection of biologically active products. These products have a key role in promoting the growth of fibroblasts and
blood vessels, as well as tissue regeneration and remodeling.
The chronic inflammatory response to biomaterials may be caused by the properties or size of the biomaterial, or its motion
within the implant site, but is usually of short duration and confined to the implant site. Due to their size disparity, the biomaterial
can rarely be engulfed by the attached inflammatory cells and can lead to frustrated phagocytosis, where the continuous release of
Fig. 2 The temporal variation in the acute inflammatory response, chronic inflammatory response, granulation tissue development, and foreign
body reaction to implanted biomaterials. The intensity and time variables depend on the extent of injury created by the implantation, as well as the
size, shape, topography, and chemical and physical properties of the biomaterial. Reproduced from Anderson JM and Shive MS (2012) Biodegrada-
tion and biocompatibility of PLA and PLGA microspheres. Advanced Drug Delivery Reviews 64: 72–82, with permission from Elsevier.
leukocyte products occurs in an attempt to degrade the biomaterial. Chronic inflammation with the presence of collections of
lymphocytes and monocytes at extended implant times, from weeks to months to years, may be an indication of long-term
infection.
Granulation tissue formation

Following biomaterial implantation and initiation of the healing process by the action of monocytes and macrophages, the forma-
tion of granulation tissue commences through the proliferation of fibroblasts and vascular endothelial cells at the implant site.
Granulation tissue may appear as early as 3–5 days following biomaterial implantation, with the characteristic features of prolif-
erating fibroblasts and small blood vessels. It is found on the surface of healing wounds and has a soft and pink granular appear-
ance. The small blood vessels are formed through neovascularization, which involves the proliferation, maturation, and
organization of endothelial cells into blood capillaries. The connective tissue in the granulation tissue is developed by fibroblasts,
which actively synthesize collagen and proteoglycans. The proteoglycans predominate in the early stages, and this later changes into
collagen (especially collagen type I) which forms the fibrous capsule. Macrophages are almost always present in granulation tissue,
but fibroblasts typically dominate.
The mode of wound healing depends on the extent or severity of the injury or defect associated with the biomaterial implan-
tation. In a clean wound with neat wound edges such as that created by surgical incision, healing proceeds with minimal infection
and tissue loss. However, in a large defect associated with extensive tissue loss, the original architecture cannot be completely recon-
stituted by the regenerating tissue, resulting in significant fibrosis and the formation of large amounts of granulation or scar tissue
that will often remain permanently.
Foreign Body Reaction

While inflammation and granulation tissue formation are common to normal wound healing and biomaterial implantation, the
foreign body reaction stage is unique to biomaterials. The foreign body reaction to biomaterials consists of foreign body giant cells
(multinucleated macrophages) and the components of granulation tissue, including fibroblasts, capillaries, and macrophages. The
nature of the foreign body reaction is determined by the form and topography of the implanted biomaterial. For example, implants
with smooth and flat surfaces such as silicone breast prostheses have a foreign body reaction composed of a layer of macrophages
one to two cells in thickness. In contrast, implants with rough surfaces such as expanded polytetrafluoroethylene vascular prostheses
have a foreign body reaction consisting of individual macrophages and foreign body giant cells on the surface, with varying degrees
of granulation tissue adjacent to the macrophage layer. A response similar to that for rough surfaces is seen for particles of implant
debris or small fragments of wear debris generated by the implant.
The foreign body reaction may be present at the tissue–implant interface for the lifetime of the implant. Multinucleated foreign
body giant cells, which are coalesced tissue macrophages derived from circulating blood monocytes, typically persist on implanted
biomaterial surfaces for decades and may contribute to implant biodegradation. There is evidence for the involvement of antiin-
flammatory Th2 helper lymphocytes in the development of the foreign body reaction to implanted biomaterials, which is mediated
by interleukin (IL)-4 and IL-13. Downstream of this, the macrophage mannose receptor has a critical role in the fusion of macro-
phages to form foreign body giant cells. Macrophages and foreign body giant cells which remain adherent on biomaterial surfaces
may continue to release cytokines or become quiescent, and their apoptosis is in part controlled by the surface chemistry of the
biomaterial. The foreign body reaction is a precursor stage to fibrous tissue encapsulation of the biomaterial.
Fibrous Capsule Formation

The end stage of the foreign body reaction and healing response to a biomaterial is often fibrous encapsulation. This involves fibro-
blasts creating a vascular and collagenous fibrous capsule that confines the implant and prevents it from interacting with the
surrounding tissue. Fibrous capsule formation is common for large impervious implants such as breast prostheses, and typically
occurs for bulk bioinert and biocompatible materials.
A number of exceptions result from the wound healing cascade for biomaterials with specific properties. Porous biocompatible
materials are not surrounded by a fibrous capsule because they allow the ingrowth of connective tissue within the pores. Porous or
nonporous bioactive materials are also not surrounded by a fibrous capsule because they interact favorably with native tissues and
encourage direct tissue apposition or bonding to the material surface. Biodegradable materials do not have a persisting foreign body
reaction or fibrous capsule because they are eventually metabolized by the body and removed through physiological processes.
Materials with toxic properties, which should not be used for biomedical applications, cause prolonged complications and ongoing
cell death and often do not proceed to the end stages of the healing process.
Influence of Biomaterial Properties on Directing Tissue Responses
All biomaterials implanted into the body will elicit tissue responses which in turn influence the function of the biomaterial. A crit-
ical component in the development of biomedical engineering devices is to select the appropriate biomaterials and processing
methods that will result in favorable tissue responses, to enhance or at least not interfere with the function of the device. Bioinert,
bioactive, and biodegradable materials each produce a different set of tissue responses, due to changes in material behavior that
influence biological interactions. The classes of biomaterials used in biomedical engineering, which can be broadly divided into
metals, polymers, and ceramics, also each exhibit specific properties that generate different tissue responses. Regardless of their clas-
sification, it is important to note that all biomaterials used for biomedical engineering applications should be biocompatible,
meaning that they should not be toxic or harmful to living tissue and should not cause immunological rejection after implantation.
Bioinert, Bioactive, and Biodegradable Materials

Bioinert materials are nontoxic, biologically inactive materials that cause the formation of a fibrous capsule with variable thickness
around the implant. Highly bioinert materials, such as alumina, zirconia, silicon nitride, and gold, can have a very thin fibrous
capsule if the implant has a tight mechanical fit, but the fibrous capsule can be hundreds of microns thick in the case of a loosely
fitted implant that enables movement. Weakly bioinert materials, such as stainless steel and nitinol, have a proportionally much
thicker fibrous capsule.
Bioactive materials are nontoxic, biologically active materials that induce the formation of a direct chemical bond between the
implant and host tissue by eliciting a biological response at the interface. These materials are often also biodegradable and are exten-
sively used in tissue engineering and regenerative medicine. They are favored by the body and can play an active role in promoting
tissue repair and regeneration at the implant site. Examples include calcium phosphates which can form a direct bond with hard
tissues such as bone, and bioactive glasses which can form a direct bond with both hard tissues and soft tissues such as muscle
and ligament.
Biodegradable materials are nontoxic, biologically degradable materials that become gradually replaced by host tissue within
a certain period of time after implantation. The material is resorbed through processes such as erosion and cell-mediated enzy-
matic degradation, and the products are removed through normal physiological processes. The formation of a thin fibrous capsule
around the material is possible depending on its bioactivity. Fast-degrading materials such as collagen and b-tricalcium phosphate
(b-TCP) generally have favorable biological interactions, but may be degraded within weeks to months and provide insufficient
support for complete tissue repair. Slow-degrading materials such as polycaprolactone (PCL) and hydroxyapatite are generally
more bioinert, often persisting for years within the implant site and may cause ongoing tissue remodeling and reorganization
around the implant.
Metals
Metals have a range of characteristics that are advantageous for biomedical engineering applications, including high tensile and
yield strengths, and high resistance to fatigue, creep, and corrosion. Metals are commonly used in the form of alloys for biomedical
engineering applications because most pure metals evoke very strong immune responses in the body. After implantation, the metal
undergoes corrosion within the body and forms metal oxides on the surface. The formation of a dense oxide layer may passivate the
metal and reinforce its corrosion resistance, such as in the case of titanium. Metal alloys that contain potentially toxic elements are
safe for use in medical applications provided that they do not corrode or generate wear particles. For example, stainless steels (Cr
and Ni) and cobalt–chrome alloys (Co and Cr) contain potentially toxic elements, but are strongly corrosion resistant and have
a long history of use in clinical practice. Nevertheless, metal ions that leach from metallic implants into the blood can cause toxicity
problems, which constitute an important safety consideration in implant design.
Metals are commonly used for load-bearing orthopedic implants and internal fixation devices, such as hip and knee replace-
ments, dental implants, cardiovascular prostheses, and surgical instruments. Stainless steels, titanium and its alloys, and cobalt-
based alloys are the most common metallic materials used in biomedical engineering. Titanium alloys in particular have been
the materials of choice for load-bearing orthopedic implants due to their superior mechanical properties, chemical stability, and
in vivo biocompatibility. High corrosion resistance is a key selection criterion for the use of metals in medical applications, as
seen in 316L stainless steel (corrosion resistance due to low carbon content), cobalt–chrome alloys (corrosion resistance due to
presence of chromium), and Ti–6Al–4V (corrosion resistance due to passivating properties). Tissue responses to medical-grade
metal implants depend largely on their surface properties. Metal implants with a smooth or polished surface tend to result in fibrous
encapsulation (Fig. 3A), while implants with a rough or functionalized surface can promote osseointegration or the direct apposi-
tion of surrounding bone (Fig. 3B).
More recently, a number of other metals have gained increasing popularity and research attention as candidate implant materials
that provide unique properties for certain applications. For example, porous tantalum implants have been fabricated with structure
and elastic modulus similar to that of natural trabecular bone, avoiding the common problems of stress shielding and insufficient
bone ingrowth experienced by conventional metal implants. Biodegradable magnesium alloys are emerging as potential candidates
for medical devices that benefit from complete resorption within a certain time period after implantation, such as screws and coro-
nary stents.
Polymers
Polymers have a long history of use in medical applications, featuring many different types with versatile properties and a wide
range of processing options. All polymers are long-chain molecules composed of small repeating monomers, and can be derived
Fig. 3 Light microscope images of titanium implants in the canine mandible at 4 weeks after implantation, showing the tissue interface for implants
having (A) a smooth surface, with fibrous capsule formation around the implant, and (B) a surface functionalized using bone morphogenetic protein
(BMP), with direct bone apposition to the implant. Hematoxylin–eosin stain, original magnification 33 . Reproduced from Xiang W, Baolin L, Yan J
and Yang X (1993) The effect of bone morphogenetic protein on osseointegration of titanium implants. Journal of Oral and Maxillofacial Surgery 51(6):
647–651, with permission from Elsevier.
from natural or synthetic sources. Most polymers are relatively bioinert in the body, unless they are decorated with biologically
active agents such as adhesion molecules or growth factors. Some polymers are biodegradable and undergo surface erosion or
bulk erosion after implantation, which ideally should create space for regenerating tissue to bridge the implant site. Their degrada-
tion rate can usually be adjusted from weeks to years by modifying the molecular weight, crystallinity, and copolymer ratio. Poly-
meric materials have been extensively used in biomedical engineering applications in the form of surgical tools, implantable devices
and coatings, catheters, vascular grafts, injectable materials, and tissue engineering scaffolds.
Natural polymers, such as collagen (from biological tissues), chitosan (from crustacean shells), and alginate (from brown algae),
are derived from natural sources. They have the advantages of possessing structures that mimic native extracellular matrix, and may
already contain biochemical cues that enhance cell attachment or migration. They have been used in biomaterial systems involving
cell encapsulation, injectable materials, and tissue engineering constructs and grafts, and are naturally accepted by the body.
However, a common disadvantage of natural polymers is their weak mechanical properties and rapid degradation after implanta-
tion, which is partly due to their ability to be degraded by cell-mediated enzymatic digestion. Due to their extraction from natural
sources, natural polymers may also exhibit large batch-to-batch variation in properties which affect their downstream functions.
Synthetic polymers are an extensive class of materials that are artificially synthesized. They may be nondegradable, such as ultra-
high molecular weight polyethylene (used in hip and knee replacements), silicone (used in tube-shaped replacements such as
trachea or gastrointestinal segments), polytetrafluoroethylene (used in blood vessel grafts), polyurethane (used in heart pace-
makers), and poly(methyl methacrylate) (used as bone cements), or biodegradable, such as polyglycolic acid, polylactic acid
and their copolymers (used as sutures and tissue engineering scaffolds), and PCL (used as long-term tissue engineering implants
and drug delivery devices). Synthetic polymers are generally bioinert and have little interaction with host tissues unless they are
functionalized with biological agents. They have a number of significant advantages for use in biomedical engineering applications,
such as easy fabrication, flexibility in processing, and tuneable chemistry to produce desired properties. They also have a controlled
chemical composition and can give highly consistent properties between batches. However, most synthetic polymers require
organic solvents for processing, which may cause a toxic reaction in the body if not completely removed. Prolonged implantation
of nondegradable synthetic polymers in the body can also result in the generation of wear particles (particularly for polyethylene)
and the release of harmful monomers (particularly for poly(methyl methacrylate)), which may trigger chronic inflammatory and
foreign body responses.
Ceramics
Ceramics are inorganic, nonmetallic materials with high compressive strength. In biomedical engineering, several classes of ceramics
have been the materials of choice for musculoskeletal tissue repair and regeneration, with applications as orthopedic implants and
implant coatings, bone cements, tissue engineering scaffolds, prostheses for dental and maxillofacial restoration, and drug delivery
systems. Commonly referred to as “bioceramics,” their main advantages for these applications are similarity in chemical composi-
tion and mechanical properties to natural hard tissues. Bioceramics may be biologically inert (alumina and zirconia), or biologically
active and often biodegradable (calcium phosphates and bioactive glasses), each with a unique set of characteristics that make them
useful for specific medical applications.
Alumina and zirconia are both highly bioinert bioceramics, with high hardness, modulus of elasticity and compressive strength
accompanied by excellent friction and wear properties. Their ability to avoid the generation of wear particles and sensitization of
tissues make them good candidates for the articulating surfaces of artificial joint replacements, as well as dental implants. However,
these materials exhibit the characteristic brittleness of ceramics and may fail under impact loading, which has been the subject of
research into toughening mechanisms.
Calcium phosphates and bioactive glasses are bioactive and often biodegradable classes of materials which have been extensively
used as bone graft substitutes and tissue engineering scaffolds for hard tissue regeneration. These materials are inherently bioactive
as they are similar in chemical composition and surface structure as the mineral phase of bone, which consists of plate-like hydroxy-
apatite crystals. After implantation, a series of surface reactions occur which result in the formation of a direct bond between the
material and native bone. These surface reactions also mediate the adsorption of extracellular matrix proteins that promote the
attachment, proliferation, and differentiation of bone-related cells, ultimately resulting in enhanced bone formation. For calcium
phosphates, their bioactive properties originate from the ability to form a carbonate apatite (CHA) layer at the bone–material inter-
face through a cell-mediated dissolution and precipitation process, which releases calcium and phosphate ions from the material
into the surrounding microenvironment and encourages the precipitation of CHA microcrystals. The tissues surrounding the mate-
rial hence become richly mineralized, creating an environment that is conducive to bone formation. For bioactive glasses, surface
reactions involving ion substitution, glass dissolution, and mineral precipitation occur within 24 h of implantation, which accel-
erate CHA layer formation on the glass surface (Fig. 4). The reactivity of the glass surface is determined by the chemical composition
of the glass. The CHA layer, which has a similar composition to bone mineral, initiates the adsorption of growth factors and
a synchronized sequence of cellular events that result in the rapid formation of new bone. Bioactive glasses are the only bioactive
materials with the ability to bond to soft tissues as well as hard tissues.
The most commonly used calcium phosphate materials for clinical bone repair are hydroxyapatite, b-TCP, and biphasic calcium
phosphate (BCP). Stoichiometric hydroxyapatite is relatively bioinert and slow-degrading, and can persist in the implant site with
little biodegradation for 5–10 years following bone reconstruction procedures. In contrast, b-TCP is relatively fast-degrading and
highly bioactive, with good ability to induce clinical bone incorporation and remodeling but may not persist within the defect
site for a sufficiently long period of time for complete bone repair to occur. BCP is a two-phase ceramic composed of hydroxyapatite
and b-TCP phases, which allows an optimal balance of bioactivity and biodegradability to be achieved between the more stable
hydroxyapatite phase and the more soluble b-TCP phase. Bioactive glasses have been used clinically as particulates for filling con-
tained bone defects, and have demonstrated excellent bone bonding and osteogenic ability that can exceed calcium phosphates,
with comparable performance to bone autografts which are the gold standard for bone reconstruction. However, the progression
from particulates to porous scaffold systems has been difficult due to the glasses losing their bioactivity after the high-temperature
Fig. 4 Scanning electron microscope image of carbonate apatite layer formed on the surface of a bioactive glass scaffold after soaking in simulated
body fluid (SBF) for 28 days. The inset reveals the formation of rod-shaped crystals of biologically similar apatite. Reproduced from Rezwan K, Chen
QZ, Blaker JJ and Boccaccini AR (2006) Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering.
Biomaterials 27(18): 3413–3431, with permission from Elsevier.
sintering process required to produce porous scaffold structures. This is due to crystallization of the glass which removes its surface
reactivity and therefore ability to directly bond with adjacent tissues. In preclinical animal models, calcium phosphates with surface
microporosity and bioactive glasses have demonstrated evidence of osteoinductivity, which is the ability to induce the homing of
stem cells from the circulation to participate in bone formation at the implant site without the incorporation of growth factors.
Summary
The implantation of a biomaterial or its prolonged contact with the body elicits a series of tissue responses that are broadly defined
by the inflammatory and wound healing responses resulting from the body’s natural reaction to a foreign material. However,
specific biomaterial properties such as composition, degradation rate, morphology, shape and size, porosity, roughness, and surface
chemistry can also influence these tissue responses and lead to significantly different healing outcomes. When designing a new
medical device that involves one or several biomaterials, the biomedical engineer must consider the tissue responses that are likely
to occur for each biomaterial to ensure the long-term safety and efficacy of the device in the human body.
Further Reading
Anderson, J. M. (2001). Biological responses to materials. Annual Review of Materials Research, 31, 81–110.
Li, J. J., Kaplan, D. L., & Zreiqat, H. (2014). Scaffold-based regeneration of skeletal tissues to meet clinical challenges. Journal of Materials Chemistry B, 2(42), 7272–7306.
Morais, J.M., Papadimitrakopoulos, F. and Burgess, D.J. Biomaterials/tissue interactions: Possible solutions to overcome foreign body response. AAPS Journal 12(2), 188–196.
Rezaie, H.R., Bakhtiari L. and Öchsner A. Biomaterials and their applications. Switzerland: Springer International Publishing.
Biomaterials, 27(18), 3413–3431.
BIOMATERIALS: BIOMATERIAL APPLICATIONS AND ADVANCED MEDICAL
TECHNOLOGIES
Biomaterials in Dentistry
Li Wu Zheng, Prince Philip Dental Hospital, The University of Hong Kong, Hong Kong
Jing Yi Wang and Ru Qing Yu, The University of Hong Kong, Hong Kong
Restorative Dentistry 278

Direct restorative Materials 279
Amalgam 279
Composites 279
Glass ionomer cements 279
Indirect Restorative Materials 279
Metals 279
Ceramics 280
Cements 280
Endodontic materials 280
Denture bases and lining materials 281
Impression materials 281
Waxes 281
Adhesive system 281
Orthodontic 282
Brackets 282
Adhesives 282
Archwires 283
Elastomeric Modules and Chains 283
Periodontics 283
Barrier Membranes 283
Bone Graft Materials 284
3D-Printed Scaffolds 284
Implants Dentistry 285
Implant Materials 285
Metals 285
Ceramics 286
Polymers and carbon compound 286
Oral and Maxillofacial Surgery 286
Autogenous Grafts 286
Bone Substitutes 287
Titanium 287
Further Reading 288
The earliest dental materials science, dating back to the early 19th century at Northwestern University, began from the investigation
of dental amalgam. For approximately a hundred years, synthetic restorative materials were the major focus in the field of dental
materials until the end of the last century, when the real potential for biological engineering of tissues and organ systems was
revealed. The comprehension of development and advances in dental biomaterials will benefit both dental practitioners and
patients in selecting appropriate materials for clinical cases and improving treatment outcomes.
Restorative Dentistry
Restorative dentistry can be traced back to ancient times. Materials used for restoration back then include cork, ivory, human teeth, and
metal foils (lead and tin), etc. Nowadays, amalgam, composites, ceramics, metals, and cements are common restorative materials.

Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Biomaterials in Dentistry 279
Direct restorative Materials

Amalgam
Amalgam has been using to restore tooth structure since the 19th century. Dental amalgam mainly contains mercury, tin, copper, and
zinc. With proper cavity preparation and manipulation of dental amalgam, this can provide adequate strength to resist masticatory
forces with quite good longevity. Although amalgam has been a great success as a restorative material, its mercury content has raised
concerns regarding the potential adverse effects such as neurotoxicity in recent decades. The exposure to mercury for dental personnel
and environment also adds to the argument of reducing the use of dental amalgam. Moreover, esthetics has become an increasingly
important aspect in the choice of materials, causing patients to be reluctant to choose amalgam’s gray color material over more esthetic
materials, which are becoming more durable. Currently, with the development of other restorative materials (i.e., composites), the use
of dental amalgam as a direct restorative material is phasing down globally, despite its low cost and good performance.
Composites
Dental composites, or resin-based composites, are synthetic materials that combine polymeric matrix with a dispersion of glass,
mineral, or resin filler particles and/or short fibers by coupling agents. Just like dental amalgam, they are used to restore tooth struc-
ture lost through trauma, caries, or other diseases. Composites can also be used as cements to cement crowns and veneers, etc. While
the amalgam is phasing out in dentistry, composites have become one of the most widely used esthetic restorative materials.
Traditional composites contain relatively large particles of ground amorphous silica and quartz, which gives them good mechan-
ical properties but makes the surface of the restoration more likely to become rough from daily abrasion. In addition, many failures
of composite restoration are seen at the interface between tooth and composite due to shrinkage or adhesive failure. To overcome
this, microfilled composites, nanofilled composites, and other hybrid composites were developed, using much smaller particles (at
the same time with a large variety in size) to fill in the matrix. With these developments, smoother surfaces are achieved, wear resis-
tance is increased, and shrinkage is decreased without compromising the mechanical and physical properties.
Composites can be classified as chemically activated (self-cure) resins and photochemically activated (light-cure) resins. The self-
cure resins are supplied as two pastes; polymerization is activated when those two pastes are mixed together. Disadvantages are that
the air may be incorporated into the mix during mixing, thus weakening the material, and the operator cannot control the working
time after mixing. The light-cure resin is supplied as a single paste using the photosensitive initiator system and a light source for
activation. It does not need mixing, which makes it stronger and less staining, and has totally controllable working time. However, it
exhibits higher marginal stress during curing and only cures within limited depth (2–3 mm). Although this incremental curing
demonstrates some advantages, the demand for bulk cure has never stopped. Some new products have claimed that the cure depth
can be up to 4 mm (the cure depth of dual cure resins is unlimited since it is a combination of chemical and light-cure technology),
but their clinical performance has not been fully assessed.
Glass ionomer cements

Glass ionomer cements conventionally are acid-base materials that have been used to esthetically restore tooth structure since the
late 20th century. The powder of a number of glass components mixes with the liquid of polyalkenoic acid to form a paste, then the
acid-base reaction starts and stiffens the paste. The mechanical property does not suit the clinical requirements enough in the begin-
ning, but slowly improves with time. One of the advantages of glass ionomers is the true bonding between materials and dentin/
enamel; thus they have been widely used for Class V restorations which have high requirements in adhesion, for Class II and Class III
restorations in deciduous teeth, for luting of crowns, and they also can be used as bases or liners. Fluoride release is another merit of
glass ionomers. The disadvantages are that they are moisture-sensitive and have relatively low strength.
The newer glass ionomers are the resin-modified glass ionomers, which were introduced in the 1980s. These are a combination
of conventional glass ionomer cement and light-cure resins to improve some characteristics of conventional glass ionomers such as
increased strength, lower solubility, and less sensitivity to moisture. However, fluoride release of resin-modified glass ionomer is
lower and the biocompatibility is not as good as that of conventional glass ionomers.
Recently, glass ionomers were combined with bioactive glass (BAG) to improve their bioactivity and regenerative capacity. These
materials might be a better choice in tooth restoration compared to conventional glass ionomers or resin-modified ones, due to
their remineralization ability.
Indirect Restorative Materials

Metals
Crowns, inlays, cast posts and cores, and partial dentures are all examples of indirect metallic restorations. They are produced in the
dental laboratory instead of being carried out in the dental chair. There is a variety of alloys used in dentistry; the main alloys include
noble and precious metal alloys and various base-metal alloys, i.e., CoeCr alloys and titanium.
Noble and precious metal alloys

Gold, platinum, iridium, ruthenium, rhodium, and osmium are considered to make up the noble metals, which are very resistant to
corrosion. Silver and palladium are usually referred to as the precious metals, which are expensive. High’gold alloys must have
a gold content > 60% and precious metal content no < 75%. They can be classified according to their gold content into four types
(I–IV): soft, medium, hard, and extra-hard. In the 1970s, alloys with reduced gold content were rising in the market because of the
280 Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Biomaterials in Dentistry
rapidly increasing price of noble metals. These alloys can be further classified into medium’gold (40%–60% gold content) and low’-
gold alloys (10%–20% gold content). Their mechanical properties are similar to the type III and IV gold alloys, but exhibit lower
ductility. The white color makes them less popular with patients, but they can be used for posts and cores since they will be covered
with other materials. Silver–palladium alloys are another kind of precious alloy that presents various properties due to their compo-
sition and are less popular than the above-mentioned ones.
Base metal alloys

There are two main kinds of base metal alloys: cobalt–chromium and titanium. Cobalt–chromium alloys have high hardness and
modulus of elasticity, but low ductility. In addition, they are relatively low cost and have very good biocompatibility. Generally,
they are a very popular choice of material in restorative dentistry.
Titanium alloys exhibit high strength, low density, and have great biocompatibility. However, casting is a significant challenge
for these alloys.
Ceramics
Ceramics is a material that is opaque and porous, thus relatively weak. Dental porcelain mainly differs from traditional ceramics in
terms of firing techniques, which make it more suitable for dental restoration. Dental porcelain has very stable chemical properties
and outstanding esthetics which are unlikely to be influenced by time. It has similar thermal conductivity and coefficient of thermal
expansion to enamel and dentine, and exhibits high compressive strength. However, the tensile strength of dental porcelain is very
low (20–60 MPa). In addition, the surface microcracks caused by various reasons during manufacturing are the starting sites of cata-
strophic fracture. These drawbacks have limited their use to low-bearing areas, which are anterior regions of both mandible and
maxilla, and made them unsuitable for multi-unit bridges.
To overcome the disadvantages of dental porcelain, three types of dental ceramics have been developed:
• Metal-ceramics (porcelain fused to metal, or PFM), combine the positive mechanical properties of cast dental alloys and
excellent esthetic property of porcelain. The choice of metals is a key element in PFM.
• Reinforced ceramic core systems, which are similar to PFM in that instead of using alloys to support the porcelain, they use
another ceramic material with high strength and toughness yet do not offer esthetic qualities.
• Resin-bonded ceramics involve the ceramic being bonded to enamel and dentine directly, and thus the support comes from its
own tooth structure by resins. Not only the increasing strength and toughness are necessary, but also the adhesive bond, which
eliminates the surface flaws of dental ceramics and thereby reduces the probability of fractures.
Cements
Cements are used in dentistry for various purposes. Some are for cavity lining or bases; others are used as luting agents to lute an
indirect restoration to a prepared tooth.
Basically, a liner is a thin layer of material (0.5 mm) placed on a prepared tooth to protect it, whereas a base acts as the dentin to
withstand the forces applying on it. It is thicker than the liner and also protects the pulp from thermal and chemical stimulates and
galvanic shock. Thus a liner or a base should have good thermally and electrically insulating properties, and not contain irritants. It
should set rapidly, exhibit enough strength to resist fracture, and not move or flow easily while the filling material is being placed.
Ideally, linings should be radiopaque so that any caries around the filling material can be seen. The liner or base must not interfere
with the setting of the filling material or affect the properties of it.
Luting cements share similar properties with linings except for the setting time. There should be enough setting time before the
final seating of the restoration. In addition, it should be strong enough to assist retention and have low solubility.
Commonly used cements are as follows:
• Zinc oxide/eugenol cements are mixtures of zinc oxide (powder) and eugenol (liquid). They are mainly used as a lining or base
under amalgam restorations and as temporary luting cements or filling materials.
• Zinc–phosphate cements have zinc oxide as the major component of the powder and phosphoric acid solution as the liquid.
They are widely used as luting cements and can also be used as linings with adjustment of the powder/liquid ratio to change the
consistency. However, they may have an irritant effect as a liner in deep cavities.
• Calcium hydroxide cements have a low strength and high solubility, and therefore are usually used as linings beneath the base of
zinc phosphate cements or other base materials, and are not suitable for luting. Nevertheless, this material does have other
properties that make it crucial to dentistry such as the fact that it can be used for pulp capping and root canal sealing.
Endodontic materials
Endodontic materials are used in endodontic treatment, which is the procedure to save the tooth when the pulp and/or periradicular
tissues are injured. These materials can be generally classified into two groups: materials used to maintain the vitality of the pulp (pulp
capping materials), and materials used to disinfect (irrigants and intracanal medicaments) and fill the pulp in root canal therapy.
Pulp capping materials should be able to induce hard-tissue formation in a superficial way, protect the pulp from further inva-
sion of bacteria, and not have side effects so that the pulp can be alive. Hard-setting calcium hydroxide cements are the most
commonly used pulp capping materials. This material causes the formation of a 1–1.5 mm thick necrosis layer in the superficial
pulp. The layer will undergo calcification eventually and it is called a dentine-bridge.
Commonly used irrigants and intracanal medicaments include 0.5% sodium hypochlorite solution (as a disinfectant or antimi-
crobial), EDTA (as a chelating agent and lubricant), chlorhexidine (antimicrobial), non-setting calcium hydroxide (as an antibac-
terial agent), and some antibiotics and anti-inflammation drugs (non-setting Ledermix paste).
The most widely used canal-filling material is gutta-percha. Gutta-percha point is one of the application of gutta-percha, warm
lateral or vertical condensation can be applied to it to soften and compact it. The other use is the hot gutta-percha application
system, which injects the heat-softened gutta-percha into the root canal system.
In addition to gutta-percha, sealer cements are also needed in root canal filling procedures. These cements include zinc oxide-
eugenol cements, calcium hydroxide-containing sealers, glass-ionomer cements, polymers, and mineral trioxide aggregate, etc. Due
to space limitations, details of these materials will not be discussed here.
Denture bases and lining materials

Nowadays, most denture bases are fabricated using acrylic resins. The materials are usually supplied as a powder and liquid. The
major component of the powder is beads of polymethylmethacrylate (PMMA), which can be added to the liquid (mainly methyl-
methacrylate monomer) to form a mixture. Shortly after the mixing, the material first has a sandy consistency, then becomes a sticky
mass, and finally comes the dough stage. The dough at this point has lost most of its tackiness and should be packed into the mold
to prevent it from becoming tough and rubbery. Acrylic denture base materials can be classified into different groups according to
their method of curing. Generally, there are heat curing materials, autopolymerizing materials, thermoplastic, light-activated, and
microwave-cured materials. With sufficient thickness, the material showed acceptable mechanical properties; however, the impact
strength and fatigue strength are relatively poor. Dimensional stability is also a problem in acrylic resins in addition to its thermal
insulated nature, which is not an advantage for denture base because it prevents the protective reflexes to stimuli in oral mucosa.
Moreover, the unreacted monomer that remained in the denture base may cause mucosal irritation and sensitization of tissues.
Denture lining materials can be roughly classified into soft acrylics and silicone rubbers. The hard reline material is to be used
when the denture base has gone through some dimensional change and become less fit so that it needs relining of the fitting surface.
The soft lining materials are to provide a cushion when the denture is under load, and a tissue conditioner is very similar except it is
softer and can only remain soft for 1–2 days, while the softness of the temporary soft lining materials can last for 1–2 months. There
also exist permanent soft lining materials used for patients who cannot bear a hard base. However, no soft lining materials are truly
permanent in practice.
Impression materials
Impression materials are used in dentistry to record the details of intraoral structures to fabricate a reproduction of teeth and soft
tissues for the construction of dental prostheses. These materials should be able to produce an accurate replica of the intraoral struc-
ture, to prevent deformation and be atraumatic when removing from undercuts; they should also have proper setting time and
biocompatibility. They can be categorized as rigid and elastic impression materials. Rigid impression materials include plaster
and compo/zinc oxide-eugenol; however, since they cannot engage the undercuts, their application is limited nowadays. Elastic
impression materials can be further divided into hydrocolloid and elastomeric impression. Hydrocolloid materials include agar,
which is reversible, and alginate, which is irreversible. Elastomeric materials include polysulfide, polyether, condensation-cured sili-
cone, and addition-cured silicone. The choice of which impression material to use in each case will depend not only on the specific
needs of each case, but also on the impression technique and tray to be used.
Alginate is currently one of the most popular impression materials. It is supplied as dust-free powders. After mixing with proper
amount of water in a rubber bowl with a spatula, it is ready for impression taking. Two to three minutes after the surface tackiness
has been lost, it can be removed from the oral cavity. However, it does not produce very accurate surface detail, and has poor dimen-
sional stability. A snap-removal technique is required to minimize permanent deformation. It is thus not recommended for the
fabrication of crowns and bridges.
The need for more satisfying impression materials promotes the development of elastomeric impression materials. These mate-
rials have a much lower chance of permanent deformation when removed and are able to reproduce the surface detail very accu-
rately, but they are hydrophobic and therefore contamination of saliva will result in loss of surface detail to some degree. Another
problem of these materials is that they all undergo setting shrinkage due to polymerization, but in general, the shrinkage is very low
(with polyether and addition-cured silicones being the lowest and condensation-cured silicones being highest).
Waxes
Waxes have a number of applications in dentistry. A major use is as pattern waxdin other words, as modeling wax and/or inlay wax.
In the process of fabrication of dentures or restorations, there is a stage which is to produce the wax pattern of the dental appliances
on the model (indirect technique) or in the mouth (direct technique). This wax pattern determines the size and shape of the appli-
ance needed. A lost-wax technique is then used to replace the waxes using alloys or polymers. The pattern wax must be able to shape
the appliance accurately, and once it is formed there should be no dimensional change. Also, the wax should be removable either by
boiling or burning, and does not leave a residue.
Adhesive system
Facilitating adhesive materials in restorative dentistry instead of mainly relying on the retention form of restorative materials has
many advantages. One of the most important advantages is the conservation of tooth structure.
Enamel bonding
The acid-etch technique is most widely used for bonding resins to enamel. In 1955, Buonocore found that by modifying the enamel
surface with the phosphoric acid solution, he could make the surface more receptive to adhesion, which led to the development of
the acid technique. The etching process increases the bonding area by creating etch pits, into which the resin is absorbed by capillary
attraction. The adhesion is mainly based on this micro-mechanical interlocking. The acid-etch technique presents excellent bonding
between the etched enamel and the resin.
Dentine bonding
Dentine is a heterogeneous tissue composed of about 70% inorganics (hydroxyapatite, HA), 20% organics (collagen), and
10% water by weight. This makes it much more difficult for bonding compared to enamel. Another problem is that it is a vital
tissue and unlikely to create a dry dentine surface, and will damage the pulp if doing so. Furthermore, the dentine is covered
by a smear layer. However, the strong demand for dentine adhesive systems has promoted the development of dentine adhe-
sive systems.
There are three principal components in dentine-bonding agents: conditioner, primer, and sealer. A conditioner is an etchant,
which is used to remove the smear layer on the dentine. The smear layer is formed because of abrasion or burr cutting. Various acids
were used as conditioners: phosphoric acid, oxalic acid, EDTA, etc. After the conditioner is applied to the dentine, it dissolves the HA
and opens the dentinal tubules, producing a demineralized layer on the surface.
The primer acts as an adhesive to bond the hydrophobic resin to the hydrophilic dentine. The primer should be able to saturate
fully into the demineralized layer, otherwise the remaining demineralized dentine will become a weak region in the restoration. The
coupling agent in the primer is carried in a volatile solvent which is for the water removal in the dentine so that a hydrophobic resin
can be bonded tightly. A sealer is the resin used to fill the cavity, which bonds to the dentine through primer.
Total-etch technique
This technique involves etching the enamel and dentine at the same time, usually with 35% phosphoric acid for 20 s.
Dentine-bonding agents can be categorized according to the number of steps used clinically as three-step, two-step, and single-
step systems. Those consisting of a dentine conditioner, primer, and the sealer are three-step systems. Two-step systems and single-
step systems are developed to make the process more efficient and easier to use. The two-step systems combine either the
conditioner and primer together (self-etching primers) or the primer and the sealer together (one-bottle bond systems). The
single-step systems are supplied in two bottles; the practitioner only needs to mix the two components and applies them to the
enamel and dentine surface for the drying and light-curing. However, the bond strength is lower compared with the aforementioned
two systems.
Apart from the application in restorative dentistry, adhesive systems can also be used for the adhesion of orthodontic brackets
using the acid-etch technique.
Orthodontic
Materials used in orthodontic field mainly include brackets, adhesives used to bond the brackets onto teeth, archwires, and elasto-
meric modules and chains.
Brackets
Orthodontic brackets are small orthodontic attachments (metal or ceramic) secured to a tooth for fastening an archwire. Each
attachment is either soldered or welded to a previously placed band enclosing the tooth, or is bonded directly onto the tooth.
Metallic brackets can be further categorized into stainless steel, non’nickel or low’nickel stainless steel, and titanium brackets. The
nickel component in traditional stainless steel has revealed genotoxic effects and may cause some allergic reaction in patients, there-
fore non’nickel or low’nickel stainless steels are substitutes for the traditional ones. This type of steel exhibits similar or higher hard-
ness but may show lower corrosion resistance. Recently, titanium has been used as an alternative to produce brackets because of its
superior biocompatibility and higher corrosion resistance. However, titanium brackets have lower hardness compared to the stain-
less steel brackets. Thus, wear presents a problem in titanium brackets.
Ceramic brackets are developed for esthetic purposes. Other esthetic materials have also been useddfor example, plastics and
polycarbonate-based materials. Plastic brackets undergo extensive creep deformation and discoloration during use, and a low hard-
ness limited their use in orthodontic area. As an alternative, ceramic brackets exhibit higher hardness and stiffness; however, they
showed a higher incidence of fracture due to brittleness and aging in the oral environment. Newly developed ceramic brackets
showed better mechanical properties and even more esthetics due to improved transparency, and are more compatible with intrao-
ral environment.
Adhesives
Adhesives used in the orthodontic field are similar to those in restorative dentistry. The acid-etch technique is commonly used for
bonding. Details can be found in the relevant section.
Archwires
Archwires should move the teeth with light and continuous forces which increase patients’ comfort and optimize the treatment
process. The material used for the archwire should have good elasticity and be elastic for weeks at least when a force is applied
on it. To meet with these criteria and objectives, several alloys have been used as orthodontic archwires. Each has its own advantage
in a particular stage of treatment, but no single alloy suits all the stages involved in a treatment.
• Stainless steel alloys

Stainless steel alloys are very strong and have high corrosion resistance; however, they have lost their popularity due to the new
materials being introduced to the archwire market, but they are still in the market because the advantage of low cost.
• Cobalt–chromium alloys
This material contains mainly cobalt and chromium, but also some iron and nickel. Not only does it show similar stiffness to stain-
less steel, but also its formability can be changed by heat treatment. In other words, it can be formed to a specific shape and be
heated to increase the resilience and strength of the material.
• Nickel–titanium alloys
This alloy has a shape memory effect and was first developed by W.F. Buehler in the 1960s. The shape memory effect means that the
nickel–titanium wires remember its original shape before deformation and have the ability to return to that shape when not loaded.
Compared with stainless steel wires, nickel–titanium wires exhibit higher strength and resilience, and a lower modulus of elasticity.
Together with the ability to recover fully from 8% strain deformation (other alloys can recover from only 1% strain deformation),
nickel–titanium wires show some superiorities as orthodontic wires: lighter forces can be applied on teeth, reducing the number of
wire changes required during the treatment. Although they also have some limitations (e.g., restricted formability), they still are
a great improvement in the history of orthodontic wires.
• Beta-titanium alloys
Compared with stainless steels, these alloys applied gentler forces on teeth during deactivation and showed higher springback. They
exhibited better formability over nickel–titanium alloys. However, their stiffness is lower, and they are not resilient enough to with-
stand the friction created during the movement of teeth.
Elastomeric Modules and Chains

The use of elastomeric modules and chains is being phased out due to the application of self-ligating brackets, but these modules are
still used to close small gaps in the anterior teeth. They should be able to be stretched easily without great loss of energy, have high
tensile strength and stiffness, and have high resilience to recover their original form fully and rapidly. Further reading is recommen-
ded for the detail of the materials.
Periodontics
In recent years, the management of periodontal diseases has evolved from simply the debridement of periodontal pockets to the
regeneration of periodontal tissues. The use of biomaterials has become crucial in the treatment of patients.
Barrier Membranes
A barrier membrane is a device originally used to prevent epithelial migration into a specific area in the guided tissue regeneration
(GTR) procedure. Barrier membranes are divided into two major types: resorbable and nonresorbable (Table 1). In order to achieve
ideal function in a surgical site, the features of a barrier membrane need to meet certain criteria including biocompatibility, tissue
integration, cell-occlusiveness, space-making, and clinical manageability.
Cellulose and ePTFE were the first non-resorbable membranes developed and used in early GTR procedures. The titanium
mesh was developed to reinforce the membrane and shaped for more space. The disadvantages of non-resorbable membranes
are that they have to be removed, which inspired the development of resorbable membranes. Nowadays most practitioners
prefer resorbable membranes, especially collagen membranes because they are biocompatible and user friendly. They are grad-
ually degraded without additional surgery once positioned into the defect site. In recent years, acellular dermal matrix, which is
human skin processed in such a way as to remove epidermis and all dermal cells, has been used in root coverage and socket
preservation. The removal of cells of this graft warrant minimal risk of rejection and inflammation after being grafted. This
grafting material acts as a bioactive scaffold for migration of fibroblasts, epithelial cells, and endothelial cells. It is also a decent
material in periodontal plastic surgeries for increasing the zone of keratinized tissue. Compared to non-resorbable membranes,
resorbable membranes are weaker in space maintaining, thus the appropriate selection of a membrane is crucial for a good
clinical outcome.
Table 1 Types of membranes for periodontal regeneration
Resorbable Non-resorbable
Synthetic membrane • Cellulose acetate filter

• Polylactic • PTFE
• Polylactic/polyglycolic • ePTFE
• PL, PG and trimethylcarbonate • Titanium mesh
• PG and TMC • Ethylene cellulose
• Polyethylene glycol • Rubber dam
Natural membrane
• Collagen
• Acellular dermal matrix
• Oxidized cellulose mesh
Bone Graft Materials

Graft materials are nowadays widely used in bone regeneration and reconstruction. Table 2 lists the main categories of graft mate-
rials apart from autograft which is beyond the scope of this chapter. Autologous grafts perform well in osteogenic, osteoconductive,
and osteoinductive cases, but require sophisticated operating skills and more surgical time.
While autografts are harvested from the patients themselves, allografts are harvested from an individual other than the patient.
Allograft bone is typically sourced from a bone bank. Allografts are basically osteoconductive and osteoinductive; however, they do
not possess the ability of osteogenesis. The advantage of allograft over autograft is that the former eliminates donor site morbidity.
However, even though the preparation of these freeze-dried bone allografts would reduce the immunogenicity of the grafts, they still
carry a risk of infection and immunoreaction. In addition, the fresh frozen bone is more prone to cause disease transmission and
rejection.
Xenografts are derived from other species such as cows, horses, coral, etc. Deproteinized bovine bone minerals are the most
generally used bone grafts to date, and are frequently used in site augmentation, sinus augmentation, ridge preservation, and mate-
rials for peri-implant defects. They are also often used in conjunction with autografts and resorbable membranes.
Alloplastic grafts are completely synthetic and they usually contain calcium and phosphate. The most used alloplast is HA, which
has a good quality of osteoconduction, hardness, and acceptability by bone. Alloplastic grafts such as calcium phosphate are often
used as an osteoconductive matrix and should be tightly packed in the adjacent host bone to maximize ingrowth. Compared to
bone grafts mentioned above, alloplastic grafts have less of an issue with limited supply and carry minimal risk of disease
transmission.
In addition to being used in oral and maxillofacial surgery, the most common application of bone grafting is in periodontics and
implant dentistry. They are either in block or particulated to adapt better to a defect.
In the development of tissue engineering, barrier membranes and graft materials have been combined with growth factors, for
example, enamel matrix derivative (EMD), to generate better outcomes. EMD is a purified acid extract from the enamel matrix of
porcine fetal tooth. It has been used in treatment of peri-implant and periodontal diseases due to its profound ability to stimulate
the soft and hard tissues surrounding the teeth, and restore cementum, periodontal ligament, and alveolar bone. There are existing
products which mix EMD and Straumann bone ceramic (Straumann Emdogain PLUS, Straumann Holding AG, Basel, Switzerland),
which proved useful in regenerative periodontal surgery such as treatment of vertical bone defects.
3D-Printed Scaffolds
Bone grafting materials are now the treatment of choice to obtain adequate bone volume in periodontal diseases and implant sites.
However, as mentioned above, these grafting materials are not flawless, and all have their pros and cons. With the development of
3D-printing technology, 3D-printed scaffolds have become an attractive alternative to bone grafts in bone augmentation, socket
preservation, and sinus augmentation. The manufactural technique of the “printing” includes inkjet printing, extrusion printing,
and laser-assisted printing with different printing methods using specific biomaterials and generating different resolutions. Using
Table 2 Types of bone graft materials
Allograft Xenograft Alloplast
Fresh frozen bone (FFB) Bovine Hydroxyapatite

Mineralized freeze-dried bone allografts (FDBA) Porcine Tricalcic phosphate
Demineralized freeze-dried bone allografts (DFDBA) Equine b-TCP and HA
Coralline hydroxyapatite Bioactive glass
Algae hydroxyapatite Polylactic acid and polyglycolic acid
computer-aided design and computer-assisted manufacturing technologies based on a CT scan of the specific defect to create
a personalized grafting scaffold and well fitted at the defect site, this technique could be a solution worth considering. There is
also great potential for the application of scaffolding matrices in periodontal tissue regeneration as a membrane and grafting mate-
rial. Although the efficacy of 3D-printed biomaterials has been demonstrated preclinically, they still have a long way to go before
being widely used in dental clinics.
Implants Dentistry
The application of dental implants can be traced back to AD 600, when humans used bamboo stakes to replace missing teeth. Since
then, dental practitioners struggled with materials until 1952, when Professor Branemark developed a threaded implant design
made of pure titanium and found that titanium apparently bonded irreversibly to living bone tissue. Subsequently, the materials
of dental implants have developed further to meet varied clinical requirements.
To produce a high-performance dental implant to replace a missing tooth, the properties of the biomaterials, including the bulk
properties, surface properties, and biocompatibility, have to be taken into consideration (Table 3).
Implant Materials
Dental implants are usually classified according to their placement situation with the tissue. There are essentially three categories:
endosseous implants, subperiosteal implants, and transosseous implants. Four classes of materialsdmetals, ceramics, polymers,
and carbon compounddare used in modern dental implants (Table 4).
Metals
Metals are currently the most popular material in dental implants and they are almost exclusively titanium based. The characteristic
properties of titanium meet many requirements in a dental implant, such as primarily high strength and high corrosion resistance,
Table 3 Properties of an implant biomaterial
Properties Function
Bulk properties
• Modulus of elasticity of 18 GPa Uniform distribution of stress
Minimizes the relative movement at implant bone interface
• High creep deformability Materials with high creep values could better tolerate high masticatory forces
• High tensile, compressive, and shear strength Prevent fractures
Improve functional stability
Improved stress transfer
• High yield strength, high fatigue strength Prevent brittle fracture under cyclic loading
• A minimum ductility of 8% Crucial for contouring and shaping of an implant
• Hardness and toughness Decrease the incidence of wear
Prevents fracture of the implants
Surface properties
• Surface tension and surface energy Determines the wettability of implant
• Surface roughness Alteration in surface roughness improve cell attachment to the implant
Biocompatibility
• Good corrosion resistance Implant bio-material should be corrosion resistant as corrosion could result in
roughening of the surface, weakening of the restoration, release of elements
from the metal or alloy, and toxic reactions
Table 4 Classification of implant materials
Types Materials
Metallic Pure titanium (CPTi)

Titanium alloys (Ti–6AL–4U)
Co–Cr alloys
Stainless steel
Precious metals
Ceramic and ceramic coated Bio-inert ceramics
Bioactive and biodegradable ceramics
Polymers PMMA, PTFE, PE, PSF
Carbon compound
which ensure good biocompatibility. Thus titanium is viewed as the material of choice in dentistry. Both commercial pure titanium
(CPTi) and Ti–6AL–4U alloy possess excellent corrosion resistance; Ti–6AL–4U alloy has a slightly higher modulus of elasticity than
pure titanium, but it is more expensive. The disadvantage of titanium material is that there might be esthetic issue due to its gray
color; in addition to that, there is an unesthetic display of metal when soft tissue recession occurs. For high load-bearing zones such
as in posterior areas with lower esthetic requirements, implant materials with high strength such as CPTi or titanium alloys should
be considered.
Stainless steel has also been used for many years; however, it is contraindicated in patients who are allergic to nickel, as it
contains 7%–8% nickel to stabilize the austenitic structure. Meanwhile, the toxicity of nickel is still a concern. Apart from that,
the low cost and good mechanical properties are its significant advantages.
Ceramics
Because of their better biologic inertness compared with metals, and their good strength, ceramics have been used as dental implant
materials. Ceramic materials are classified into two types: inert ceramics, which are non-reactive materials such as aluminum oxide
(AL2O3), carbon, and zirconia (ZrO2); and bioactive and biodegradable ceramics such as glass ceramics, BAG, and calcium phos-
phate ceramics (CPCs).
Inert ceramics from aluminum, zirconium oxides, and titanium have been used for endosteal plate form, root form, and pin type
of dental implants. This type of material is high in compressive, tensile, and bending strength, with better physical characteristics
such as similar color as natural tooth, and minimal thermal electrical conductivity. However, its inherent brittleness has limited its
application. Modern dentistry has placed more emphasis on bioactive and biodegradable ceramics as these materials possess similar
constituents to those of normal tissue, with excellent biocompatibility, and a similar modulus of elasticity to bone. They could be
gradually resorbed and even replaced by tissue over time. As a matter of fact, they are primarily used as scaffolds or to coat metallic
implants because of their good tissue bonding property.
Bioactive and biodegradable ceramics, such as calcium phosphate ceramics (CPCs), have attracted attention in the development
of implant materials. Calcium phosphate materials have been applied as bone augmentation and replacement materials as well as
in implant materials. They could be formed to serve as structural support under relatively high-magnitude loading conditions for
implant applications, or as alloplastic grafts and mixed with drugs, collagen, and growth factors such as bone morphogenic proteins.
In addition, CPC coating on metallic surfaces has been used in endosteal and subperiosteal dental implant designs to improve the
longevity and surface biocompatibility of an implant. Mixtures of HA, tricalcium phosphate (TCP), and teracalcium phosphate can
be either plasma sprayed or coated to produce a bioactive surface on an implant. In fresh extraction site or a newly grafted site, HA-
coated implants seem a better choice for its greater implant bone interphase and higher shear bond strength.
Polymers and carbon compound

Polymeric implants were used in the past few decades. Due to their low mechanical strength and susceptibility to fracture, they are
nowadays only used as adjuncts to enable stress distribution along with implants. Carbon compounds have an excellent quality of
biocompatibility as well as a modulus of elasticity similar to that of bone. However, their low compressive strength results in their
application only as coatings on metallic and ceramic materials.
Oral and Maxillofacial Surgery
In terms of maxillofacial diseases, the most crucial concern, apart from treatment of the actual disease, is the esthetic requirements
and functional demands of the oral and maxillofacial region. The need to reconstruct the facial region for the reason of tumor resec-
tion, trauma congenital disorders, or even esthetic need is one of the main challenges of maxillofacial surgeons. Autologous bone or
soft tissue grafts are routinely used in reconstruction design nowadays. However, the donor site morbidity, the medical condition of
the patient to tolerate a major surgery, the special training and sophisticated technical requirements of a surgeon, and the relatively
high rate of infection and graft resorption are always concerns. Apart from the widely used autologous grafts, many other bioma-
terials have been used alone or in combination with bone grafts. As mentioned above, allografts, xenografts, and alloplastic mate-
rials have been used as bone substitutes in periodontics and implant surgeries to restore small defects. However, will these
biomaterials be an appropriate choice of treatment for major head and neck reconstruction?
Autogenous Grafts
To date, autogenous bone is the gold standard for maxillofacial reconstruction. An autogenous graft is prepared from a healthy part
of the patient’s own bone and grafted onto the defect area. A graft of cortical bone ensures a good structural support and a reduced
resorption, while a graft of cancellous bone ensures early revascularization. The types of autogenous grafts include free bone graft,
particulate cancellous bone marrow graft, pedicle composite flaps, microvascular free-flap transfer, bone marrow aspirates, and
plasma-rich protein. Among these, microvascular free-flap transfer performs noticeably well in major defect reconstruction since
it is vascularized with adequate blood supply and excellent in functional and esthetic reconstruction. However, the morbidity asso-
ciated with the harvesting procedures of these autogenous grafts, the limited availability when the volume of the defect is huge, and
the prolonged operation time have all inspired the continuous seeking of alternatives.
Bone Substitutes
Bone substitutes include allografts, xenografts, and alloplastic grafts. Allografts are costly and proved to have a high rate of resorp-
tion with poor mechanical properties due to chemical and radiation pre-treatment. Therefore, allografts are limited to small and
medium defects, and are not suitable for reconstruction of critical-size defects in the maxillofacial region.
Xenografts are less costly than allografts. They gained popularity in reconstruction of small bony defects because of their slow
resorption property. A common example of a xenograft is a kind of natural porous bone mineral called Bio-OssÔ (Geistlich, Wolhusen
Switzerland), which is derived from bovine bone and is commonly used in periodontics. However, as with allografts, xenografts are
not suitable for the reconstruction of critical-size defects due to their poor mechanical properties and lack of vascularization.
Alloplastic grafts contain a wide range of materials (Table 2), some of which have been used for bone regeneration. Ceramics such
as HA and calcium sulfate have been used in the restoration of periodontal defects and small correction of maxillofacial regions such as
sinus augmentation, but they still possess some limitations when it comes to repairing major defects in the maxillofacial region.
While ceramics slowly find their way to repair small defects in maxillofacial surgery, BAG has also proved its usefulness and
bright future in the reconstruction of small cranial-facial bone defects. BAG is a silicate glass-based material which undergoes
a specific surface reaction. It forms a bond with hard and soft tissues when implanted. BAG demonstrates controlled resorption
in optimal time, efficient bioactivity, and the ability to modulate cell migration, with a cortical bone-like modulus of elasticity.
However, similar to ceramics, it is not suited for reconstruction of continuity defects of jaw bones due to the lack of the required
mechanical properties. It has been applied for contour refinements as inlay for frontal bone, nasal dorsum, mandibular angle, and
alveolar ridge augmentation.
Polymeric materials used in maxillofacial surgery are mainly synthetic polymers. Although natural polymers such as collagen (used
in periodontal regeneration in combination with other grafting materials), alginate, hyaluronic acid, peptide hydrogen, and chitosan
are biocompatible, they could only be used in combination with other materials because of their disadvantage in being water soluble.
Synthetic polymers could be prefabricated as well as contoured with a burr in a surgery. Prefabricated polymers that have played a vital
role in maxillofacial surgery include hard-tissue replacement polymers, PMMA, and porous polyethylene (MEDPOR; Stryker, Kalama-
zoo, Mich., USA). PMMA, as the first substitute used for adult cranial reconstruction, presented a 9.5% infection rate in cranial recon-
struction. However, in recent years it has also been used as a framework in combination with bioglass fabricated into a customized
porous implant to repair calvarial and midface bony defects. This promising material proved good functional and esthetic outcomes
with no observation of long-term complications. Porous polyethylene implants are also commercially available and have been used
for nasal and malar augmentation, orbital floor reconstruction, and calvarial defect reconstruction. They are dense implants with a pore
size of 100–250 mm. However, these materials still have limitations such as the risk of infection, exposure, and extrusion.
Resorbable poly(L-lactide) (PLLA) and polyglycolide polymers (PGA) have also been widely used in maxillofacial surgeries.
Their combination product copolymers, which take in the most positive characteristics of them both, are poly(lactide-co-glycolide)
(PLGA) and PLLA-PGA; these are widely used as bioresorbable plates and screws for the fixation of bones. These resorbable plates
have gained popularity in treatment of pediatric mandibular complex fractures, since they function well in realignment and stable
positioning of rapidly healing fracture segments with no secondary removal operations needed. They have also been used in internal
fixation of adult maxillofacial fracture in certain anatomy sites when strength is not much of a concern. The limitations of these
resorbable materials are their limited ability to withstand masticatory forces and the chances of inflammation when the materials
begin to degrade. In addition, PGA and PLGA have been combined with HA or b-TCP to form a composite scaffold in an attempt to
increase the degradation time and improve the material’s mechanical properties.
Titanium
Titanium not only has outstanding performance in implant dentistry, it is also a common choice for maxillofacial surgeon for its
desirable mechanical properties and good biocompatibility. It is used as a bone tray for mandibular reconstruction, for cranioplas-
ties, orbital floor implants, condylar reconstruction, and titanium plates and screws for internal fixation of fractures. Its high
mechanical properties and lightness render it a good choice when used as fixation plates/screws for major force-bearing fracture
sites such as mandibular angle fractures. This metallic material has also been used in combination with polymeric materials
such as polyethylene; the combined material possesses not only the mechanical strength offered by titanium but also the porous
biocompatible surface offered by polyethylene. Titanium has the advantage of visibility on post-op with minimal distortion in MRI
images, yet the other side of the coin is that it produces artifacts and interfere with the interpretation of MRI images just like other
metallic objects. Other limitations of titanium are that the thermal conductivity of the metal would bring discomfort to patients in
cold weather, and the gray color would be an esthetic issue when soft tissue is thin and pervious to light.
The functional and esthetic reconstruction in the maxillofacial region has always been a vital task for surgeon and researchers.
The application of biomaterials for maxillofacial repair and reconstruction are summarized in Table 5.
Tissue Engineering
Biomaterials have been widely used in almost all fields of dentistry; however, none of them is able to completely restore or replace
the structure and function of missing tissues. The burning needs in restoration and reconstruction in clinic inspire and promote the
Table 5 A brief summary of biomaterial application in maxillofacial repair and reconstruction
Maxillofacial region Repair/reconstruction requirement Materials commonly used Alternatives
Extra-oral
Maxillofacial fracture Strong mechanical strength, stable Titanium plates/screws –
positioning
Pediatric complex fracture Stable positioning, biocompatibility Resorbable plates/screws Titanium plates/screws
Maxillofacial bone defect Reconstruction, protection Autogenous grafts Titanium mesh, polymers, injectable
cement, tissue engineering
Facial augmentation Restoration of bone volume and Autogenous grafts, titanium, bioactive Resorbable polymer, polymer face
contour, esthetic glass, polymers filler, 3D scaffolds
Intra-oral
Ridge augmentation Restoration of function and esthetic, Titanium, deproteinized bovine bone Bioactive glass, injectable cement
implant placement minerals ceramic, tissue engineering
Maxillary sinus lift Implant placement Mineral composite Tissue engineering
Temporomandibular joint Function, esthetic Cast cobalt–chromium–molybdenum –
replacement alloy, titanium with polyethylene
Modified from Deb, S. (2015). Biomaterials for oral and craniomaxillofacial applications. S. Karger AG.
development of tissue engineering, a brand new technique using a combination of scaffolds, cells, and biologically active molecules
to assemble functional constructs that restore, maintain, or improve damaged tissues for medical or dental purposes. Tissue engi-
neering evolved from the field of biomaterials, but has been growing in scope and importance and is now an independent field. The
great potential of tissue engineering in dentistry has advanced the study and clinical trials in periodontal regeneration, dental pulp
regeneration, and maxillofacial reconstruction; however, the imperfect technique, high cost, risk of biological contamination, and
ethical issues are concerns that need be solved before the engineered tissues can be widely used in clinics.
Further Reading
Agarwal, S., Gupta, A., Grevious, M., & Reid, R. R. (2009). Use of resorbable implants for mandibular fixation: A systematic review. Journal of Craniofacial Surgery, 20(2),
331–339.
Anusavice, K. J., Shen, C., & Rawls, H. R. (2013). Phillips’ science of dental materials. London, UK: Elsevier Health Sciences.
Asa’ad, F., Pagni, G., Pilipchuk, S. P., et al. (2016). 3D-printed scaffolds and biomaterials: Review of alveolar bone augmentation and periodontal regeneration applications.
International Journal of Dentistry, 2016, 1239842.
Carpena Lopes, G., Narciso Baratieri, L., de Andrada, C., Mauro, A., & Vieira, L. C. C. (2002). Dental adhesion: Present state of the art and future perspectives. Quintessence
International, 33(3).
Craig, R. G., & Powers, J. (2002). Restorative dental materials, 11th edn. St. Louis: Mosby.
Darby, I. (2011). Periodontal materials. Australian Dental Journal, 56(Suppl. 1), 107–118.
Deb, S. (2015). Biomaterials for oral and craniomaxillofacial applications. S. Karger AG, Basel.
Dhuru, V. B. (2004). Contemporary dental materials. Oxford: Oxford University Press.
Eliades, T. (2007). Orthodontic materials research and applications: Part 2. Current status and projected future developments in materials and biocompatibility. American Journal of
Orthodontics and Dentofacial Orthopedics, 131(2), 253–262.
Jones, J. R. (2015). Reprint of: Review of bioactive glass: From Hench to hybrids. Acta Biomaterialia, 23(Suppl), S53–82.
Lyngstadaas, S. P., Wohlfahrt, J. C., Brookes, S. J., et al. (2009). Enamel matrix proteins; old molecules for new applications. Orthodontics & Craniofacial Research, 12(3),
243–253.
McCabe, J. F., & Walls, A. W. (2013). Applied dental materials. Chichester: John Wiley & Sons.
Misch, C. E. (2008). Contemporary implant dentistry. St. Louis: Mosby Incorporated.
Payne, K. F., Balasundaram, I., Deb, S., et al. (2014). Tissue engineering technology and its possible applications in oral and maxillofacial surgery. British Journal of Oral &
Maxillofacial Surgery, 52(1), 7–15.
Profeta, A. C., & Huppa, C. (2016). Bioactive-glass in oral and maxillofacial surgery. Craniomaxillofacial Trauma and Reconstruction, 9(1), 1–14.
Sam, G., & Pillai, B. R. (2014). Evolution of barrier membranes in periodontal regenerationd"Are the third generation membranes really here?”. Journal of Clinical and Diagnostic
Research, 8(12). Ze14–17.
Van Noort, R., & Barbour, M. E. (2013). Introduction to dental materials4: Introduction to dental materials. Elsevier Health Sciences.
von Recum, A. F. (1998). Handbook of biomaterials evaluation: Scientific, technical and clinical testing of implant materials. London, UK: Taylor & Francis, 2nd edn.
Biomaterials in Ophthalmology
Rachel L Williams, Hannah J Levis, Rebecca Lace, Kyle G Doherty, Stephnie M Kennedy, and Victoria R Kearns, University of
Liverpool, Liverpool, United Kingdom
Introduction 289
Refraction 289
Contact Lenses 289
Intraocular Lenses 290
Space Filling 291
Vitreous Substitutes 291
Scleral Buckles 292
Orbital Implants 292
Flow Control 292
Electric Stimulation 293
Tissue Regeneration 294
Natural Scaffolds 295
Biological Polymers 296
Synthetic Polymers 296
Conclusion 297
References 297
Introduction
Innovations in biomaterials science and engineering have the potential to make a significant contribution to the development of
treatments for ophthalmic diseases and thus to reduce the burden of vision loss on the global community. The loss of vision
has major social and economic consequences not only to the individual but also to society at large, and with the increasing age
of the population, this will have greater consequences. There are functional requirements of the eye and visual system that allow
vision to occur, and various diseases and trauma can disrupt these processes. Biomaterials have a role in addressing several of these
functional problems such as refraction, space filling, flow control, electric stimulation, and tissue regeneration. This article will
discuss the biomaterials used in each of these categories.
Refraction
The cornea is the transparent window that allows light to enter the eye. It also has a major role in focusing light, providing about
80% of the eye’s refractive power, with the rest being achieved by the lens. To overcome problems with the ability of the eye to
refract the light and focus it onto the retina, biomaterials have been used in the form of contact lenses and intraocular lenses.
Contact Lenses
Contact lenses not only are traditionally used to correct refractive error but also can be used to assist wound healing as a bandage
contact lens or, alternatively, as a drug reservoir. The material properties that must be considered for contact lens selection are high
oxygen permeability so oxygen can reach the cornea and prevent vascularization, high water content for tear film wettability and
comfort, and resistance of protein/lipid/mucus deposits on the contact lens (Refojo, 1996). There are four main groups of materials
used for contact lenses; these can be classified as either “hard” or “soft.” Soft flexible contact lenses include hydrogels (based on
poly(hydroxyethylmethacrylate) (pHEMA)), silicones, and silicone hydrogels. Gas-permeable materials (usually made from fluo-
rosilicone acrylates) are rigid and classed as hard contact lenses. The largest proportion of contact lenses on the market today are
silicone hydrogel at 64%. These lenses combine different ratios of each material to achieve both the benefits of the high oxygen
permeability from the silicone and increased wear comfort from the hydrogel (Caló and Khutoryanskiy, 2015; Kirchhof et al.,
2015). Soft contact lenses have also been used for bandage contact lenses; these aim to prevent necrosis after surgery and facilitate
wound healing by protecting the eye from external assault and sources of infection; they also keep the ocular surface hydrated and
relieve pain by isolating friction during blinking. An example of when these are routinely used is after keratoprosthesis (Carreira
et al., 2014; Thomas et al., 2015). A bandage contact lens that offered a therapeutic antimicrobial effect could be beneficial as
a replacement for conventional antimicrobial eye drops in treating infectious keratitis. Gallagher et al. have demonstrated this
with a hydrogel synthesized from poly-ε-lysine with additionally bound biomolecules to achieve a hydrogel with optimized

290 Biomaterials: Biomaterial applications and advanced medical technologies j Biomaterials in Ophthalmology
mechanical and antimicrobial properties (Gallagher et al., 2016). Another potential application for contact lenses is their use in
drug delivery to the front of the eye; this would be beneficial as there is a low bioavailability of ophthalmic eye drop (about
5%) due to the high drainage via the tear duct or down the cheek. One of the challenges is to achieve a sustained therapeutic release
of the drug over a period of time. Simple methods involve soaking commercial hydrogel contact lenses in solutions containing
drugs; however, the release profile of the drug is uncontrolled, leading to an initial high overdosing period over a few hours fol-
lowed by a long underdosing period, making them unsuitable for long-term release. An alternative method is to immobilize drugs
onto the surface of hydrogel contact lenses that may require modification using polyethylene glycol (Kirchhof et al., 2015; Xinming
et al., 2008). Other methods to trap drugs and release them from contact lenses include molecular imprinting, colloid encapsula-
tion, and polymeric nanoparticles (Dixon et al., 2015; Maulvi et al., 2016). Ciolino et al. examined the properties of a copolymer of
poly(hydroxyethylmethacrylate) and methacrylic acid (pHEMA/MAA) contact lens that encapsulated a poly(lactic-co-glycolic) acid
(PLGA) film that incorporated the glaucoma drug latanoprost. They demonstrated that as the PLGA drug film degraded, the contact
lens delivered a therapeutic amount of the drug over a 4-week period in vivo without any signs of cytotoxicity (Ciolino et al., 2014).
Drug delivery in the eye, and the biomaterials used in general, is a large topic and will not be covered further in this article.
Intraocular Lenses
Cataracts are the leading cause of preventable blindness worldwide. During surgery, the cloudy lens is removed from its capsular bag
and replaced by a polymeric intraocular lens (IOL). Over the years various polymers and designs have been used for IOLs, in an
attempt to prevent postoperative complications associated with scarring. The polymers used can be grouped into three main groups:
hydrophobic acrylic (e.g., phenylethyl methacrylate (PEMA) and phenylethyl acrylate (PEA)); hydrophilic acrylic (e.g., poly hydrox-
yethylmethacrylate (pHEMA)); and silicone (e.g., poly dimethylsiloxane (PDMS)). Due to the nature of the surgery, the chosen
material should be foldable, so it can be inserted into an incision of 3–4 mm diameter or less to minimize the damage caused
by the surgery and postoperative scarring. How the IOL responds to the native tissue plays a key role in scarring. Postoperative
complications occur when residual lens epithelial cells migrate onto the previously cell-free posterior capsule in which the lens
is housed. Once here, these cells dedifferentiate into fibroblast-like cells causing the posterior capsule to wrinkle and disrupt the
path of light to the back of the eye (Apple et al., 1992) (Fig. 1). This is known as posterior capsule opacification (PCO). The inci-
dence of PCO varies between studies, both the IOL material (hydrophobic vs hydrophilic) and design (sharp- or round-edged IOLs)
can play a part in this (Auffarth et al., 2004; Yuen et al., 2006). Although IOL material and design choice may slow down the devel-
opment of PCO, it does not prevent it. Rønbeck et al. demonstrated in a 12-year postoperative review of three IOLs including
a round-edged heparin surface-modified poly(methyl methacrylate) IOL, a round-edged silicone IOL, and a sharp-edged hydro-
phobic acrylic IOL that there was no significant difference in the incidence of PCO after 12 years between the acrylic and silicone
IOL regardless of edge shape. There was, however, a significant difference short term (5-year postsurgery), in which patients with
acrylic IOLs had significantly less PCO than those with silicone IOLs (Rønbeck and Kugelberg, 2014). With an increasing ageing
population, other methods need to be investigated to prevent the long-term effects of PCO; one possible strategy could be the
use of IOLs as a drug delivery system (see Liu et al., 2013).
Injectable gel-like polymers, based on polysiloxane, have been investigated as an alternative to conventional IOLs. The gel struc-
ture can be injected inside the capsule bag, leaving it intact. The gel conforms to the shape of the capsular bag, while the capsular bag
supports the gel. These gels have the added benefit of accommodation, similar to the natural lens, which is not fully possible with
Fig. 1 Photograph of a donor eye with intraocular lens implant and early posterior capsule opacification formation. Early fibrosis (white arrows) was
observed as scar tissue at the periphery and the development of Soemmering’s ring (red arrow).
Biomaterials: Biomaterial applications and advanced medical technologies j Biomaterials in Ophthalmology 291
the conventional IOLs (de Groot et al., 2001). By varying Young’s modulus of polysiloxane gels to 0.8 kPa, similar to the native
human lens (1 kPa), lenses can undergo changes in refractive power during equatorial stretching, which simulates accommodation
and have a higher lens power than the age-matched natural lenses (Koopmans et al., 2003). However, the incidence of PCO remains
a problem and in addition, Nd:YAG laser capsulotomy, which is the removal of the opaque posterior capsule via a laser, cannot be
performed as this would affect the accommodation capabilities of the soft lens. Alternative methods to eliminate PCO have been
investigated, for example, aggressive cytotoxic chemicals to kill remaining LECs prior to injecting the gel-like lens (van Kooten et al.,
2006); however, due to the level of aggression needed to kill all remaining LECs, further complications of cornea opacification have
been observed (Koopmans et al., 2011).
Space Filling
Vitreous Substitutes
Replacement of the vitreous humor is required if the native vitreous body needs to be removed, either to facilitate surgical treatment
for retinal detachment or if the vitreous body itself is compromised due to other conditions including infection, tumor, or trauma
(Kleinberg et al., 2011). Vitreous substitutes also have the potential to be used as drug delivery devices. The vitreous humor is
predominantly composed of interpenetrating networks of collagen fibrils and hyaluronan molecules (with various other compo-
nents, including cells) forming a clear hydrogel. It provides support to the surrounding structures, absorbs mechanical trauma,
and is involved in the circulation and regulation of oxygen, metabolites and nutrients. The composition and structure of the vitreous
humor and vitreoretinal interface has been described in detail by Sebag (1998, 1992).
Silicone oils (Giordano and Refojo, 1998) were first used to treat retinal detachment in the 1960s and have been more
commonly used since the availability of vitrectomy. They are currently the only class of vitreous substitute available for long-
term use (it is usually removed within 3–6 months, although there are patients with permanent silicone oil (Morphis et al.,
2012) but are used much more in Europe than in the United States (D’Amico, 2016). The conventional belief is that their primary
mode of action is to block the flow of fluid through the retinal breaks, excluding the inflammatory factors that can lead to the forma-
tion of scar tissue that can cause the retina to distort and detach (Wong and Williams, 2005). The use of silicone oil has, however,
been associated with the development of cataract (Heimann et al., 2008) and corneal endothelial graft failure (Friberg and Guibord,
1999). Early formulations caused problems relating to the presence of low-molecular-weight components (Pastor et al., 1998); all
currently available products are highly purified to remove these components.
Silicone oils have a propensity to emulsify. This is likely to be due to a combination of high shear forces, insufficient interfacial
tension between the oil and aqueous phases in the eye, and the presence of proteins that both lower the interfacial tension and
stabilize emulsified droplets. The formation of emulsions affects the passage of light but, more significantly, is associated with
the development of complications such as glaucoma (Ichhpujani et al., 2009) and migration into the anterior chamber (Light,
2006). It has been suggested that emulsification is underreported, as droplets that can be observed clinically are relatively large
compared with those that can be measured from samples retrieved from patients but studied in vitro (Chan et al., 2015). A number
of strategies have been developed to attempt to reduce emulsification, including the use of oils with higher shear viscosity (Scott
et al., 2005; Chan et al., 2011); there are no randomized clinical trials, however, showing superior emulsification resistance of
5000 mPa$S oil over 1000 mPa$S oil. A more recent strategy has been the development of silicone oils with increased extensional
viscosity, achieved by adding a small percentage of a high-molecular-weight component to standard 1000 mPa$S oil (Williams
et al., 2010). These oils, which are in clinical use, also experience shear thinning, making them relatively easy to inject (Williams
et al., 2011).
Silicone oils have a lower density than the aqueous solutions that fill the remainder of the cavity; thus, they are unsuitable for
treatment of pathology in the lower part of the vitreous cavity. A range of “heavy” silicone oils have been developed, and a few are in
clinical use (Heimann et al., 2008). They are based on standard silicone oil with the addition of perfluorohexyloctane or a partially
fluorinated olefin. The resulting oils, which have densities of 1.06 and 1.02 g/cm3, respectively, are reported to result in anatomical
success, but complications such as emulsification exist (Ozdek et al., 2011; Wickham et al., 2010). A more recent development is the
combination of the addition of perfluorohexyloctane and the high-molecular-weight additive, resulting in a “heavy oil” that should
have increased emulsification resistance (Caramoy et al., 2015), although a large body of clinical evidence is not yet available for
this tamponade. An alternative strategy, which aims to take advantage of the light and oxygen diffusion properties of silicone oil
while mimicking the natural structure of vitreous and vitreoretinal interface, is to contain the silicone oil (or other fluid) within
a silicone rubber capsule within the eye (Lin et al., 2012; Yang et al., 2014). This technique has been tested in clinical trials, although
has not been widely adopted.
Hydrogels (Swindle-Reilly et al., 2016; Chirila et al., 1998; Su et al., 2015) are the most widely researched alternative to silicone
oils, because of their potential to more closely mimic the structure of the vitreous humor. Hydrogels should be designed to have
appropriate physical, chemical, and biological properties, including the ability to gel in situ; however, to date, none has made it into
clinical practice. Unlike silicone oils, they are not able to exert a significant tamponade effect (Liang et al., 1998), so are limited to
conditions that require space filling and as drug delivery devices. Many of them are reported to degrade once implanted into the
vitreous cavity. Hyaluronan- and collagen-based gels have been widely investigated, but even following the addition of other
components and cross-linking, these materials do not have the required physical properties and have a lower density than water,
limiting their use for tears in the upper area of the vitreous cavity or are still susceptible to degradation in vivo (Liang et al., 1998;
Schramm et al., 2012; Pruett et al., 1979; Barth et al., 2016). Work on various other natural and synthetic polymers, including poly-
acrylamide (Santhanam et al., 2016), polyethylene glycol (Annaka et al., 2011), and chitosan (Yang et al., 2008), has been reported.
Scleral Buckles
Scleral buckles are devices that are placed within or on the sclera and are used to treat retinal breaks that are associated with retinal
detachment. The principle is that by displacing the sclera, vitreoretinal traction is reduced, and fluid, that includes inflammatory
factors, is moved away from the retinal breaks. Although it remains a successful treatment, with clinical outcomes comparable
with other treatments (Khan et al., 2015), it is being used less frequently. Devices based on silicone are the only materials that
are currently used clinically. They may be solid structures, often bands between 2 and 5 mm wide (Schepens and Acosta, 1991),
or sponges. One of the advantages of silicone is that it does not encourage the attachment of tissue, which can cause complications
if the implant needs to be removed. Furthermore, they are sufficiently tough and pliable to allow the surgeon to manipulate them
during implantation. The tendency of other materials to allow tissue attachment is one of the reasons why they are not clinically
successful. Hydrogels, biologically derived polymers and degradable polymers have insufficient mechanical strength for the
required duration (Baino, 2010).
Orbital Implants
It is sometimes necessary to remove the eye of the patient, for example, following cancer, severe infection, or trauma. An orbital
implant (Baino and Potestio, 2016; Sami et al., 2007) can be used to restore the resulting cavity, which is important for aesthetic
and psychological reasons. Orbital implants can be classified as integrated or nonintegrated. From a materials perspective, this can
be linked to whether the implant surface is porous or not, although some are designed to allow tissue ingrowth on the posterior
surface while usually having a smooth anterior surface. A recent Cochrane review (Schellini et al., 2016) has been unable to deter-
mine whether either strategy is better. Polymethyl methacrylate is in clinical use to produce orbital implants of various designs
(Baino and Potestio, 2016) as it has a good track record and is cheap. Polyethylene, particularly in porous form with a smooth ante-
rior surface or coating to reduce irritation to surrounding tissue such as the conjunctiva and to allow connection of extraocular
muscles, is also used (Karesh et al., 1994; Jung et al., 2012). Porous silicone (Son et al., 2012) and expanded polytetrafluoroethylene
have been investigated (Dei Cas et al., 1998), but are not used clinically.
Ceramic implants (Baino and Vitale-Brovarone, 2015), both hydroxyapatite and alumina, are used in various clinically available
implants (Suter et al., 2002; De Potter et al., 1994; Jordan et al., 2003), both allowing desirable tissue ingrowth. Promising early
results have also been reported for patients receiving a bioactive glass–ceramic implant (Crovace et al., 2016). Optimizing micro-
structure and topography may be important in determining and enhancing the clinical response (Mawn et al., 1998; Patel et al.,
2010), although this is not yet widely studied. Composites of the earlier materials have been investigated, although many have
not been successful in patients. In particular, bioactive ceramics have been used as coatings to encourage tissue ingrowth. Those
that have made it into clinical use include one based on hydroxyapatite and silicone, and another based on porous polyethylene
and 45S5 bioglass (Ma et al., 2011).
Flow Control
Normal eye pressure ranges from 12 to 22 mmHg. The intraocular pressure is maintained in the normal range by the flow of
aqueous humor, which is produced by the ciliary body, from the back of the eye into the anterior chamber through the pupil. It
then flows out of the eye through the trabecular meshwork into Schlemm’s canal and is absorbed into the bloodstream. The produc-
tion and flow of the aqueous humor is an active, continuous process that is needed for the health of the eye. If the flow is restricted,
pressure builds up in the eye, which can cause damage to the optic nerve, leading to vision loss.
Glaucoma is usually characterized by a high intraocular pressure that may be due to the reduction of fluid flow through the
trabecular meshwork or in the collector channels or venous plexus further downstream. In many cases, glaucoma can be treated
with eye drops that cause a lowering of the intraocular pressure either by reducing the amount of aqueous humor produced or
by increasing its outflow. In some situations, however, this is not sufficient or eyedrops are no longer functional, and alternative
strategies are needed to ensure the pressure remains in the normal range. In general, this involves surgery to produce a channel
through the obstructed tissue to allow outflow of the fluid. Minimally invasive glaucoma surgery has been designed to make glau-
coma surgery simpler to perform with less trauma to the eye and a more easily reproducible technique so that the procedure is avail-
able to all ophthalmologists rather than just glaucoma specialists (Manasses and Au, 2016). This has resulted in several different
designs of glaucoma microstents that can be classified by the targeted outflow destination: into Schlemm’s canal, the suprachoroidal
space, or the subconjunctival space. The iStent, which is 1 mm long and has a lumen of 120 mm, is designed to bypass the trabecular
meshwork and allow outflow directly into Schlemm’s canal. It is made from a heparin-coated titanium alloy (Ti6AI4V), and it is
possible to insert two or three at the time of cataract surgery. Placement of the istent in conjunction with cataract surgery in
mild to moderate glaucomatous eyes has been shown to cause a moderate reduction in IOP and reduce the dependency of the
patient on medication (Manasses and Au, 2016). The Hydrus Microstent is similarly a trabecular meshwork bypass device. It is
much longer at 8 mm and is designed to follow the curve of Schlemm’s canal preventing its compression. It is manufactured
from NiTi alloy and is fenestrated along its length to aid outflow of aqueous into Schlemm’s canal. One year clinical results show
that this can maintain a reduced IOP, rarely achieving less than 15 mmHg, and a reduction in use of medication over at least
12 months. The Cypass stent is designed to bypass the trabecular meshwork and provides a direct outflow channel into the supra-
choroidal space. It is made from polyimide and is 6.35 mm in length with a 300 mm lumen with fenestrations along its length to
facilitate outflow. Like the Schlemm’s canal devices, IOP is rarely observed below 15 mmHg and generally resides above 16 mmHg.
The iStent Supra uses a similar approach and is a 4 mm tube made from fenestrated polyethersulfone with a Ti sleeve.
The more traditional way to release fluid from the anterior chamber is into the subconjunctival space. There are two glaucoma
microstents that are designed to take advantage of this route and bypass all potential outflow obstructions. The XEN 45 is manu-
factured from porcine-derived gelatin cross-linked with glutaraldehyde. It is a 6 mm long tubular structure with a lumen of 45 mm
that has been designed specifically to control the rate of fluid flow to maintain the correct IOP.
The InnFocus MicroShunt (Fig. 2) similarly drains into the subconjunctival space. It has been through various design iterations
to ensure as little trauma to the tissues and a lumen size that minimizes hypotony (Pinchuk et al., 2017). The final design resulted in
a stent that is 8.5 mm long with a 70 mm diameter lumen. The material that it is manufactured from was a key part of the design
process. The objective was to design a synthetic thermoplastic elastomer that is biostable and caused less of an inflammatory
response than conventional materials. The material is synthesized at InnFocus and is a triblock copolymer of a soft polyisobutylene
central block with glassy polystyrene end blocks called poly(styrene-block-isobutylene-block-styrene) or SIBS. It has enhanced
biocompatibility and long-term stability resulting in less inflammation than other materials. A lowering of the IOP
to 14 mmHg was recorded after 3 years in 95% of the patients (Manasses and Au, 2016).
Electric Stimulation
Damage to retinal tissue via various disease mechanisms has led to research to attempt to augment or replace the function of
damaged tissues using retinal implants. Patients in many of the clinical trials that have been run to date suffer from retinitis pig-
mentosa (RP), a group of hereditary diseases that damage the photoreceptor cells of the outer retina but leave the nerve cells of
the inner retina intact. Retinal implant devices provide electric stimulation to these remaining nerve cells that transmit the signals
along their axons, which form the optic nerve, to the visual cortex in the brain.
The devices aim to capture light and convert this to an electric pulse that is delivered to nerve cells, particularly the ganglion cells.
These electric impulses cause patients to perceive flashes of light called phosphenes. Many groups are investigating various different
approaches to light or image capture, processing and conversion to electric impulses, and electrode design and location (Maghami
et al., 2014; Ha et al., 2016). The Alpha IMS, developed by Retinal Implant AG based in Reutlingen, Germany, uses a CMOS active
pixel sensor array that is implanted subretinally, beneath the transparent retina, replacing the defunct photoreceptor cells. It received
CE marking for sale in Europe in 2013, and the latest version, the Alpha AMS, obtained CE marking in 2016. The devices contain
a photodiode-amplifying microelectronics electrode set within each pixel of the array. Incident light on the photodiode is converted
and amplified into an electronic pulse and is delivered via the electrodes to nearby nerve cells. The circuitry is created using a CMOS
process. This is encapsulated by an insulating material, such as a polymer, that will prevent fluids in the eye from shorting the
circuitry and thus causing device failure. Electrodes are connected to the circuitry and made of conducting noncorrosive materials,
such as TiN or IrO (Graf et al., 2009). Work by this group in 1999 investigated the biocompatibility of various semiconductor and
electrode materials including SiO2, Si3N4, TiN, and Ir (Guenther et al., 1999). Rat retinal cells were seeded onto the materials, and
biocompatibility was determined by examining cell attachment and growth. Cells grew on all materials, and although there were
fewer cells on TiN compared with Ir, it appears that this material is still used for electrodes. The chip is mounted on a metallized
Fig. 2 Diagram of the InnFocus MicroShunt in situ in the anterior chamber angle. Provided by InnFocus Inc.
polyimide ribbon cable that exits the eye and extends toward an induction coil located behind the ear, similar to a cochlear implant.
The coil must be hermetically sealed, usually in silicone or ceramic, similar to other implants. Power is wirelessly transferred from an
external supply to this coil and via the ribbon cable to the chip. Polyimide has previously been demonstrated as a biocompatible,
flexible substrate for electrode arrays (Klinge et al., 2001; Seo et al., 2004; Richardson et al., 1993).
The Argus II (Fig. 3), developed by Second Sight based in Sylar, CA, the United States, is an epiretinal device that has been
granted permission to be marketed in the United States and Europe. It consists of a pair of glasses with a mounted camera that
is connected to a small processing unit. Images are captured and processed outside the eye, and then, information and power
are transmitted by transcranial induction to a paired coil and electronics unit that is held in place outside of the eye by a scleral
band. The scleral band and antenna are encased in silicone (Second Sight Medical Products, 2013), which functions similarly to
scleral buckles mentioned previously. The electronics unit is hermetically sealed and sends information and electric pulses, via
a ribbon cable that passes through the eye, to an electrode array attached to the inner surface of the retina. The conducting wires
of the electrode cable are encased in polyimide, but the end of cable and the electrode array are coated with silicone (Second Sight
Medical Products, 2013). Electric impulses are delivered to the retina via platinum electrodes. The electrode array is held in position
with a spring-loaded titanium alloy retinal tack (de Juan et al., 2013).
One of the drawbacks of the systems outlined earlier and those similar is the fact that information is captured outside the eye and
needs to be transferred, along with power, from an external source to the inside of the eye. A device that was contained completely
within the eye at the site of operation would be beneficial as image capture would move with the patients’ eye, and not their head as
with external mounted image capture devices and would not necessarily require materials to pass through different layers of tissue.
Optoelectronic polymers are materials that could produce these benefits and are currently being investigated for their use in electric
stimulation of the retina. These polymers create an electric impulse when struck by photons of light but are much more flexible
compared to stiff silicon-based photovoltaic materials. Work using regioregular poly(3-hexylthiophene) blended with or without
phenyl-C61-butyric acid methyl ester as a semiconducting layer and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) as
a conductive layer has shown promising results. They have been demonstrated to restore light sensitivity to diseased explanted
rat retinas (Ghezzi et al., 2013) and are stable for at least 5 months when implanted into rat retinas (Antognazza et al., 2016). These
materials show promise, but much more work is required.
Tissue Regeneration
The regeneration of damaged tissue using tissue engineering strategies is being investigated for tissues throughout the eye. The two
major areas are the ocular surface, involving the cornea and conjunctiva, and the retinal pigment epithelial layer under the retina.
The precise functional requirements are different in either case, but there are also many similarities.
In cases of corneal damage, the current treatment is replacement of the dysfunctional tissue with a corneal transplant; however,
due to the high demand for tissue, there is a shortage of donors, and so tissue engineering approaches are needed to provide alter-
native options. The cornea is composed of five layers (Fig. 4A): the outermost epithelial layer with its underlying Bowman’s
membrane; the stroma whose regular arrangement of collagen fibers is responsible for the transparency of the cornea; and finally,
the most posterior layer, the monolayer of corneal endothelial cells sitting on the Descemet’s membrane. The cornea is continuous
with the tough, opaque sclera; therefore, it plays a key role in maintenance of the protective outer layer of the eye but must be trans-
parent to allow light to reach the retina. The conjunctiva (Fig. 4B) is a transparent membrane that covers the inner surface of the
eyelids and extends over the sclera to meet the limbus at the periphery of the cornea. It is a stratified, nonkeratinized epithelium that
serves as a barrier to protect underlying tissue but also as a mucous membrane. Goblet cells within the epithelium secrete mucins
that maintain the tear film protecting the cornea from infection and desiccation. One of the striking features of the conjunctiva is the
flexibility of the continuous tissue over the large and cavernous regions of the eyelids.
Fig. 3 A cartoon of an implanted Argus II epiretinal implant, developed by Second Sight. Information and power are transmitted to the electronics
case via the antenna. From the electronic case, electric impulses are sent to various electrodes on the array, depending upon the information
received. The electrode array is pinned to the internal surface of the retina.
Fig. 4 (A) Schematic showing the five layers of the cornea; outermost epithelium, Bowman’s layer, stroma with interspersed keratocytes, Desce-
met’s membrane and most posterior endothelial layer that contacts the aqueous humor of the anterior chamber (B) conjunctiva comprises a non-
keratinized, stratified squamous epithelium interspersed with goblet cells.
Disease and disorder can affect each of the components of the ocular surface, and the latest surgical techniques often target the
specific layer affected. Therefore, recent tissue engineering strategies have followed the same path with most attempting to combine
an ex vivo expanded cell population with a suitable material for transplant that meets the particular requirements of the damaged
tissue.
Retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) cause damage to the retinal
pigment epithelium. The retinal pigment epithelium is a monolayer of cells that sits beneath this neural retina on a thin basement
membrane known as Bruch’s membrane that separates it from the blood vessels in the choroid. The retinal pigment epithelial (RPE)
monolayer of cells is the crucial component of the support tissue of the retina and is known to play a key role in maintaining its
normal functions. In particular, the RPE cells phagocytose the spent outer segments of the photoreceptors. Damage to, or loss of RPE
cells can have serious consequences, particularly as its ability to support the overlying photoreceptors will be compromised leading
to irreversible vision loss. Cell transplantation of RPE cells offers a potential therapy to prevent photoreceptor loss. Successful cell
transplantation requires the precise delivery, appropriate cell organization, alignment, integration, and differentiation of cells to
form a functional epithelial monolayer.
Natural Scaffolds
The complex structure of the corneal layers and cellular basement membranes pose a significant challenge to replicate. Some regen-
erative medicine strategies aim to circumvent this problem by simply reusing these perfected structures as starting scaffolds. Corneal
stroma comprises predominantly type I collagen fibers arranged in a precise orthogonal fashion interspersed with keratocyte cells.
Decellularization of stromal tissue retains the precise structure of the scaffold allowing it to be transplanted in cases of stromal scar-
ring with reduced risk of rejection (Choi et al., 2011).
A commonly trialed natural material in ocular surface tissue engineering is amniotic membrane, the innermost layer of the fetal
membrane. This is because it has some very useful characteristics such as anti-angiogenic and antiinflammatory properties, its rela-
tive elasticity and flexibility, and its favorable basement membrane features that are conducive to epithelial cell growth (Rahman
et al., 2009). For these reasons, it has been used in conjunctival reconstruction, as a carrier/substrate for corneal epithelial layer
transplantation and also as a substrate for corneal endothelial cell expansion. As is the case with any biological tissue, there is signif-
icant donor variation in terms of thickness and related degradation rate and the presence of anti-inflammatory cytokines. In addi-
tion, the tissue preparation protocols are not standardized between centers, which make clinical data relating to its use in tissue
engineering difficult to interpret as the membrane composition may differ widely.
An alternative to tissue engineering using a biomaterial substrate is injected cell therapy, which is currently being explored as
a potential therapy for damaged corneal endothelium (Okumura et al., 2012). This approach involves simply injecting a cell suspen-
sion into the anterior chamber and allowing the monolayer to form in situ on the posterior corneal surface. There are some concerns
about this approach regarding the ultimate destination of the injected cells at aberrant ocular sites. Therefore, due to the relative
simplicity of the corneal endothelium, attempts have been made to engineer a scaffold-free endothelial cell sheet ex vivo using a ther-
moresponsive poly (N-isopropylacrylamide) (pNIPAAm) substrate for cell growth. At 37 C, the surface is hydrophobic, and cells
can adhere, but when the temperature is lowered to 20 C, the polymer chains of pNIPAAm hydrate to form an expanded structure
detaching the cells as an intact sheet without the need to use enzymatic digestion (Tang et al., 2012). The major advantage of a scaf-
fold-free sheet is that they have been associated with fewer inflammatory responses that are typically observed in the biodegradation
of natural scaffolds; however, delivery of the intact sheet to the site of transplant still remains a significant challenge.
In the subretinal position, injection of RPE cell suspensions and tissue grafts have resulted in poor cell survival and low integra-
tion of cells, with disorganized and incorrectly localized grafts and difficulty in retaining the cells in the targeted site (Tomita et al.,
2005; MacLaren et al., 2006; Klassen et al., 2004). However, this continues to be a strategy employed in current clinical trials (Kim-
brel and Lanza, 2015). Transplantation of RPE cells onto alternative natural material including decellularized Bruch’s membrane,
amniotic membrane, Descemet’s membrane, and lens capsule (Hartmann et al., 1999; Lee et al., 2007; Turowski et al., 2004) that
mimic the mechanical properties of natural tissue has been investigated with varying levels of success. Limitations with these
approaches arise due to the limited expansion of cells and availability of donor tissue. Scaffold-free approaches using pNIPAAm,
similar to that described earlier for corneal endothelium, have been also reported (Yaji et al., 2009).
Biological Polymers
Collagen is widely used in corneal tissue engineering since the basement membranes of both the corneal epithelial and endothelial
layers, as well as the stroma, are abundant in collagens including types IeV. Many research groups have developed stromal tissue
equivalents using animal-derived collagen (Levis et al., 2010, 2013), but an alternative strategy has been reported that uses recombi-
nant type III human collagen cross-linked with carbodiimide. This material was designed to mimic the collagenous extracellular
matrix of the stroma to stimulate in situ repopulation of the graft with keratocytes and corneal nerves. The material has sufficient
tensile strength and elasticity and allows keratocytes to repopulate the graft over time in addition to maintaining a tear film on the
surface (Liu et al., 2008). These hydrogels have since been trialed in clinical studies with four patients all displaying stable corneal
regeneration 4 years after implantation (Fagerholm et al., 2014). These materials have also been investigated for conjunctival epithe-
lial and goblet cell growth (Table 1) (He et al., 2016).
Other natural materials that have been used for ocular surface repair are chitosan, silk fibroin, and fibrin. The latter forms the
basis of Holoclar, the first stem cell-based medicinal product to be approved for use in Europe. Human limbal stem cells are culti-
vated on a fibrin (thrombin and fibrinogen) membrane and transplanted onto the ocular surface to repair damage in cases where
the native limbal stem cell population has been destroyed by chemical or thermal burns. The fibrin substrate supports the expansion
and growth of a healthy epithelial layer and, after transplantation, slowly degrades leaving the stem cells to seed the regrowth of
a transparent cornea. One additional advantage of the fibrin gels is that its constituents can be produced from autologous plasma
(Pellegrini et al., 2016).
In vitro studies have shown that collagen scaffolds are capable of supporting RPE cell adherence and proliferation, with pheno-
typic characteristics of differentiated RPE cells (Malafaya et al., 2007; Karwatowski et al., 1995). In vivo studies in rabbits have
shown biocompatibility with no immune rejection or inflammation and the ability to phagocytose photoreceptors (Thumann
et al., 1997).
Synthetic Polymers
Synthetic materials have an advantage over natural materials because they can be produced with a fully defined composition and
designed features and structures. Both poly-L-lactic acid (PLLA) and poly-DL-lactic-co-glycolic acid (PLGA) have been tested as
Table 1 Conjunctival cells grown on various biopolymer and synthetic polymer substrates (He et al., 2016)
Material Production method Cell interaction Mechanical properties
Recombinant human collagen Hydrogel Good viability Variable degradation rates
Majority of cells CK7þ (goblet)

>90%
Recombinant human collagen 2- Hydrogel Good viability Variable degradation rates
methacryloylxyethyl phosphorylcholine
>90%
Arginine-glycine-aspartic acid (RGD)-modified Film Good viability Variable degradation rates
fibroin (Bombyx mori)
>90%
Poly-D-lysine (PDL)-coated fibroin (B. mori) Film Good viability Variable degradation rates

>80%
Collagen Electrospun N/A Too fragile
Poly (acrylic acid)(PAA) Electrospun Reasonable viability Control over structure and chemical
composition
>90% Difficult to handle
Poly (caprolactone)(PCL) Electrospun Very low cell viability Control over structure and chemical
composition
Poly (vinyl alcohol) (PVA) Electrospun No cell attachment Control over structure and chemical
composition
potential substrates for corneal endothelial cell monolayer transplantation, as well as polyvinylidene fluoride coated with type IV
collagen. Confluent monolayers formed on PLLA and PLGA substrates and degradation could be controlled by varying the mono-
mer ratios to allow the carrier to remain intact for an interval sufficient for cells to lay down their own basement membrane (Had-
lock et al., 1999). However, current surgical treatments commonly graft an endothelial layer with a thin (< 150 mm) piece of stroma
attached to the posterior corneal surface. This stromal layer persists long term, and vision is still restored, so it is debatable as to
whether a degradable carrier is required.
The requirements for conjunctiva reconstruction differ from that of the cornea. The substitute must consist of a stable elastic
matrix, but does not necessarily need to be transparent, which allows for a wider variety of materials to be investigated. Maintaining
a mixed population of goblet and epithelial cells in a conjunctival tissue equivalent is an important consideration. It is reported that
while biopolymers such as recombinant human collagen and coated silk have variable degradation rates, their cell compatibility
and handleability was superior to that of the synthetic polymers tested (Table 1) (He et al., 2016).
PLGA/PLLA scaffolds have been evaluated for their ability to support RPE cell delivery and survival and shown to promote inte-
gration and differentiation of the cells (Tomita et al., 2005; Lavik et al., 2005). Electrospinning of PLGA-generated 3-D nanofibrous
scaffolds with mechanical properties similar to Bruch’s membrane and when coated with collagen I were able to support human
RPE cells, with a correctly orientated monolayer of cells (Warnke et al., 2013). Fibrous PLA scaffold improved retinal ganglion
cell survival and guided retinal axon orientation onto rat retinal explants in vitro (Kador et al., 2013). However, when the PLA/
PLGA scaffolds were thick (150–250 mm), they resulted in retinal detachments in rodent models and were an unsuitable scaffold
for subretinal transplantation. In addition, in some studies, degradation of PLGA/PLLA (Tomita et al., 2005; Lavik et al., 2005; Warf-
vinge et al., 2005) caused a buildup of acidic degradation products within the subretinal space leading to inflammation, fibrosis,
and cell death (Warfvinge et al., 2005; Sundback et al., 2005). PCL, which degrades more slowly than PLGA/PLLA, showed no
increase in acidity within the subretinal space (Tao et al., 2007; Grayson et al., 2004). In vitro studies showed an increase in cell
attachment and organization with a decrease in markers of early progenitors and an increase in photoreceptor markers (Steedman
et al., 2010). Thin (5–6 mm) PCL scaffolds that were highly permeable resulted in minimal physical distortion and good perme-
ability (Tao et al., 2007) and were shown to be highly compatible with the subretinal space in mice and pigs. They supported
RPE cells (Steedman et al., 2010), and porous scaffolds with 1 mm continuous pores supported a functional monolayer of fetal
human RPE cells that expressed mature markers, increased pigmentation, cell density, barrier function, polarized growth factor
secretion, and metabolite transport (McHugh et al., 2014). Improving the hydrophilicity and surface morphology of PCL scaffolds
using plasma treatments or alkaline hydrolysis increased the biocompatibility, wettability, cell adhesion, and viability of the cells
and led to a functional RPE cell layer (Shahmoradi et al., 2017). Hybrid electrospun scaffolds fabricated with PCL, silk fibroin, and
gelatin generated thin porous scaffolds that sustained a RPE morphological phenotype without signs of inflammation (Xiang et al.,
2014).
Some researchers have suggested that biostable scaffolds may be advantageous in the long term by providing support for the
transplanted cells. This is because biodegradable substrates may eventually leave the cells in direct contact with compromised
Bruch’s membrane and because the local response to degradation products may have negative effects. Biostable polymers investi-
gated include parylene, polyethylene terephthalate, and polyimide (Ilmarinen et al., 2015; Pennington and Clegg, 2016; Stanzel
et al., 2014). Surface-modified polytetrafluoroethylene (ePTFE), which is biocompatible, biostable, porous, and flexible and has
good mechanical properties for surgical handling, has also demonstrated the ability to support a functional layer of RPE cells
(Kearns et al., 2012; Krishna et al., 2011).
Conclusion
A wide range of biomaterials from decellularized tissue, biological polymers, synthetic polymers, and metals to ceramics have been
used in ophthalmic applications. The functional requirement of the biomaterial must be considered in each application and in the
eye the array of requirements is also broad, including the ability to refract the light coming into the eye, passive or active space
filling, controlling fluid flow out of the eye, electric stimulation of the neural retina or tissue regeneration. For some applications,
the use of biomaterials has been well established for many years, for example, as contact or intraocular lenses or silicone oil tam-
ponade agents to treat retinal detachments. In other applications, however, the optimal biomaterial is not yet defined, such as in
relation to tissue engineering scaffolds for subretinal RPE or corneal endothelial cell transplantation, and in these areas, further
studies are required.
References
Annaka, M., et al. (2011). Design of an injectable in situ gelation biomaterials for vitreous substitute. Biomacromolecules, 12, 4011–4021.
Antognazza, M. R., et al. (2016). Characterization of a polymer-based, fully organic prosthesis for implantation into the subretinal space of the rat. Advanced Healthcare Materials, 5,
2271–2282.
Apple, D. J., et al. (1992). Posterior capsule opacification. Survey of Ophthalmology, 37, 73–116.
Auffarth, G. U., et al. (2004). Comparison of Nd:YAG capsulotomy rates following phacoemulsification with implantation of PMMA, silicone, or acrylic intra-ocular lenses in four
European countries. Ophthalmic Epidemiology, 11, 319–329.
Baino, F. (2010). Scleral buckling biomaterials and implants for retinal detachment surgery. Medical Engineering & Physics, 32, 945–956.
Baino, F., & Potestio, I. (2016). Orbital implants: state-of-the-art review with emphasis on biomaterials and recent advances. Materials Science and Engineering: C, 69, 1410–1428.
Baino, F., & Vitale-Brovarone, C. (2015). Ceramics for oculo-orbital surgery. Ceramics International, 41, 5213–5231.
Barth, H., Crafoord, S., Andréasson, S., & Ghosh, F. (2016). A cross-linked hyaluronic acid hydrogel (Healaflow®) as a novel vitreous substitute. Graefe’s Archive for Clinical and
Experimental Ophthalmology, 254, 697–703.
Caló, E., & Khutoryanskiy, V. V. (2015). Biomedical applications of hydrogels: a review of patents and commercial products. European Polymer Journal, 65, 252–267.
Caramoy, A., et al. (2015). Development of emulsification resistant heavier-than-water tamponades using high molecular weight silicone oil polymers. Journal of Biomaterials
Applications, 30, 212–220.
Carreira, A. S., et al. (2014). New drug-eluting lenses to be applied as bandages after keratoprosthesis implantation. International Journal of Pharmaceutics, 477, 218–226.
Chan, Y. K., et al. (2011). Emulsification of silicone oil and eye movements. Investigative Ophthalmology & Visual Science, 52, 9721–9727.
Chan, Y. K., Cheung, N., Chan, W. S. C., & Wong, D. (2015). Quantifying silicone oil emulsification in patients: Are we only seeing the tip of the iceberg? Graefe’s Archive for Clinical
and Experimental Ophthalmology, 253, 1671–1675.
Chirila, T. V., Hong, Y., Dalton, P. D., Constable, I. J., & Refojo, M. F. (1998). The use of hydrophilic polymers as artificial vitreous. Progress in Polymer Science (Oxford), 23,
475–508.
Choi, H. J., et al. (2011). Efficacy of pig-to-rhesus lamellar corneal xenotransplantation. Investigative Ophthalmology & Visual Science, 52, 6643–6650.
Ciolino, J. B., et al. (2014). In vivo performance of a drug-eluting contact lens to treat glaucoma for a month. Biomaterials, 35, 432–439.
Crovace, M. C., Souza, M. T., Chinaglia, C. R., Peitl, O., & Zanotto, E. D. (2016). Biosilicate® d A multipurpose, highly bioactive glass-ceramic. In vitro, in vivo and clinical trials.
Journal of Non-Crystalline Solids, 432(Part A), 90–110.
D’Amico, D. J. (2016). Different preferences between United States and European vitreoretinal surgeons: Personal observations. Current Opinion in Ophthalmology, 27, 196–200.
Dei Cas, R., et al. (1998). Gore-Tex as an orbital implant material. Ophthalmic Plastic & Reconstructive Surgery, 14, 425–431.
Dixon, P., et al. (2015). Therapeutic contact lenses: A patent review. Expert Opinion on Therapeutic Patents, 25, 1117–1129.
Fagerholm, P., et al. (2014). Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold. Biomaterials, 35, 2420–2427.
Friberg, T. R., & Guibord, N. M. (1999). Corneal endothelial cell loss after multiple vitreoretinal procedures and the use of silicone oil. Ophthalmic Surgery and Lasers, 30, 528–534.
Gallagher, A. G., et al. (2016). A novel peptide hydrogel for an antimicrobial bandage contact lens. Advanced Healthcare Materials, 5, 2013–2018.
Ghezzi, D., et al. (2013). A polymer optoelectronic interface restores light sensitivity in blind rat retinas. Nature Photonics, 7, 400–406.
Giordano, G. G., & Refojo, M. F. (1998). Silicone oils as vitreous substitutes. Progress in Polymer Science, 23, 509–532.
Graf, H. G., et al. (2009). High dynamic range cmos imager technologies for biomedical applications. IEEE Journal of Solid-State Circuits, 44, 281–289.
Grayson, A. C., et al. (2004). Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-
delivery microchip. Journal of Biomaterials Science. Polymer Edition, 15, 1281–1304.
de Groot, J. H., et al. (2001). Injectable intraocular lens materials based upon hydrogels. Biomacromolecules, 2, 628–634.
Guenther, E., Tröger, B., Schlosshauer, B., & Zrenner, E. (1999). Long-term survival of retinal cell cultures on retinal implant materials. Vision Research, 39, 3988–3994.
Ha, S., et al. (2016). Towards high-resolution retinal prostheses with direct optical addressing and inductive telemetry. Journal of Neural Engineering, 13. Article No. 056008.
Hadlock, T., Singh, S., Vacanti, J. P., & McLaughlin, B. J. (1999). Ocular cell monolayers cultured on biodegradable substrates. Tissue Engineering, 5, 187–196.
Hartmann, U., Sistani, F., & Steinhorst, U. H. (1999). Human and porcine anterior lens capsule as support for growing and grafting retinal pigment epithelium and iris pigment
epithelium. Graefes Archive for Clinical and Experimental Ophthalmology, 237, 940–945.
He, M., et al. (2016). Artificial polymeric scaffolds as extracellular matrix substitutes for autologous conjunctival goblet cell expansion. Investigative Ophthalmology & Visual Science,
57, 6134–6146.
Heimann, H., Stappler, T., & Wong, D. (2008). Heavy tamponade 1: A review of indications, use, and complications. Eye, 22, 1342–1359.
Ichhpujani, P., Jindal, A., & Jay Katz, L. (2009). Silicone oil induced glaucoma: A review. Graefe’s Archive for Clinical and Experimental Ophthalmology, 247, 1585–1593.
Ilmarinen, T., et al. (2015). Ultrathin polyimide membrane as cell carrier for subretinal transplantation of human embryonic stem cell derived retinal pigment epithelium. PLoS ONE,
10, e0143669.
Jordan, D. R. M. D., Gilberg, S. M. D., & Mawn, L. A. M. D. (2003). The bioceramic orbital implant: Experience With 107 Implants. Ophthalmic Plastic & Reconstructive Surgery, 19,
128–135.
de Juan, E., Spencer, R., Barale, P.-O., da Cruz, L., & Neysmith, J. (2013). Extraction of retinal tacks from subjects implanted with an epiretinal visual prosthesis. Graefe’s Archive
for Clinical and Experimental Ophthalmology, 251, 2471–2476.
Jung, S. K., Cho, W. K., Paik, J. S., & Yang, S. W. (2012). Long-term surgical outcomes of porous polyethylene orbital implants: A review of 314 cases. British Journal of
Ophthalmology, 96, 494–498.
Kador, K. E., et al. (2013). Tissue engineering the retinal ganglion cell nerve fiber layer. Biomaterials, 34, 4242–4250.
Karesh, J. W., Dresner, S. C., & Dutton, J. J. (1994). High-density porous polyethylene (Medpor) as a successful anophthalmic socket implant. Ophthalmology, 101, 1688–1696.
Karwatowski, W. S., et al. (1995). Preparation of Bruch’s membrane and analysis of the age-related changes in the structural collagens. The British Journal of Ophthalmology, 79,
944–952.
Kearns, V., et al. (2012). Plasma polymer coatings to aid retinal pigment epithelial growth for transplantation in the treatment of age related macular degeneration. Journal of
Khan, M. A. M. D., Brady, C. J. M. D., & Kaiser, R. S. M. D. (2015). Clinical management of proliferative vitreoretinopathy: An update. Retina, 35, 165–175.
Kimbrel, E. A., & Lanza, R. (2015). Current status of pluripotent stem cells: Moving the first therapies to the clinic. Nature Reviews. Drug Discovery, 14, 681–692.
Kirchhof, S., Goepferich, A. M., & Brandl, F. P. (2015). Hydrogels in ophthalmic applications. European Journal of Pharmaceutics and Biopharmaceutics, 95(Part B), 227–238.
Klassen, H. J., et al. (2004). Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Investigative
Ophthalmology & Visual Science, 45, 4167–4173.
Kleinberg, T. T., Tzekov, R. T., Stein, L., Ravi, N., & Kaushal, S. (2011). Vitreous substitutes: A comprehensive review. Survey of Ophthalmology, 56, 300–323.
Klinge, P. M., et al. (2001). Immunohistochemical characterization of axonal sprouting and reactive tissue changes after long-term implantation of a polyimide sieve electrode to the
transected adult rat sciatic nerve. Biomaterials, 22, 2333–2343.
Koopmans, S. A., Terwee, T., Barkhof, J., Haitjema, H. J., & Kooijman, A. C. (2003). Polymer refilling of presbyopic human lenses in vitro restores the ability to undergo
accommodative changes. Investigative Ophthalmology & Visual Science, 44, 250–257.
Koopmans, S. A., Terwee, T., & van Kooten, T. G. (2011). Prevention of capsular opacification after accommodative lens refilling surgery in rabbits. Biomaterials, 32, 5743–5755.
van Kooten, T. G., et al. (2006). Development of an accommodating intra-ocular lensdIn vitro prevention of re-growth of pig and rabbit lens capsule epithelial cells. Biomaterials,
27, 5554–5560.
Krishna, Y., et al. (2011). Expanded polytetrafluoroethylene as a substrate for retinal pigment epithelial cell growth and transplantation in age-related macular degeneration. British
Journal of Ophthalmology, 95, 569–573.
Lavik, E. B., Klassen, H., Warfvinge, K., Langer, R., & Young, M. J. (2005). Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal
progenitors. Biomaterials, 26, 3187–3196. http://www.sciencedirect.com/science/article/pii/S0142961204007719.
Lee, H. I., et al. (2007). The efficacy of an acrylic intraocular lens surface modified with polyethylene glycol in posterior capsular opacification. Journal of Korean Medical Science,
22, 502–507.
Levis, H. J., Brown, R. A., & Daniels, J. T. (2010). Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture. Biomaterials, 31, 7726–7737.
http://www.sciencedirect.com/science/article/B7726TWB-7750NH7708G-7727/7722/64200e64278cfc64207b64206da64278af64205c64200a64205a64206c64702d.
Levis, H. J., Massie, I., Dziasko, M. A., Kaasi, A., & Daniels, J. T. (2013). Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture.
Liang, C., et al. (1998). An evaluation of methylated collagen as a substitute for vitreous and aqueous humor. International Ophthalmology, 22, 13–18.
Light, D. J. (2006). Silicone oil emulsification in the anterior chamber after vitreoretinal surgery. OptometrydJournal of the American Optometric Association, 77, 446–449.
Lin, X. M. D. P., et al. (2012). Preliminary efficacy and safety of a silicone oil-filled foldable capsular vitreous body in the treatment of severe retinal detachment. Retina, 32,
729–741.
Liu, W., et al. (2008). Recombinant human collagen for tissue engineered corneal substitutes. Biomaterials, 29, 1147–1158.
Liu, Y. C., Wong, T. T., & Mehta, J. S. (2013). Intraocular lens as a drug delivery reservoir. Current Opinion in Ophthalmology, 24, 53–59.
Ma, X., Schou, K. R., Maloney-Schou, M., Harwin, F. M., & Ng, J. D. (2011). The porous polyethylene/bioglass spherical orbital implant: A retrospective study of 170 cases.
Ophthalmic Plastic & Reconstructive Surgery January/February, 27, 21–27.
MacLaren, R. E., et al. (2006). Retinal repair by transplantation of photoreceptor precursors. Nature, 444, 203–207. https://doi.org/210.1038/nature05161.
Maghami, M. H., Sodagar, A. M., Lashay, A., Riazi-Esfahani, H., & Riazi-Esfahani, M. (2014). Visual prostheses: The enabling technology to give sight to the blind. Journal of
Ophthalmic and Vision Research, 9, 494–505.
Malafaya, P. B., Silva, G. A., & Reis, R. L. (2007). Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Advanced
Drug Delivery Reviews, 59, 207–233.
Manasses, D. T., & Au, L. (2016). The new era of glaucoma micro-stent surgery. Ophthalmology and Therapy, 5, 135–146.
Maulvi, F. A., Soni, T. G., & Shah, D. O. (2016). A review on therapeutic contact lenses for ocular drug delivery. Drug Delivery, 23, 3017–3026.
Mawn, L. A., Jordan, D. R., & Gilberg, S. (1998). Scanning electron microscopic examination of porous orbital implants. Canadian Journal of Ophthalmology, 33, 203–209.
McHugh, K. J., Tao, S. L., & Saint-Geniez, M. (2014). Porous poly(epsilon-caprolactone) scaffolds for retinal pigment epithelium transplantation. Investigative Ophthalmology &
Visual Science, 55, 1754–1762.
Morphis, G., et al. (2012). Retrospective review of 50 eyes with long-term silicone oil tamponade for more than 12 months. Graefe’s Archive for Clinical and Experimental
Okumura, N., et al. (2012). ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. The American Journal of Pathology,
181, 268–277.
Ozdek, S., Yuksel, N., Gurelik, G., & Hasanreisoglu, B. (2011). High-density silicone oil as an intraocular tamponade in complex retinal detachments. Canadian Journal of
Ophthalmology/Journal Canadien d’Ophtalmologie, 46, 51–55.
Pastor, J. C., Zarco, J. M., Del Nozal, M. J., Pampliega, A., & Marinero, P. (1998). Clinical consequences of the use of highly purified silicone oil: Comparative study of highly and
less purified silicone oil. European Journal of Ophthalmology, 8, 179–183.
Patel, M., et al. (2010). Cyclic acetal hydroxyapatite nanocomposites for orbital bone regeneration. Tissue Engineering – Part A, 16, 55–65.
Pellegrini, G., et al. (2016). From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU. Regenerative Medicine, 11, 407–420.
Pennington, B. O., & Clegg, D. O. (2016). Pluripotent stem cell-based therapies in combination with substrate for the treatment of age-related macular degeneration. Journal of
Ocular Pharmacology and Therapeutics.
Pinchuk, L., et al. (2017). The development of a micro-shunt made from poly(styrene-block-isobutylene-block-styrene) to treat glaucoma. Journal of Biomedical Materials Research.
Part B, Applied Biomaterials, 105, 211–221.
De Potter, P., Shields, C. L., Shields, J. A., & Singh, A. D. (1994). Use of the hydroxyapatite ocular implant in the pediatric population. Archives of Ophthalmology, 112, 208–212.
Pruett, R. C., Schepens, C. L., & Swann, D. A. (1979). Hyaluronic acid vitreous substitute: A six-year clinical evaluation. Archives of Ophthalmology, 97, 2325–2330.
Rahman, I., Said, D. G., Maharajan, V. S., & Dua, H. S. (2009). Amniotic membrane in ophthalmology: Indications and limitations. Eye (Lond), 23, 1954–1961.
Refojo, M. F. (1996). Ophthalmic applications. In B. D. Ratner, A. S. Hoffman, F. J. Schoen, & J. E. Lemons (Eds.), Biomaterials science: An introduction to materials in medicine.
United States of America: Academic Press.
Richardson, R. R., Jr., Miller, J. A., & Reichert, W. M. (1993). Polyimides as biomaterials: Preliminary biocompatibility testing. Biomaterials, 14, 627–635.
Rønbeck, M., & Kugelberg, M. (2014). Posterior capsule opacification with 3 intraocular lenses: 12-year prospective study. Journal of Cataract & Refractive Surgery, 40, 70–76.
Sami, D., Young, S., & Petersen, R. (2007). Perspective on orbital enucleation implants. Survey of Ophthalmology, 52, 244–265.
Santhanam, S., Liang, J., Struckhoff, J., Hamilton, P. D., & Ravi, N. (2016). Biomimetic hydrogel with tunable mechanical properties for vitreous substitutes. Acta Biomaterialia, 43,
327–337.
Schellini, S., El Dib, R., Silva, L. R. E., Farat, J. G., Zhang, Y., & Jorge, E. C. (2016). Integrated versus non-integrated orbital implants for treating anophthalmic sockets. Cochrane
Database of Systematic Reviews, 11. https://doi.org/10.1002/14651858.CD010293.pub2. Article No. CD010293.
Schepens, C. L., & Acosta, F. (1991). Scleral implants: An historical perspective. Survey of Ophthalmology, 35, 447–453.
Schramm, C., et al. (2012). The cross-linked biopolymer hyaluronic acid as an artificial vitreous substitute. Investigative Ophthalmology & Visual Science, 53, 613–621.
Scott, I. U., et al. (2005). Outcomes of complex retinal detachment repair using 1000- vs 5000-centistoke silicone oil. Archives of Ophthalmology, 123, 473–478.
Sebag, J. (1992). Anatomy and pathology of the vitreo-retinal interface. Eye, 6, 541–552.
Sebag, J. (1998). Macromolecular structure of the corpus vitreus. Progress in Polymer Science, 23, 415–446.
Second Sight Medical Products (2013) Argus II Retinal Prosthesis System Surgeon Manual. Document number: 09001-004. Second Sight, Sylmar.
Seo, J.-M., et al. (2004). Biocompatibility of polyimide microelectrode array for retinal stimulation. Materials Science and Engineering: C, 24, 185–189.
Shahmoradi, S., et al. (2017). Controlled surface morphology and hydrophilicity of polycaprolactone toward human retinal pigment epithelium cells. Materials Science and
Engineering: C, 73, 300–309.
Son, J., Kim, C.-S., & Yang, J. (2012). Comparison of experimental porous silicone implants and porous silicone implants. Graefe’s Archive for Clinical and Experimental
Stanzel, B. V., et al. (2014). Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space. Stem Cell
Reports, 2, 64–77.
Steedman, M. R., Tao, S. L., Klassen, H., & Desai, T. A. (2010). Enhanced differentiation of retinal progenitor cells using microfabricated topographical cues. Biomedical
Microdevices, 12, 363–369.
Su, X., et al. (2015). Recent progress in using biomaterials as vitreous substitutes. Biomacromolecules, 16, 3093–3102.
Sundback, C. A., et al. (2005). Biocompatibility analysis of poly(glycerol sebacate) as a nerve guide material. Biomaterials, 26, 5454–5464. http://www.sciencedirect.com/science/
article/pii/S0142961205001547.
Suter, A. J., Molteno, A. C. B., Bevin, T. H., Fulton, J. D., & Herbison, P. (2002). Long term follow up of bone derived hydroxyapatite orbital implants. British Journal of
Swindle-Reilly, K. E., Reilly, M. A., & Ravi, N. (2016). Biomaterials and Regenerative Medicine in Ophthalmology (Second Edition) (pp. 101–130). Duxford, UK: Woodhead
Publishing.
Tang, Z. L., Akiyama, Y., & Okano, T. (2012). Temperature-responsive polymer modified surface for cell sheet engineering. Polymers, 4, 1478–1498.
Tao, S., et al. (2007). Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space.
Lab on a Chip, 7, 695. http://pubs.rsc.org.ezproxy.liv.ac.uk/en/Content/ArticleLanding/2007/LC/b618583e.
Thomas, M., Shorter, E., Joslin, C. E., McMahon, T. J., & Cortina, M. S. (2015). Contact lens use in patients with Boston keratoprosthesis type 1: Fitting, management, and
complications. Eye and Contact Lens, 41, 334–340.
Thumann, G., Schraermeyer, U., Bartz-Schmidt, K. U., & Heimann, K. (1997). Descemet’s membrane as membranous support in RPE/IPE transplantation. Current Eye Research, 16,
1236–1238.
Tomita, M., et al. (2005). Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells, 23, 1579–1588. http://onlinelibrary.
wiley.com/doi/1510.1634/stemcells.2005-0111/abstract.
Turowski, P., et al. (2004). Basement membrane-dependent modification of phenotype and gene expression in human retinal pigment epithelial ARPE-19 cells. Investigative
Ophthalmology & Visual Science, 45, 2786–2794.
Warfvinge, K., et al. (2005). Retinal progenitor cell xenografts to the pig retina: Morphologic integration and cytochemical differentiation. Archives of Ophthalmology, 123, 1385–
1393. http://archopht.ama-assn.org/cgi/content/abstract/1123/1310/1385.
Warnke, P. H., et al. (2013). Primordium of an artificial Bruch’s membrane made of nanofibers for engineering of retinal pigment epithelium cell monolayers. Acta Biomaterialia, 9,
9414–9422.
Wickham, L., Tranos, P., Hiscott, P., & Charteris, D. (2010). The use of silicone oil-RMN3 (Oxane HD) as heavier-than-water internal tamponade in complicated inferior retinal
detachment surgery. Graefe’s Archive for Clinical and Experimental Ophthalmology, 248, 1225–1231.
Williams, R. L. P., Day, M. P., Garvey, M. J. P., English, R. P., & Wong, D. F. (2010). Increasing the extensional viscosity of silicone oil reduces the tendency for emulsification.
Retina, 30, 300–304.
Williams, R. L., et al. (2011). Injectability of silicone oil-based tamponade agents. British Journal of Ophthalmology, 95, 273–276.
Wong, D., & Williams, R. (2005). In B. Kirchhof, & D. Wong (Eds.), Vitreo-retinal surgery (pp. 147–161). Berlin, Heidelberg: Springer Berlin Heidelberg.
Xiang, P., et al. (2014). A novel Bruch’s membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells. Biomaterials, 35, 9777–9788.
Xinming, L., et al. (2008). Polymeric hydrogels for novel contact lens-based ophthalmic drug delivery systems: A review. Contact Lens and Anterior Eye, 31, 57–64.
Yaji, N., Yamato, M., Yang, J., Okano, T., & Hori, S. (2009). Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model. Biomaterials, 30, 797–803.
Yang, H., Wang, R., Gu, Q., & Zhang, X. (2008). Feasibility study of chitosan as intravitreous tamponade material. Graefe’s Archive for Clinical and Experimental Ophthalmology,
246, 1097–1105.
Yang, W., et al. (2014). Preliminary study on retinal vascular and oxygen-related changes after long-term silicone oil and foldable capsular vitreous body tamponade. Scientific
Reports, 4, 5272.
Yuen, C., Williams, R., Batterbury, M., & Grierson, I. (2006). Modification of the surface properties of a lens material to influence posterior capsular opacification. Clinical &
Experimental Ophthalmology, 34, 568–574.
Biomaterials in Orthopaedics
Emmanuel Gibon and Stuart B Goodman, Stanford University, Stanford, CA, United States
Introduction 301
Polyethylene 301
Ceramics 302
Metals 303
Conclusion 305
References 305
Introduction
Biomaterials in orthopedic surgery cover a broad range of different devices used in different procedures and for different goals.
Among these procedures, total joint replacements (TJRs) of the hip and knee are probably the most successful operations. TJRs
are projected to dramatically increase in number and cost for society (Kurtz et al., 2007). Studies have shown that for orthopedic
surgery alone, the underlying industry is expected to grow to $41.1 billion by 2016 (Richards et al., 2012). Currently, the majority of
the studies analyzing the survivorship of total hip arthroplasty (THA) show data ranging above 90% after 15 years (El Masri et al.,
2010; McLaughlin and Lee, 2006). Similarly, the long-term outcome of total knee arthroplasty (TKA) has shown survivorship after
15 years ranging from 81.7% to 98.14% (Attar et al., 2008; Epinette and Manley, 2007; Melton et al., 2012). However, this outcome
has not come without a great deal of research and development and both successes and failures. Indeed, different implants demon-
strate a variable degree of durability (Keurentjes et al., 2014). Considering TJRs, biomaterials currently used include metals and their
alloys, polymers, and ceramics. In TJRs, surgeons aim to replace the joint itself, that is, articular surfaces and the adjacent bone. To
reach these goals, metal alloys are used to replace the bone that is located underneath the articular cartilage, whereas the articular
surfaces are usually replaced either by polyethylene, ceramics, or in some cases metals. Implants are fixed to the bone with poly-
methyl methacrylate (PMMA) also called “bone cement” or without cement using “press-fit cementless” implants. The aim of
this article is to discuss the biological response to bearing materials used in orthopedic surgery.
Polyethylene
Metal on polyethylene (MoP) is the most commonly reported bearing used in the United States with up to 51% according to Bozic
et al. (2009). Since the original failures when using the polymer polytetrafluorethylene by Sir John Charnley, the biomaterial of
choice for one of the bearing surfaces has included polyethylene (PE). However, periprosthetic bone loss and implant loosening
due to the chronic inflammatory and foreign body reaction to polyethylene wear particles limited implant durability (Gallo
et al., 2013a,b). Therefore, after the first generation of “conventional” polyethylene also called “ultra-high molecular weight poly-
ethylene (UHMWPE),” a second generation of PE was developed to improve wear and mitigate the production of wear particles. Hip
stimulator studies showed that UHMWPE subjected to 10 Mrad of g-irradiation significantly produced fewer particles than 5 Mrad,
g-inert, or conventional UHMWPE (Ries et al., 2001). Second-generation highly cross-linked polyethylene (HXLPE) (Goodman
et al., 2009) includes the doping of the antioxidant vitamin E within the PE or repeated treatment with heating and annealing
of the polymer (Essner et al., 2005). Wear particles are always generated during regular usage of joint replacements. Each individual
takes around 500,000–2000,000 steps each year, and hundreds of thousands to millions of wear debris are generated with each step
(Goodman and Ma, 2010). Generation of wear debris is dramatically increased with increased femoral head roughness (Wang et al.,
1998). Roughening of the femoral head was shown to increase wear by an order of magnitude resulting in a two- to threefold
increase in the wear rate that could be predicted by the following equation: k ¼ 7.21 106 (Ra)0.42 (mm3/Nm), with K being
the specific wear rate and Ra the centerline average roughness of the femoral head. The consequences of such an excessive amount
of generated particles might be harmful and led to periprosthetic osteolysis and potential aseptic loosening of the implants. Camp-
bell et al. (1995) have shown that PE particles are needlelike in shape and are generally < 1 mm in size. PE wear particles migrate
within the entire periprosthetic bed (Schmalzried et al., 1992), known as “the effective joint space.” Production of PE particles leads
to a nonspecific macrophage-mediated foreign body reaction (Goodman, 2007). Macrophages become activated either by phago-
cytosis (Xing et al., 2002) of PE particles or simply by cell membrane contact without phagocytosis. Activation occurs through recep-
tors present in the outer cell membrane (CD11b, CD14, Toll-like receptors, etc.). The Toll-like receptors (TLRs) are known to
function in the innate immune response (Tuan et al., 2008). TLR2 and TLR4 have been found to be critical for particle-induced
osteolysis (Valladares et al., 2014). TLRs are activated by different types of stimuli and act through an adapter protein called myeloid
differentiation primary response gene 88 (MyD88) to induce activation of nuclear factors such as nuclear factor kappa-B (NFkB).
Recently, Lin et al. (2016) using a well-established small-animal model of continuous PE particle delivery (Ma et al., 2008, 2009;

302 Biomaterials: Biomaterial applications and advanced medical technologies j Biomaterials in Orthopaedics
Ortiz et al., 2008) showed that NFkB decoy oligodeoxynucleotide (ODN) mitigates wear-particle-associated bone loss. Moreover,
NFkB decoy ODN reversed the loss of bone mineral density in the distal femur exposed to particles. Activation of NFkB in macro-
phages then triggers an inflammatory cascade leading to the release of various pro-inflammatory cytokines (Wang et al., 2010;
Shanbhag et al., 2007) (IL-1, IL-6, and TNF-a), growth factors (macrophage colony-stimulating factor-1), and chemokines (MIP-
1a and MCP-1) that would ultimately lead to systemic recruitment and local infiltration of more macrophages to the area of inflam-
mation (Ren et al., 2010, 2011). Retrieval studies have found high levels of pro-inflammatory factors released in the surrounding
tissues of failed arthroplasty groups (Wang et al., 2010; Goodman et al., 1998). Locally activated macrophages undergo polarization
toward the M1 (pro-inflammatory) phenotype (Mantovani et al., 2013). M1 macrophages produce primarily pro-inflammatory
mediators including TNF-a, IL-1, and IL-6 and express inducible nitric oxide synthase (iNOS) (Murray et al., 2014). Subsequently,
locally and systemically recruited activated macrophages differentiate into multinucleated giant cells and osteoclasts leading to
bone resorption around implants within a foreign body reaction (Goodman et al., 1989) (Fig. 1). Particle generation remains
an issue; however, newer highly cross-linked PE is now significantly better than conventional PE (Scemama et al., 2017; Langlois
et al., 2015).
Ceramics
Ceramics are considered “hard bearing surfaces.” Current ceramics used for joint replacement are actually composites of two of
ceramicsdalumina (AL2O3) and zirconia (ZrO2) in which alumina is the primary or continuous phase (70%–95% composition)
and zirconia is the secondary phase (30%–5% composition) (Kurtz et al., 2014)dand are called either “alumina-toughened
zirconia” (ATZ) or “zirconia-toughened alumina” (ZTA). Zirconia or alumina alone is no longer used. Ceramic-on-ceramic
(CoC) bearings currently represent 14% of bearing surface usage in the United States (Bozic et al., 2009). However, a ceramic
head can also articulate with a PE liner. The reason for ceramic-on-polyethylene (CoP) bearing surfaces use is twofold. First,
with this bearing combination, surgeons can avoid squeaking from CoC bearing surfaces. Second, with a ceramic head (instead
Joint replacement
with polyethylene
“MoP”
Polyethylene (PE) particles
size: < 1 μm
Macrophage =
key cell
MΦ activation
Cell contact:
Phagocytosis
TLRs, CD11b, CD14
MΦ activation
Systemic
recruitment
MCP-1 MΦ polarization
Cytokines/chemokines
MIP-1α
/ROS release
TNF-α
IL-1,6 M1 = inflammatory response
MSCs
chemotaxis
Foreign-body
reaction,
granuloma
Differentiation into osteoclast

Bone defect
Fig. 1 Biological response to PE particles. Production of PE particles leads to a nonspecific macrophage-mediated foreign body reaction. IL, inter-
leukin; MF, macrophage; MCP-1, monocyte-chemoattractant molecule 1; MGC, multinucleated giant cell; MIP-1, macrophage inflammatory protein 1;
MSCs, mesenchymal stem cells; TLRs, Toll-like receptors; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-alpha.
Biomaterials: Biomaterial applications and advanced medical technologies j Biomaterials in Orthopaedics 303
of a metal femoral head) on a metal taper, surgeons avoid so-called trunnionosis caused by corrosion at the head–neck junction.
Moreover, in vivo studies have shown polyethylene wear rates in CoP bearings to be lower than rates in MoP bearings (Wang
et al., 2013; Meftah et al., 2013) albeit it remains controversial (Kawate et al., 2009). A drawback of CoP bearings compared with
MoP bearings is the price, with CoP being more expensive than MoP. Recent work by Carnes et al. (2016) showed that CoP bearings
might not be worth the cost given the economic burden for society. Hatton et al. (2002) characterized alumina wear debris by
analyzing tissues retrieved at revision surgery. Interestingly, their work compared tissues from CoP implants and MoP implants.
The authors found alumina wear debris to have a bimodal size range distribution. The particles were either nanometric (5–
90 nm) or micrometric (0.05–3.2 mm). Furthermore, histomorphometric analysis showed that tissues retrieved from CoC implants
had significantly less macrophages and giant cells than MoP implants but significantly more neutrophils. Ding et al. (2012) chal-
lenged RW 264.7 macrophages with ceramic and titanium particles. They used three distinct sizes of ceramic particles (0.2–
1.2 mm, 1.2–10 mm, and > 10 mm). Both ceramic and titanium particles increased the expression of TNF-a in a time-dependent
manner. However, ceramic particles provoked a significantly lower production of the pro-inflammatory cytokine TNF-a. The authors
also investigated the effect of the sizes of the particles. Smaller ceramic particles (0.84 mm) are more bioactive and more pro-
inflammatory as they induced significantly higher mRNA and protein expression of TNF-a and RANKL than larger particles. Similarly,
Zhang et al. (2011) challenged osteoblast-like cells (MG-63) and murine RAW 264.7 macrophages with ceramic particles (AL2O3 and
ZrO2) of a nanometric size (40–50 nm). The cytotoxic assay performed with the MG-63 cells showed no adverse effects at any time
points (24, 48, and 72 h). Moreover, ZrO2 particles significantly increased the intrinsic alkaline phosphatase (ALP) activity of MG-63
cells at higher concentration (500 ppm), whereas AL2O3 particles had the same effect but at lower concentrations (115 ppm). Inter-
estingly, the smallest AL2O3 particles were more potent to increase ALP activity. Tsaousi et al. (2010) used primary human fibroblasts
to test the cytotoxicity and genotoxicity of AL2O3 particles. Particles used were either 0.2 nm mean size or 2 mm mean size or fiber
sized (0.9 mm in diameter and 12.03 mm in length). The authors found no significant differences in cell viability between control and
ceramic-treated cells, at all doses and time points studied. Moreover, AL2O3 particles were only weakly genotoxic (for high doses,
involving chromosome loss and tetraploidy). Further studies have shown limited biological reaction to alumina particles and
a weak local inflammatory reaction (Germain et al., 2003; Gutwein and Webster, 2004; Catelas et al., 1998, 1999a,b; Lucarelli
et al., 2004). Ceramic particles appear to be low in number and well tolerated and elicit a minor cellular response (Fig. 2).
Metals
Metal-on-metal (MoM) bearings are also “hard-on-hard” bearings. With the idea of decreasing the production of wear debris and
periprosthetic osteolysis, MoM bearings became popular among the orthopedic community about one decade ago. This resurgence
was supported by studies showing that the volumetric wear released by MoM articulations can be up to 100-fold lower than with
MoP bearings (Greenwald and Garino, 2001; St John et al., 2004). Bozic et al. showed that between October 2005 and December
2006, MoM bearings were the second most used articulations in the United States comprising up to 35% of primary THA (Bozic
et al., 2009). MoM bearings reached a peak in 2008 making up to 40% of all primary THA procedures in the United States. However,
unexpected issues started to be reported with MoM bearings, and the Food and Drug Administration recalled many implants (Ng
et al., 2011; Hug et al., 2013). MoM bearings are made of an alloy containing cobalt (Co) (62%), followed by chromium (Cr)
(27%–30%) and a small amount of molybdenum (Amanatullah et al., 2016). Studies have shown that metal particles or
Joint replacement
with ceramic bearings
“CoC”
Ceramic (Al2O3 and ZrO2) particles
size: 5–90 nm or 0.05–3.2 μm (bimodal distribution)
Activation
CoC bearings: TLRs Phagocytosis
mild inflammatory response
ALP activity
RANKL
TNF-α
Fig. 2 Biological response to ceramic particles. Production of ceramic particles leads to a more mild biological response. MF, macrophage; NFkB,
nuclear factor-kappa-B; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-alpha; ALP, alkaline phosphatase; RANKL, receptor activator of nuclear
factor-kappa-B ligand.
particulates generated are very small ranging from 20 to 90 nm (Doorn et al., 1998; Ingham and Fisher, 2005). Furthermore, ions of
Co and Cr released from MoM bearing surfaces can travel via lymph and blood to various sites throughout the body including bone
marrow, lymph nodes, the spleen, the liver, and the heart (Case et al., 1994; Langkamer et al., 1992; Urban et al., 2000). The effect of
metal particles and ions has been widely studied. DNA can be damaged by Co and Cr through oxidative stress (Cannizzo et al.,
2011). Similarly, free radicals released by Cr can cause breakage in DNA strands, and both Cr and Co ions can induce an inhibition
of DNA repair pathways leading to defective gene expression (Daley et al., 2004; Polyzois et al., 2012). Cells exposed to Co and Cr
had a decrease in DNA synthesis (Hodges and Chipman, 2002). Chromosomal aberrations and translocations have also been re-
ported (Dunstan et al., 2008; Doherty et al., 2001). With these different studies reporting genetic aberrations induced by Co and Cr
released by MoM joint, one may ask if there is an increased risk of developing cancer. In 40 tumor bioassays in which Co and Cr ion
concentrations were significantly higher than those in hip implants, none reported a significant increase in systemic cancer (Chris-
tian et al., 2014). A Finnish study also looked at 2000 patients roughly 15 years after a first-generation MOM articulation and found
that there was no increase in the incidence of cancer. The authors concluded that factors other than the TJR surgery are responsible
for the development of cancer in hip implant patients (Visuri et al., 1996). Similarly, studies of patients with more modern metal-
on-metal articulations failed to suggest an increased risk of cancer (Smith et al., 2012).
Another adverse reaction induced by MoM bearing is metal hypersensitivity. Metal hypersensitivity (or metal allergy) to jewelry
is found in roughly 10%–15% of the general population (Thyssen et al., 2007; Thyssen and Menné, 2010). However, metal ions
released by MoM joints by themselves are too small to mount a hypersensitivity reaction. The immune response is elicited by dena-
turized protein (e.g., albumin) bound to the ions and forming haptens (Adala et al., 2011). The key cell in the biological reaction to
metallic particles and ions is the lymphocyte (T lymphocyte) (Hallab et al., 2001). More specifically, the immune response is a cell-
mediated type IV delayed hypersensitivity response (Hensten-Pettersen, 1993), as opposed to PE particles where it is a nonspecific
nonantigenic immune response (foreign body reaction). The local tissue reaction associated with metallic by-products is now well
established and is referred to as adverse local tissue reaction (ALTR), which is a contributing factor to THA failure (Prieto et al.,
2014). ALTR is often associated with pain in the groin, hip, thigh, and buttock (Pivec et al., 2014). ALTR has been categorized
in three major types: aseptic lymphocyte-dominated vasculitis-associated lesions (ALVaL), pseudotumors, and osteolysis (Fig. 3):
• ALVaL describes a periprosthetic histological inflammatory reaction that is similar to a type IV hypersensitivity response in the
soft tissue surrounding metal-on-metal implants (Kolatat et al., 2015; Lawrence et al., 2014).
Joint replacement
with metal bearings Metal particulates
“MoM” size: 40–50 nm Fibroblast
+ Cr and Co ions ROS
MCP-1
Metalloproteinase
MSCs cytokines, PGE2

GM-CSF
MΦ
Denaturized proteins
(haptens)
Lipid
peroxidation
Inhibition of
osteoblastic differentiation
Lymphocyte
proliferation Osteoblast toxicity
Osteoclast
chemotaxis DNA flaws
Osteolysis
Hypersensitivity reaction:
type IV delayed
Pseudotumor
Fig. 3 Biological response to metal particulates and ions. Production of metal particulate and ions leads to a nonspecific immune response and in
some cases to a type IV (cell-mediated) hypersensitivity reaction. GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP-1, monocyte-
chemoattractant molecule 1; MSC, mesenchymal stem cell; PGE2, prostaglandin E2; ROS, reactive oxygen species.
• Pseudotumors are large, cystic, or solid masses that are often seen in patients with ALTR. The rate of development of a pseu-
dotumor is controversial, and the literature reports rates ranging from 1% to 60% (Pandit et al., 2008; Hart et al., 2012; van der
Weegen et al., 2013).
• Osteolysis is the destruction of the bone, resulting from osteoclast resorption (Ries and Link, 2012). Swedish studies showed
that osteolysis was the cause of revision surgery for 75% of patients with metal-on-metal articulation (Malchau et al., 2002;
Dattani, 2007).
Conclusion
Biomaterials in orthopedics are numerous, and their effects on the human body have been extensively studied. Newer generations of
highly cross-linked PE have seen great success, whereas some hard-on-hard bearings such as metal on metal and alumina on
alumina have been withdrawn. Future directions and current research aim to develop bioactive, “smart” materials that are well toler-
ated and cause less local inflammation, facilitate bone integration, and limit infection. With younger patients now receiving joint
replacements and the overall aging of our population in general, developing safe, durable, and cost-effective bearing surfaces is of
paramount importance. Surgeons, engineers, manufacturers, and regulatory agencies must work together to achieve these goals.
References
Adala, R., Chakravarthy, M., Srinivas, V., & Pai, S. (2011). Orthopaedic surgery in a patient with metal sensitivity. Journal of Cutaneous and Aesthetic Surgery, 4(1), 67–68.
Amanatullah, D. F., Sucher, M. G., Bonadurer, G. F., Pereira, G. C., & Taunton, M. J. (2016). Metal in total hip arthroplasty: wear particles, biology, and diagnosis. Orthopedics,
39(6), 371–379.
Attar, F. G., Khaw, F.-M., Kirk, L. M. G., & Gregg, P. J. (2008). Survivorship analysis at 15 years of cemented press-fit condylar total knee arthroplasty. Journal of Arthroplasty,
23(3), 344–349.
Bozic, K. J., Kurtz, S., Lau, E., Ong, K., Chiu, V., Vail, T. P., Rubash, H. E., & Berry, D. J. (2009). The epidemiology of bearing surface usage in total hip arthroplasty in the United
States. Journal of Bone and Joint Surgery. American Volume, 91(7), 1614–1620.
Campbell, P., Ma, S., Yeom, B., McKellop, H., Schmalzried, T. P., & Amstutz, H. C. (1995). Isolation of predominantly submicron-sized UHMWPE wear particles from periprosthetic
tissues. Journal of Biomedical Materials Research, 29(1), 127–131.
Cannizzo, E. S., Clement, C. C., Sahu, R., Follo, C., & Santambrogio, L. (2011). Oxidative stress, inflamm-aging and immunosenescence. Journal of Proteomics, 74(11),
2313–2323.
Carnes, K. J., Odum, S. M., Troyer, J. L., & Fehring, T. K. (2016). Cost analysis of ceramic heads in primary Total hip arthroplasty. Journal of Bone and Joint Surgery. American
Volume, 98(21), 1794–1800.
Case, C. P., Langkamer, V. G., James, C., Palmer, M. R., Kemp, A. J., Heap, P. F., & Solomon, L. (1994). Widespread dissemination of metal debris from implants. Journal of Bone
and Joint Surgery. British Volume, 76(5), 701–712.
Catelas, I., Huk, O. L., Petit, A., Zukor, D. J., Marchand, R., & Yahia, L. (1998). Flow cytometric analysis of macrophage response to ceramic and polyethylene particles: Effects of
size, concentration, and composition. Journal of Biomedical Materials Research, 41(4), 600–607.
Catelas, I., Petit, A., Marchand, R., Zukor, D. J., Yahia, L., & Huk, O. L. (1999a). Cytotoxicity and macrophage cytokine release induced by ceramic and polyethylene particles in vitro.
Journal of Bone and Joint Surgery. British Volume, 81(3), 516–521.
Catelas, I., Petit, A., Zukor, D. J., Marchand, R., Yahia, L., & Huk, O. L. (1999b). Induction of macrophage apoptosis by ceramic and polyethylene particles in vitro. Biomaterials,
20(7), 625–630.
Christian, W. V., Oliver, L. D., Paustenbach, D. J., Kreider, M. L., & Finley, B. L. (2014). Toxicology-based cancer causation analysis of CoCr-containing hip implants: A quantitative
assessment of genotoxicity and tumorigenicity studies. Journal of Applied Toxicology, 34(9), 939–967.
Daley, B., Doherty, A. T., Fairman, B., & Case, C. P. (2004). Wear debris from hip or knee replacements causes chromosomal damage in human cells in tissue culture. Journal of
Bone and Joint Surgery. British Volume, 86(4), 598–606.
Dattani, R. (2007). Femoral osteolysis following total hip replacement. Postgraduate Medical Journal, 83(979), 312–316.
Ding, Y., C-q, Q., Y-r, F., Xu, J., & Huang, D.-S. (2012). In vitro comparison of the biological activity of alumina ceramic and titanium particles associated with aseptic loosening.
Biomedical Materials (Bristol, England), 7(4), 045019.
Doherty, A. T., Howell, R. T., Ellis, L. A., Bisbinas, I., Learmonth, I. D., Newson, R., & Case, C. P. (2001). Increased chromosome translocations and aneuploidy in peripheral blood
lymphocytes of patients having revision arthroplasty of the hip. Journal of Bone and Joint Surgery British Volume, 83(7), 1075–1081.
Doorn, P. F., Campbell, P. A., Worrall, J., Benya, P. D., McKellop, H. A., & Amstutz, H. C. (1998). Metal wear particle characterization from metal on metal total hip replacements:
Transmission electron microscopy study of periprosthetic tissues and isolated particles. Journal of Biomedical Materials Research, 42(1), 103–111.
Dunstan, E., Ladon, D., Whittingham-Jones, P., Carrington, R., & Briggs, T. W. R. (2008). Chromosomal aberrations in the peripheral blood of patients with metal-on-metal hip
bearings. Journal of Bone and Joint Surgery. American Volume, 90(3), 517–522.
El Masri, F., Kerboull, L., Kerboull, M., Courpied, J. P., & Hamadouche, M. (2010). Is the so-called “French paradox” a reality? Long-term survival and migration of the Charnley-
Kerboull stem cemented line-to-line. Journal of Bone and Joint Surgery. British Volume, 92(3), 342–348.
Epinette, J. A., & Manley, M. T. (2007). Hydroxyapatite-coated total knee replacement: Clinical experience at 10 to 15 years. Journal of Bone and Joint Surgery. British Volume, 89-
1, 34–38.
Essner, A., Herrera, L., Yau, S.-S., Wang, A., Dumbleton, J., & Manley, M. (2005). In Sequentially crosslinked and annealed UHMWPE CR knee wearTransactions of the 51st Annual
Meeting of the Orthopaedic Research Society. Washington, DC.
Gallo, J., Goodman, S. B., Konttinen, Y. T., & Raska, M. (2013a). Particle disease: Biologic mechanisms of periprosthetic osteolysis in total hip arthroplasty. Innate Immunity, 19(2),
213–224.
Gallo, J., Goodman, S. B., Konttinen, Y. T., Wimmer, M. A., & Holinka, M. (2013b). Osteolysis around total knee arthroplasty: A review of pathogenetic mechanisms. Acta
Germain, M. A., Hatton, A., Williams, S., Matthews, J. B., Stone, M. H., Fisher, J., & Ingham, E. (2003). Comparison of the cytotoxicity of clinically relevant cobalt-chromium and
alumina ceramic wear particles in vitro. Biomaterials, 24(3), 469–479.
Goodman, S. B. (2007). Wear particles, periprosthetic osteolysis and the immune system. Biomaterials, 28(34), 5044–5048.
Goodman, S. B., & Ma, T. (2010). Cellular chemotaxis induced by wear particles from joint replacements. Biomaterials, 31(19), 5045–5050.
Goodman, S. B., Chin, R. C., Chiou, S. S., Schurman, D. J., Woolson, S. T., & Masada, M. P. (1989). A clinical-pathologic-biochemical study of the membrane surrounding loosened
and nonloosened total hip arthroplasties. Clinical Orthopaedics and Related Research, (244), 182–187.
Goodman, S. B., Huie, P., Song, Y., Schurman, D., Maloney, W., Woolson, S., & Sibley, R. (1998). Cellular profile and cytokine production at prosthetic interfaces. Study of tissues
retrieved from revised hip and knee replacements. Journal of Bone and Joint Surgery. British Volume (London), 80(3), 531–539.
Goodman, S. B., Gomez Barrena, E., Takagi, M., & Konttinen, Y. T. (2009). Biocompatibility of total joint replacements: A review. Journal of Biomedical Materials Research. Part A,
90(2), 603–618.
Greenwald, A. S., & Garino, J. P. (2001). Alternative bearing surfaces: The good, the bad, and the ugly. Journal of Bone and Joint Surgery American Volume, 83-A(Suppl. 2 Pt 2),
68–72.
Gutwein, L. G., & Webster, T. J. (2004). Increased viable osteoblast density in the presence of nanophase compared to conventional alumina and titania particles. Biomaterials,
25(18), 4175–4183.
Hallab, N. J., Mikecz, K., Vermes, C., Skipor, A., & Jacobs, J. J. (2001). Orthopaedic implant related metal toxicity in terms of human lymphocyte reactivity to metal-protein
complexes produced from cobalt-base and titanium-base implant alloy degradation. Molecular and Cellular Biochemistry, 222(1–2), 127–136.
Hart, A. J., Satchithananda, K., Liddle, A. D., Sabah, S. A., McRobbie, D., Henckel, J., Cobb, J. P., Skinner, J. A., & Mitchell, A. W. (2012). Pseudotumors in association with well-
functioning metal-on-metal hip prostheses: A case-control study using three-dimensional computed tomography and magnetic resonance imaging. Journal of Bone and Joint
Surgery. American Volume, 94(4), 317–325.
Hatton, A., Nevelos, J. E., Nevelos, A. A., Banks, R. E., Fisher, J., & Ingham, E. (2002). Alumina-alumina artificial hip joints. Part I: A histological analysis and characterisation of
wear debris by laser capture microdissection of tissues retrieved at revision. Biomaterials, 23(16), 3429–3440.
Hensten-Pettersen, A. (1993). Allergy and hypersensitivity. In B. F. Morrey (Ed.), Biological, material, and mechanical considerations of joint replacements (pp. 353–360). New York:
Raven Press.
Hodges, N. J., & Chipman, J. K. (2002). Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human
A549 lung carcinoma cells. Carcinogenesis, 23(1), 55–60.
Hug, K. T., Watters, T. S., Vail, T. P., & Bolognesi, M. P. (2013). The withdrawn ASR™ THA and hip resurfacing systems: How have our patients fared over 1 to 6 years? Clinical
Orthopaedics and Related Research, 471(2), 430–438.
Ingham, E., & Fisher, J. (2005). The role of macrophages in osteolysis of total joint replacement. Biomaterials, 26(11), 1271–1286.
Kawate, K., Ohmura, T., Kawahara, I., Tamai, K., Ueha, T., & Takemura, K. (2009). Differences in highly cross-linked polyethylene wear between zirconia and cobalt-chromium
femoral heads in Japanese patients: A prospective, randomized study. Journal of Arthroplasty, 24(8), 1221–1224.
Keurentjes, J. C., Pijls, B. G., Van Tol, F. R., Mentink, J. F., Mes, S. D., Schoones, J. W., Fiocco, M., Sedrakyan, A., & Nelissen, R. G. (2014). Which implant should we use for
primary total hip replacement? A systematic review and meta-analysis. The Journal of Bone and Joint Surgery. American Volume, 96(Suppl. 1), 79–97.
Kolatat, K., Perino, G., Wilner, G., Kaplowitz, E., Ricciardi, B. F., Boettner, F., Westrich, G. H., Jerabek, S. A., Goldring, S. R., & Purdue, P. E. (2015). Adverse local tissue reaction
(ALTR) associated with corrosion products in metal-on-metal and dual modular neck total hip replacements is associated with upregulation of interferon gamma-mediated
chemokine signaling. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society, 33(10), 1487–1497.
Kurtz, S., Ong, K., Lau, E., Mowat, F., & Halpern, M. (2007). Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030. Journal of Bone
and Joint Surgery. American Volume, 89(4), 780–785.
Kurtz, S. M., Kocagoz, S., Arnholt, C., Huet, R., Ueno, M., & Walter, W. L. (2014). Advances in zirconia toughened alumina biomaterials for total joint replacement. Journal of the
Mechanical Behavior of Biomedical Materials, 31, 107–116.
Langkamer, V. G., Case, C. P., Heap, P., Taylor, A., Collins, C., Pearse, M., & Solomon, L. (1992). Systemic distribution of wear debris after hip replacement. A cause for concern?
Journal of Bone and Joint Surgery. British Volume, 74(6), 831–839.
Langlois, J., Atlan, F., Scemama, C., Courpied, J. P., & Hamadouche, M. (2015). A randomised controlled trial comparing highly cross-linked and contemporary annealed
polyethylene after a minimal eight-year follow-up in total hip arthroplasty using cemented acetabular components. The Bone & Joint Journal, 97-B(11), 1458–1462.
Lawrence, H., Deehan, D., Holland, J., Kirby, J., & Tyson-Capper, A. (2014). The immunobiology of cobalt: Demonstration of a potential aetiology for inflammatory pseudotumours
after metal-on-metal replacement of the hip. The Bone & Joint Journal, 96-B(9), 1172–1177.
Lin, T.-H., Pajarinen, J., Sato, T., Loi, F., Fan, C., Córdova, L. A., Nabeshima, A., Gibon, E., Zhang, R., Yao, Z., & Goodman, S. B. (2016). NF-kB decoy oligodeoxynucleotide
mitigates wear particle-associated bone loss in the murine continuous infusion model. Acta Biomaterialia.
Lucarelli, M., Gatti, A. M., Savarino, G., Quattroni, P., Martinelli, L., Monari, E., & Boraschi, D. (2004). Innate defence functions of macrophages can be biased by nano-sized
ceramic and metallic particles. European Cytokine Network, 15(4), 339–346.
Ma, T., Huang, Z., Ren, P. G., McCally, R., Lindsey, D., Smith, R. L., & Goodman, S. B. (2008). An in vivo murine model of continuous intramedullary infusion of polyethylene
particles. Biomaterials, 29(27), 3738–3742.
Ma, T., Ortiz, S. G., Huang, Z., Ren, P., Smith, R. L., & Goodman, S. B. (2009). In vivo murine model of continuous intramedullary infusion of particlesdA preliminary study. Journal
of Biomedical Materials Research. Part B, Applied Biomaterials, 88(1), 250–253.
Malchau, H., Herberts, P., Eisler, T., Garellick, G., & Söderman, P. (2002). The Swedish Total hip replacement register. The Journal of Bone and Joint Surgery. American Volume,
84-A(Suppl. 2), 2–20.
Mantovani, A., Biswas, S. K., Galdiero, M. R., Sica, A., & Locati, M. (2013). Macrophage plasticity and polarization in tissue repair and remodelling. Journal of Pathology, 229(2),
176–185.
McLaughlin, J. R., & Lee, K. R. (2006). The outcome of total hip replacement in obese and non-obese patients at 10- to 18-years. Journal of Bone and Joint Surgery. British
Volume, 88(10), 1286–1292.
Meftah, M., Klingenstein, G. G., Yun, R. J., Ranawat, A. S., & Ranawat, C. S. (2013). Long-term performance of ceramic and metal femoral heads on conventional polyethylene in
young and active patients: A matched-pair analysis. Journal of Bone and Joint Surgery. American Volume, 95(13), 1193–1197.
Melton, J. T. K., Mayahi, R., Baxter, S. E., Facek, M., & Glezos, C. (2012). Long-term outcome in an uncemented, hydroxyapatite-coated total knee replacement: A 15- to 18-year
survivorship analysis. Journal of Bone and Joint Surgery. British Volume, 94(8), 1067–1070.
Murray, P. J., Allen, J. E., Biswas, S. K., Fisher, E. A., Gilroy, D. W., Goerdt, S., Gordon, S., Hamilton, J. A., Ivashkiv, L. B., Lawrence, T., Locati, M., Mantovani, A., Martinez, F. O.,
Mege, J.-L., Mosser, D. M., Natoli, G., Saeij, J. P., Schultze, J. L., Shirey, K. A., Sica, A., Suttles, J., Udalova, I., van Ginderachter, J. A., Vogel, S. N., & Wynn, T. A. (2014).
Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity, 41(1), 14–20.
Ng, V. Y., Arnott, L., & McShane, M. A. (2011). Perspectives in managing an implant recall: Revision of 94 Durom Metasul acetabular components. Journal of Bone and Joint
Surgery. American Volume, 93(17). e100(1–5).
Ortiz, S. G., Ma, T., Epstein, N. J., Smith, R. L., & Goodman, S. B. (2008). Validation and quantification of an in vitro model of continuous infusion of submicron-sized particles.
Journal of Biomedical Materials Research. Part B, Applied Biomaterials, 84(2), 328–333.
Pandit, H., Glyn-Jones, S., McLardy-Smith, P., Gundle, R., Whitwell, D., Gibbons, C. L. M., Ostlere, S., Athanasou, N., Gill, H. S., & Murray, D. W. (2008). Pseudotumours associated
with metal-on-metal hip resurfacings. The. Journal of Bone and Joint Surgery. British Volume, 90(7), 847–851.
Pivec, R., Meneghini, R. M., Hozack, W. J., Westrich, G. H., & Mont, M. A. (2014). Modular taper junction corrosion and failure: How to approach a recalled total hip arthroplasty
implant. The Journal of Arthroplasty, 29(1), 1–6.
Polyzois, I., Nikolopoulos, D., Michos, I., Patsouris, E., & Theocharis, S. (2012). Local and systemic toxicity of nanoscale debris particles in total hip arthroplasty. Journal of Applied
Toxicology: JAT, 32(4), 255–269.
Prieto, H. A., Berbari, E. F., & Sierra, R. J. (2014). Acute delayed infection: Increased risk in failed metal on metal total hip arthroplasty. Journal of Arthroplasty, 29(9), 1808–1812.
Ren, P. G., Huang, Z., Ma, T., Biswal, S., Smith, R. L., & Goodman, S. B. (2010). Surveillance of systemic trafficking of macrophages induced by UHMWPE particles in nude mice by
noninvasive imaging. Journal of Biomedical Materials Research. Part A, 94(3), 706–711.
Ren, P. G., Irani, A., Huang, Z., Ma, T., Biswal, S., & Goodman, S. B. (2011). Continuous infusion of UHMWPE particles induces increased bone macrophages and osteolysis.
Clinical Orthopaedics and Related Research, 469(1), 113–122.
Richards, R. G., Moriarty, T. F., Miclau, T., McClellan, R. T., & Grainger, D. W. (2012). Advances in biomaterials and surface technologies. Journal of Orthopaedic Trauma, 26(12),
703–707.
Ries, M. D., & Link, T. M. (2012). Monitoring and risk of progression of osteolysis after total hip arthroplasty. Journal of Bone and Joint Surgery. American Volume, 94(22),
2097–2105.
Ries, M. D., Scott, M. L., & Jani, S. (2001). Relationship between gravimetric wear and particle generation in hip simulators: Conventional compared with cross-linked polyethylene.
Journal of Bone and Joint Surgery. American Volume, 83-A(Suppl. 2 (Pt 2)), 116–122.
Scemama, C., Anract, P., Dumaine, V., Babinet, A., Courpied, J. P., & Hamadouche, M. (2017). Does vitamin E-blended polyethylene reduce wear in primary total hip arthroplasty: A
blinded randomised clinical trial. International Orthopaedics, 41(6), 1113–1118.
Schmalzried, T. P., Jasty, M., & Harris, W. H. (1992). Periprosthetic bone loss in total hip arthroplasty. Polyethylene wear debris and the concept of the effective joint space. Journal
of Bone and Joint Surgery. American Volume, 74(6), 849–863.
Shanbhag, A. S., Kaufman, A. M., Hayata, K., & Rubash, H. E. (2007). Assessing osteolysis with use of high-throughput protein chips. Journal of Bone and Joint Surgery. American
Volume, 89(5), 1081–1089.
Smith, A. J., Dieppe, P., Porter, M., Blom, A. W., & National Joint Registry of England and Wales. (2012). Risk of cancer in first seven years after metal-on-metal hip replacement
compared with other bearings and general population: Linkage study between the National Joint Registry of England and Wales and hospital episode statistics. BMJ (Clinical
Research ed.), 344, e2383.
St John, K. R., Zardiackas, L. D., & Poggie, R. A. (2004). Wear evaluation of cobalt-chromium alloy for use in a metal-on-metal hip prosthesis. Journal of Biomedical Materials
Research. Part B, Applied Biomaterials, 68(1), 1–14.
Thyssen, J. P., & Menné, T. (2010). Metal allergy–a review on exposures, penetration, genetics, prevalence, and clinical implications. Chemical Research in Toxicology, 23(2),
309–318.
Thyssen, J. P., Linneberg, A., Menné, T., & Johansen, J. D. (2007). The epidemiology of contact allergy in the general population–prevalence and main findings. Contact Dermatitis,
57(5), 287–299.
Tsaousi, A., Jones, E., & Case, C. P. (2010). The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles. Mutation Research, 697(1–2), 1–9.
Tuan, R. S., Lee, F. Y., TK, Y., Wilkinson, J. M., & Smith, R. L. (2008). What are the local and systemic biologic reactions and mediators to wear debris, and what host factors
determine or modulate the biologic response to wear particles? Journal of the American Academy of Orthopaedic Surgeons, 16(Suppl. 1), S42–8.
Urban, R. M., Jacobs, J. J., Tomlinson, M. J., Gavrilovic, J., Black, J., & Peoc’h, M. (2000). Dissemination of wear particles to the liver, spleen, and abdominal lymph nodes of
patients with hip or knee replacement. Journal of Bone and Joint Surgery. American Volume, 82(4), 457–476.
Valladares, R. D., Nich, C., Zwingenberger, S., Li, C., Swank, K. R., Gibon, E., Rao, A. J., Yao, Z., & Goodman, S. B. (2014). Toll-like receptors-2 and 4 are overexpressed in an
experimental model of particle-induced osteolysis. Journal of Biomedical Materials Research. Part A, 102(9), 3004–3011.
van der Weegen, W., Brakel, K., Horn, R. J., Hoekstra, H. J., Sijbesma, T., Pilot, P., & Nelissen, R. G. H. H. (2013). Asymptomatic pseudotumours after metal-on-metal hip
resurfacing show little change within one year. The Bone & Joint Journal, 95-B(12), 1626–1631.
Visuri, T., Pukkala, E., Paavolainen, P., Pulkkinen, P., & Riska, E. B. (1996). Cancer risk after metal on metal and polyethylene on metal total hip arthroplasty. Clinical Orthopaedics
and Related Research, 329(Suppl. S2), 80–89.
Wang, A., Polineni, V. K., Stark, C., & Dumbleton, J. H. (1998). Effect of femoral head surface roughness on the wear of ultrahigh molecular weight polyethylene acetabular cups.
Journal of Arthroplasty, 13(6), 615–620.
Wang, C.-T., Lin, Y.-T., Chiang, B.-L., Lee, S.-S., & Hou, S.-M. (2010). Over-expression of receptor activator of nuclear factor-kappaB ligand (RANKL), inflammatory cytokines, and
chemokines in periprosthetic osteolysis of loosened total hip arthroplasty. Biomaterials, 31(1), 77–82.
Wang, S., Zhang, S., & Zhao, Y. (2013). A comparison of polyethylene wear between cobalt-chrome ball heads and alumina ball heads after total hip arthroplasty: A 10-year follow-
up. Journal of Orthopaedic Surgery and Research, 8, 20.
Xing, S., Waddell, J. E., & Boynton, E. L. (2002). Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro. Microscopy
Research and Technique, 57(6), 523–529.
Zhang, Y. F., Zheng, Y. F., & Qin, L. (2011). A comprehensive biological evaluation of ceramic nanoparticles as wear debris. Nanomedicine: Nanotechnology, Biology, and Medicine,
7(6), 975–982.
Cell Encapsulation and Delivery
Stefani Mazzitelli and Claudio Nastruzzi, University of Ferrara, Ferrara, Italy
Cell Encapsulation for Immunoisolation 308

Biomaterials for Cell Encapsulation 309
Bioencapsulation Procedures 310
Cell Encapsulation by Gas Driven Mono-jet, Vibrating Nozzle and Jet Cutter 311
Cell Encapsulation by Microfluidics 311
Cross Junction Microfluidic Chips 313
T-Junction Microfluidic Chips 313
Microcapillary Coaxial Device Method 314
Preparation of Fibrous Scaffolds 314
Further Reading 315
Glossary
Alginate Alginate is a natural polysaccharide that comprises from 30% to 60% of brown algae (on dry weight basis). It is an
anionic polymer which is present in cell walls of algae, where through binding with minerals from seawater it forms a viscous
gum structure (named as “jelly bodies”). Alginate and its derivatives have been utilized as hydrocolloids in a variety of
applications such as food additives, pharmaceuticals, cosmetics and textile manufacturing.
Biopolymer Macromolecules are formed by living organisms. Biopolymers can be polynucleotides (such as the nucleic acids
DNA and RNA), polypeptides (that is, proteins) or polysaccharides (that is, polymeric carbohydrates). These consist of long
chains of repeating, covalently bonded units, such as nucleotides, amino acids or monosaccharides.
Cell encapsulation A process in which cells or cell aggregates are surrounded by a coating materials to give small devices
(typically in the dimensional range of hundreds of microns) with diverse useful properties. The process is used to incorporate
food ingredients, enzymes, cells or other materials on a micro metric scale. A simple form of cell encapsulation device is
microcapsule, a sphere with an uniform wall around it. The material inside the microcapsule is referred to as the core, internal
phase, or fill, whereas the wall is generally called shell, coating, or membrane.
Microfluidics It is a multidisciplinary science dealing with the manipulation and control of fluids, usually in the range of
microliters to picoliters, in a network of channels with dimensions typically from tens to hundreds of micrometers.
Microfluidics is a science at the intersection of engineering, physics, chemistry, biochemistry, nanotechnology, and
biotechnology, with practical applications in the design of systems in which low volumes of fluids are processed to achieve
multiplexing, automation, and high-throughput screening.
Cell Encapsulation for Immunoisolation
Cell encapsulation involves the design and application of specific procedures for immunoisolating cells into appropriate scaffolds.
It represents a major advance in cell based therapy as it avoids constraints associated with cell sources, making both allogenic and
xenogenic cells putative alternatives to the scarcely available autologous cells.
Cell based therapies require therefore the combined presence of a biomaterial based scaffold and selected cells to create
a biocompatible device that, once transplanted, isolates cells from the host’s immune system, eliminating or reducing the require-
ment for immunosuppressant systemic drug administration. In this respect, the biomaterial plays a fundamental role in cell encap-
sulation for an efficient implantation of cells, providing functional groups to anchor cells and offering an environment in which the
transplanted cells can survive, migrate to target sites and effectively function.
Cells are combined with biomaterials following two main strategies: (a) “conventional” seeding of cells in preformed scaffolds
or (b) embedding cells into scaffold during the scaffold production (i.e. cell encapsulation procedure).
If compared to conventional seeding strategies, the encapsulation of cells in 3D scaffolds provides a better control on prolifer-
ation, cell morphology, and precise geometric constrains. The seeding of cells often leads to an inhomogeneous distribution of cells
along the scaffold surface/structure, due to cell aggregation in clusters, thus leading to heterogeneous properties of final construct.
The success of different encapsulation approaches is mostly related to properties of biomaterial used for scaffold production.
Biomaterials provide indeed the support to cells to adhere, differentiate and/or grow both in vitro culture or in vivo after implan-
tation. Materials employed to produce the scaffolds must be compatible with both the cellular components of transplanting tissue
and cells of hosts and they should possess a porous structure for the effective transport of nutrients and metabolites.

Biomaterials: Biomaterial applications and advanced medical technologies j Cell Encapsulation and Delivery 309
Intuitively, the best encapsulation material should be similar to the extracellular matrix (ECM) of target tissue in its native state.
Unfortunately, the enormously complex molecular architecture and function of ECM make it extremely difficult to be mimicked.
Therefore, a variety of biomaterials with different physical and chemical properties have been proposed and utilized. For instance,
materials able to form hydrogels are particularly attractive as scaffolding materials.
Hydrogels possess indeed great potential for cell encapsulation thanks to their distinct physical properties. They provide an
appropriate environment that is mechanically and structurally similar to the topology of native ECM, supplying adhesion sites
to the cells. Moreover, hydrogels can be generally obtained by mild reaction conditions and can be administered by minimally inva-
sive protocols.
Biomaterials for Cell Encapsulation
The essential function of a biomaterial for cell encapsulation is to provide a temporary 3D structure for cells. Criteria for scaffold
selection include controlled biodegradability, suitable mechanical strength and appropriate surface chemistry. Another important
feature of scaffold is related to its porosity which should be designed to enable nutrition, proliferation and integration into host
tissue/organ.
At the present, different scaffold dimensions and shapes have been proposed for cell encapsulation, generally defined as macro
and microdevices, the latter generally in shapes as particles, fibers, flat sheets and disks (see Fig. 1). Among microdevice class, micro-
particles represent one of the most widely known and studied systems; over the years, therefore, various alternative cell encapsula-
tion technology for the spherical immunoisolation have been described.
Microcapsules with a spherical shape are considered beneficial as sphericity provides an optimal surface-to-volume ratio for effi-
cient protein and nutrient diffusion, improving cell viability and functions compared to other scaffold geometries. Furthermore,
small microcapsule dimensions, usually with a diameter comprised between 250 and 750 mm, facilitate the implantation at
different body sites through small diameter catheters.
The microcapsule forming materials are indeed expected to permit diffusion of nutrients and molecules including oxygen and
growth factors essential for cellular metabolism, proliferation, differentiation and morphogenesis, while excluding entrance of all
high molecular weight molecules such as immunoglobulins and immune system cells.
(A)
medium
core
cells
shell
micro-particles
increasing device size
(B)
micro-fibre
(C)
macrodevice
(milli-cylinder)
Fig. 1 Scheme of hydrogel biomaterial for cell encapsulation protocols. The scaffolds can be tailored to possess different geometrical features (i.e.,
dimensions), namely: microparticles (A), microfibers (B) or macrodevices (C).
310 Biomaterials: Biomaterial applications and advanced medical technologies j Cell Encapsulation and Delivery
Microcapsules are generally constituted of hydrogel matrix forming a highly hydrated microenvironments, similar to those of
their native tissue; in addition matrix permeability provides a high degree of diffusion for low-molecular mass biochemical and
physical stimuli for cellular processes such as differentiation, proliferation, and migration. Hydrogels are prepared by mild proce-
dures, via thermo-, ion-, or photo-induced processes, leading to an uniform distribution of cells into the gel matrix. These features
result in a high biocompatibility associate to minimal host-cell adhesion and protein adsorption phenomena.
The success of therapeutic approaches based on cell encapsulation requires a detailed characterization of the biomaterials
employed and of the cell–material and material-host tissue interactions, with special regard to the biocompatibility and immuno-
genicity of the cell-microcapsule assembly.
Alginate or other polysaccharides with similar properties, such as pectins, carrageenans, xanthans, chitosans and natural and
semisynthetic celluloses, occupy a prominent position for producing hydrogels intended as biomaterials for cell encapsulation.
Microcapsular shape greatly influences the in vivo performances, therefore irregular geometries, such as fused or partially fused
capsules or tear shaped capsules result, after implantation, in the formation of capsular fibrotic overgrowth. Other microcapsular
defects such as presence of cracks or surface fissures, together with an irregular (rough or waved) surface, can also cause an obvious
immunological in vivo response.
Dimension and morphology represent also critical characteristics that influence the microcapsule performances, including: cell
loading capacity, diffusive properties, method of implantation and biocompatibility.
Porosity and pore size and structure are other important factors as they impact nutrient supply to transplanted cells. To regen-
erate highly vascularized organs such as liver, porous scaffolds with large void volumes and large surface-area-to-volume ratios are
desirable for maximal cell seeding, growth, and vascularization. Small-diameter pores are preferred to yield high surface area per
unit volume so long as the pore size is greater than the diameter of a cell in suspension (typically 10–15 mm).
Finally, the mechanical behavior is a critical feature for microcapsules since they are usually exposed to several mechanical
stresses during the processing, post processing (i.e., washing step), implantation and in vivo permanence. In this respect, the
mechanical and elastic properties can be tailored to tune the microcapsule properties by adjusting the polymer source and concen-
tration, processing methods, formulation and gelling conditions.
Alginates, which are by far the most frequently used biomaterial in the field of cell microencapsulation, are a family of
unbranched binary copolymers of 1 / 4 linked b-D-mannuronic acid (M) and a-L-guluronic acid (G), of widely varying compo-
sitions and sequential structures.
The composition has a great effect on alginate properties including gelling behavior, permeability, mechanical resistance, in vivo
stability and biocompatibility.
When dispersed in water, alginates form a colloidal dispersion, which gels ionotropically by the addition of divalent cations (i.e.,
Ca2 þ, Sr2 þ, or Ba2 þ). The most important asset of alginate is indeed the ability to form gels under extremely mild conditions
without chemicals or at specific pH. The polymer cross-linking occurs following the exchange of sodium ions from the guluronic
acids with the divalent cations resulting in a chain-chain association that constitutes the junction zones of so-called “egg box
model.”
The buckled chain of guluronic acid units is shown as a two-dimensional analogue of a corrugated egg-box with interstices in
which divalent ions may pack and be coordinated. Since hydrogel formation happens following selectively linkage between the
carboxylic moieties on the G blocks of alginate and cations, high ratio of G:M outcomes in stiff gels.
Alginate forms mechanical and chemical stabile gel with a defined pore size and a narrow pore-size distribution. Unfortunately,
the above features do not pertain at all the commercially available alginates; for cell encapsulation procedure, only ultrapure algi-
nates should be considered and an efficient purification process should be accomplished to obtain starting materials containing low
number of contaminants including toxic, pyrogenic and immunogenic substances.
Alginate microcapsules are generally prepared by dripping chosen cell/alginate suspension into a divalent crosslinking solution.
Gelling bath with Ca2 þ or Ba2 þ are generally preferred for cell encapsulation procedures in reason of the higher biocompatibility.
After preparation, the gelified microcapsules are usually coated by a cationic polyelectrolyte. The coating is performed to slow down
the swelling and in vivo degradation, however it may cause immunological reactions and fibrotic growth; this latter can subse-
quently decrease the therapeutic efficacy of the entrapped cells sharply reducing the diffusive properties of alginate beads.
In this respect, the use of the positively charged polyelectrolytes for the capsular coating (i.e., poly-L-lysine) can lead to a more
intense overgrowth compared to negative ones. This behavior was attributed to activated macrophages that preferentially adhere to
positive charged surfaces.
Ba2 þ ions have also been proposed, as alternative to calcium, for the alginate cross-linking. It has been proved that Ba2 þ ions
provide stronger gels, allowing the transplantation of microcapsules without the need of the coating procedure. As drawback,
barium is sometimes believed to be toxic but recent studies have shown that when used at low concentration, with short time
of gelling and intensive rinsing of the beads, no barium leakage and cytotoxic effect were observed.
Bioencapsulation Procedures
As previously mentioned, numerous natural and synthetic polymers have been explored for microencapsulation design, but algi-
nates have been the most used polymers for bioencapsulation due to easy gelling properties and biocompatibility.
Hydrogel based microparticles, for cell encapsulation, can be prepared by different procedures, as schematized in Fig. 2,
including emulsion method (Fig. 2A), vibrating nozzle procedure (Fig. 2B), gas driven mono-jet (Fig. 2C) or jet cutter (Fig. 2D).
Irrespectively of the preparation strategy, the microparticle formation consists of three main steps, as below summarized.
Step 1. Preparation of a suspension of viable cells in an aqueous colloidal alginate dispersion (the polymer is usually employed
at a concentration ranging from 1% to 3%, w/v).
Step 2. Generation of alginate droplets which represent microparticle precursors; this step is preferably achieved by a controlled
procedure resulting in dimensionally homogenous microdroplets.
Step 3. The consolidation of the droplets by a gelation process with final formation of soft hydrogel based microparticles.
Cell Encapsulation by Gas Driven Mono-jet, Vibrating Nozzle and Jet Cutter
The encapsulation procedure based on the use of a gas driven mono-jet device (also known as coaxial bead generator), represents
also one widely applied approach for the production of microcapsules for cell encapsulation.
Different encapsulation hardware is commercially available for the production of alginate microbeads in a controllable manner.
The general principle of the device is based on a coaxial air stream that blows droplets from a needle tip into a gelling bath. A typical
instrument is composed of a gas driven mono-jet device connected to a precision pump for the alginate feeding (syringe or peri-
staltic pumps) and to a gas flask (usually nitrogen) equipped with a flow meter, providing gas for the atomization of alginate
stream. Generated microdroplets are then consolidated to give the microparticles by a gelation procedure normally based on
calcium or barium ion solutions. The feeding cell suspension is continuously stirred to prevent cell clumping, which could lead
to inhomogeneous distribution of cells in the microparticles.
Typically step 2 represents the most critical phase of the processes. During preparation process a stream of liquid alginate (con-
taining the cells) is forced through a nozzle, generating the individual droplets by gravity or with the aid of various mechanisms.
The alginate droplet break-off, at the nozzle tip is achieved by an air-jet directed to the forming droplet while in the case of
vibrating nozzle procedure by vibrations or a mechanical cutting in jet cutter encapsulation (emulsion methods do not pertain
to this discussion).
Varying the experimental settings such as the nozzle diameter, the alginate pumping rate and the applied air-flow or vibration
frequency, the droplet diameter can be adjusted. Unfortunately, all the aforementioned fabrication methods can lack of reproduc-
ibility in macro-scale environments and they are prone to the production of large and polydispersed microparticles, often charac-
terized by an irregular surface.
In this respect, aside the well described microparticle fabrication procedures in recent years, new microfluidic based techniques
have been described in response to the need of preparing extremely homogenous microparticles.
Cell Encapsulation by Microfluidics
Microfluidics is a new developing technological field dealing with the handling of fluids in micro/nano environments (i.e., the
microfluidic chips). This technology has recently found various applications including chemical synthesis, biochemical assays,
drug screening.
In the case of cell encapsulation, the use of microfluidic chips can overcome some of the drawbacks associated to conventional
encapsulation methods, resulting in the continuous formation of microparticles with an elevate control of their size and
morphology.
Moreover, microchip channels represent a controllable environment that does not need particular sterilization procedures,
making the fabrication process suitable for cell encapsulation.
Therefore, microfluidics provides a route to encapsulate cells in hydrogel microdevices with a superior control of the process of
microencapsulation.
Microfluidic strategies for cell encapsulation typically involve the initial formation of an emulsion constituted of aqueous poly-
mer droplets dispersed in a nonmiscible phase (i.e., an oil phase), followed by the consolidations of the droplets. Depending on the
chemical structure of the polymer, the liquid droplets are hardened by different procedures including the previously mentioned
ionic or thermal gelation (Fig. 3).
Homogeneity of microparticle dimension is strictly related to the emulsification step (i.e., the homogeneity of the microdrop-
lets). Optimal microfluidic devices should be indeed able to produce a regular and stable stream of droplets with precise volume
and rates. To this end, microfluidic chips have been designed based on different channel geometries such as: (a) cross junction
(Fig. 4A), (b) T-junction (Fig. 4B) and (c) microcapillary coaxial devices (Fig. 4C).
These different geometries share a common mechanism: the phase to be dispersed (water phase) is injected into a microchannel,
where it encounters the immiscible oil phase coming from another inlet(s). The junction where the two phases encounter is
designed to optimize the reproducibility of droplet production. Furthermore, the geometry of the junction, the relative flow rates
and the physical properties of the fluids, such as viscosity and interfacial tension, lead to droplet pinch off.
Notably, size of the formed droplets (into the microchannels) can be controlled by adjusting the flow parameters, therefore
microparticles of any size can be virtually obtained. Microparticles or different geometries (e.g., disk-shape particles) can be
(A) (B)
Emulsion method Vibrating nozzle procedure
cells in control
polymer syringe panel
pump
mineral
oil
charge
generator
microparticle
consolidation
vibrating
nozzle
w/o emulsion micro
droplets
(C)
gelling bath
BaCl2
Gas driven mono-Jet
alginate
microbeads
syringe pump
(D)
Jet cutter
cells in
polymer
cells in
sodium electric polymer
alginate inlet motor
atomizing air
nozzle
inlet
fuid jet
micro
microdroplets droplets
rotating
cutter
gelling bath
BaCl2
gelling bath
BaCl2
alginate
microbeads
alginate
microbeads
Fig. 2 Schemes showing some procedures typically employed for production of microparticles for cell encapsulation: emulsion method (panel A),
vibrating nozzle procedure (panel B), gas driven monojet (panel C) and jet cutter (panel D).
liquid droplets
multiphasic flow
(emulsion)
consolidation
solid particles
thermal ionic
gelation cross-linking
suspension
agarose alginate
microparticles microparticles
Fig. 3 Alternative gelling strategies (i.e., thermal gelation and ionic cross-linking) for hardening/consolidation of liquid droplets generated in micro-
fluidic channels.
generated customing the droplet consolidation inside the channel. Moreover, properly varying hydrodynamic parameters droplets
with interesting multiphase morphologies such as Janus microparticles can be also obtained.
In the case of alginate, the consolidation of microdroplets into microparticles is accomplished in different ways; when the algi-
nate droplets reside in the microchannels, the consolidation process is defined as “internal gelation.” This procedure involves the
dispersion of an insoluble (or slightly soluble) calcium salt (e.g., calcium carbonate) into alginate solution. At the pH reduction by
the surrounding continuous phase, Ca2 þ ions are released from the insoluble calcium salt, allowing the crosslinking of the alginate
in microparticles. However, the starting of gelation process inside the channel can lead to the formation of clogs inside the chip, by
the aggregation of the particle of insoluble calcium salt, or to alginate droplets not completely gelified due to the short resident time
into the channel. On the contrary, the external gelation is based on the gelation outside the chip by transferring the obtained algi-
nate droplets into a crosslinking solution where a uniform gelation occurs.
Cross Junction Microfluidic Chips

The principle of a cross junction microchip is to create a stream of dispersed phase, later periodically broken up by a flow focusing
with a second, immiscible carrier fluid, acting as continuous phase. When these two phases come in contact, droplets in a drop-by-
drop fashion are generated by the shear force imposed by the stream of the continuous phase. The formation of droplets occurs,
indeed, by a balance between the interfacial tension and the shear of the continuous phase, depending on fluid flow rate, viscosity
and microchannel dimensions. In cross junction chip many different geometrical variants are available but in all channel geometries
the dispersed phase is pumped through the central inlet and the second immiscible fluid through the two lateral inlets. In order to
obtain hydrogel microparticles, the uniform droplets formed at the cross junction are subsequently hardened by different gelling
mechanisms.
T-Junction Microfluidic Chips

Although very simple in design, the T-junction represent a largely employed geometry for microfluidic devices used for microparticle
production. In T-junction chip, the dispersed phase meets at 90 degrees, in a T-shaped junction, the continuous phase. The forming
droplet of the dispersed phase obstructs the channel as it grows, resulting in a restriction of the flow of the continuous phase around
it. This reduction in the gap through which the continuous phase can flow causes to a dramatic increase in the dynamic pressure
which squeezes the dispersed phase leading to the droplet formation.
Manipulating relative flow rates of the two fluids, the precise control of droplet size can be reached together with the synchro-
nization of alternately formed droplets by two T-junctions. T-junction, for its simplicity, represent the most effective geometry for
(A)
external
phase
cross-junction device
microdroplet
internal
phase
external
phase
(B)
internal
T-junction device
phase
microdroplet
external
phase
(C)
coaxial device
external
phase
internal
phase
Fig. 4 Alternative strategies for the production of microdroplets in microfluidic devices. Geometries currently used are: cross-junction (A) T-junction
(B), and microcapillary coaxial (C).
dispersing droplets in a continuous phase. Moreover, coalescence probability of two consecutive droplets is low, depending greatly
on flow rate, as droplets tend to flow out to the main channel one by one.
Regarding material fabrication, microfluidic devices with T-junction geometry are generally constituted of glass or polydimethyl-
siloxane (PDMS).
Microcapillary Coaxial Device Method

Capillary microfluidic devices are microfluidic systems assembled generally from the alignment of glass tubes: a cylindrical internal
tube with a rectangular or cylindrical outer channels. The key advantages of glass microfluidic devices reside in the highly resistance
to chemicals and the possibility to create three dimensional flows. Moreover, the glass surface can be hydrophobic or hydrophilic
after specific treatment with surface modifiers. Generally, to build a microcapillary device a cylindrical glass capillary with an outer
diameter of about 1 mm is used. The capillary is later heated and pulled to obtain a gradually narrowing shape with a fine orifice.
The latter is then inserted into another glass capillary with square cross-section to assemble the final microfluidic device. To achieve
the coaxial alignment of the two glass capillaries (the rounded and the squared ones) the outer diameter of the rounded capillary
has the same dimensions of inner diameter of the squared one. By pumping one fluid through the circular capillary and another
immiscible fluid through the square capillary, flowing in the same direction a coaxial flow is obtained.
On the contrary, when the two immiscible fluids are introduced from the two ends of the same square capillary but in opposite
directions a flow-focusing mechanism is achieved. The outer fluid hydrodynamically focuses the liquid stream of the inner fluid
through the small orifice of the rounded capillary, provoking the generation of extremely regular droplets. The droplet size can
be tuned by adjusting the fluid flow rates.
Preparation of Fibrous Scaffolds

Scaffolds in a fibrous form could be an appealing substitute to spherical microparticles, representing an example of scaffold with
controlled physical and architectural features for tissue engineering applications or cell delivery. This statement derives from the fact
that fibrous scaffold could enable the guided growth, alignment and migration of cells and they could find many potential appli-
cations as small vascular grafts, nerve conduits, artificial kidney tubules as well as cell release vehicles.
Over the years, different technologies have been developed to create fibers with well-defined physical and architectural features
including melt spinning wet spinning and electrospinning. Many of these approaches, however, have limitations on the control of
the morphology, dimension, or direction of alignment of obtained fibers.
Moreover, when hydrogels and cells pass through conventional nozzles they are subjected to high shear forces causing cytotoxic
effects. Conversely, the shear stress developed in microfluidic channel is significantly reduced during fiber production due to the
envelop of a laminar core by a sheath flow. Furthermore, microfluidics, due to small channel dimensions and efficient mixing,
allows a precise control of the dimensional and morphological characteristics of the produced microfibers, providing new routes
for in situ fabrication of fibrous scaffold.
Further Reading
Nicodemus, G. D., & Bryant, S. J. (2008). Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Engineering Part B Review, 14, 149–165.
Orive, G., Santos, E., Poncelet, D., Hernández, R. M., Pedraz, J. L., Wahlberg, L. U., De Vos, P., & Emerich, D. (2015). Cell encapsulation: Technical and clinical advances. Trends
in Pharmacological Sciences, 36, 537–546.
Krishnan, R., Alexander, M., Robles, L., Foster, C. E., 3rd, & Lakey, J. R. (2014). Islet and stem cell encapsulation for clinical transplantation. Review of diabetic Studies, 11,
84–101.
Silva, R., Fabry, B., & Boccaccini, A. R. (2014). Fibrous protein-based hydrogels for cell encapsulation. Biomaterials, 35, 6727–6738.
Kang, A., Park, J., Ju, J., Jeong, G. S., & Lee, S. H. (2014). Cell encapsulation via microtechnologies. Biomaterials, 35, 2651–2663.
Mazzitelli, S., Capretto, L., Quinci, F., Piva, R., & Nastruzzi, C. (2013). Preparation of cell-encapsulation devices in confined microenvironment. Advanced Drug Delivery Review, 65,
1533–1555.
Rokstad, A. M., Lacík, I., de Vos, P., & Strand, B. L. (2014). Advances in biocompatibility and physico-chemical characterization of microspheres for cell encapsulation. Advanced
Drug Delivery Review, 68, 111–130.
Hunt, N. C., & Grover, L. M. (2010). Cell encapsulation using biopolymer gels for regenerative medicine. Biotechnology Letters, 32, 733–742.
Drug Delivery Systems and Controlled Release
Nicholas J Kohrs, Thilanga Liyanage, Nandakumar Venkatesan, Amir Najarzadeh, and David A Puleo, University of Kentucky,
Lexington, KY, United States
Introduction 317
History and Progression of Drug Delivery Systems 317
Topical Delivery 318
Transdermal Delivery 319
GI Delivery 319
Nasal Delivery 319
Mucoadhesive Delivery 319
Parenteral Delivery 319
Implantable Carriers 319
Solid Dosage Forms 319
Tablets and Capsules 320
Microparticles and Nanoparticles 320
Films 320
Mechanisms of Controlled Drug Delivery 320
Diffusion-Controlled Release 321
Erosion-Controlled Release 321
Targeted Release 321
Active targeting 321
Passive targeting 322
Stimuli-Responsive Release 322
pH 322
Temperature 322
Drug Delivery Materials 322
Polyesters 324
Poly(ortho esters) 324
Polyanhydrides 325
Polyamides 325
Polymeric prodrugs 325
Biopolymers 325
Lipids 326
Surfactants 326
Ceramics 326
Hydroxyapatite systems 326
Biphasic calcium phosphate systems 326
Bioglasses 327
Metallic Nanoparticles 327
Future Materials and Systems 327
Carbon Nanotubes 327
Microchips 328
3-D Scaffolds 328
3D Printing 328
Conclusion 328
Further Reading 328
Glossary
Condensation polymerization A process in which two low-molecular-weight subunits combine to form a molecule with
a higher molecular weight, typically generating by-products such as water or alcohol.
Copolymer A higher-molecular-weight substance formed by polymerizing two or more monomers together.
Cross-linking Act of bonding two or more polymers together.

Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Drug Delivery Systems 317
Crystallinity Referring to the degree of structural order of a material, crystalline (highly ordered) or amorphous (highly
disordered).
Emulsification The process of mixing together two or more liquids in which one liquid forms microscopic droplets in the
other.
Hydrolysis Process in which water breaks down chemical bonds.
Ring-opening polymerization Process of opening cyclic monomers and covalently joining them to produce a higher-
molecular-weight chain.
Solution polymerization Polymerization process in which the monomer, solvent, and initiator are mixed together prior to
polymerization.
Abbreviations
3DP Three-dimensional printing
BCNU Bis-chloroethylnitrosourea
CNT Carbon nanotubes
EPR Enhanced permeability and retention
FDA Food and Drug Administration
GI Gastrointestinal
HA Hydroxyapatite
LCST Lower critical solution temperature
PCL Poly(ε-caprolactone)
PEG Poly(ethylene glycol)
PLA Poly(D,L-lactic acid)
PLGA Poly(D,L-lactic-co-glycolic acid)
PLLA Poly(L-lactic acid)
RGD Arg-Gly-Asp
SiRNA Small interfering RNA
Tg Glass transition temperature
Introduction
Drugs have been used throughout history to aid in the treatment of illnesses that plagued the populace of the period. During this
time frame, modifications were made to drugs and their associated delivery systems, improving their bioavailability and efficacy.
Fast-forwarding to modern times, drug delivery systems now also play a major role in wound healing and tissue regeneration, allow-
ing for antibiotics, growth factors, hormones, and other therapeutic agents to be released in a controlled manner to the affected
areas. In addition to treating systemic conditions, the need for controlled and localized delivery of drugs has never been more perti-
nent for site-specific applications. Fig. 1 shows how uncontrolled release permits variability in drug concentration, which can lead to
either toxic or ineffective drug levels. Controlled release aims to prevent this variability and maintain an appropriate therapeutic
dose for the desired duration.
Remarkable material innovations, system formulations, and strategies have been developed for a myriad of local and systemic
applications, such as heart disease, diabetes, wound healing, and cancer treatments. This article summarizes the history and progres-
sion of drug delivery systems, solid dosage forms, mechanisms of controlled release, and materials used in controlled drug delivery
systems.
History and Progression of Drug Delivery Systems
The history of controlled release originates from ancient times when drugs were either applied directly to the injury as a paste or
poultice or administered orally, such as a medicinal wine. As history progressed, so too did the methods for drug delivery. Current
318 Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Drug Delivery Systems
TOXIC LEVEL
DRUG CONCENTRATION
UNCONTROLLED
RELEASE
CONTROLLED
RELEASE
MINIMUM EFFECTIVE
CONCENTRATION
INEFFECTIVE LEVEL
TIME
Fig. 1 Uncontrolled release versus controlled release. Controlled release reduces undesirable fluctuations of drug concentration in systemic circula-
tion, thus diminishing side effects and improving treatment outcomes.
systems and methods of drug administration include gastrointestinal (GI), transdermal, topical, nasal, mucoadhesive, parenteral,
and implantable carrier systems. Table 1 summarizes advantages and disadvantages of the different approaches.
Topical Delivery
Topical systems focus on localized delivery to the top layers of the integumentary system, that is, the epidermis and dermis. Delivery
of antiseptic, antifungal, or anti-inflammatory agents, along with emollients, provides immediate treatment and protection to the
different layers of the skin. Topical systems attempt to locally accumulate drugs, via chemical permeation enhancers, deep within
the strata of the skin. Systemic effects are still possible but are hindered due to the slow diffusion of the drug into the surrounding
vasculature.
Table 1 Advantages and disadvantages of common drug delivery approaches
Approach Advantages Disadvantages
Transdermal/topical 1.Sustained delivery 1.Possible skin reaction

2.Avoids first-pass metabolism 2.Delayed drug action
3.Reduced dosing frequency 3.Variability in drug absorbance rates due to application
4.Steady absorption location
5.Noninvasive 4.Adhesion failure
Gastrointestinal 1.Convenient 1.Gastric retention time
2.Simple design 2.Requires high levels of gastric liquid and food
3.Safest administration route 3.Drugs with solubility and stability issues in highly acidic
4.Low cost environment cannot be used
4.First-pass metabolism
Nasal 1.Rapid absorption 1.Possible nasal irritation
2.Quick drug action 2.Potential toxicity of absorption enhancers
3.Avoids first-pass metabolism 3.Difficult to halt after administered
Mucoadhesive 1.Prolonged residence times 1.Potential for damage to mucosa
2.Localization of treatment 2.Patient discomfort
3.High drug flux at absorbing tissues
4.Avoids first-pass metabolism
Parenteral 1.Highest bioavailability 1.Pain
2.Immediate physiological response 2.Potential injection site infection
3.Avoids first-pass metabolism 3.Patient aversion to needles
4.Dosing control 4.Difficult to halt after administered
Implantable 1.Eliminates need for patient compliance 1.Invasive
2.Avoids first-pass metabolism 2.Limited loading capacity
3.Potential for zero-order release 3.Biocompatibility considerations
4.Surgical removal at therapy termination
5.Possible device failure and drug dumping
Transdermal Delivery
The ease of starting, stopping, and restarting treatment makes transdermal and topical delivery convenient pathways. Transdermal
delivery aims to rapidly introduce therapeutic agents into systemic circulation. This is a difficult task for certain drugs due to the
protective integumentary system. Drug transport, across the skin, can be accomplished by chemical permeation enhancers,
allowing < 400 kDa-sized drugs to pass, or electroporation, opening pores to allow passage of 4000 kDa-sized drugs. Transdermal
treatment aims to increase drug bioavailability by avoiding first-pass metabolism.
GI Delivery
Delivery of dosage forms, that is, pills, tablets, and capsules, through the GI pathway is the most convenient and commonly
employed pathway for administering drugs. Depending on patient compliance, appropriate drug concentrations can be delivered
daily, allowing for an extended time period of drug–tissue contact. This pathway is limited, however, by first-pass metabolism,
wherein the drug is degraded or metabolized in the GI tract or liver prior to reaching systemic circulation, leading to decreased ther-
apeutic blood concentrations and longer time to achieve said concentrations.
Nasal Delivery
The nasal pathway is a logical choice for treating issues affecting the nose or paranasal sinus, such as sinusitis and rhinitis, and the
ease of administration and quick transport into systemic blood circulation makes nasal delivery convenient for systemic conditions,
such as drug overdose, diabetes insipidus, and prolonged labor, where therapeutic agents are needed rapidly. The large surface area,
high permeability, and close proximity to blood vessels allow for a bypass of first-pass metabolism and rapid attainment of systemic
therapeutic drug levels. This route of administration is limited by solubility and drug size, making it unattainable for most large or
hydrophobic drugs.
Mucoadhesive Delivery
Mucoadhesive systems allow for intimate contact between dosage form and the mucosa, resulting in localized release, increased
residence time, rapid adsorption, and high drug flux at the site of adherence to provide high bioavailability. These systems can
take the form of tablets, films, microparticles, and gels for application to sites such as the oral cavity, eye conjunctiva, nasal cavity,
vagina, and GI tract.
Parenteral Delivery
Parenteral delivery is defined by the US Food and Drug Administration (FDA) as drug administration by injection, infusion, and
implantation or by some other route other than the alimentary canal. Parenteral injections and infusions allow for systemic
drug delivery and can be divided into several categories, including intravenous, intramuscular, intraarticular, epidural, intrathecal,
intraosseous, and subcutaneous injections. Because this approach avoids physical barriers such as the skin or mucosa and first-pass
metabolism, parenteral delivery has high levels of drug bioavailability and quick drug action.
Implantable Carriers
Implantable carrier systems, such as implantable rods, osmotic pumps, ceramics and glasses, and synthetic polymers, allow for both
site-specific administration and systemic distribution of drugs. Advantages of implantable devices include elimination of need for
patient compliance, lower dosage required due to avoidance of first-pass metabolism, and ability to tune drug release kinetics. Drug
release can be actively or passively controlled, depending on the type of implantable system. For instance, implantable drug pumps,
such as an insulin pump, can be programmed to administer an appropriate dose at a specific time or measureable level. Passive
releasing systems, such as silicone-based birth control, allow for diffusion from the implant to provide long-term sustained release.
Starting from simple solutions, pastes, and pills to deliver therapeutic agents, drug delivery and controlled release have devel-
oped into a complex field combining aspects of materials science and engineering, pharmacology, anatomy, and physiology. Many
different dosage forms have been developed to treat specific tissues and locations.
Solid Dosage Forms
The development of dosage forms has had a profound impact on the efficacy of controlled release systems. Insight to the physico-
chemical properties of a drug enables selection of certain materials and mechanism to obtain the desired release kinetics. Without
a proper delivery system, adverse consequences, for example, premature release, tissue toxicity, and device failure, can occur. A
variety of dosage forms have been developed to account for the different physiological and mechanical stresses that these systems
will experience. Current solid dosage forms include tablets and capsules, micro- and nanoparticles, and films.
Tablets and Capsules

Tablets and capsules are the dosage forms of choice when it comes to drug delivery via the GI tract because they can be self-
administered and deliver an accurate dose. Tablets and capsules are designed to readily degrade once within the proper physiolog-
ical environment, whether the stomach or intestines. These dosage forms are created either by compressing a granulated drug into
a desired shape (tablets) or by encapsulating the drug into a gelatin casing (capsules). Current formulations use a variety of pro-
cessing steps to both improve delivery and sustain release of their loaded agents. For example, some formulations use enteric coat-
ings to prevent degradation until the dosage form has entered the small intestine, while other formulations use laser-drilled holes to
enhance drug transport from the dosage form. Yet, others employ different materials to alter the physicochemical properties, for
example, mucoadhesivity, of the dosage form.
Microparticles and Nanoparticles

Nano- and microparticles are increasingly used in controlled release systems, and the difference in size has numerous effects. Nano-
scale carriers offer enhanced versatility when compared with particles of larger size owing to their small sizes and improved disper-
sibility. In addition, circulation time of nanoparticulate vehicles is prolonged, and their size enables easier uptake for intracellular
delivery of drugs. Nanoscale carriers are increasingly modified for attachment targeting ligands or for “stealth” capabilities by conju-
gation with poly(ethylene glycol) (PEG).
Microspheres and nanospheres can employ many different materials, for example, polymers, glasses and ceramics, and metals,
each with different processing techniques. Polymers are most prevalently used in the formulation of microspheres for drug
delivery. Ceramics, glasses, and metals are gaining interest because they can be bioactive and have better mechanical properties.
Typically, ceramics, metals, and bioactive glasses are loaded via adsorption, whereas polymeric particles are formulated via
emulsification.
Emulsions rely on the hydrophobicity or hydrophilicity of drug molecules to partition into the respective phase. For hydro-
phobic drugs, an oil-in-water (O/W) single emulsion is used because the oil phase dissolves the therapeutic agent for emulsification.
For hydrophilic drugs, either a water-in-oil (W/O) single emulsion or a water/oil/water (W1/O/W2) double emulsion is used. In
either of these latter cases, the therapeutic agent is dissolved in an aqueous solution. Particles are collected for storage after solvent
evaporation. Particle size is determined by multiple factors, including organic solvent, polymer concentration, surfactant, and emul-
sification energy. Large surface area-to-volume ratios expose a greater percentage of the loaded drug to the external phase, which can
lead to adverse outcomes, such as small particles having a lower maximal drug loading and a sizable loss of payload.
Films
Film delivery systems are typically flexible sheets of polymeric matrices incorporating the therapeutic agent. Interest in polymeric
films as dosage forms has increased as an alternative approach to conventional dosage forms. Films can be used for systemic or local
action via several approaches, for example, oral, topical, GI, and transdermal delivery. An example with significant clinical impact is
found in drug-eluting stents. These devices encompass thin polymeric films deposited onto the metallic struts of a stent to release
a drug, such as paclitaxel or sirolimus, to control cell proliferation and prevent restenosis of the blood vessel.
Film formulations offer several advantages, including convenient administration through noninvasive routes, that is, trans-
dermal or oral mucoadhesive patches, and ease of handling during manufacture and transportation. Commonly used techniques
for the preparation of films include solvent casting, photopolymerization, hot-melt extrusion, and three-dimensional printing
(3DP).
In solvent casting, a solution of polymer and drug is dispensed into a suitable mold, and the solvent is allowed to evaporate,
which leaves a drug-loaded polymeric film. The rheological properties of the polymeric mixture affect the drying rate, film thickness,
morphology, and content uniformity of the films. Hot-melt extrusion is a substitute method that is especially useful when an
organic solvent system must be avoided. A mixture of pharmaceutical ingredients is melted and charged through a die to obtain
homogeneous matrices. 3DP is briefly described in the section “Future Materials and Systems.”
Microparticles, nanoparticles, films, and other drug delivery systems each have advantages and disadvantages. Different proper-
ties can be achieved by combining two or more of these systems together to confer the advantages of one class of vehicle on another.
This approach has been used, for example, with microparticles in films, nanoparticles in a cross-linked hydrogel matrix, micropar-
ticles in scaffolds, and many other combinations. All steps, including the synthesis method, encapsulation process, or final surface
modification for targeted delivery, determine the characteristics of these systems and their main goal, that is, the controlled release
of bioactive agents.
Mechanisms of Controlled Drug Delivery
Drug delivery systems are used to enhance the therapeutic effect of the drug at the target site by protecting the drug under physi-
ological conditions, controlling its release, increasing or reducing systemic circulation, and assisting in reaching the site of action.
These functions are achieved by complex and diverse mechanisms that work individually, in combination, or sequentially.
Polymers, ceramics, glass, metals, and metal alloys are often used as vehicles in the drug delivery systems. Control of drug release
from dosage forms is multifactorial, involving diffusion of drug out of or water into the system, dissolution of the drug, partitioning,
system/matrix swelling, degradation, or erosion.
Diffusion-Controlled Release
Most polymeric drug delivery systems are diffusion-controlled, where the drug diffuses from the vehicle along a concentration
gradient and the release is either Fickian or non-Fickian. Glass transition, also known as second-order transition, is a characteristic
nonphase transition that occurs at a temperature specific to each polymer. Below the glass transition temperature (Tg), polymers
turn rigid, hard, and dimensionally stable and are considered to be in a glassy state, but above Tg, they turn soft and are said to
be in a rubbery state. Polymers in a rubbery state allow better infiltration of the surrounding medium, allowing dissolution and
subsequent diffusion of the drug. When diffusivity of the drug is independent of concentration, the release is Fickian, but if diffusion
depends on time and concentration, then the release is non-Fickian.
Two kinds of diffusion-controlled systems are monolithic and reservoir-based (Fig. 2A). In monolithic systems, the drug is
uniformly distributed within the vehicle. The rate of diffusion depends on the diffusion coefficient of the molecule, and it decreases
as a function of time because the concentration of drug in the core decreases with time. In reservoir systems, the diffusion coefficient
is controlled by the rate-limiting membrane, because the drug partitions from the core into the membrane and then diffuses from
the membrane due to a concentration gradient. When saturation of the core drops, diffusion from the membrane decreases.
Erosion-Controlled Release
Polymers can be degraded actively (by enzymes) or passively (by hydrolysis), resulting in surface or bulk erosion (Fig. 2B). Surface
erosion is a heterogeneous process wherein degradation of the polymer happens at only the surface and the rate is proportional to
the surface area. Drug release in surface-eroding systems is often correlated with a predictable erosion rate, which is considered ideal
for many drug delivery applications. Erosion begins from the outside and progresses inward. In most cases, thicker systems have
longer erosion times, and hydrophilic polymers degrade faster compared with hydrophobic materials. With bulk-eroding systems,
degradation is homogenous throughout the material, and the size of the system remains constant in most cases. The drug is released
in three stages: burst release from the surface, release from initial degradation of the polymer, and release of residual drug during
complete degradation/homogeneous erosion of the polymer. Polyanhydrides and polyesters are typical examples for the surface-
and bulk-eroding systems, respectively. Hydrophobic or medium-insoluble drugs are generally released by erosion.
Targeted Release
Targeted drug delivery, postulated as a “magic bullet” in the 1990s, is widely studied to deliver drug to a specific tissue or organ to
achieve a desired therapeutic outcome without activating side effects. Efficiency of this approach depends on three important
factors: specific ligand that is characteristic to the tissue of interest, effective drug for the condition to be treated, and appropriate
mechanism for delivery of the drug. Targeted delivery systems are achieved by two different mechanisms, including ligand–receptor
interaction (active) and physiological changes (passive).
Active targeting
Active targeting involves conjugating the drug/carrier system to a specific ligand that guides the drug to the target site. The ligand
acts as a “homing signal” that improves selective drug delivery to a specific organ. Antibodies are commonly conjugated to drugs
(A) (B)
Surface Bulk
Erosion Erosion
Monolithic
Time
Reservoir
Time
Fig. 2 (A) Schematic representation of monolithic and reservoir drug delivery system. (B) Changes in the polymeric delivery system during surface
and bulk erosion.
because of their specificity. For example, some of the promising studies include a prostate-specific membrane antigen antibody
J591 conjugated to a polymer-containing drug that is used to target prostate cancer cells. Folate receptors are also widely targeted
because of their overexpression in most cancer tissues, and folate-conjugated doxorubicin polymer aggregates actively target the
folate receptor in cancer cells and inhibit tumor progression. Similarly, Arg-Gly-Asp (RGD) peptide is used to target integrin avb3
receptors that are overexpressed in neovasculature and activated endothelial cells to deliver nucleic acids constructed with poly-
mer aggregates (siRNA–polyethyleneimine–polyethylene glycol–RGD) to achieve tumor-specific and also gene-specific targeted
therapy.
Passive targeting
Passive targeting refers to drug accumulation at the target site via systemic circulation without any specific ligand. Accumulation is
achieved through physiological changes, such as the dense vasculature with loose or defective architecture associated with most
solid tumors. The enhanced permeability and retention (EPR) effect is a molecular-weight phenomenon in which high-
molecular-weight molecules are retained in the dense vasculature due to poor lymphatic drainage. This characteristic leaky vascular
architecture is widely used to deliver the drug selectively to the tumor. For example, increased retention of the low-molecular-weight
antitumor protein neocarzinostatin was achieved by increasing its weight by conjugation with poly(styrene-co-maleic anhydride).
This effect has also been studied with other antitumor drugs, such as doxorubicin encapsulated in PEG-coated liposomes, which
increase circulation and localization within the tumor.
Stimuli-Responsive Release
The release pattern of the drug delivery system depends not only on the stimulus but also on the material from which the drug
is released. Responsive polymers undergo a conformational change responding to specific physiological stimuli. There are two
types of responsive drug delivery systems, including open-loop (externally regulated) and closed-loop (self-regulated) systems.
Open-loop systems are nonpolymeric drug release systems, where the delivery of drug is predetermined, for example, an
insulin pump. In the case of closed-loop systems, drug delivery is determined by physiological conditions, such as pH and
temperature.
pH
The characteristic differences in pH at various sites, for example, GI tract, blood, and intracellular environment, along with the acidic
conditions associated with most cancers and infected tissues, make pH an attractive stimulus for responsive polymers. pH-
responsive polymers are polyelectrolytes with weak acidic or basic groups that undergo a change in their ionization state by accept-
ing or donating protons. Changes in the ionization state result in conformational changes and swelling behavior of the polymer that
consequently modulate drug delivery. For example, studies on chitosan/heparin nanoparticles tested against Helicobacter pylori
protect the drug from gastric secretion (the positive surface charge is stable at pH 1.2–2.5), infiltrate through the mucus layer
(pH 4.5–7), and disintegrate at the infection site (nanoparticles are deprotonated at pH 7) to release the drug. Similar studies
have shown that hydrogels with 2,4,6-trimethoxybenzaldehyde groups exhibit a hydrophobic nature at pH 7.4 and transition to
hydrophilic at pH 5, releasing a drug found to be effective against lung cancer.
Temperature
Certain polymers exhibit phase transition at a specific temperature that alters solubility. Changes in the solvation state are attributed
to the inter- and intramolecular hydrogen bonding of the polymer. This transition can be used for drug release from polymers that
expand or collapse at body temperature. Poly(N-isopropylacrylamide) is a common thermoresponsive polymer that is widely tested
for drug delivery applications. Below the lower critical solution temperature (LCST) of 32–33 C, water molecules bond to the poly-
mer network to cause swelling, which enables loading of drugs within the polymer. Above the LCST, a hydrophilic to hydrophobic
transition causes the polymer network to collapse and become insoluble, thereby releasing all the water molecules and entrapped
drug during transition.
Drug Delivery Materials
Materials used in controlled release systems can be classified into many categories, but this section will focus on both biodegradable
materials, for example, biodegradable polymers, lipids, and ceramics, and nonbiodegradable materials such as magnetic nanopar-
ticles. Nonbiodegradable materials can be used in therapeutic applications only if the material can be recovered or excreted after
drug release, for example, intraocular implants, buccal patches, and chemotherapy nanoparticles. Most therapeutic applications
use a biocompatible material that will degrade within the body and whose degradation products are not cytotoxic. Due to these
constraints, the study of synthetic polymers has increased and has yielded several successful systems. Table 2 summarizes the advan-
tages and disadvantages of different materials, while Table 3 lists the examples of commercially available products that employ
these materials.
Table 2 Drug delivery material advantages and disadvantages
Material Advantages Disadvantages
Polyesters 1.Simple synthesis 1.Bulk erosion causes nonlinear degradation kinetics

2.Physical and chemical properties can be tuned 2.Acidic degradation products can cause irritation
3.Easily fabricated into a variety of dosage forms
Poly(ortho esters) 1.Zero-order release kinetics 1.Synthetically complex
2.Surface erosion is slow 2.Need for excipients to accelerate degradation
Polyanhydrides 1.Easy synthesis by low-cost resources by one-step 1.Hydrolytic instability. Requires storage under cold,
synthesis. No need of any purification step moisture-free conditions
2.Degrade into nontoxic carboxylic acids at a predictable 2.Low mechanical strength and film or fiber-forming
rate properties
3.Degradation can be tuned
Polyamides 1.The carboxyl and amino groups provide synthetic 1.Immunogenic
handles for further functionalization of the polymer 2.Poor mechanical properties
backbone 3.Require enzymes to degrade
Polymeric prodrugs 1.High drug loading 1.Synthetically complex
2.Prolonged and localized release
Chitosan 1.Mucoadhesive property suitable for use in both oral and 1.Slow enzymatic degradation
nasal formulations
2.Ability to condense DNA to form complexes to form
nonviral gene delivery vehicles
Collagen 1.The most abundant biomaterial 1.Mildly immunogenic
2.Can be processed into different forms
Lipids 1.Pharmaceutical stability 1.Low encapsulation efficiency
2.Ability to deliver hydrophilic and lipophilic drugs 2.Premature membrane degradation
3.Long drug circulation times 3.Low solubility
4.Difficult to scale up
Hydroxyapatite 1.Tunable morphology leading to higher drug loading 1.Brittle
2.Localized delivery (implanted) 2.Quick clearance (of nanoparticles)
3.Loading limited by absorption and electrostatic
interactions
Biphasic calcium phosphate 1.Tunable dissolution rates and mechanical properties 1.Brittle
2.Synthesis comparatively straightforward, easy, and 2.Low loading efficiency
inexpensive
3.Localized delivery (implanted)
Bioglass 1.Delivers hydrophilic drugs 1.Poor mechanical properties
2.Ability to carry high dose of drug in nanoparticles
3.Regenerative properties with therapeutic effects
Metal nanoparticles 1.Can penetrate biological barriers 1.Potential toxicity
2.Biocompatible and inert (depending on material) 2.Removal issues
3.High drug loading 3.Susceptible to physical instability
Table 3 Examples of commercially available drug delivery systems using different materials
Material Commercially available products
Polyesters Lupron Depot®, Decapeptyl®, Risperdal Consta®,

Eligard®
Poly(ortho esters) Alzamer®
Polyanhydrides Gliadel®, Septacin®
Polymeric prodrugs TransCon Growth Hormone, PolyAspirin
Collagen INFUSE®
Lipids Lipoquin®, Neoral®, Kaletra®, Sustiva®
Calcium phosphates KaliLight
Metallic nanoparticles Ferumoxytol
Synthetic Polymers
Synthetic polymers consist of long chains of repeating molecules that are designed with a variety of chemical and physical properties
in mind. Most of these materials possess ester, anhydride, or amide linkages that enable degradation by hydrolysis or enzymatic
activity in vivo. Biodegradability makes it possible to administer the controlled release system without the need for a second
procedure to remove the exhausted delivery vehicle. Because of the large variety available, synthetic polymers are the most widely
used materials for developing controlled release systems.
Polyesters
Polyesters are the most commonly studied biodegradable polymeric materials. They can be easily prepared by ring-opening poly-
merization of the corresponding cyclic lactone monomer or by condensation polymerization. They mainly degrade via hydrolysis of
ester bonds. Polyester-based biodegradable polymers include poly(D,L-lactic acid) (PLA), poly(glycolic acid) (PGA), poly(D,L-lactic-
co-glycolic acid) (PLGA), and poly(ε-caprolactone) (PCL). PLA is prepared by polymerizing lactic acid monomers or by
ring-opening polymerization of cyclic lactide monomer. Lactic acid has an asymmetrical a’carbon, which gives rise to R and S enan-
tiomeric forms commonly referred to as the D and L forms of lactic acid.
The extra methyl group in the side chain makes PLA more hydrophobic compared with its chemically similar analogue PGA.
Poly(L-lactic acid) (PLLA), which is prepared using all L-lactic acid as the monomer unit, has a glass transition temperature of
60–65 C and a melting temperature of 175 C. Due to its good tensile strength, high modulus ( 4.8 GPa), and slow degradation
( years) due to high crystallinity of PLLA polymers, they are considered appropriate for load-bearing applications, such as ortho-
pedic fixation (e.g., Phantom Suture AnchorÒ (DePuy)). The amorphous racemic PLA polymer that consists of both D and L form
of lactic acid has a glass transition temperature of 55–60 C, lower tensile strength ( 1.9 GPa), and relatively fast degradation time
( months) compared with PLLA. Note that degradation is highly dependent on the molecular weight of the corresponding poly-
meric material, its composition, and the processing method, with higher-molecular-weight polymers exhibiting slower degradation.
The amorphous form of PLA has applications in drug delivery and scaffolding materials for tissue regeneration because of its low
strength, lower crystallinity, and comparatively faster degradation profile. PLA is used in dosage forms such as microparticles or
nanoparticles for delivery of drug molecules, peptides/proteins, and nucleic acids. To alter crystallinity and degradation kinetics,
lactic acid is often copolymerized with other monomers, oligomers, or polymers, for example, glycolide, caprolactone, and PEG,
to prepare di- or triblock copolymers.
PGA is also obtained by ring-opening polymerization of the cyclic diester of glycolic acid known as a glycolide. PGA shows low
solubility in common organic solvents (e.g., dichloromethane, tetrahydrofuran, acetone, chloroform, and ethyl acetate) due to its
hydrophilicity. With a melting temperature of 225 C, PGA is a hard, tough, crystalline polymer. Due to its fiber-forming properties,
PGA was used as the first synthetic absorbable suture material (DEXONÒ). Poor solubility, high melting point, and high rate of
degradation limit its uses in drug delivery.
Copolymerization of lactic and glycolic acids gives rise to the polymeric material commonly known as PLGA. Properties of PLGA
polymers can be easily tuned by controlling the ratio of lactide (L) to glycolide (G) (L is more hydrophobic due to the a-methyl
group). For example, PLGA polymers with 50:50, 75:25, and 85:15 (L/G) have degradation timescales of 1–2, 4–5, and 5–6 months
under in vitro conditions (isotonic saline, pH 7.4, and at 37 C). The degradation products produced may lead to an increase in
acidity that can cause irritation at the implant site. This can be overcome by incorporating basic salts to control the pH. PLGA
has been used to form various drug delivery vehicles, such as microspheres and nanospheres, nanofibers, and films, for controlled
release of therapeutic agents. For example, Lupron DepotÒ is a PLGA microsphere-based drug delivery system that releases gonad-
otropin to treat prostate cancer.
The nontoxic, water-soluble PEG can be introduced into PLGA to form PLG–PEG diblock (AB) and PLGA–PEG–PLGA (ABA)
triblock, multiblock, or branched block copolymers exhibiting different physicochemical properties, including microphase separa-
tion, crystallinity, water solubility, and biodegradability. The presence of PEG in the backbone results in a significantly more hydro-
philic and protein-resistant polymer, allowing longer circulation time in the body. The amphiphilic PLGA–PEG block copolymers
can also form micelles used for delivering hydrophobic drugs. The size and morphology of micelles can be fine-tuned by adjusting
the chemical composition, total molecular weight, and ratio of the PLGA and PEG block lengths. Various hydrophobic drugs, such
as the chemotherapeutic paclitaxel, have been incorporated into these micelles.
Another commonly used degradable aliphatic polyester is PCL. PCL is synthesized by ring-opening polymerization of the six-
membered ring ε-caprolactone using a catalyst such as stannous octoate. Due to extremely slow degradation rates, PCL is often
used as a long-term scaffold or drug/vaccine delivery material, for example, the contraceptive device CapronorÒ. PCL has a glass
transition temperature of approximately 60 C, making it semirigid at room temperature. Block copolymers are synthesized by
copolymerizing PCL with other monomers or polymers, for example, PLGA and PLA, to decrease the degradation time. Hydrophilic
block segments, such as PEG, can be conjugated to the hydrophobic PCL portion to prepare micelles. For example, PCL–PEG
diblock copolymer micelles were used to formulate doxorubicin for targeted drug delivery.
Poly(ortho esters)
This class of polymers is synthesized by transesterification or by addition of polyols to diketene acetals. The orthoester linkages that
connect polymer segments can undergo rapid hydrolysis, but the bulk of the polymer is hydrophobic and minimizes the water
penetration. Therefore, hydrolysis and the resulting drug release occur only at the surface of the material (surface erosion). The
pH sensitivity of orthoester bonds allows adjustment of degradation rate; introducing acidic excipients, for example, suberic
acid, to the polymer matrix accelerates degradation, while neutral and basic excipients stabilize the polymer matrix by neutralizing
the acidic by-products formed through hydrolysis. The recent modification of the poly(ortho ester) backbone by incorporating gly-
colide or lactide avoids the need for excipients, because once hydrolyzed, the resulting lactic or glycolic acid monomers can catalyze
orthoester bond breakage. As an example, a poly(ortho ester) synthesized from 1,10-decanediol and 1,10-decanediol dilactide was
used to prepare a tetracycline delivery system for treating periodontal disease.
Polyanhydrides
Polyanhydrides are mainly prepared by melt condensation of dicarboxylic acid with an acetic anhydride at a high temperature under
vacuum. The most widely studied polyanhydrides are based on sebacic acid, adipic acid, and terephthalic acid. The anhydride bonds
hydrolytically cleave and form water-soluble products. The hydrophobic polymer backbone prevents water penetration into the
bulk of the polymer, which causes the polymer to degrade by surface erosion. Consequently, the polymer maintains nearly zero-
order degradation kinetics while retaining its structural integrity.
Degradation can be tuned by introducing hydrophobic or hydrophilic monomers into the polymer backbone. Hydrophobic
monomers stabilize the anhydride bond over hydrolysis. Relatively less hydrophobic aliphatic polyanhydrides based on sebacic
acid degrade fast (within days), while polymers based on more hydrophobic aromatic (p-carboxyphenoxy)hexane take years to
degrade. Poly(anhydrides) have many applications and can be fabricated into microparticles, nanoparticles, wafers, etc. For
example, poly[(carboxy phenoxy propane)-(sebacic acid)] is used for controlled release of the chemotherapeutic agent BCNU to
treat brain cancer (GliadelÒ). Salicylic acid-based polyanhydrides are able to stimulate new bone formation.
Polyamides
The most common polyamides are poly(amino acids). Compared with polyesters and polyanhydrides, polyamides are more stable
toward hydrolytic cleavage due to the amide linkage but instead rely on enzymes for degradation. The degradation rate can be
manipulated by hydrophilicity of the amino acids. The most widely used polyamino acids are poly(g-glutamic acid), poly(3-L-
lysine), poly(aspartic acid), and their derivatives. These polymers consist of ionizable pendant groups, such as carboxyl and amino
groups. The functional groups provide synthetic handles for further functionality of the polymer backbone, for example, glycosy-
lated poly(g-glutamic acid) used for liver-specific targeted drug delivery.
Polymeric prodrugs
In these polymers, the bioactive molecule, that is, drug, serves as a repeating monomer unit, or it can be attached as a pendant
group. Incorporation of bioactive molecules into the polymer backbone allows for higher drug loading, controlled release, and
easy processing. The most widely studied polymeric prodrugs, based on salicylate, are prepared through melt condensation or solu-
tion polymerization. These polymers degrade by surface erosion and have controlled and extended release. Ibuprofen, ketoprofen,
and naproxen have been used as pendant groups on the polymer backbone. Rather than adding pendants, a polysimvastatin pro-
drug has been synthesized as a diblock copolymer of simvastatin monomers linked to different molecular-weight PEG moieties to
control the hydrophilicity. The PEG moiety initiates the ring-opening polymerization through simvastatin lactone ring in the pres-
ence of a stannous octoate catalyst or other catalysts. Release of simvastatin is highly dependent on the PEG moiety incorporated
into the polymer backbone.
Biopolymers
Biopolymers are used in drug delivery applications due to their availability and biocompatibility. These materials include protein-
based polymers, such as collagen, albumin, and gelatin, and polysaccharide-based polymers, such as agarose, alginate, carrageenan,
hyaluronan, dextran, chitosan, and cyclodextrins. Natural polymers have limitations in batch-to-batch reproducibility, uncontrolled
rates of hydration, broad molecular-weight distributions, and susceptibility to microbial contamination. The two most widely
studied protein- and polysaccharide-based polymers are collagen and chitosan, respectively.
Collagen
As the most abundant protein in the body, collagen is a major component of musculoskeletal tissues, the skin, and blood vessels.
Collagen can be easily cross-linked using chemicals, such as aldehydes, carbodiimides, hexamethylene diisocyanate, and epoxy
compounds; elevated temperatures; or high-energy irradiation. Cross-linked collagen is used as scaffolds for tissue engineering
and drug delivery vehicles. The drug release profile from collagen matrices can be manipulated by varying the physical properties,
such as density, porosity, and degradation kinetics. Cross-linked collagen has also been used as a vehicle for delivery of absorbed/
adsorbed bioactive proteins, such as recombinant human bone morphogenetic protein-2 (INFUSEÒ (Medtronic)).
Chitosan
Chitosan is a linear polysaccharide consisting of glucosamine and N-acetyl glucosamine units. In vitro degradation can occur by
chitosanase, lysozyme, and papain, but in vivo degradation occurs mainly via lysozyme. Chitosan’s degradation kinetics mainly
depend on the degree of acetylation and crystallinity; an increase in the degree of deacetylation causes an increase in crystallinity
and a decreased degradation rate. Chemical modifications of chitosan can significantly affect its degradation rate. For example,
inclusion of bulky side groups disrupts its strong hydrogen-bonding network and accelerates degradation. Due to the strong inter-
action with the negatively charged mucous membrane and low solubility in water, chitosan is widely used in mucoadhesive
systems.
Chitosan also shows both anti-inflammatory and antibacterial properties, which are beneficial in wound-healing applications.
Chitosan is also used in the production of injectable thermosensitive carriers. Chitosan can be easily blended with other polymers or
fabricated into nanoparticles, microspheres, gels, or fibers for drug delivery applications. Due to its ability to form electrostatic
complexes with DNA, extensive research is ongoing to develop chitosan-based gene delivery systems.
Lipids
Lipids are a large and diverse class of biological substances including surfactants, phospholipids, waxes, and oils. These materials do
not readily solubilize in water and are typically composed of a long hydrocarbon tail attached to a polar head group. Due to molec-
ular interactions in aqueous solutions, the formation of lipid bilayers is energetically favorable for them to sequester their hydro-
carbon tails from water. This allows formulation of systems such as vesicles, multilamellar/unilamellar liposomes, micelles, and
other membranes for use in physiological environments.
Lipid systems have the ability to encapsulate hydrophobic and hydrophilic drugs, maintain longer drug circulation times, reduce
systemic toxicity, and allow for targeting via ligand conjugation. Loading depends on the number of lipid layers and size of the
carrier. Multiple layers present the opportunity to load multiple drugs, create different release profiles, or provide extra layers of
protection. Most clinically used liposomes are 50–300 nm in size. In addition to allowing different sized drugs or proteins to be
loaded into the system, the size range reduces uptake in the liver, which provides the liposomes a better chance of remaining in
circulation.
Delivered via parenteral injections, liposomal systems can actively target, via ligand conjugation, or passively target, via aggre-
gation, their desired tissues, thus lowering cytotoxic or excess drug concentrations in healthy tissues. Release occurs by aggregation
and destabilization of the lipid membrane. This method of release is energetically favorable compared with fusing with the cells.
Environmental factors, such as pH and temperature, affect stability of the lipid membrane, thereby causing issues of premature
release and short half-life. While encapsulation of hydrophilic drugs can be accomplished, it remains difficult and is often associated
with poor encapsulation efficiency.
Surfactants
Surfactants are a subgroup of lipid-like materials that have the ability to lower the surface tension at an interface. At low concen-
trations, surfactant molecules remained separate, but once past a critical concentration, they begin to aggregate into small particles
called micelles. Depending upon the hydrophile to lipophile balance, different colloidal systems can be formed. Two such systems
are O/W micelles and W/O reverse micelles. Micelles and reverse micelles are typically one of the smaller delivery systems, having
particle sizes ranging from 10 to 100 nm and 1 to 10 nm, respectively. However, encapsulation is not the only use of surfactants in
drug delivery. Facilitation of drug transport across the respiratory wall and enhanced skin penetration by target molecules depends
on the formal charge of the surfactant head group. The charge on this head group can interact with the membrane and alters its
structure to hydrate, expand, or electrically change the membrane, resulting in drug transport.
In recent years, 90% of new drugs have been classified as poorly soluble in water. Surfactants enhance the permeation of drug
delivery systems across physiological barriers, leading to rapid and efficient delivery of proteins and insoluble drugs to their targeted
tissues. One adverse effect is the poor thermodynamic stability of low-molecular-weight surfactants in solution, which causes
premature dissociation of the surfactant membrane prior to reaching the targeted tissues. While this system has flaws, delivery
via surfactants allows for increased bioavailability, distribution, and permeabilization while also reducing adverse reactions.
Ceramics
Ceramics are inorganic, nonmetallic materials formed by ionic and covalent bonding of metallic and nonmetallic elements. Their
microstructure varies from crystalline (ceramics) to amorphous (glasses). In biomedical applications, the materials are often used to
replace diseased, deteriorating, or damaged bone, with some also being used in soft tissues. Being nonimmunogenic and compat-
ible with most incorporated therapeutic agents, these materials are suitable candidates for drug delivery systems.
Hydroxyapatite systems
Hydroxyapatite (HA) is a pure, synthetic version of bone mineral. It is osteoconductive and biocompatible, and depending on pro-
cessing, the material can possess similar compressive strength and nanostructure as bone. Processing to generate high surface area-
to-volume ratio allows for sorption of therapeutic agents onto and into the matrix. Manipulation of the porosity and pore structure
allows for controlled loading and release kinetics.
Commercial products such as nanoXIM release therapeutic agents from HA scaffolds and microspheres by diffusion. HA has an
associated initial burst followed by a prolonged release. The initial burst comes from differences in concentration between the scaf-
fold and interstitial fluid. With decreased drug concentration, the gradient between the scaffold and interstitial fluid drops, slowing
release and maintaining a release until most of the drug has been delivered. One consideration in choosing this system is the rate of
dissolution. HA systems are designed to remain in the body for an extended period of time. As such, HA would not be suitable for an
application that needs relatively quick degradation.
Biphasic calcium phosphate systems

Biphasic calcium phosphates (BCPs) are a mixture of HA and b-tricalcium phosphate (b-TCP). The ratio of HA/TCP plays a key role
in the bioactivity and dissolution rate of the material. This combination of stable HA and resorbable b-TCP allows for controllable
resorption and gives BCP its mechanical properties. Like most ceramics, BCP exhibits an inherent brittleness and inability to
mechanically support load-bearing areas. Attempts have been made to create composite materials that increase the ability of
BCPs to support load-bearing areas. Drug loading and release from BCPs depend upon the percent porosity, morphology, and
purity of the material. The release kinetics of BCP are similar to those of HA, with an initial burst followed by a slowed rate of
release. A tailorable pore structure and bioactive properties make BCP a versatile ceramic and a suitable option for antibiotics,
hormones, insulin, cancer drugs, etc.
Bioglasses
Bioglass is a family of bioactive glasses composed of silicon dioxide, sodium oxide, calcium oxide, and phosphorous pentoxide.
This material yields a surface pore structure, 5–20 nm, with excellent surface area, pore volume, cytocompatibility, and ability to
induce apatite formation. As with HA and BCP, clinical applications of bioglasses have focused on osteogenesis. From this came
an increase in research on the loading of therapeutic agents to help enhance the regeneration of the surrounding tissues. Loading
depends upon the crystallinity, porosity, and morphology of the implant, whereas release is dictated by the rate of degradation,
typically ranging from a few hours to several weeks. This variability grants control over how quickly the loaded agents will release.
Bioglass degrades via hydrolysis of the silica matrix to form Si(OH)4 and silanol. These products first cause surface pH levels to rise,
leading to further hydrolytic degradation and nucleation of carbonated HA in the silica gel surface, which contributes to the devel-
opment of new bone.
Until recently, bioglass materials were not clinically used as drug delivery systems but as a material to aid in bone regeneration.
However, as of recent, there has been success with therapeutically loaded bioglasses. In a clinical study, an enamel matrix protein
derivative was loaded into bioglass and has resulted in the formation of new cementum with associated periodontal ligament and
enhanced mineralization around the bioglass particles.
Metallic Nanoparticles
Metallic delivery systems have potential for gene and drug delivery applications. These systems can vary from simple nanoparticles
to complex nanoshells and nanocages. Nanoscale particles have the unique ability to more easily pass through biological barriers,
for example, blood–brain barrier and cell membranes. Having release occur inside the cell promotes higher therapeutic efficacy than
obtained via free drug absorption. Once the magnetic nanoparticles are injected near the targeted area, a high-powered magnetic
field is turned on to magnetically direct the nanoparticles to the desired location. Nanoparticles release via diffusion, and direct
conjugation of the drug and nanoparticle can be weak requiring the action of organic linkers such as amines, carboxylic acid, alde-
hyde, or thiol. These linkers allow for active targeting of cancer-specific markers that upon binding will allow release to occur. For
passive release, aggregations of nanoparticles begin to attach or penetrate the targeted cells/tissues and release their payload via
diffusion. In the early 2000s, clinical trials began to be initiated and have shown metallic nanoparticles to be successful carrier
systems. Currently, a system of paclitaxel and gold nanoparticles (Aurmine (CytImmune Sciences, Inc.)) is being developed to
increase the effectiveness of chemotherapy.
Because the magnetic field is generated outside the body and its effective strength decreases with distance, targeting locations
deep within the body is difficult. Another issue to consider is the toxic by-products left from the drug delivery system after release.
Metal nanoparticles must be excreted from the body via alternative routes, that is, defecation and urination.
Future Materials and Systems
Current drug delivery systems have made significant strides to improve issues such as targeting, loading, release, and circu-
lation times. Future technologies will enable the creation of controlled release formulations that further improve upon these
successes. However, there is a need not only to further study nanoparticles but also to look for new materials and new
systems as means for drug delivery. Several technologies are being developed with ultimate goal of becoming
commercialized.
Carbon Nanotubes
Carbon nanotubes (CNTs) are structures comprising long graphene tubes with either single or multiple walls. CNTs can be up to
several microns long and have diameters from 0.4–3 to 2–100 nm for single and multiwall structures, respectively. This system has
drawn considerable attention due to its structural, electronic, and mechanical properties; thermal stability; high surface area; and
size. One downfall of a larger surface area is the increased chance for protein opsonization.
Drugs, proteins, and bioactive molecules are loaded onto or into CNTs via functionalization of the surface by methods of
covalent and noncovalent bonding. Toxicity depends upon several factors, such as functionalization, purity, length, size, diam-
eter, surface chemistry, and route of dispersion. Excess CNT accumulation, which can occur in different parts of the body such as
the lung, heart, testes, and brain, leads to oxidative stress and cellular toxicity. Other researchers, however, suggest that once func-
tionalized, such as with polycations, CNTs do not pose a significant threat to the body. Toxicity of CNTs remains a hotly debated
topic.
Microchips
Implantable systems have been around for many years, but controlled release from these implants depends upon which material is
used, how the system is processed, and the system mechanisms of release. Researchers have begun using implantable microchips to
obtain precisely controlled release patterns rather than rely on standard mechanisms of release, that is, diffusion or degradation. The
microchip contains drug-filled microreservoirs that are covered with a thin gold film. Once in place, release can be activated wire-
lessly by applying a voltage to rupture the reservoir membrane and release the loaded contents. Because this system is program-
mable, it allows control over the dose the patient receives and the time at which drug is released. Currently, this device is being
clinically tested for delivering parathyroid hormone to osteoporosis patients.
3-D Scaffolds
3-D scaffolds are designed to be implanted, support the surrounding tissue while ingrowth and regeneration occur, and degrade
over time. Different systems and materials have been developed for mechanical and physiological environments pertinent to
specific applications, including the skin, cartilage, bone, and blood vessels. Ongoing development is attempting to provide
controlled release of therapeutic agents from the scaffold to promote tissue regeneration or remodeling. In these systems,
controlled release depends on modifying either the rate of degradation or the swelling behavior of the given scaffold to allow
release to occur.
3D Printing
Rapid prototyping (RP) techniques, including 3DP, were originally created to allow for quick fabrication of new devices during
product development. In using computer-generated 3-D models, companies are able to quickly build, test, and analyze different
models. A variety of RP methods have found several uses for biomedical applications.
Germane to the present topic of drug delivery, three main techniques are being currently studied: laser-based systems, nozzle-
based deposition systems, and printing-based inkjet systems. Selective laser sintering and stereolithography (SLA), two prominent
strategies of laser-based systems, use a laser to either sinter or polymerize different materials. Fused deposition modeling, a nozzle-
based system, extrudes hot thermoplastics layer by layer onto a loading platform in which the final product is formed. 3DP, an ink-
jet system, involves the use of particles and binding agent to form the product.
Computer-aided design allows for a geometrically precise, layer-by-layer design of the delivery system. Both loading and release
of therapeutic agents can be controlled by altering the porosity and structure of the dosage form. 3DP allows for specific drug load-
ings that were otherwise unobtainable by traditional methods. Each system has limitations, however. Laser-based systems are
limited by material because it must be able to withstand the extreme temperatures and potential denaturation of the fabrication
process. Another limitation, mainly with SLA, is issues of scarce biocompatible resin materials, cytotoxic photoinitiators, and
entrapment of unreacted monomer and residual photoinitiator. Fused deposition modeling is limited by the type of thermoplastic,
as the polymer must have good melt viscosity with low enough viscosity to be extruded from the nozzle but viscous enough to
maintain the printed shape.
Conclusion
Drug delivery systems are designed to achieve a desired therapeutic effect at the target tissues without systemic toxicity. These
systems are engineered to load different types of hydrophilic or hydrophobic drugs and enhance transportation across physiological
barriers, without any compromise in bioactivity and while maintaining controlled release of therapeutic agents. Controlled release
improves patient compliance by reducing the invasive procedures and oral drug intake.
Controlled release technologies have increased the quality and longevity of life for patients. Many ailments, such as cancer, dia-
betes, tobacco addiction, and tissue infections, have been ameliorated or cured through the use of drug delivery technologies.
Currently, research and clinical trials are attempting to treat diseases once thought incurable with cutting-edge controlled release
systems. Future formulation strategies and materials used in the development of new controlled release systems will be at the fore-
front of disease treatment.
Further Reading
Agnihotri, J., Saraf, S., & Khale, A. (2011). Targeting: New potential carriers for targeted drug delivery system. International Journal of Pharmaceutical Sciences Review and
Research, 8(2), 117–123.
Bae, Y. H., & Park, K. (2011). Targeted drug delivery to tumors: Myths, reality and possibility. Journal of Controlled Release, 153(3), 198–205.
De Jong, W. H., & Borm, P. J. A. (2008). Drug delivery and nanoparticles: Applications and hazards. International Journal of Nanomedicine, 3(2), 133–149.
Eltorai, A. E. M., et al. (2016). Microchips in medicine: Current and future applications. BioMed Research International, 2016, 1743472.
Fang, J., Nakamura, H., & Maeda, H. (2001). The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the
effect. Advanced Drug Delivery Reviews, 63(3), 136–151.
Gujral, S., & Khatri, S. (2013). A review on basic concept of drug targeting and drug carrier system. International Journal of Advances in Pharmacy, Biology and Chemistry, 2(1),
130–136.
Heller, J. (1993). Polymers for controlled parenteral delivery of peptides and proteins. Advanced Drug Delivery Reviews, 10(2), 163–204.
Huang, X., & Brazel, C. S. (2001). On the importance and mechanisms of burst release in matrix-controlled drug delivery systems. Journal of Controlled Release, 73(2–3),
121–136.
Jain, S., Jain, V., & Mahajan, S. C. (2014). Lipid based vesicular drug delivery systems. Advances in Pharmaceutics, 2014, 574673.
Lian, T., & Ho, R. J. (2001). Trends and developments in liposome drug delivery systems. Journal of Pharmaceutical Sciences, 90(6), 667–680.
McBain, S. C., Yiu, H. H., & Dobson, J. (2008). Magnetic nanoparticles for gene and drug delivery. International Journal of Nanomedicine, 3(2), 169–180.
Middleton, J. C., & Tipton, A. J. (2000). Synthetic biodegradable polymers as orthopedic devices. Biomaterials, 21(23), 2335–2346.
Miller, R. A., Brady, J. M., & Cutright, D. E. (1977). Degradation rates of oral resorbable implants (polylactates and polyglycolates): Rate modification with changes in PLA/PGA
copolymer ratios. Journal of Biomedical Materials Research, 11(5), 711–719.
Rastogi, V., et al. (2014). Carbon nanotubes: An emerging drug carrier for targeting cancer cells. Journal of Drug Delivery, 2014, 670815.
Schmaljohann, D. (2006). Thermo- and pH-responsive polymers in drug delivery. Advanced Drug Delivery Reviews, 58(15), 1655–1670.
Tan, M. L., Choong, P. F. M., & Dass, C. R. (2010). Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery. Peptides, 31(1),
184–193.
Tan, M. L., Choong, P. F. M., & Dass, C. R. (2010). Recent developments in liposomes, microparticles and nanoparticles for protein and peptide drug delivery. Peptides (New York,
NY, United States), 31(1), 184–193.
Torchilin, V. P. (2001). Structure and design of polymeric surfactant-based drug delivery systems. Journal of Controlled Release, 73(2–3), 137–172.
Torchilin, V. P. (2005). Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery, 4(2), 145–160.
Tsung, J., & Burgess, D. J. (2012). Biodegradable polymers in drug delivery systems. In J. Siepmann, R. A. Siegel, & M. J. Rathbone (Eds.), Fundamentals and applications of
controlled release drug delivery (1st edn, pp. 107–123). New York: Springer US.
Uhrich, K. E., Cannizzaro, S. M., Langer, R. S., et al. (1999). Polymeric systems for controlled drug release. Chemical Reviews (Washington, DC), 99(11), 3181–3198.
Verron, E., et al. (2010). Calcium phosphate biomaterials as bone drug delivery systems: A review. Drug Discovery Today, 15(13), 547–552.
Wu, C., & Chang, J. (2012). Mesoporous bioactive glasses: Structure characteristics, drug/growth factor delivery and bone regeneration application. Interface Focus, 2(3),
292–306.
Electrospinning and Electrospray for Biomedical Applications
Qilong Zhao, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Introduction 331
Biomedical Requirements for Fibrous or Particulate Devices 331
Electrospinning 332
History, Principle, and Apparatus 332
Advanced Electrospinning Techniques 333
Materials for Electrospinning 334
Influencing Factors 335
Tissue Engineering Scaffolds and Applications 336
Fibrous Drug Delivery Systems and Applications 337
Biosensors 338
Other Biomedical Applications 339
Electrospray 340
Principle and Apparatus for Electrospray 340
Electrosprayed Solid Microparticles and Applications 341
Electrosprayed Core-Shell Structured Microparticles for Biomolecule Delivery 341
Electrosprayed Microparticles for Cell Delivery 342
Combining Electrospinning and Electrospray for Fabricating Novel Medical Devices 342
Acknowledgments 344
Further Reading 344
Glossary
Aptamer Oligonucleotide or peptide molecules binding to a specific target molecule.
Antibiotic A type of drug used for killing or inhibiting the growth of bacteria.
Collagen A main structural protein of extracellular matrix and connective tissues with a triple helix structure.
Extracellular matrix Nanofibrous networks formed by extracellular molecules secreted by cells that provide structural and
biochemical support to the surrounding cells.
Gelatin An irreversibly hydrolyzed form of collagen.
Glycosaminoglycan A series of long unbranched and highly polar polysaccharides consisting of a repeating disaccharide unit.
Progenitor cell A type of cell enabling limited self-renewal and having the capability to differentiate into a specific type of cell.
Stem cell A type of cell enabling self-renewal as also differentiation into specialized cell types.
Nomenclature
AgNP Silver nanoparticle
AuNP Gold nanoparticle
bFGF Basic fibroblast growth factor
BMP Bone morphogenetic protein
Ca–P Calcium phosphate
EDTA Ethylene diamine tetraacetic acid
EGF Epidermal growth factor
FDA Food and drug administration
GelMA Gelatin methacryloyl
HA Hyaluronic acid

Biomaterials: Biomaterial applications and advanced medical technologies j Electrospinning and Electrospray 331
IGF Insulin-like growth factor

NGF Nerve growth factor
PAN Polyacrylonitrile
PDGF Platelet-derived growth factor
PEO Poly(ethylene oxide)
PHBV Poly(hydroxybutyrate-co-hydroxyvalerate)
PLA Poly(lactic acid)
PLACL Poly(lactide-co-caprolactone)
PLGA Poly(lactic-co-glycolic acid)
PM2.5 Particulate matter 2.5 mm in size
PS Polystyrene
PU Polyurethane
PVA Poly(vinyl alcohol)
PVDF Poly(vinylidene fluoride)
PVP Poly(vinyl pyrrolidone)
RGD Arginine/glycine/aspartic acid
SEM Scanning electron microscope
SERS Surface-enhanced Raman scattering
SF Silk fibrin
TEM Transmission electron microscope
TGF Transforming growth factor
Introduction
Electrospinning and electrospray are facile electrohydrodynamic (EHD) techniques governed by similar principles, which use elec-
trostatic forces to overcome the surface tension of charged liquids. With the emergence of nanotechnology in the 1990s, electrospin-
ning and electrospray have become popular research topics in the academic world as well as in the R&D of fibrous and particulate
products for various applications. Electrospinning enables high-efficiency fabrication of ultrathin fibers (from nanofibers with
diameters of tens of nanometers to microfibers with diameters of hundreds of micrometers), while electrospray provides continuous
production of small-diameter particles (from nanoparticles with diameters of tens of nanometers to microparticles with diameters
of hundreds of micrometers).
In a typical electrospinning or electrospray process, an electrostatic force is applied to overcome the surface tension of a charged
liquid which normally comes out of a syringe with a metallic needle connected to a high-voltage power supply. The electrostatic force
stretches or breaks up the charged liquid to be viscoelastic filaments or jets, resulting finally in dry fibrous or particulate products after
the evaporation of solvent in the liquid (if the liquid is a polymer solution) during the journey towards a grounded fiber or particle
collector. A broad range of materials can be processed by electrospinning or electrospray. The morphology and structure (diameter,
surface morphology, interior structure, etc.) and properties of electrospun or electrosprayed products can be effectively controlled,
which makes electrospinning and electrospray very promising for a variety of applications, ranging from the energy field to biomed-
ical and healthcare applications. Owing to the ease of conducting electrospinning or electrospray and also the unique characteristics
of electrospun or electrosprayed products, electrospinning and electrospray techniques are widely investigated and used in the
biomedical field. Electrospun or electrosprayed products can be used for tissue engineering, drug delivery, wound dressing,
biosensor, filtration, mask, etc. (Fig. 1). Continuous investigations into electrospinning and electrospray will certainly assist the prog-
ress of biomedical research and generate improved and advanced biomedical devices for the rapidly growing healthcare market.
Biomedical Requirements for Fibrous or Particulate Devices
Biomedical devices need to possess appropriate properties and provide required functions for targeted applications. Fibrous and
particulate materials fabricated by electrospinning and electrospray have their respective advantages as biomedical devices. For
332 Biomaterials: Biomaterial applications and advanced medical technologies j Electrospinning and Electrospray
Fig. 1 Biomedical applications of fibrous and particulate products fabricated via electrospinning and electrospray, respectively.
tissue engineering applications, porous scaffolds resembling the structure and features of native ECM are advantageous for cell
attachment and proliferation. In spite of variations in composition and structure, ECMs are normally composed of intermeshed
nanofibers of proteins (collagen, elastin, etc.) and glycosaminoglycans with different fiber assemblies, offering structural and
biochemical support for cells of the tissue. Consequently, producing mechanically stable artificial scaffolds with nanofibrous
and porous structures is an important goal in the tissue engineering field. Although some other methods such as molecular self-
assembly and phase separation are studied for making nanofibrous tissue engineering scaffolds, many drawbacks, particularly
the low efficiency in scaffold manufacture, prevent them from being widely adopted in the tissue engineering field. As a simple,
versatile and effective technique for nanofiber production, electrospinning has shown its superiority in making nanofibrous and
porous tissue engineering scaffolds. Compared to the molecular self-assembly and phase separation techniques, electrospinning
is capable of processing a broader range of materials to nanofibers and requires only simple control or device for fiber alignment.
Therefore, a variety of nanofibrous scaffolds with different compositions, morphologies and architectures are fabricated via electro-
spinning for different tissue engineering applications.
Through electrospinning and electrospray, solidified fibrous and particulate materials with diameters down to nanometer range
can be directly formed from liquid precursors through a simple one-step procedure. Furthermore, electrospinning and electrospray
are amiable to incorporating bioactive agents in fibers and particles. These distinctive features make these two techniques highly
attractive for encapsulating bioagents (drug, growth factor, gene, etc.) for their controlled delivery. Good drug delivery vehicles
should have excellent capability for drug loading, provide controlling drug release and protect the bioactivity of delicate bioagent
(e.g., growth factor). Electrospinning and electrospray are capable of processing both water-phased solutions and oil-phased solu-
tions, resulting in highly efficient encapsulation of hydrophilic bioagents and hydrophobic bioagents, respectively. And the ease to
control the composition, morphology, and structure of electrospun and electrosprayed products through using different electro-
spinning and electrospray techniques makes it relatively easy to control the release of bioagents in specific spatiotemporal manners.
With appropriate modification, electrospinning and electrospray can achieve efficient biomolecular and even live mammalian cell
encapsulation in fibers and particles for their subsequent controlled release.
Fibrous and particulate biomaterials can also find other biomedical applications such as biosensor, filtration, and personal
healthcare products. Nanofibers and nanoparticles, due to their high surface-area-to-volume ratios and other useful properties,
are excellent for high-efficiency adsorption and enrichment of various species such as biomolecules to be detected, thereby possess-
ing high potential as biosensors with improved detection sensitivity and wide detection range. Electrospun nanofibrous meshes can
also be excellent materials for removing unwanted biomolecules as well as pollutants with good air and water permeation rates,
exhibiting their potential as high-efficiency, cost-effective filtration membranes and personal protective masks/surgical masks
with light weight, high flexibility, and good mechanical stability.
Electrospinning
History, Principle, and Apparatus
An early investigation conducted by Lord Rayleigh in 1882 revealed the amount of charges required for overcoming the surface
tension of a liquid drop. Inspired by this study, the devices which used electrostatic stretch to spray liquids into small droplets
were invented by Cooley and Morton in 1902–1903. They were the early explorations of the electrospray technique. Formhals
in 1934 applied for the first patent, demonstrating the process and apparatus of electrospinning for producing artificial threads.
However, electrospinning remained as a neglected area for a long time until the early 1990s when research on electrospinning
produced different polymer fibers with diameters down to nanometers, which revitalized this old technique. Since late 1990s,
studies on the manufacture of nanofibrous biomaterials by electrospinning for potential biomedical applications increased rapidly.
Over the past 10 years, the number of publications on electrospinning has been growing exponentially, reaching > 3000 in 2016,
with the majority of these publications reporting different biomedical applications of electrospun materials.
Electrospinning shows convenience, simplicity, and versatility for making nanofibers and nanofibrous products (non-woven,
aligned, stacked, etc.) through the use of a variety of materials. The simple principle and apparatus for electrospinning make it
easy to perform and control, offering the possibility to fabricate nanofibrous materials with high efficiency as well as versatility
to meet customers’ requirements. In a typical electrospinning process (Fig. 2), a high-voltage power supply, a syringe filled with
a precursor solution (normally a polymer solution) and equipped with a metallic needle, a syringe pump, and a ground collector
are used, where the metallic syringe needle is connected to the high-voltage power supply for generating an electrostatic field. Drop-
lets of the precursor solution flowing out the needle tip are firstly charged and then stabilized by the force between the applied elec-
trostatic force and the surface tension of the solution. The excess of the electrostatic force over the surface tension stretches and
deforms the charged droplets at the needle tip, forming a stable cone-shaped structure, namely, “Taylor cone”. A single and rapidly
whipping viscoelastic jet with decreased diameter and elongated length is generated from the tip of Taylor cone, which then splits
into many small jets in accordance with the charge repulsion effects after traveling a small distance. The small jets are further
stretched in the electrostatic field, finally resulting in dry fibrous products after the evaporation of the solvent (which has been
used to make the solution for electrospinning) in the trajectory of jets towards the collector. Non-woven mats can be formed
through such a simple electrospinning process. The ease manufacture of nanofibers offers electrospinning a bright perspective
for a broad range of biomedical applications such as tissue engineering, drug delivery, biosensor, wound dressing, etc. Electrospun
aligned fibers and other structures can be obtained using different (and specially designed) fiber collection devices.
Advanced Electrospinning Techniques

In addition to the ability and ease to form nanofibers, other significant advantages of electrospinning include high adaptability and
ease for modification. After some specific modification(s), advanced electrospinning techniques have been developed, which are
capable of making nanofibrous products to meet specific requirements of different biomedical applications.
Conventional electrospinning using a polymer solution normally results in solid (i.e., non-porous) nanofibers with smooth
surface or nanoporous surface (which is caused by the rapid evaporation of solvent in the polymer solution). While a stable
water-in-oil emulsion is electrospun, nanofibers with core-shell structures are fabricated. Electrospinning using an emulsion is
termed emulsion electrospinning. For biomedical applications, nanofibers made via emulsion electrospinning usually have
aqueous cores, which are suitable reservoirs for the encapsulation and protection of bioactive molecules. Therefore, emulsion elec-
trospinning has been intensively studied for making nanofibrous delivery vehicles for the controlled release of drugs, growth factors,
genes, etc. Another electrospinning technique for producing core-shell structured nanofibers is coaxial electrospinning, in which
a coaxial spinneret is employed with the inner capillary being fed with an aqueous solution (containing bioactive agent) and
the outer capillary being fed with a polymer solution. During emulsion electrospinning, two immiscible solutions are spun out
of the simple spinneret (commonly a metallic needle) and are both stretched by electrostatic force. The solution with a relatively
high conductivity (usually the inner aqueous solution) is the driving liquid to transfer the tangential electrostatic force through
Fig. 2 A schematic diagram showing the experimental setup for electrospinning.

viscosity on the liquid interface and to deform the merged droplets to become small jets with aqueous interior, finally resulting in
core-shell structured nanofibers upon sufficient stretch. Compared to emulsion electrospinning, coaxial electrospinning minimizes
the contact between the “water phase” and “oil phase”, providing enhanced protection for the bioactive molecules to be delivered,
whereas emulsion electrospinning displays jetting instability. With appropriate modification, modified coaxial electrospinning even
enables the encapsulation and delivery of live mammalian cells, which is termed cell electrospinning. In one study of cell electro-
spinning, the outer capillary and the inner capillary of a coaxial spinneret are fed, respectively, with a CaCl2 aqueous solution and
a mixture of an aqueous alginate sodium solution and an aqueous cell suspension. Cell-encapsulated hydrogel microfibers are
made after the crosslinking of alginate by Ca2 þ ions. As-spun cell-encapsulated hydrogel microfibers exhibit tunable fiber diameter
and good cell viability.
Fiber arrangement/pattern is a key topographical feature of electrospun scaffolds, which can be made/created using specially
designed devices or following physics laws/principles. Scaffolds made by conventional electrospinning normally show randomly
arranged nanofibers. Aligned nanofibers can be produced by using either a special fiber collector (e.g., a high-speed rotating
drum collector or a parallel electrode collector) or a directional magnetic field (which requires adding magnetic nanoparticles
into the electrospinning solution). Detailed studies on the effects of processing parameters of electrospinning on fiber alignment
when using a high-speed rotating collector indicate the major effects of rotating speed and polymer solution conductivity on the
alignment of nanofibers (Fig. 3). Electrospun scaffolds with aligned nanofibers resemble the ECM of some human tissues such
as cardiac tissue and ligament/tendon, which provide contact guidance on the cells grown on the scaffolds and give topographical
cues for directing the elongation and cytoskeleton development of cells. Electrospinning employing a point electrode and a ring
electrode as the collector can produce scaffolds with radically aligned nanofibers, which are potentially useful for wound closure
as the migration of skin cells can be promoted by these scaffolds.
Electrospun nanofibrous scaffolds normally have small interconnected pores, which is a shortcoming with regard to cell infil-
tration and vascularization. It has been determined that a scaffold with an average pore size of at least 20 mm will enable cell infil-
tration and the diameters of fibers need to be larger than 4 mm. However, the increase of fiber diameter normally results in
unsatisfactory cell attachment since the ECM-like nanofibrous architecture is changed. Electrospinning techniques for increasing
the pore size and porosity of electrospun nanofibrous scaffolds are thus investigated. Methods such as particulate-leaching-associ-
ated electrospinning, wet electrospinning (i.e., collecting electrospun fibers in a liquid bath, followed by a post-electrospinning
freeze-drying treatment), cryogenic electrospinning (i.e., using a freezing collector for fiber collection), and selective removal of
sacrificial electrospun fibers or electrosprayed microparticles are developed. For example, using the removal of sacrificial fiber
approach, removing water-soluble gelatin fibers from gelatin and PLGA bicomponent scaffolds fabricated by dual-source dual-
power electrospinning results in scaffolds with significantly enlarged micropores and well-preserved nanofibrous architecture
(Fig. 4). However, the increase of pore size and porosity by using these electrospinning techniques sometimes causes new problems
such as decreased structural stability and low mechanical properties of electrospun scaffolds.
Inspired by additive manufacturing, an advanced electrospinning technique that can produce fibrous scaffolds with accurately
deposited fibers and designed porous architecture in a programed manner is established, which is termed melt electrospinning. It
employs molten polymer for electrospinning and an automated stage as the collector. Melt electrospinning makes 3D scaffolds by
the programed stacking of fibers, which addresses the problems in conventional electrospinning such as the difficulty to form thick
scaffolds with large interconnected pores and well-defined fiber pattern. But the range of materials suitable for melt electrospinning
is limited and bioactive species such as cells and bioactive molecules that are susceptible to high temperature damages cannot be
incorporated into scaffolds during melt electrospinning.
Materials for Electrospinning

The range of materials suitable for electrospinning is wide, including synthetic polymers, natural polymers, ceramics, and compos-
ites. Among them, polymers are the most frequently used materials for electrospinning. Water-insoluble synthetic polymers such as
Fig. 3 An SEM image showing the morphology of electrospun aligned PHBV nanofibers formed on a high-speed rotating cylinder.
Fig. 4 Morphology of nanofibrous scaffolds fabricated by dual-source dual-power electrospinning: (A) bicomponent scaffold consisting of PLGA
fibers and gelatin fibers (1:1) and (B) PLGA scaffold with enlarged pore size after post-electrospinning water-immersion treatment.
PS, PAN, PVDF, and PU can be electrospun into fibers for different applications. Water-soluble synthetic polymers such as PVA, PVP,
and PEO can also be made into nanofibers via electrospinning, which can be nanofibrous carriers for bioactive agents or used as
template or sacrificial fibers to form fibrous products with complex structures. Biodegradable synthetic polymers, such as PCL,
PGA, PLA, and their copolymers (e.g., PLGA and PLACL), have different advantages in the biomedical field. Electrospun tissue engi-
neering scaffolds using these biodegradable polymers resemble the nanofibrous feature of native ECM and degrade through hydro-
lysis into nontoxic products those can be subsequently cleared by metabolic pathways with no or very limited side-effect on the
human body. However, the quick degradation of some of these polymers leads to local acidic microenvironment and provokes
inflammation. Scaffolds fabricated through the electrospinning of natural polymers such as chitosan, collagen, gelatin, and hyalur-
onic acid (HA) show improved biocompatibility for tissue engineering applications whereas some problems such as poor mechan-
ical properties exists. Unlike most of natural polymers, SF exhibits high mechanical strength, which can be directly used for some
applications requiring high mechanical loading or stability such as bone tissue engineering. However, making solutions by dissolv-
ing of SF for electrospinning is a challenge. Some modified natural polymers such as norbornene- or methacrylate-modified hya-
luronic acid and GelMA can be electrospun into nanofibrous hydrogel scaffolds with a post-electrospinning photo-crosslinking
treatment. Unlike normal electrospun hyaluronic acid or gelatin scaffolds with chemical crosslinking where the nanofibrous archi-
tecture of scaffolds is affected by chemical crosslinking, photo-crosslinked electrospun hyaluronic acid or GelMA hydrogel scaffolds
show well-preserved nanofibrous structure with good swelling behavior.
Ceramic fibers with diameters ranging from hundreds of nanometers to tens of micrometers can be fabricated via electrospin-
ning. For making ceramic fibers, either a ceramic particles-dispersed polymer solution with an appropriate ceramic particle concen-
tration and solution rheology or a suitable precursor enabling in situ chemical synthesis of ceramic by sol–gel, coprecipitation or
other methods is needed for electrospinning. When using a ceramic particles-dispersed polymer solution for electrospinning, a post-
electrospinning annealing treatment is required for the removal of polymer component and for the sintering of ceramic, resulting
finally in ceramic fibers. To date, a variety of fibrous ceramic products (BaTiO3, Fe3O4, hydroxyapatite, etc.) have been fabricated by
electrospinning for their potential applications in the energy, sensor, and healthcare fields.
Electrospinning can also produce composite fibers, including metal-polymer composites and ceramic–polymer composites,
when a metallic particle-dispersed polymer solution or a ceramic particles-dispersed polymer solution is used. For example, fibrous
silver nanoparticle-incorporated polymer scaffolds are electrospun, which have anti-bacterial property and are intended for tissue
engineering and wound dressing applications. Biodegradable scaffolds consisting of ceramic-polymer composite fibers that contain
bioceramic nanoparticles such as hydroxyapatite, calcium phosphate, etc. are made via blend electrospinning. They exhibit excellent
bioactivity and good mechanical properties for bone tissue engineering.
Influencing Factors
There are many factors influencing electrospinning and electrospun products, which can be categorized into three areas: polymer
solution properties, processing parameters, and environmental factors. Polymer solution properties include polymer molecular
weight, polymer solution concentration, viscosity of polymer solution, solution conductivity, solvent property, etc. The effects of
different polymer solution properties are sometimes difficult to be isolated and studied because some of these parameters are highly
related. For example, changing the conductivity of a polymer solution may also change its viscosity. Processing parameters in elec-
trospinning include solution feeding rate, applied voltage and field strength, nozzle configuration, etc. Environmental factors refer
to temperature, humidity, air velocity, etc. All of these factors have effects, small or large, on the electrospinning process and conse-
quently determine the morphology and structure of electrospun fibers.
For most polymers, a polymer with different average molecular weights that are within a reasonable range, as-spun fibers fabri-
cated from the polymer with a higher average molecular weight generally tend to form less beads on fibers and have a more uniform
fibrous structure. For example, the electrospun scaffolds made from chitosan with an average molecular weight of 30 kDa show
many beads, while the use of chitosan with an average molecular weight of 398 kDa results in uniform fibers. But not all polymers
follow this “law” on fiber morphology. For example, PEO fibers electrospun from PEO with different molecular weights exhibit little
difference between them. Solution viscosity (also polymer solution concentration, which is highly related) is a dominant polymer
solution property affecting the electrospinning process. In general, a very low viscosity of the polymer solution (which also means
very low polymer solution concentration) leads to a very high surface tension of the polymer solution, which cannot be overcome
by electrostatic force and hence no solidified fibrous products can be collected. With a gradual increase in viscosity of the polymer
solution (i.e., increasing the polymer solution concentration and decreasing the surface tension of the polymer solution), polymer
particles or beads start to form. When the viscosity of polymer solution further increases to be above a critical point, a stable electro-
spinning process is established, resulting in the production of fibrous products. For polymer solutions with a very high viscosity
(i.e., at a high polymer solution concentration), droplets of the polymer solution at the tip of the syringe needle are easy to dry
out before jets are initiated and hence electrospinning cannot be performed. A stable electrospinning process requires viscosity
(and polymer solution concentration) to be in an appropriate range. Within this range, the increase in viscosity (and polymer solu-
tion concentration) usually results in the increase of diameters of as-spun fibers. Conductivity is another key polymer solution prop-
erty influencing the electrospinning process. Usually, the increase of polymer solution conductivity or charge density leads to
relatively high mobility of ions and increasing whipping instability of polymer jets during electrospinning. Strong and homoge-
neous electrostatic stretch tends to occur and thin jets tend to form, thereby resulting in uniform fibers with relatively small diam-
eters. For example, the addition of a volatile salt, pyridium formiate, into a PLA solution can significant increase the conductivity of
the polymer solution and subsequently minimize the formation of beads on fibers. Likewise, the use of solvents with different
polarities, for example, alcohol and tetrachloromethane, can also modulate the conductivity of a polymer solution, resulting in
fibrous product with a uniform structure and that with concomitant beads, respectively. Solvent properties such as the boiling point
of the solvent determine the evaporation rate of solvents during electrospinning and can affect the morphology of electrospun
fibers, resulting in either smooth surface fibers or nanoporous surface fibers.
Applied voltage and electrical field strength are similar parameters, which indicate the strength of electrostatic force, are also
major processing parameters affecting the electrospinning process. During electrospinning, the applied voltage (field strength)
has significant effects on tuning the shape of Taylor cone. The increase in applied voltage (field strength) results in relatively small
Taylor cone and relatively high surface tension, which may increase the difficulty to initialize jetting and the frequency of bead
formation. However, the increase in applied voltage (field strength) enhances electrostatic stretch at the same time. Consequently,
the influence of the field strength on the diameter of electrospun fiber is not predictable. For polymers such as PLA, PLGA, and PVA,
high applied voltage (field strength) results in large diameter electrospun fibers, whereas the trend for other polymers such as cellu-
lose acetate is the opposite. As for electrospray, some electrosprayed microparticles (e.g., alginate microparticles) with the smallest
diameter can be formed in a medium range of applied voltage (field strength), while an increase and decrease in applied voltage
(field strength) out of this range causes an increase in diameter of as-sprayed microparticles. The polymer solution feeding rate
is another processing parameter that can affect electrospinning even though the influence may not be large. Usually, a relatively
low feeding rate results in the ease of solvent evaporation, thereby leading to small diameter electrospun fibers. In electrospray,
the diameter of as-sprayed microsphere and polymer solution feeding rate generally follows the same relationship.
The influences of environmental factors, including temperature and humidity, on electrospinning should not be ignored. Since
the increase in temperature may slightly decrease the viscosity of a polymer solution, relatively thin fibers are sometimes electro-
spun. For electrospinning performed at a sufficiently low temperature (e.g., on ice or into liquid nitrogen), which is termed cryo-
genic electrospinning, the evaporation of solvent can be significantly delayed, which induces solid-liquid phase separation and
hence the generation of tunable porous surface morphologies of as-spun fibers. The increase in humidity has similar effects on
the solvent evaporation and pore formation on the fiber surface. It also increases the difficulty to generate desirable uniform fibers.
Tissue Engineering Scaffolds and Applications

As a simple, effective, and versatile method to form ECM-like nanofibrous scaffolds, electrospinning has been extensively investi-
gated for making many types of tissue engineering scaffolds since late 1990s. These nanofibrous scaffolds have shown great poten-
tial and also usefulness for assisting the regeneration and reconstruction of different types of human body tissues and organs,
ranging from tissues such as bone, skin, and blood vessels to organs such as liver. Early research revealed that electrospun nano-
fibrous scaffolds of biodegradable polymers could closely mimic the hierarchical architecture of native ECM and facilitate good
attachment and proliferation of cells. Later, the potential of highly biocompatible electrospun scaffolds for engineering many types
of human body tissues with relatively simple structures such as bone, skin, and blood vessels was shown and is now generally real-
ized. With the assistance of electrospun nanofibrous scaffolds, the repair or regeneration of some avascular tissues such as cartilage
and tendon/ligament which have limited self-healing or self-renewal capability becomes reality.
In both in vitro and in vivo situations, electrospun tissue engineering scaffolds as artificial extracellular microenvironments can
offer specific structural, mechanical, and biochemical cues for anchoring cells. For example, electrospun scaffolds with specific
topography have significant influences on behaviors of attached cells, where cells response to the fiber orientation, cell morphogen-
esis, and cell migration are directed. Electrospun scaffolds of aligned nanofibers can induce the anisotropic elongation, spreading,
and arrangement of cells along to the fiber direction, showing their benefits for nerve, tendon/ligament, and smooth muscle tissue
engineering. Fiber diameter is also a key structural factor affecting cell behaviors on electrospun scaffolds. With decrease in fiber
diameter, electrospun scaffolds normally exhibit increasing surface-area-to-volume ratio, showing significant advantages in 3D
cell attachment, whereas cell attachment on microfibrous scaffolds is sometimes on single fibers that is more in a 2D manner, which
is different from the native cell-ECM organization. For engineering some tissues requiring the load-bearing ability such as bone,
cartilage, and tendon/ligament, suitable mechanical properties, particularly mechanical strength, are required for electrospun tissue
engineering scaffolds. The composite approach, with the incorporation of reinforcing component such as ceramic particles having
relatively high mechanical strength, is also used for fabricating mechanically reinforced tissue engineering scaffolds. The control
over fiber alignment is also effective for making electrospun scaffolds with anisotropic mechanical properties. The crucial point
for the mechanical properties of electrospun scaffolds is their compatibility with the target/host tissues. For some cells from soft
tissues, electrospun scaffolds with relatively low mechanical stiffness result in favorable cell attachment and cell spreading, whereas
scaffolds consisting of fibers with high rigidity lead to relatively poor cell response. Normally, topographical cues and mechanical
properties of electrospun scaffolds work together to contribute to the major impact on cell cytoskeleton development, where cells
can sense the physical and mechanical signals and respond by changing their adhesion and morphogenesis, subsequently exhibiting
specific phenotype and differentiation potential. Biochemical cues, such as surface chemistry, incorporation and delivery of bioac-
tive agents, etc., are also critical factors influencing the performance of electrospun tissue engineering scaffolds. Electrospun scaf-
folds with specific surface functionalization can enhance cell attachment by ways of hydrophobic interaction (e.g., having
specific chemical group terminals that promote the interaction with cell membrane), avidin–biotin covalent coupling, affinity
bonding (i.e., functionalized by specific ligands such as antibody or aptamer that can recognize their targets on cell membrane),
etc. For example, a commonly employed method for enhancing cell attachment is to functionalize electrospun scaffolds with cyclic
RGD sequences or to incorporate components containing RGD motifs (e.g., collagen and gelatin) since RGD can be recognized by
almost one-half of cell integrins to readily form stable RGD-integrin ligament-receptor pairs. Biochemical properties of electrospun
scaffolds also have influences on cell phenotype and differentiation, particularly for progenitor cells and stem cells. For example, the
incorporation of bioactive bioceramics (e.g., Ca–P including hydroxyapatite, TCP, and BCP) in scaffold fibers is effective for
enhancing the osteogenic properties of electrospun scaffolds in directing mesenchymal stem cell functions for bone tissue engi-
neering. Another direct but highly effective way for improving the biological activity of electrospun scaffolds is to incorporate
specific growth factor(s) in electrospun fibers via emulsion electrospinning or coaxial electrospinning, where growth factor mole-
cules are encapsulated in the aqueous core of core-shell structured fibers with well-preserved bioactivity for their later in vitro or
in vivo release in a specific spatiotemporal manner. Controlled local delivery of specific growth factor(s) (Table 1) has provided
significant benefits for a range of tissue engineering applications (e.g., bone, skin, blood vessel, cartilage, nerve, and tendon/liga-
ment). For mesenchymal stem cells, similar electrospun nanofibrous scaffolds encapsulated with different types of growth factors
(e.g., BMPs and NGF) are able to stimulate cell differentiation towards different cell lineages (e.g., osteogenic cells by BMP and
neuronal cells by NGF, respectively). In many cases, the delivery of multiple growth factors is required. For example, in blood vessel
tissue engineering, the sequential delivery of VEGF and PDGF from bilayer electrospun scaffolds consisting of different types of
biodegradable polymers with different degradation rates promotes the regeneration and remodeling of endothelial layer and
smooth muscle layers of blood vessel, respectively.
Currently, it is still a great challenge to construct complex electrospun scaffolds for the regeneration of complex human body
tissues/organs such as liver, kidney, gastrointestinal tract, cardiac tissue, etc. Good vascularization in newly formed tissue by regen-
erative medicine is essential for the survival of cells and successful tissue remodeling of engineered complex tissues. A promising
method of concurrent emulsion electrospinning and coaxial cell electrospray has been developed recently. Endothelial cells can
be placed inside the nanofibrous matrix of scaffolds that encapsulate VEGF and provide its controlled and sustained release, which
shows the potential to build 3D vascularized cell-laden constructs for engineering complex human body tissues.
Fibrous Drug Delivery Systems and Applications

Electrospun multifunctional nanofibrous scaffolds providing controlled release of growth factors for tissue engineering applications
have been presented in the last section. Electrospinning is indeed an excellent technique for producing fibrous delivery systems for
a variety of cargos, ranging from small molecule drugs, biomacromolecules, genes, and even live mammalian cells. Different strat-
egies, including adsorption and encapsulation, can be used to load different types of cargos into/onto electrospun fibers.
Table 1 Growth factors for use in tissue engineering
Growth factor Major functions Major applications
bFGF Promoting proliferation of many cell types Skin, musculoskeletal system

BMP-2 Stimulating chondrogenic and osteogenic differentiation of MSCs Bone, cartilage
BMP-7 Stimulating bone and cartilage maturation Bone, cartilage
EGF Promoting proliferation of mesenchymal, glial, and epithelial cells Skin
IGF Promoting proliferation of many cell types Cartilage, tendon/ligament
NGF Promoting neurite outgrowth and neural cell survival Nerve
PDGF Promoting proliferation of connective tissue, glial, and smooth muscle cells Musculoskeletal system
TGF-b Anti-inflammatory, promoting wound healing, and inhibiting macrophage and Cartilage, skin
lymphocyte proliferation
VEGF Regulating endothelial cell proliferation, angiogenesis, and vascular permeability Blood vessel
Owing to their high surface-area-to-volume ratio, electrospun nanofibers are naturally suitable for loading bioagents via adsorp-
tion. Bioactive agents such as small molecule drugs, biomolecules, and inorganic nanoparticles can be loaded onto electrospun
fibers through different adsorption strategies, including physical adsorption and chemical bonding. Chemical bonding usually
causes permanent immobilization of bioagents onto electrospun fibers, resulting in difficulties to release bioagents and sometimes
in the damages or dysfunctions of immobilized biomolecules arising from their conformational changes. Physical adsorption
through hydrogen bonding, hydrophobic/hydrophilic interactions or electrostatic interactions are popular for loading bioagents,
particularly biomolecules as their bioactivity is not obviously or significantly affected. However, using surface adsorption to load
bioagents has inherent problems in the amount of loaded bioagents (as compared to the encapsulation approach) and release
behavior of bioagents (again, as compared to the encapsulation approach), thereby limiting their scope of applications.
Blend electrospinning is a straightforward way to encapsulate bioagents in electrospun fibers as bioagents are directly suspended
in the polymer solution to form a blend solution for electrospinning and subsequently encapsulated in resultant fibers. Blend elec-
trospinning has been popularly for fabricating fibrous delivery vehicles for small molecule drugs (antibiotics, anti-inflammation
drugs, anti-cancer drugs, etc.). Inorganic nanoparticles can also be encapsulated in electrospun fibers through blend electrospinning.
For example, as shown earlier in this chapter, bone tissue engineering scaffolds of composite fibers containing homogeneously
dispersed bioceramic nanoparticles can be fabricated by blend electrospinning, which provide sustained release of Ca2 þ ions
and possess desirable osteogenic properties for promoting bone regeneration. AgNP-encapsulated fibrous scaffolds can also be
made via simple blend electrospinning, which possess anti-bacterial property with the release of silver ions and hence can be
used for wound treatment. Blend electrospinning requires the use of a polymer solution in which bioagents are dispersed. This
significantly restricts its application for delivering delicate biomolecules such as growth factors, enzymes, and genes. In order to
avoid the denaturation/degradation of biomolecules to be delivered, polymers requiring toxic organic solvents for making polymer
solutions cannot be used to form fibrous delivery vehicles. While water-soluble polymers such as gelatin, PVA, and PEO can be used
in blend electrospinning for forming fibers to deliver these biomolecules, specific post-electrospinning crosslinking treatment is
necessary for these polymer fibers for subsequent handing and applications, which may cause damages too to encapsulated biomol-
ecules. Another drawback of blend electrospinning is that encapsulated molecules tend to migrate to near the surface of fibers
during electrospinning due to the charge repulsion effect, leading to initial burst release of bioagents in the in vitro and in vivo
situations.
Emulsion electrospinning and coaxial electrospinning are two frequently techniques for creating fibers for biomolecule delivery,
employing a simple spinneret, and a coaxial spinneret, respectively, as both techniques can effectively minimize the contact between
biomolecules and toxic organic solvent in polymer solutions with the help of core-shell structured fibers. Despite the small differ-
ences in the manufacturing procedures and also the difference in core-shell structures of resultant fibers, emulsion electrospinning
and coaxial electrospinning produce biomolecule-encapsulated fibrous scaffolds which have similar biomolecule release profiles
that are governed by the same release mechanisms. These fibrous delivery vehicles normally exhibit two-stage release profiles,
that is, initially rapid release and subsequently sustained release, which are mainly controlled by biomolecule diffusion in the initial
stage and polymer degradation in the late stage. The initial rapid release is undesirable and increases the risk of cancer. Since biomol-
ecules (e.g., growth factors) usually carry a certain type of electrical charges (positive or negative) in the physiological environment,
strategies such as incorporating biodegradable polyelectrolytes bearing a specific type of electrical charges or emulsion electrospin-
ning using power suppliers with different polarities (positive applied voltage or negative allied voltage) have therefore been inves-
tigated for modulating the release of biomolecules through electrostatic interaction. These strategies can work well, effectively
modulating growth factor release to be steady and sustained under specific conditions and therefore providing appropriate stimuli
on cell functions (Fig. 5).
Coaxial electrospinning, as compared to emulsion electrospinning, involves less diffusion between two separated liquids (i.e.,
the water phase and oil phase) during electrospinning and therefore may cause less disturbance/damage to biomolecules. It has
been shown that coaxial electrospinning is feasible for producing non-viral fibrous delivery vehicles, providing successful gene
(e.g., plasmid DNA) delivery. With good protection on bioactive substances to be delivered, coaxial electrospinning can be even
be used for the encapsulation and delivery of live mammalian cells. This is demonstrated in a study where a sodium alginate solu-
tion containing suspended cells and a CaCl2 aqueous solution are fed respectively to the inner capillary and the outer capillary of
a coaxial spinneret for electrospinning, resulting in cell-encapsulated hydrogel microfibers.
Biosensors
The simplicity and versatility of electrospinning for fabricating nanofibrous meshes of a variety of materials make it attractive for
preparing biosensors. For example, electrospinning of a viscous solution containing PVA, glucose, prehydrolyzed tetramethyl ortho-
silicate and horseradish peroxide enzymes is conducted for fabricating nonwoven meshes consisting of enzyme-encapsulated silica
nanofibers. Nanofibers in the meshes have nanoporous surface that is caused by the glucose template, enabling sufficient contact
between the encapsulated enzymes and hydrogen peroxide. These nanofibrous meshes, as potential biosensors, provide effective
and reliable determination of hydrogen peroxide and simultaneously exhibit good mechanical flexibility, thermal stability, and
reusability. Electrospun nanofibrous meshes are also investigated as non-enzymatic amperometric biosensor for hydrogen peroxide
determination. In this study, polyurethane nanofibers filled with carbon nanotubes and AgNPs are fabricated via blend electrospin-
ning, resulting in hybrid nanofibrous meshes with enhanced electrocatalytic activity on hydrogen peroxide, which is a desirable elec-
trochemical sensor for hydrogen peroxide detection with an excellent sensitivity and a wide linear range. In another study, through
Fig. 5 Emulsion electrospun PLGA scaffolds fabricated at different applied voltages (positive or negative) for controlling VEGF release: (A) electrical
charge retention by different scaffolds, (B) in vitro release behavior of VEGF for scaffolds made at different applied voltages, (C) confocal microscopy
of vascular endothelial cells cultured on two types of scaffolds, showing the effect of polarity of applied voltage on VEGF release and consequently
cell behavior.
electrospinning of a PVP/SnCl2/AgNO3 solution which is followed by sintering, Ag/SnO2 composite nanotubes are formed. Meshes
consisting of these Ag/SnO2 composite nanotubes exhibit sensitive amperometric response to hydrogen peroxide with a wide linear
range and a low detection limit, making them promising electrochemical sensors for hydrogen peroxide. Electrospun fibrous
membranes are also investigated as biosensors for immunoassay. For example, electrospun PCL nanofibrous membranes are
able to effectively adsorb antibody proteins via hydrophobic interactions and then sufficiently capture target antigens for immuno-
assay. With the assistance of repeated fold-press procedures, immunoassay based on these free-standing electrospun membranes can
obtain significantly amplified fluorescence signals, leading to reliable detection of human serum albumin with a good linear range
and a low detection limit. Another example is an electrospun membrane of a special polymer with the anti-fouling property, which
shows clear advantages for immunoassay. This nanofibrous structure allows sufficient binding and immobilization of antibodies
and its anti-fouling property remarkably reduce the non-specific adsorption of proteins, which together contribute to the fast
and reliable detection of target proteins (25% shorter detection time as compared to conventional immunoassay methods, and
a wide linear detection range from) together with optimized signal-to-noise ratio. Electrospun membranes with specific function-
alization can be developed as free-standing and flexible SERS substrates for molecular detection with extremely high sensitivity. For
example, blend electrospinning of a PVA/AgNPs solution produces nanofibers with particular assembly and arrangement of AgNPs,
which can provide reliable detection of reporter molecules at a very low dosage by SERS.
Other Biomedical Applications

Electrospun fibrous structures are also investigated and developed for other applications, such as wound dressing and air/water
filtration. There are already some commercial products on the market. Electrospun meshes have been extensively investigated as
wound dressings since they can be effective barriers for inhibiting bacteria but allowing air and water permeation, thereby offering
a favorable environment for wound healing. In order to have the anti-infection property for electrospun wound dressings, various
strategies are explored. Polymers naturally having the anti-bacterial property such as chitosan and PVP are electrospun into wound
dressings which are capable of inhibiting bacteria/fungal adhesion to some extent. However, to achieve active antibacterial property
for electrospun wound dressings, anti-bacterial agents such as antibiotics and AgNPs are incorporated into fibers for their controlled
released later. Between the use of antibiotics and AgNPs, electrospun wound dressings with incorporated AgNPs can achieve more
durable anti-infection effect. Through electrospinning of an emulsion consisting of a silver ion-containing polymer solution and an
aqueous PVP solution, fibrous AgNP-incorporated polymer scaffolds with evenly distributed AgNPs in fibers are formed by the post-
electrospinning in situ reduction of silver ions by PVP (Fig. 6). Other considerations for wound dressings include rapid hemostasis
and accelerating wound closure, which can also be fulfilled by electrospun functional wound dressings. For example, electrospin-
ning of a medical adhesive, cyanoacrylate, results in wound dressings consisting of ultrathin cyanoacrylate nanofibers, which have
the ability to immediately stop aortic bleeding within dozens of seconds. In other studies, wound dressings fabricated by emulsion
electrospinning with sustained releases of growth factors facilitating specific cell migration and growth are effective in treating some
wounds that are difficult to heal by ordinary treatments as in the case of chronic wounds of diabetic people.
Electrospun nanofibrous membranes having relatively small interconnected pores are not good for cell infiltration but they are
suitable candidates as filtration membranes for water/air purification. Electrospun membranes can be improved by incorporating
active agents or be modified by other methods for enhancing the filtration of toxins/contaminants. For example, electrospun
membranes consisting of PS nanofibers coated by a mesoporous silica layer are made for water purification purpose. A frequently
employed method is to functionalize or to modify the nanofibers of electrospun filtration membranes with reactive groups such as
oximes, cyclodextrins, and chloramines that are able to bind and detoxify hazardous agents. In recent years, air pollution, partic-
ularly the problems arising from PM2.5 air pollutants, is critical public health issue in China and also in some other developing
countries. Electrospun polymer membranes (e.g., polyamide-6) with nano-sized pores have been proven effective as high-
efficiency air filters for PM2.5. Electrospun superhydrophilic polyacrylonitrile/silica nanofibrous membranes have a high moisture
vapor transmission rate and are excellent in PM2.5 capture even in a high humidity environment, exhibiting their potential for use in
personal protective masks.
Electrospray
Principle and Apparatus for Electrospray
Electrospray is an electrohydrodynamic technique similar to electrospinning. It is governed by similar principle and usually uses
identical apparatus, that is, a high-voltage power supply, a syringe filled with a precursor solution (normally a polymer solution)
and equipped with a metallic needle, a syringe pump controlling the feeding rate of the solution and a ground collector. During
electrospray, a stable Taylor cone is also formed, which is stabilized by the liquid surface tension, electrostatic force and gravity.
Electrostatic force acts as the major force against the surface tension of emitting droplets and deforms them to spherically shaped
jetting beads. Compared to electrospinning, the degree of electrostatic stretch over the surface tension is relatively low during
electrospray, leading to the formation of particulate products (nanoparticles or microparticles) instead of fibrous products.
Consequently, a polymer solution with a relatively low polymer concentration (and hence low viscosity) is normally used in
electrospray. But a very low viscosity of the solution will result in the whipping mode and also still liquid droplets when they
arrive in the collector, whereas a relatively high viscosity of the solution will lead to intermittent formation of fibers. Polymer
Fig. 6 A TEM image of the fiber internal structure of an AgNP-incorporated emulsion electrospun polymer scaffolds, showing well-dispersed AgNPs
in the fiber.
Fig. 7 A schematic diagram showing the experimental setup for simple electrospray (left) and coaxial electrospray (right).
solution concentration (and viscosity) needs to be in a specific range for electrospray and for determining the geometry of electro-
sprayed products.
Electrospray can be categorized into two modes, that is, simple electrospray and coaxial electrospray, with the use of different
types of spinneret (either a simple spinneret or a coaxial spinneret) (Fig. 7). Coaxial electrospray is more complicated because two
immiscible liquids are involved. In the coaxial electrospray process, two immiscible liquids (usually one “oil phase” and one “water
phase”) are merged out of spinneret to form conically shaped cone-jet under the excess force between the tangential electrostatic
force and solution surface tension. Owing to different charge densities of the two immiscible liquids, different tangential electro-
static forces are exerted on them, where the liquid with relatively short electrical relaxation time (usually the water phase) bears the
high tangential electrostatic force and therefore acts as the major driving phase deforming the compound jets during coaxial electro-
spray. Coaxial electrospray with the inner driving liquid are favorable for attaining the stable cone-jet mode, resulting in uniform
core-shell structured particles with aqueous cores. But coaxial electrospray is not the only method for fabricating core-shell struc-
tured particles. By using a water-in-oil emulsion in simple electrospray, core-shell structured microparticles with multiple cores
can also be produced. Due to its simplicity and high versatility in fabricating microparticles with tunable structures, electrospray
has great potential for drug delivery applications.
Electrosprayed Solid Microparticles and Applications

Simple electrospray of a single-phase polymer solution usually results in non-porous (“solid” or “monolithic”) nanoparticles or
microparticles. Due to the high surface-area-to-volume ratio of electrosprayed solid particles and the relatively low shear stress
exerted during electrospray as compared to electrospinning, electrospray has been extensively investigated for fabricating carriers
for drug delivery, as well as other bioactive agents. Bioagents can be loaded in/on electrosprayed solid particles through either
encapsulation or adsorption. For achieving efficient and high loading of bioagents by adsorption, electrosprayed nanoparticles
are preferred owing to their very high surface-area-to-volume ratio. Bioagents, usually small molecule drugs, can be loaded onto
the surface of nanoparticles through weak interactions (hydrogen bonding, electrostatic interaction, etc.) or layer-by-layer assembly.
Regardless of adsorption mechanisms for loaded drugs, relatively low loaded amount and low loading efficiency are inherent prob-
lems, and drugs adsorbed on nanoparticles are easily to desorb, which normally provides release profiles with an initial burst
release. Using the encapsulation approach, bioagents are firstly dissolved or dispersed in a polymer solution for electrospray, which
either directly make bioagent-incorporated solid nanoparticles/microparticles or initially produce bioagent-containing droplets that
are subsequently gelled/solidified by crosslinking in a particle collection bath. Both routes can achieve high drug loading amount
and high encapsulation efficiency, for example, nearly 100% encapsulation efficiency for doxorubicin by electrosprayed PLGA
microparticles. Drug-incorporated electrosprayed nanoparticles/microparticles meet different therapeutic requirements, for
example, antibiotic-releasing electrosprayed PLGA microparticles for anti-infection purpose and paclitaxel-releasing electrosprayed
PLGA microparticles for cancer therapy. However, the simple electrospray method for incorporating drugs also has shortcomings.
Difficulties occur for dispersing some drugs in particulate carriers, which arise from the difference in the hydrophilicity/hydropho-
bicity between the drug and the carrier polymer. Another issue is the direct contact between delicate bioagents (e.g., growth factors)
and the organic solvent in the polymer solution, which causes damages to biomolecules.
Electrosprayed Core-Shell Structured Microparticles for Biomolecule Delivery

Electrosprayed core-shell structured microparticles are very useful for the delivery of bioactive agents such as growth factors and
genes. The fabrication of electrosprayed core-shell structured microparticles involves bi-phased liquids, one being the water phase
and the other the oil phase. The bioagent is contained in the water phase (which is not necessarily water; it can be an aqueous solu-
tion) and the polymer solution acts as the oil phase. Such structural arrangement provides good protection for bioagents and a high
retention level (approaching 70%) for their bioactivity. Another advantage of using core-shell structured microparticles as delivery
vehicles is the release profiles of biomolecules can be effectively tailored. Compared to the normally diffusion-controlled release
kinetics of bioagents from solid particles, the release kinetics of bioagents from core-shell structured microparticles are more compli-
cated, which are determined by the diffusion of encapsulated bioagents, swelling, and degradation of the carrier polymer, and core-
shell structure of the microparticles. For example, both emulsion electrospray and coaxial electrospray can produce core-shell
Fig. 8 PLGA microparticles fabricated by emulsion electrospray and coaxial electrospray, respectively: (A) TEM images showing the internal struc-
ture of emulsion electrosprayed (left) and coaxial electrosprayed (right) microparticles, (B) in vitro VEGF release profiles for different microparticles.
structured PLGA microparticles, showing multi-compartmented cores and one single core, respectively (Fig. 8a). Consequently, the
release characteristics of biomolecules (e.g., VEGF) from these two types of microparticles are different (Fig. 8b), meeting the
requirement for different therapeutic purposes.
Electrosprayed Microparticles for Cell Delivery

The research to deliver live cells using electrosprayed microparticles for therapeutic purposes emerges in recent years. Cell delivery
methods must meet stringent requirements for biocompatible materials and cell-friendly fabrication processes. An early study indi-
cates the suitability of electrospray for cell encapsulation and delivery. In this study, rabbit aorta smooth muscle cells are successfully
encapsulated in electrosprayed PEO microdroplets, achieving a high cell survival rate (70%). PDMS solution and aqueous solutions
of ECM protein that have suitable viscosity are also used to make particulate carriers for mammalian cells. Using cell electrospray
techniques, the encapsulation and delivery of many types of cells, including matured somatic cell isolated from different tissues
(heart, skin, blood vessels, etc.) and stem cells, are investigated. Results show excellent cell viability and no significant changes
for stem cell functions. A recent investigation studies a new cell electrospray technique that enables cell encapsulation in solid
hydrogel microparticles. Sodium alginate is used as the carrier polymer which is crosslinked post-electrospray by Ca2 þ ions for
generating cell-encapsulated hydrogel microparticles (Fig. 9). Cells encapsulated in electrosprayed hydrogel microparticles exhibit
high viability (> 90%), particularly for core-shell structured hydrogel microparticles fabricated by coaxial cell electrospray. Impor-
tantly, these electrosprayed cell-encapsulated microparticles have sufficient mechanical integrity, providing both the ease for
handling and enhanced protection for the cells even under subsequent harsh device fabrication processes. Cells released from
the electrosprayed hydrogel microparticles also exhibit high cell viability.
Combining Electrospinning and Electrospray for Fabricating Novel Medical Devices

Electrospinning and electrospray have respective advantages in different biomedical areas. An integration strategy to combine elec-
trospinning and electrospray can lead to formation of novel medical devices for different applications. Concurrent electrospinning
Fig. 9 A microscopic image of cell-encapsulated alginate hydrogel microparticles made by cell electrospray: cells are labeled by using a Live/Dead
cell staining kit (live cell: green; dead cell: red).
and electrospray is thus investigated for fabricating drug- or biomolecule-releasing scaffolds for tissue engineering. Although electro-
spun scaffolds alone with the capability to deliver specific drugs or biomolecules can be made and used, nanofibrous scaffolds
embedded with homogeneously distributed electrosprayed microparticles can have high drug or biomolecule loading and hence
provide enhanced therapeutic effects.
To solve the cell infiltration problem in conventional electrospun tissue engineering scaffolds, concurrent electrospinning and
electrospray can be used to fabricate nanofibrous scaffolds embedded with cell-encapsulated biodegradable microspheres, resulting
in cell-laden scaffolds that resemble the native cell-ECM organization. Early studies on the concurrent fabrication method use direct
electrospray of cell suspensions, which incurs several problems for cells. A new concurrent emulsion electrospinning and coaxial cell
electrospray technique has now been then developed. It firstly place structurally stabled electrosprayed cell-encapsulated hydrogel
microparticles into the nanofibrous polymer scaffolds. The cells are subsequently released in the scaffolds through selective disrup-
tion of the hydrogel microparticles, which forms 3D cell-scaffold constructs with well-preserved cell viability. The nanofibrous scaf-
folds in the meantime can be emulsion electronspun scaffolds containing chosen growth factor. Such advanced scaffolds effectively
stimulate cell functions by providing both biomimetic structural and mechanical cues of the scaffolds and biochemical signals with
the sustained release of specific growth factor from the scaffolds.
Concurrent electrospinning and electrospray can be used to create novel medical devices. For example, novel tissue engineering
scaffolds embedded with theranostics are investigated for post-surgery cancer patients. Theranostics are multifunctional nanodevi-
ces that simultaneously provide diagnostic and therapeutic functions for cancer. They are now intensively investigated as future
medical technologies. Current treatment for cancer includes surgery, chemotherapy, and radiotherapy. After surgical removal of
the tumor, cancer patients face tissue dysfunction and cancer recurrence. Advanced multifunctional medical devices for promotion
tissue regeneration and for detecting and treating recurrent cancer are therefore developed. In a recent investigation, AuNP-based
theranostics can be encapsulated in coaxial electrosprayed core-shell structured polymer microspheres for their controlled release.
Through concurrent electrospinning and electrospray, the AuNP theranostic-encapsulated microspheres are embedded in nanofi-
brous PLLA scaffolds. Controlling the biodegradation of microspheres enables controlled release of theranostics in the scaffolds,
making the scaffolds multifunctional for cancer patients. While the nanofibrous scaffolds assist local tissue regeneration, the
released AuNP-based theranostics detect and kill cancer cells. Such a comprehensive treatment holds great promise for postoperative
cancer patients.
Concluding Remarks
The simplicity and versatility of electrospinning and electrospray for respectively producing fibrous products and particulate prod-
ucts for a variety of biomaterials make these techniques highly attractive in the biomedical field. With advances in electrospinning
and electrospray techniques, fibrous and particulate products possessing well-defined composition, size, morphology, and structure
can be made, leading to numerous promising medical devices with distinctive but controllable characteristics and properties for
meeting the requirements of different biomedical applications. Electrospun scaffolds with different nanofiber assemblies
mimicking the features of native ECM of different human body tissues and with high surface-area-to-volume ratios have been exten-
sively investigated, demonstrating their great potential and effectiveness in tissue engineering, drug delivery, biosensor and many
other applications. Electrosprayed nanoparticles and microparticles have also been investigated as delivery vehicles for the
controlled release of drugs, biomolecules, genes, and even live cells. Through integrating electrospinning with electrospray, novel
multifunctional medical devices can be made, such as novel tissue engineering scaffolds embedded with theranostics for post-
surgery cancer patients. Even though great successes have been made by developing and using electrospinning and electrospray
in the biomedical field, the potential of these techniques has not been fully realized and there are still many issues that need to
be dealt with for currently electrospun or electrosprayed products. A future development of electrospinning and electrospray
may go in the direction of combining them with other manufacturing technologies for fabricating better medical devices and novel
medical devices for millions of patients.
Acknowledgments
Min Wang thanks The University of Hong Kong, Hong Kong Research Grants Council and the National Natural Science Foundation of China and
Qilong Zhao thanks China Postdoctoral Science Foundation, Guangdong Innovative and Entrepreneurial Research Team Program and the Funda-
mental Research Program of Shenzhen for funding their research in the biomaterials and tissue engineering field.
Further Reading
Bosworth, L., & Downes, S. (2016). Electrospinning for tissue regeneration (1st ed.). Cambridge: Woodhead Publishing.
Brown, T. D., Dalton, P. D., & Hutmacher, D. W. (2011). Direct writing by way of melt electrospinning. Advanced Materials, 23(47), 5651–5657.
Chakraborty, S., Liao, I. C., Adler, A., & Leong, K. W. (2009). Electrohydrodynamics: A facile technique to fabricate drug delivery systems. Advanced Drug Delivery Reviews, 61(12),
1043–1054.
Cui, W. G., Zhou, Y., & Chang, J. (2010). Electrospun nanofibrous materials for tissue engineering and drug delivery. Science and Technology of Advanced Materials, 11(1),
014108.
Greiner, A., & Wendorff, J. H. (2007). Electrospinning: A fascinating method for the preparation of ultrathin fibers. Angewandte Chemie International Edition, 46(30), 5670–5703.
Jiang, T., E. Carbone, J., Lo, K. W. H. and Laurencin, C. T. (2015). Electrospinning of polymer nanofibers for tissue regeneration. Progress in Polymer Science, 46: 1–24.
Khorshidi, S., Solouk, A., Mirzadeh, H., Mazinani, S., Lagaron, J. M., Sharifi, S., & Ramakrishna, S. (2016). A review of key challenges of electrospun scaffolds for tissue-
engineering applications. Journal of Tissue Engineering and Regenerative Medicine, 10(9), 715–738.
Liu, W., Thomopoulos, S., & Xia, Y. (2012). Electrospun nanofibers for regenerative medicine. Advanced Healthcare Materials, 1(1), 10–25.
Macagnano, A., Zampetti, E., & Kny, E. (2015). Electrospinning for high performance sensors (1st ed.). Switzerland: Springer International Publishing.
Mitchell, G. R. (2015). Electrospinning: Principles, practice and possibilities (1st ed.). Cambridge: Royal Society of Chemistry.
Peng, S., Jin, G., Li, L., Li, K., Srinivasan, M., Ramakrishna, S., & Chen, J. (2016). Multi-functional electrospun nanofibres for advances in tissue regeneration, energy conversion &
storage, and water treatment. Chemical Society Reviews, 45(5), 1225–1241.
Pham, Q. P., Sharma, U., & Mikos, A. G. (2006). Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Engineering, 12(5), 1197–1211.
Pisignano, D. (2013). Polymer nanofibers: Building blocks for nanotechnology (1st ed.). Cambridge: Royal Society of Chemistry.
Rnjak-Kovacina, J., & Weiss, A. S. (2011). Increasing the pore size of electrospun scaffolds. Tissue Engineering Part B Reviews, 17(5), 365–372.
Sun, B., Long, Y. Z., Zhang, H. D., Li, M. M., Duvail, J. L., Jiang, X. Y., & Yin, H. L. (2014). Advances in three-dimensional nanofibrous macrostructures via electrospinning.
Progress in Polymer Science, 39(5), 862–890.
Szentivanyi, A., Chakradeo, T., Zernetsch, H., & Glasmacher, B. (2011). Electrospun cellular microenvironments: Understanding controlled release and scaffold structure. Advanced
Drug Delivery Reviews, 63(4–5), 209–220.
Uyar, T., & Kny, E. (2017). Electrospun materials for tissue engineering and biomedical applications (1st ed.). Cambridge: Woodhead Publishing.
Wang, C., & Wang, M. (2014). Electrospun multifunctional tissue engineering scaffolds. Frontiers of Materials Science, 8(1), 3–19.
Wendorff, J. H., Agarwal, S., and Greiner, A. (2012). Electrospinning: Materials, processing, and applications. (1st ed.). Chichester: Wiley.
Zhao, Q. L., & Wang, M. (2017). Smart multifunctional tissue engineering scaffolds. In Q. Wang (Ed.), Smart materials for tissue engineering: Applications (pp. 558–595).
Cambridge: Royal Society of Chemistry.
Zhong, S., Zhang, Y., & Lim, C. T. (2012). Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: A review. Tissue Engineering Part B Reviews, 18(2),
77–87.
Gene Delivery and Clinical Applications
Mahboob Morshed, Independent University, Dhaka, Bangladesh
Ezharul Hoque Chowdhury, Monash University, Clayton, VIC, Australia
Introduction 345
Transport Vehicles for Genetic Materials 346
Adenoviral Vectors 346
Adeno-Associated Viral Vectors 346
Retroviral Vectors 346
Nonviral Vectors 346
Strategies for long-term and tissue-specific expression of nonviral DNA 347
Lipid-based DNA 347
Polymeric DNA vectors 347
Determinants of polyplex stability 347
Determinants of stability of lipid-based complexes 347
Passive targeting and endothelial escape 347
Active targeting and uptake by target cells 348
Endosomal escape 348
Nuclear targeting 348
Gene Therapy Applications 348
Gene Therapy of Hereditary Diseases 349
X-linked severe combined immune deficiency 349
Hemophilia B 349
X-linked adrenoleukodystrophy 349
b-thalassemia 349
Gene Therapy for Cardiovascular Diseases 349
Gene Therapy for Neurodegenerative Diseases 349
Gene Therapy for Human Immunodeficiency Virus 349
Gene Therapy for Viral Hepatitis 350
Gene Therapy for Cancer 350
p53 gene 350
TK gene 350
TNF-related apoptosis-inducing ligand gene 350
p21 gene 351
Proinflammatory cytokine genes 351
Angiotensin gene 351
References 351
Introduction
Gene therapy is reversing of pathological processes through introduction of protein-coding or noncoding nucleic acids (DNA or
RNA) in order to provide the missing protein function(s), correct the abnormal function(s), or silence the overexpression of
protein(s). A gene is transcribed within the nucleus of a cell into an mRNA, which is subsequently transported to cytoplasm and
translated into a protein or polypeptide. Cell functions are predominantly carried out by proteins and as a result mutation in a single
or multiple genes could lead to suppression or overexpression of protein(s) with consequential perturbation of cellular functions.
Many human diseases are caused by germline mutation(s), giving rise to inherited (genetic) diseases, or somatic mutations, causing
acquired diseases, such as cancer. Introducing a functional gene ex vivo which involves extraction and culture of a specific cell type
from a patient, genetic modification of the cultured cells and finally, transfer of the genetically altered cells into the same patient,
and in vivo which means administration of the gene into the patent via a suitable route, such as intravenous or intramuscular or
intranasal route, is a powerful approach to treat various critical human diseases at the genetic level, with many preclinical and clin-
ical studies currently undergoing. The ultimate purpose of gene delivery into the target cells is to provide a new function or restore
the missing function or suppress an unwanted function, by expressing new protein(s), or silencing harmful protein(s). Like
a plasmid carrying a protein-coding gene(s), a short hairpin RNA (shRNA), which provides long-term effects on gene-silencing
compared to a small interfering RNAs (siRNAs), is transcribed as an RNA transcript in the nucleus from plasmid DNA and subse-
quently transported to the cytoplasm. The exogenous nucleic acid or functional gene is either inserted into the genome of viral

346 Biomaterials: Biomaterial applications and advanced medical technologies j Gene Delivery and Clinical Applications
particles (viral vectors) or in the plasmid DNA used as a naked form or in association with a synthetic or semisynthetic particles
(nonviral vectors). Long-term gene expression enables to avoid the repeated administration of gene therapeutics, while short-
term expression is considered relatively safer.
Transport Vehicles for Genetic Materials
Since nucleic acids are vulnerable to degradation and unable to be transported across the cell membrane to reach the desirable site of
target cells, they often require nanocarriers for being safely carried, overcoming the extracellular and intracellular barriers. Despite
possessing advantages and disadvantages, both viral and nonviral vectors have been extensively explored as nucleic acid carriers in
many preclinical and clinical trials of gene therapy.
Adenoviral Vectors
Employed in approximately 25% of all gene therapy trials, adenoviral vector is the most clinically used vector that can efficiently
transduce both dividing and nondividing cells. Following cellular uptake usually promoted by interaction between the virus fiber
“knob” and cellular coxsackievirus–adenovirus receptor, and subsequent nuclear translocation, the viral genome remains episomal,
replicating in synchrony with the host genome. As a result, when the vector carrying a therapeutic protein-coding gene was trans-
ferred into relatively quiescent tissues, such as the brain, liver, or muscle, the therapeutic protein, such as coagulation factors, a1
antitrypsin, or erythropoietin, was stably produced throughout the lifetime of a mouse (Roth et al., 1996). Since the vector can
induce severe toxicity owing to an immediate innate immune response and a secondary antigen-dependent response, the second-
and third-generation adenoviral vectors have been produced with less toxicity than the first-generation vectors, by deleting addi-
tional viral genes. The majority of gene therapy trials (> 400) carried out or undergoing with human adenoviral vectors aims at
treating various cancers. Among the large number of different transgenes incorporated into the adenoviral genome for clinical trials,
cystic fibrosis transmembrane conductance regulator (CFTR), E. coli cytosine deaminase, granulocyte macrophage colony stimu-
lating factor (GM-CSF), human immunodeficiency virus (HIV) proteins (gag, pol, nef), human IL-2, human IL-12, human inter-
feron (IFN), human fibroblast growth factor 4 (FGF-4), heat shock protein 70 (HSP 70), hepatitis C virus (HCV) nonstructural
proteins, ornithine transcarbamylase, p53, thymidine kinase (TK), tumor necrosis factor (TNF), prostate-specific antigen, malaria
circumsporozoite protein, Her-2, and vascular endothelial growth factor (VEGF) are worth mentioning (Wold and Toth, 2013).
Adeno-Associated Viral Vectors

Adeno-associated virus (AAV), one of the most attractive gene therapy vectors, usually interacts with heparan sulfate proteoglycans
on cell membrane as the first step for cellular internalization. Like an adenovirus, it also induces strong immune responses, infects
both dividing and nondividing cells, and remains episomal without being integrated into host chromosome. The short genome size
(4 kb) of the virus poses a limitation in carrying a long foreign gene. In clinical trials for familial lipoprotein lipase (LPL) deficiency,
intramuscular injection of an AAV1 vector encoding the gain-of-function LPLS447X variant led to persistent gene expression, with
consequentially sustained decrease in the incidence of pancreatitis. Considering the efficacy as well as the safety profile, AAV1-
LPLS447X vector received marketing approval in 2012 in the European Union as the first approved gene therapy product in Western
nations under the name “alipogene tiparvovec” (Glybera). In another trial, administration of an AAV1 vector with ATP2A2 gene
encoding sarcoplasmic/endoplasmic reticulum calcium ATPase 2 was found to improve the key outcomes in the patients with
advanced heart failure (Kotterman and Schaffer, 2014).
Retroviral Vectors
Only lentiviruses among the retroviruses are capable of replicating in nondividing cells, making them attractive for transducing
human cells comprising both dividing and nondividing cells. In addition, as a member of retroviruses, they can stably integrate
their genomes into the host chromosome, which although enables sustained gene expression, simultaneously increases the risk
of proto-oncogene activation and cancer development. Genetic engineering enables production of targetable lentiviral vectors
through fusion of a ligand protein or antibody to viral glycoproteins. In order to promote gene expression in target cells, in addition
to ensuring the specific uptake via the receptor–ligand engagement, lentiviral vectors with a tissue-specific promoter can be used to
target specific cell type. Lentiviral vectors made defective for recombination with host chromosome can promote short-term trans-
gene expression in nondividing cells while reducing insertional mutagenesis. These vectors were used in preclinical animal studies
for correction of hemophilia B, b-thalassaemia, Parkinson’s disease (PD), cystic fibrosis, sickle cell anemia, and spinal muscular
atrophy.
Nonviral Vectors
Despite being highly carcinogenic and immunogenic, in 70% of gene therapy clinical trials viral vectors have been used. On the
other hand, although much inefficient compared to the viral counterpart, nonviral vectors are currently being sought considering
Biomaterials: Biomaterial applications and advanced medical technologies j Gene Delivery and Clinical Applications 347
their increased safety profile and capacity to deliver larger genetic material. Nonviral physical methods including gene gun, electro-
poration, hydrodynamic delivery, sonoporation, and magnetofection are generally less applicable to systemic delivery and there-
fore, a range of synthetic or semisynthetic nonviral vectors have been developed.
Strategies for long-term and tissue-specific expression of nonviral DNA

Following nuclear transport, plasmids that are routinely used as nonviral expression vectors carrying the gene(s) of interest remain
episomal, thus reducing the risk of insertional mutagenesis. To control the intensity and the duration of transgene expression, viral
enhancers and promoters derived from cytomegalovirus (CMV), respiratory syncytial virus, and simian virus 40 (SV40), and consti-
tutive mammalian promoters, such as the human ubiquitin C and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1),
are frequently used as the regulatory sequences of the therapeutic genes. Additionally, there are numerous cis-acting sequences
including various polyadenylation signals introns and scaffold/matrix attachment regions (S/MARs) with ability to increase the
level and the duration of transgene expression. DNA size and topology can also influence the gene expression level with the small
covalently closed circular plasmid DNA causing greater levels of transgene expression than the large or linearized plasmids. A variety
of transposition systems, such as recombinases phiC, PiggyBac, and Sleeping Beauty, can be included to prolong the gene expression
by integrating the transgene into the chromosome. Finally, usage of tissue-specific promoters or enhancers, such as the alpha-
fetoprotein (AFP) enhancer or albumin (ALB) promoter, which regulates gene expression exclusively in the liver, can minimize
the unwanted transgene expression in nontarget tissues, and thus increase the overall delivery efficacy and reduce the off-target
effects (Yin et al., 2014).
Lipid-based DNA
Cationic liposomes are the most commonly used nonviral vectors for plasmid DNA delivery. Various cholesterol or PEG-modified
cationic liposomal formulations were tested clinically, such as DOTAP–cholesterol for delivery of fus1 tumor suppressor gene and
GL67A–DOPE–DMPE–PEG for delivery of CFTR (pGM169) gene in patients with non-small-cell lung cancer and cystic fibrosis,
respectively (Yin et al., 2014).
Polymeric DNA vectors

Cationic polymers constitute another attractive class of nonviral DNA vectors partly owing to their chemical diversity and potential
for surface functionalization. PEI and its variants having a high charge density at reduced pH values are effective in condensation
and endosomal escape of DNA, leading to efficient intracellular transgene delivery. The transgene delivery efficacy and cytotoxicity
of PEI strongly depend on its molecular weight and structural complexity (linear vs. branched forms). A range of modifications were
undertaken for better performance of PEI; for example, block co-polymers of PEG and PEI for improved stability and biocompat-
ibility, degradable disulfide-crosslinked PEIs for reduced toxicity, and alkylated PEI for increased potency (Yin et al., 2014). Intra-
venous injection of PEI–DNA polyplexes was shown to enhance transgene delivery into the lungs of mice. PEI was studied for local
gene therapy of various cancers, including bladder, ovarian and pancreatic cancers, multiple myeloma, B cell lymphoma, and
pancreatic ductal adenocarcinoma in humans. A PEG–PEI–cholesterol lipopolymer carrying interleukin-12 (IL-12) plasmid is
under clinical investigation for immunotherapy of ovarian and colorectal cancers.
Determinants of polyplex stability

Cationic polymers are commonly used to complex with nucleic acids having negatively charges phosphate backbone, forming poly-
plexes, the stability of which depends on the ratio of positive to negative charges (N/P). For example, linear PEI/plasmid complexes
were less stable than branched PEI/plasmid complexes (Madani et al., 2011). In order for systemic delivery, polyplex nanomicelles
can be formed through self-assembly of amphiphilic block copolymers carrying a cationic polymer segment which forms the core by
condensing with nucleic acid, and a hydrophilic polymer chain, such as PEG on the surface facing the aqueous solution to prevent
destabilization of the resultant complexes by hindering nonspecific interactions with blood components, such as nuclease and
opsonins.
Determinants of stability of lipid-based complexes

The stability of lipoplexes, which are formed by ionic interactions between cationic lipids and anionic nucleic acids, usually depends
on the ionic strength under which the lipoplexes are prepared. Thus, a lipoplex formulated under low ionic strength solutions will
be destabilized releasing the nucleic acid payloads, after being exposed to physiological saline and serum. Although the existence of
PEG on the surface of lipoplexes confers a steric barrier at the lipoplex surface, blocking a variety of interactions with molecular and
cellular components in blood and tissues, the ultimate transfection activities of the surface-modified lipoplexes could be hampered.
The stability of lipo-polyplexes which are formed by addition of liposomes to preformed polyplexes is mainly determined by the
outer liposome shell independent of pDNA and cationic polymer in the polyplexes, whereas the stability of stable nucleic-acid-lipid
particle, which consists of a lipid bilayer including a mixture of cationic and neutral lipids (cholesterol and fusogenic lipids) and
a PEG-lipid coating, is mainly dependent on the outer liposomal shell.
Passive targeting and endothelial escape

Due to the enhanced permeability and retention effect (EPR), nanoparticles with appropriate size could escape the tumor blood
capillaries composed underdeveloped, leaky endothelium and be retained in the tumor tissues for days and even weeks due to
the lack of lymphatic drainage. In addition, acting as vasodilators, nitric oxide (NO), prostaglandins, and bradykinin were reported
to enhance the EPR effect in the tumor by increasing its vascular permeability. In fact, the surface properties and diameter of protein
corona-decorated particles have notable influence on the outcome of passive targeting. Particles circulating in blood should be
larger than 40 kDa with half-lives to be sufficiently high so as to exert the EPR effect. In addition, particles with high positive charges
can bind nonspecifically to the luminal surface which is negatively charged due to the presence of sulfated and carboxylate sugar
moieties, resulting in rapid clearance from the circulation.
Active targeting and uptake by target cells

Covalent or noncovalent attachment of a targeting ligand on the surface of nanoparticles enables them to recognize the specific
antigens or receptors on target cells which subsequently engulf the particles through endocytosis. The targetability and efficiency
of the uptake are governed by the interactions between the nanoparticle surface groups and the plasma membrane antigens/recep-
tors, which in turn depend on density of the ligands and the antigens/receptors present on a nanoparticle and a cell, respectively. The
diverse moieties investigated so far as targeting ligands include carbohydrates (e.g., galactose), monoclonal antibodies (e.g., anti-
Her2, anti-EGFR), peptides (e.g., Arg-Gly-Asp or RGD), proteins (e.g., lectins, transferrin), vitamins (e.g., vitamin D), and aptamers
(e.g., RNA aptamers against HIV glycoprotein).
Endosomal escape
The superior transgene expression efficacy of viral vectors over the nonviral vectors is partly due to the ability of viral proteins to
facilitate the endosomal escape, following cellular uptake of the viral particle. Thus, in case of adenoviruses, acidic pH of endo-
some induces conformational changes in viral capsid, leading to endosomal membrane lysis. There are two major strategies
undertaken for nonviral vectors to facilitate the endosomal escape: inclusion of fusogenic peptides within the vectors as
a virus-mimicking strategy to induce endosomal low pH-induced conformation change of the peptide, leading to fusion
with the endosomal membrane; and implementation of “proton sponge” effect with PEI, for instance, by neutralizing the endo-
somal acidic pH with its excess uncharged amines, eventually bringing in chloride into endosomes, causing osmotic swelling
and finally, rupturing the endosomal membrane. In addition, in case of cationic liposomes, the interactions between positive
charges of the liposomes and anionic phospholipids of the endosomal membrane cause some of the anionic lipid to displace in
a “flip-flop” mechanism and diffuse into the lipoplex, forming a charge neutral ion pair with the cationic lipids and resulting in
release of the liposomal contents into the cytosol. The inclusion of neutral helper lipids as a constituent of liposome can accel-
erate the fusion of lipid layers by switching from a lamellar to a hexagonal phase as the pH of endosome drops. The presence of
PEG in the lipoplex surface could impair the phase transition process, ending up with an incomplete endosomal escape
(Chowdhury, 2016).
Nuclear targeting
Most eukaryotic DNA viruses and some RNA viruses can gain access to the nucleus of their host cells via nuclear membrane pore
(NMP) having an internal channel diameter of 25 nm. Intact virions with a diameter smaller than that of the NMP can either
translocate through the NMP by recruiting appropriate nuclear import receptors, or undergo conformational changes to allow their
outer surface to interact with channel components, thus resulting in the release of their genome in the host nucleus. However,
virions significantly larger than the maximum diameter of the NMP are required to partially disassemble or uncoat themselves either
in the cytosol or after docking at the NMP before transferring their genomes into the nucleus. Since NMP permits passive transfer of
linear DNA fragments up to 300 bp, although dividing cells (e.g., cancer cells) permit entry of plasmid DNA into the nucleus once
the nuclear membrane is disrupted during mitosis, nondividing cells do not allow trafficking of molecules larger than 40–45 kDa
through NMP unless they possess nuclear localization signals (NLSs) (Chowdhury, 2009). Nanoparticles could be modified with
the NLSs originally derived from the Simian virus 40 large T antigen to enter the nucleus following cellular uptake. However, even
though a nanocarrier with bound DNA can successfully reach the nucleus, the DNA must be released from the carrier for undergoing
transcription in the nucleus. The plasmid that reaches the cytosol after cellular uptake and release from both the endosome and the
carrier will be prone to nuclease-mediated degradation during its waiting for the breakdown of nuclear membrane in case of
dividing cells (Chowdhury, 2009). For nuclear translocation of a plasmid DNA, the DNA can be covalently linked to a NLS peptide,
or specific DNA sequences, such as a 366-bp DNA containing the SV40 origin of replication and early promoter can be incorporated
into the DNA by recombinant DNA technology, prior to the complexation of the DNA with a nanocarrier that ensures the cytosolic
release of the DNA cargo (Chowdhury, 2009).
Gene Therapy Applications
For treating both genetic and acquired human diseases, plasmid DNA carrying gene(s) of interest can be administered either in vivo
via different routes, or ex vivo by genetically modifying the autologous cells derived from a patient and reintroducing them in the
patient’s body. Ex vivo gene therapy was particularly attempted for the monogenic diseases of blood cells, such as sickle cell anemia
or b-thalassemia.
Gene Therapy of Hereditary Diseases

X-linked severe combined immune deficiency
X-linked severe combined immune deficiency (X-SCID), a combined cellular and humoral immunodeficiency caused by mutations
in interleukin 2 receptor, gamma (IL2RG) gene with almost complete absence of T and natural killer lymphocytes, is fatal in the first
2 years of life without reconstitution of the immune system via bone marrow transplantation or gene therapy. Although autologous
transplantation of retrovirally transduced bone marrow cells expressing the IL2RG gene resulted in a functional immune system in
the children with X-SCID, 5 out of the 20 patients subsequently developed leukemia as a result of integration of the retroviral DNA
into the patient’s genome and the eventual activation of the LIM domain only 2 proto-oncogene in those hematopoietic cell-derived
cells (Chowdhury, 2016).
Hemophilia B
A mutation in factor IX gene causes a deficiency in factor IX clotting factor, consequentially prolonging bleeding after an injury in
patients with hemophilia B. Although administration of a single-dose of AAV2 vectors carrying the factor IX gene in order for its
liver-based delivery and expression, led to a long-term correction of hemophilia B in mice and dogs, preexisting antibodies against
the capsid of the AAV2 serotype, or a CD8þ T cell response to the capsid led to short-term expression of factor IX with poor ther-
apeutic outcome in a patient with hemophilia. However, peripheral infusion of an AAV8 vector expressing a codon-optimized
human factor IX transgene led to long-term expression of factor IX, thus improving the bleeding phenotype in patients with severe
hemophilia B (Chowdhury, 2016).
X-linked adrenoleukodystrophy
Mutations in the ABCD1 gene encoding peroxisomal membrane protein, ALDP X-linked adrenoleukodystrophy (X-ALD gene
product) disrupt the transmembrane transport of very long-chain fatty acids and thereby cause a severe lipid storage disorder
with brain demyelination in patients with X-ALD. Lentiviral vector-mediated transfer of ABCD1 gene in the autologous hematopoi-
etic stem cells (HSCs) from the patents with X-ALD showed the outcome apparently comparable to the allogeneic hematopoietic
cell transplantation (Chowdhury, 2016).
b-thalassemia
Mutations in beta-globin chains result in the reduced production of hemoglobin in patients with b-thalassemia. Transfer of the
b-globin gene with the help of a lentiviral vector into the HSCs of an 18-year-old patient of b-thalassemia led to 10% of the patient’s
blood cells having the normal hemoglobin, due to the integration of the b-globin gene without having any insertional mutagenesis
(Chowdhury 2016).
Gene Therapy for Cardiovascular Diseases

Renin–angiotensin system (RAS) plays a pivotal role in development of atherosclerosis and hypertension, leading to congestive
heart failure, myocardial infarction, and kidney damage. In RAS, angiotensinogen (AGT) acts as the precursor in liver for synthesis
of angiotensin II which increases blood pressure (BP), promotes progression of atherosclerosis, generates a larger amount of super-
oxides and reactive oxygen species, and impairs endothelium-dependent dilation by reducing NO production. Silencing of AGT
mRNA transcript by nanoparticle-facilitated delivery of specific shRNA was shown to markedly reduce expression of AGT and
Ang II in rats, resulting in the decline in BP with the atherosclerotic lesions markedly attenuated. Furthermore, an effective lowering
of high BP and attenuation of the pathophysiology were observed in different experimental models of hypertension by overexpress-
ing vasodilators including atrial natriuretic peptide, endothelial nitric oxide synthase, kallikrein, and adrenomedullin (Chowdhury,
2016).
Gene Therapy for Neurodegenerative Diseases

Progressive loss of neurons and gradual appearance of disable neurological symptoms characterize the neurodegenerative diseases
that predominantly include Huntington’s disease (HD), Alzheimer’s disease (AD), and PD. Silencing of mutant human huntingtin
in the striatum and cerebellum of HD mice with a specific shRNA carried by an AAV caused a significant pathological and behavioral
improvement as well as a reduction in the size and number of neuronal inclusions. On the other hand, lentiviral delivery of shRNA
targeting BACE1 in a transgenic mouse model of AD significantly reduced Ab production, amyloid plaques, and neuronal death,
with improved learning and memory. Knockdown of a-synuclein, a principle protein component of Lewy bodies, has been
proposed as a potential therapeutic option for the treatment of PD (Chowdhury, 2016).
Gene Therapy for Human Immunodeficiency Virus

HIV decreases and weakens the immune system, paving the way for opportunistic infections and development of acquired immu-
nodeficiency syndrome, by binding to and destroying CD4 þ T lymphocytes. Targeting the cellular components involved in the viral
infection process, such as CD4 (the primary receptor) by which HIV enters the cells, could be a promising strategy. The first clinical
trial (NCT00569985; 04047) was based on a lentiviral vector (rHIV7-shI-TAR-CCR5RZ) that was used to transduce autologous,
CD34 þ hematopoietic progenitor cells, expressing a shRNA targeted to an exon of the HIV-1 genes tat/rev (shI) to destroy the viral
mRNA, a decoy for the HIV TAT-activated RNA (TAR) to antagonize the viral transactivation, and a ribozyme targeting the host T cell
CCR5 cytokine receptor (CCR5RZ) (a coreceptor) to block viral entry (Deng et al., 2014).
Gene Therapy for Viral Hepatitis

HCV infection is a major cause of chronic liver disease. Single-stranded HCV genome could be an attractive target for potential
RNAi-based therapeutics. Specifically designed shRNAs (or siRNAs) can be delivered by either viral or nonviral vectors to target
the 50 UTR and 30 UTR, the most conserved regions of the HCV RNA with significant functional importance in the viral life cycle,
NS3 helicase which unwinds the viral genomic RNA during replication, NS3 serine protease which contributes to HCV polyprotein
maturation, and NS5A, a pleiotropic protein having key roles in both viral RNA replication and modulation of the host cell’s phys-
iology (Motavaf et al., 2012).
Gene Therapy for Cancer

Cancer, a leading cause of mortality worldwide, is caused by uncontrolled cell division owing to mutational and epigenetic changes
that lead to overexpression or suppression of cellular genes. Metastasis of cancer cells happens due to the downregulation of cell
adhesion receptors, upregulation of cell motility-enhancing receptors and activation of membrane metalloproteases. Gene therapy
for cancer could be classified into three major categories: tumor suppressor gene replacement therapy, immune gene therapy, and
enzyme- or prodrug-based therapy. On the other hand, shRNAs (and siRNAs) have been extensively used to silence antiapoptotic
genes, such as bcl-2, proangiogenic growth factor genes, such as fibroblast growth factor (bFGF), VEGF, growth factor receptor genes,
such as epidermal growth factor receptor (EGFR), estrogen receptor, human epidermal growth factor receptor-2 (HER2), and VEGFR
receptor, multidrug transporter genes, such as MDR1 and MRP1 genes in many preclinical studies, to inhibit tumor cell growth,
angiogenesis, metastasis, and chemo-resistance.
p53 gene
p53 belongs to the class of tumor suppressors, having crucial roles in preventing tumor development by ensuring cell cycle progres-
sion, DNA repair, and induction of apoptosis against cellular stress and damage, and is therefore known as “guardian of genome.” In
addition, p53 plays pivotal roles in enhancing the therapeutic effects of antiangiogenesis therapy, chemotherapy, and radiotherapy.
Systemic delivery of liposome-p53 DNA complex was reported to reduce the size of the primary tumors and prevent the relapse and
metastases of a malignant human breast cancer in nude mice. In addition, intratumoral injection of biodegradable poly(b-amino
ester) polymer-carrying p53 gene in xenograft model of SCLC resulted in significant inhibition of tumor growth. GendicineÔ is
a p53 adenovirus approved for clinical use in China for the treatment of head and neck squamous cell cancer in combination
with radiotherapy (Bakhtiar et al., 2014).
TK gene
Herpes simplex virus thymidine kinase (HSVtk), the most commonly used TK gene for cancer gene therapy, was successfully deliv-
ered individually as well as in combination with ganciclovir (GCV) (a prodrug) in vivo. Enhanced antitumor efficacy of HSV-1tk/
GCV prodrug system was observed in baby hamster kidney (BHK) tumor grown as xenografts in severe combined immunodefi-
ciency disease (SCID) mice, following delivery with sindbis viral vector. Conversion of prodrug GCV into the active form by the
expressed HSVtk promoted the bystander effects responsible for killing surrounding untransducted tumor cells. In a similar
approach, repeated delivery of HSVtk gene using hemagglutinating virus of Japan (HVJ)-liposomes in the induced liver tumors, fol-
lowed by GCV treatment significantly inhibited the tumor growth and markedly improving the survival of animals. In clinical trials,
intratumoral injection of adenovirus-associated HSV-tk gene and subsequent intravenous administration of GCV led to an overall
mean survival of 70.6 weeks without significant safety issues, compared to 39.0 weeks for the standard therapy group in glioblas-
toma multiforme (Bakhtiar et al., 2014).
TNF-related apoptosis-inducing ligand gene

TNF-related apoptosis-inducing ligand (TRAIL), a cytokine belonging to the TNF superfamily, binds to its receptors, R1 and R2,
forming the death-inducing signaling complex which in turn directs cleavage and activation of caspases for inducing apoptosis.
TRAIL is considered to be a promising gene therapeutic in cancer treatment considering its specificity in inducing apoptosis and
toxicity in cancer cells. A nonviral vector, PEI600-Cyd, prepared by linking low-molecular-weight polyethylenimine (PEI) with
b-cyclodextrin (b-CD) was used to introduce the TRIAL gene to mesenchymal stem cells (MSCs) which was later utilized as a deliv-
ering agent in vivo to significantly reduce the tumor size. In another study, a low-molecular-weight PEI10 k modified with myristic
acid (MC) was complexed with TRAIL gene to form MC-PE10 k/DNA nanoparticles which were subsequently used to deliver the
TRAIL gene to the brain, with an improvement in median survival time (Bakhtiar et al., 2014). TRAIL gene-carrying MSCs
(MSCsTRAIL) were delivered along with 5-flourouracil to xenografts of HCT116 colon cancer cells, resulting in significant tumor
regression compared to the single-agent treatment (Bakhtiar et al., 2014). The combined delivery of doxorubicin and pTRAIL
gene using PEI-CD (cyclodextrin) was demonstrated to be efficient with higher retention time for drugs, achieving good therapeutic
effects in inhibiting the tumor growth with significantly prolonged survival of tumor-bearing mice (Fan et al., 2012). c(RGDyK)–
poly(ethylene glycol)–polyethyleneimine (RGD–PEG–PEI) nanoparticles were used in a similar study for the codelivery of TRAIL
gene and paclitaxel, producing better antiglioblastoma effects in vivo by overcoming blood–brain barrier and blood–tumor barrier
(Zhan et al., 2012).
p21 gene
A vital regulator of cell cycle progression, p21, can block cell-cycle progression from the G1 to S phase through inactivation of cyclin/
CDK activity. Retroviral vector-mediated delivery of p21 gene was shown to reduce breast tumor growth in vivo by reducing expres-
sion of cyclins D1 and E and Cdks 2, 4, and 6 (Bakhtiar et al., 2014).
Proinflammatory cytokine genes

Survival and sustainable growth of a tumor is supported by lack of expression of recognizable tumor antigens, inability of the
expressed tumor antigens to adequately stimulate the immune system or downregulation of the immune response by the tumor
itself. Tumor expression of proinflammatory cytokine(s), such as GM-CSF or fms-like tyrosine kinase 3 receptor ligand (Flt3L)
and subsequent immunization with the tumor lysate containing tumor-associated antigens, could dramatically increase the number
of potent antigen-presenting dendritic cells (DCs) in blood. For example, vaccination with the CT26 colon carcinoma cell lysate and
the Flt3L-encoding adenoviral vector (pAdFlt3L) injected subcutaneously prevented the tumor growth in a BALB/c mouse model
through significant expansion of DCs (Riediger et al., 2013).
Angiotensin gene
An octapeptide hormone, angiotensin II, plays a key role in the rennin–angiotensin system by binding with angiotensin II type 1 or
type 2 receptors. Angiotensin II type 2 (AT2R) is known to inhibit cell proliferation and stimulate apoptosis. A marked reduction in
tumor growth was observed following bolus administration of HIV-1 TAT peptide-based nanoparticles with encapsulated AT2R or
TNF-related apoptosis-inducing ligand (TRAIL) pDNA. A single intratumoral injection of defective adenovirus expressing a secreta-
ble angiostatin K3 molecule from the CMV promoter (AdK3) into rat C6 glioma or human MDA-MB-231 breast carcinoma (estab-
lished in athymic mice) resulted in a significant arrest of tumor growth with suppression of neovascularization in the tumors
(Bakhtiar et al., 2014).
In addition, adenoviral delivery of the genes for selective interleukin (IL), such as IL-2 and IL-12, interferon (IFN), such as IFN-a,
IFN-b, and IFNg, and Fas ligand was shown to exert potent antitumor effects in different tumor models.
References
Bakhtiar, A., Sayyad, M., Rosli, R., Maruyama, A., & Chowdhury, E. H. (2014). Intracellular delivery of potential therapeutic genes: Prospects in cancer gene therapy. Current Gene
Therapy, 14(4), 247–257.
Chowdhury, E. H. (2009). Nuclear targeting of viral and non-viral DNA. Expert Opinion on Drug Delivery, 6(7), 697–703.
Chowdhury, E. H. (2016). Nanotherapeutics: From laboratory to clinic. Boca Raton, FL: CRC Press.
Deng, Y., Wang, C. C., Choy, K. W., et al. (2014). Therapeutic potentials of gene silencing by RNA interference: Principles, challenges, and new strategies. Gene, 538(2), 217–227.
Fan, H., Hu, Q. D., Xu, F. J., et al. (2012). In vivo treatment of tumors using host-guest conjugated nanoparticles functionalized with doxorubicin and therapeutic gene pTRAIL.
Kotterman, M. A., & Schaffer, D. V. (2014). Engineering adeno-associated viruses for clinical gene therapy. Nature Reviews. Genetics, 15(7), 445–451.
Madani, S. Y., Naderi, N., Dissanayake, O., Tan, A., & Seifalian, A. M. (2011). A new era of cancer treatment: Carbon nanotubes as drug delivery tools. International Journal of
Nanomedicine, 6, 2963–2979.
Motavaf, M., Safari, S., & Alavian, S. M. (2012). Therapeutic potential of RNA interference: A new molecular approach to antiviral treatment for hepatitis C. Journal of Viral Hepatitis,
19(11), 757–765.
Riediger, C., Wingender, G., Knolle, P., et al. (2013). Fms-like tyrosine kinase 3 receptor ligand (Flt3L)-based vaccination administered with an adenoviral vector prevents tumor
growth of colorectal cancer in a BALB/c mouse model. Journal of Cancer Research and Clinical Oncology, 139(12), 2097–2110.
Roth, J. A., Nguyen, D., Lawrence, D. D., et al. (1996). Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nature Medicine, 2(9), 985–991.
Wold, W. S., & Toth, K. (2013). Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Current Gene Therapy, 13(6), 421–433.
Yin, H., Kanasty, R. L., Eltoukhy, A. A., et al. (2014). Non-viral vectors for gene-based therapy. Nature Reviews. Genetics, 15(8), 541–555.
Zhan, C., Wei, X., Qian, J., et al. (2012). Co-delivery of TRAIL gene enhances the anti-glioblastoma effect of paclitaxel in vitro and in vivo. Journal of Controlled Release, 160,
630–636.
Materials for Exoskeletal Orthotic and Prosthetic Systems
Man Sang Wong, Babak Hassan Beygi, and Yu Zheng, The Hong Kong Polytechnic University of Hong Kong, Hung Hom, Hong
Kong
Background of Orthotic and Prosthetic Systems 352

Ankle-Foot Assembly 355
Shank 355
Socket and Socket Interface 355
Suspension 355
Knee Units 356
Upper Limb Prostheses 356
Materials 357
Casting Materials and Fabrication Methods 357
Gypsum plaster or plaster of paris (POP) 357
Fiber glass bandage 358
Foam box impression 358
Alginate 358
Leather 358
Textile Fibers and Fabrics 358
Natural Fibers 358
Synthetic Fibers 358
Metals 359
Steel 359
Aluminum 359
Titanium 359
Wood 359
Plastics 360
Elastomers 364
Adhesives 365
Surface Finishing 365
3D Printing 365
Summary and Possible Future Direction 366
Further Reading 366
List of Relevant Websites 367
Glossary
Cranial remodeling orthosis An orthosis prescribed to correct the asymmetry of the skull in infants.
Ionomer A copolymer made by bonding of electrically neutral components and the ionized units.
Maxillofacial prostheses Any prosthesis fabricated to replace the missing structure of face such as eye, ear and nose due to
congenital or acquired impairment.
Polycaprolactone A biodegradable polyester with a low melting temperature around 60 C.
Polymer Any large molecule, natural or synthetic which is composed of repeated subunits.
Polyolefins Any group of polymers produced from a simple Olefin (alkene) with the formula of CnH2n as subunit.
Background of Orthotic and Prosthetic Systems
Orthopedic technology targets all devices applied externally to the user’s body to substitute a missing body part (exoprosthesis) or
to replace a missing function (orthosis). For clarification, the discussion on endoprosthesis, an implanted internal prosthesis is
beyond the topic of this article and the term prosthesis in following pages will stand for exoprostheses.
In general, the goal of the appliance is to improve the user’s mobility and ability to perform daily functional activities. Orthotics
and prosthetics (O&P) refer to the science and the field of knowledge that deals with orthoses and prostheses design and

Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Materials for Exoskeletal Orthotic 353
application. The orthotist is a person who designs and fabricates the orthotic devices. Similarly, a prosthetist’s profession is related
to the manufacturing and fitting the artificial limb as well as the education of an amputee, a person with missing limb to restore his
or her functional abilities. Unique expertise of orthotists and prosthetists in patient assessment, design, and materials offers patients
an increased level of mobility and independence.
Exoskeletons, also called exoskeletal suits are wearable frameworks alongside of human limbs which are the integrated products
of orthotics/prosthetics and robotics. Exosuits are powered by a system of electronic servomotors, pneumatics, levers, hydraulics,
and sensors that allow the limb to extend, enhance or even substitute the human function, the functions that might be helpful
for disabled people (Fig. 1).
Orthotic prescription is developed to achieve specific goals by applying biomechanical principles through orthotic design and
material and component selection. Orthotic goals are classified into four groups:
B To protect a joint
B To assist a joint movement
B To stabilize a joint by stopping or limiting motion
B To help manage a skeletal deformity by preventing, supporting or correcting of abnormality (e.g., scoliosis orthoses, cranial
remodeling orthoses)
The application of word orthosis is preferred to phrases like brace and splint, although they might be used interchangeably. A stan-
dardized nomenclature including an acronym composed of the first letter of the joints encompassed within the orthosis is used for
naming and easier communication (Figs. 2–4).
The general classifications of orthoses and prostheses have been summarized in Tables 1 and 2, respectively.
Orthotic devices can be classified according to the type of materials used as rigid, semi rigid or soft in prefabricated or custom-
made designs in a variety of procedures. Previously, orthoses were fabricated from metal bands and bars, called conventional
Fig. 1 Application of exoskeleton in disabled people.

354 Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Materials for Exoskeletal Orthotic
Fig. 2 Spinal orthoses made from plastics, metals and foamed plastics.
Fig. 3 AFOs made from plastics, composites with or without hinges.
Fig. 4 Combination of HKAFOs and upper structures such as TLSOs.

Table 1 General classification of orthoses
Lower limb orthoses Upper limb orthoses Spinal orthoses
FO (foot orthosis) HO (hand orthosis) SO (sacral orthosis)

AFO (ankle foot orthosis) WHO (wrist hand orthosis) LSO (lumbo sacral orthosis)
KAFO (knee ankle foot orthosis) EWHO (elbow wrist hand orthosis) TLSO (thoraco lumbo sacral orthosis)
HKAFO (hip knee ankle foot orthosis) SEWHO (shoulder elbow wrist hand orthosis) CTLSO (cervico thoraco lumbo sacral orthosis)
KO (knee orthosis) EO (elbow orthosis) CO (cervical orthosis)
HO (hip orthosis) SO (shoulder orthosis)
Table 2 General classification of amputation levels and corresponding prostheses
Lower extremity Upper extremity
Hip disarticulation Shoulder disarticulation

Transfemoral (above knee) Transhumeral (above elbow)
Knee disarticulation Elbow disarticulation
Transtibial (below knee) Trans radial (below elbow)
Ankle disarticulation Wrist disarticulation
Partial foot Partial hand
orthoses; However with introduction of new materials, the majority of orthoses were replaced by plastics/composites or a combi-
nation of both metal and plastic/composite materials as it can be represented in current prosthetic designs.
Any lower extremity prosthesis includes a socket with a liner or interface, a method of suspension, and a foot. Transfemoral pros-
theses also incorporate a knee mechanism.
Materials application in prosthetic technology is subdivided into main following domains of lower limb and upper limb
components:
Ankle-Foot Assembly
The distal foundation of the lower limb prosthesis is the foot-ankle assembly. All prosthetic feet should provide a stable base of
support for the amputee and absorb the shocks imposed during the walking.
Shank
The linking component located between the foot-ankle assembly and the distal portion of the socket is the shank segment. Shank
transmits the wearer’s weight from the superstructure to the foot.
The type of prosthesis is referred to as either an endoskeletal or an exoskeletal; Exoskeletal types include those prostheses in
which a rigid shank characterizes the structure; On the other hand, most prostheses, today use an endoskeletal type or modular
system with a central tube structure (pylon) that provides the slight adjustment of prosthesis alignment.
Modular pylons are covered with resilient cosmetic foam that enhances the appearance of the prosthesis similar to sound-side
limb.
Exoskeletal shanks are made of rigid expandable foam which is laminated externally to match the wearer’s contralateral leg.
Socket and Socket Interface

The socket is the part that contacts the wearer’s skin. It allows the transmission of forces and moments through the prosthesis to the
floor.
Most transtibial prostheses are furnished with a combination of insert (soft socket) or liner and prosthetic socks to distribute the
stress around the limb and increase the comfort and cushioning.
Transfemoral sockets are generally made of a combination of a flexible socket encompassed in a rigid laminated plastic frame.
Suspension
Suspension is a component that prevents the prosthesis from slipping off the amputation limb during walking.
Old-fashion suspension designs (e.g., a leather thigh corset with side metal joints) are rarely used in the age of advanced
technology.
There are a variety of belts, strap (e.g., supracondylar strap) and knee sleeves that are also implemented for suspension purpose.
Fig. 5 (Left) Below knee endoskeletal prosthetic designs with different suspension methods, (right) details of suspension with metal pin locking
system.
A metal pin at the end of prosthetic liner which engages into a locking mechanism in socket or a lanyard method (e.g., appli-
cation of a nylon fabric cord) is considered as suspension methods through the distal end of the socket (Fig. 5).
Knee Units
Knee units are mass-produced components and have several functions including the body weight support and provision of
controlled movement.
Knee mechanism may be simply mechanical in single axis or polycentric design or may function with the use of hydraulic or
pneumatic mechanism.
By using a liquid medium (usually silicone oil or magnetized rheological fluid), the hydraulic controlled knee mechanisms will
facilitate the patient to experience variable walking speed. Some of these hydraulic systems can be driven by incorporating the
microprocessors to possess a computer-driven control (Fig. 6).
There are some knees and ankle designs including electromechanical motors interlinked with microprocessors which are able to
move knee and ankle, a mechanism which might be quite helpful in stair ascending.
Upper Limb Prostheses

The upper extremity prosthesis basically consists of a socket, a method of suspension, a power source, plus elbow and shoulder
joints depending on level of amputation. There are two basic types of upper extremity prostheses: body-powered and myoelectric.
Fig. 6 Transfemoral prostheses with fluid-control knee units.

Essentially, a body-powered prosthesis uses body movements, harnessed with control straps and cables to mobilize the terminal
device. The terminal device may be a hook or a hand.
In Myoelectric prostheses, the implanted electrodes in the prosthetic socket play a key role in detection of signals of contracted
muscles in residual limb. The signal will be interpreted in controller to open and close the terminal device.
To design an orthotic or prosthetic device, many features should be kept into the consideration such as specific goals and require-
ments of the patient, weight of device, cosmetics, durability, ease of putting on (donning) and taking off (doffing) and ventilation to
prevent skin maceration.
Unfortunately, no single material has an ideal set of properties for application in O&P. The type of material influences the design
characteristics as well as the effectiveness of the orthosis for a given patient. As an example, the skin exerts water and gases; if they get
collected at the surface of an orthotic or prosthetic appliance, they may cause dermatological problems. Selection of appropriate
lining materials with enough perforation properties and special consideration such as silver treatment as well as ease of cleanliness
may enhance the quality of final products. Similarly, choosing the right material in active patients or those who will involve in sport
activities will decrease the risk of appliance failure and potential damages to the user.
Orthotic and prosthetic services will address a wide range of complications including the neurological and musculoskeletal
disorders occurring in children, adults and elderly people. Following, a description of the most common materials will be provided.
Materials
O&P have always derived advantage from advances in technology and materials. The partial substitution of traditional prosthetic
and orthotic materials, such as wood, aluminum, and leather by modern materials, such as thermoplastics and advanced compos-
ites, has fostered design innovation and resulted in significant improvements in function, durability and appearance of modern
prostheses and orthoses.
Despite the development of new materials, traditional ones are still in wide use. Metals, wood, leather, fabrics, thermoplastics,
thermosetting composites, foamed plastics, and elastomers are the principle materials commonly used in current orthopedic
industry.
Most orthoses contain several materials with miscellaneous mechanical properties. The materials may be sewn, riveted, or glued
to one another.
To understand recommended design and fabrication procedures, it is important to have a basic knowledge of the properties of
materials. Some of the material properties are listed and defined in Table 3.
In addition to materials’ inherent mechanical properties, certain physical characteristics of the materials also affect the material
behavior and appliance function such as thickness and shape of the material used in appliance.
This article presents an overview of the common materials used in O&P.
Casting Materials and Fabrication Methods

There are a variety of manufacturing technologies to fabricate and obtain an orthopedic appliance. Many individual patient solu-
tions begin with conventional fabrication of plaster casts (negative impression) and the manufacture of positive model. Following,
some of the most common used materials for casting are discussed.
Gypsum plaster or plaster of paris (POP)

POP has two functions in orthotic and prosthetic field; POP bandage is used to generate a negative cast of body part. POP powder is
then blended with water to make a liquid plaster poured into the negative cast to create a positive mold resembling the patient’s
limb.
Table 3 Materials properties
Properties Definition
Strength The ability of a material to sustain the external force before the breakage
Ductility The ability of a material to undergo permanent change of shape without rupture
Stiffness The amount of deformation (e.g., bending or compression) that happens when the
load is applied to a material
Elasticity (resilience) The ability of a material to recover the original dimension after unloading
Hardness The resistance of a material to penetration in response to a compression load. The
measure of material hardness is referred to as its durometer
Density A material weight per volume
Toughness The ability of a material to withstand a shock force
Corrosion The ability of a material to resist degradation in response to exposure to chemicals
Durability (fatigue resistance) The ability of a material to resist the cyclic loading without breaking
POP bandage consists of calcium sulfate hemihydrate generally on a cotton bandage. Addition of water allows the POP to
conform to the limb before the completion of crystallization process and plaster setting.
In case of big molds, cast fillers in forms of sand might be mixed into the plaster to lower the positive cast weight.
Fiber glass bandage

Fiber glass bandage includes knitted glass fibers impregnated with polyurethane (PUR) resin; similar to POP, Fiberglass bandages
need to be immersed in water to be activated. This bandage can be used for light supporting limb casting; however they cannot make
the same formability as POP bandages do.
Foam box impression

The foam box is considered as an alternative to plaster casting for accurately capturing the plantar surface of patient’s in semi-
weight bearing impressions. Closed-cell and moisture-proof Phenolic foam (made by curing a foaming phenolic resin) will
be served for replication of the foot imprint. This foot shape later will be duplicated by pouring the liquid plaster into the
foam box.
Alginate
Alginate (a natural polysaccharide) is a powder used for taking impression with good surface details. Mixing with water, it makes
a creamy gelatinous form used for detailed cast taking of hands, fingers, feet, and maxillofacial prostheses. It will provide greater
accuracy than plaster and remains elastic once cured.
Alternative to physical negative casting, Computer-Assisted Design and Computer-Assisted Manufacturing (CAD-CAM) tech-
niques are used. With the technology, an optical or laser scanner captures the surface geometry of the body segment. This digital
impression is then rectified virtually and used to direct milling machinery to fabricate a rectified positive model from a carving block
of PUR.
Once the positive model is created, either via casting and hand fabrication or digital scanning/carving, the device is then fabri-
cated in a conventional manner of vacuum forming or lamination over the model.
In case of orthoses with metal uprights, factory-made uprights are individually conformed to body contour and then assembled
to custom-made segment which is produced by either vacuum-forming or lamination.
In an attempt to eliminate the role of positive cast in the procedure of orthotic fabrication, low temperature plastics have been
used for many years to be molded directly on the limb.
In some technologies, the concept of positive model as a foundation for fabrication of orthoses and prostheses has been totally
replaced by direct manufacture of the product from the scanning digital file of the segment.
CNC-controlled milling machines can carve materials such as Ethylene Vinyl Acetate (EVA) and cork with different hardness to
create a custom-design foot insole.
3D printing is another technology which possesses the direct production of wide range of orthopedic products.
Leather
Leather is fabricated from animal skin and hides processed tanning treatment. The final properties of the leather rely on the tanning
procedure and the type of hide used in the fabrication of a product. Strength, stretch, formability, and water vapor permeability are
the characteristics of leather which makes it valuable for O&P. Leather is used for components such as suspension straps, belts, and
thigh corset in conventional hinged lower limb prostheses. Thinner leather can be used as lining material to cover the metal bands
and bars in conventional lower limb orthoses, as well as insoles. Cowhide is served as very strong leather and therefore it can be used
for straps and the upper portion of shoes.
Textile Fibers and Fabrics

Natural and synthetic polymer fabric materials are commonly-used fabrics in orthoses and prostheses.
Natural Fibers
Cotton fiber which is taken from the capsules of the cotton plant is perspiration absorbent and tear-resistant. Wool has excellent
resilience and high heat-retaining capacity and can absorb the humidity.
Synthetic Fibers
Synthetic Fibers might be polymeric-base or mineral-one. Mineral fibers include fiberglass and Carbon which will be explained later.
Synthetic polymer fabrics used in orthoses include polyester and polyamide (Nylon and Aramid).
Fabrics are widely used for prosthetic socks, tubular stockinette, straps, lining along with other materials, harnesses for upper-
limb prostheses as well as fastening and base material in less rigid orthotic supports such as fabric stabilizing spinal corsets. The
greatest use is for prosthetic socks and stockinette. They are commonly made of wool, cotton, or blend of these natural fibers
combined with nylon, acrylics or other synthetic materials. Fabrics may incorporate rubber to create an elastic cloth applicable
for straps used in harness system of upper limb prostheses.
A very popular use of nylon is hook and loop (Velcro) which are used for frequent engage in strapping to secure an appliance in
place in an easier way to buckles and laces.
Webbing straps which are strong flat strips of woven fabric might be made from nylon or polyester.
Felt, a nonwoven fabric made from textile fibers such as polyester or wool can create a fine surface, and serve as a filling material
in lamination or be used for padding purpose.
Nylon fabrics nowadays use as a covers material for neoprene products as well as outer layer of many prosthetic liners.
Nylon in the form of cord and cable is applied in lanyard suspension system and body-powered prosthetic control, respectively.
Cosmetic nylon stockinette will improve the final appearance of covered modular prosthesis.
Metals
Metals are used for manufacturing of prosthetic modular components such as adaptors, clamps, and joints as well as uprights and
joints in orthotic designs. To bond the materials, steel, aluminum and copper rivets as well as tubular rivets, buckles and loops
(made from nickel plated steel) and brass eyelet are implemented to show a minor but important procedure in constructing of
an appliance. However, metals mostly used in the fabrication of orthoses and prostheses can be categorized into three groups: steel,
aluminum, and titanium.
Steel
Iron is never used in pure state. Any iron-based alloy material is generally called as steel. Stainless steel is a steel alloy that contains
nickel and chromium to enhance the resistance of alloy to corrosion and oxidation. Because of properties such as durability, corro-
sion, fatigue-resistance and high strength, stainless steels are used frequently within O&P. The obvious disadvantage is the weight of
product.
Stainless steel is widely used in factory-made orthotic and prosthetic joints, support uprights, band material, springs and bear-
ings of lower limb orthoses, shoe attachments (stirrup) in conventional lower limb orthoses and spring bands for corset construc-
tion in metal frame spinal orthoses. Also, testing screws which are used for connection of orthoses during the trial phase are made
from steel.
Aluminum
Aluminum alloyed with copper and manganese is well suited for O&P due to low weight and high resistance to corrosion. The
primary alloy used in O&P is aluminum alloy 2024 which provides a shiny well-finished component.
Two main types of aluminum include the wrought and cast alloys. Cast aluminum is usually used for prefabricated prosthetic
components such as finger assembly in hand prostheses.
In case of structural purposes such as prosthetic pylon tubes, orthotic uprights, and upper extremity joints, wrought aluminum
alloys are preferred to cast ones.
In general, aluminum is used in applications in which the device is subjected to lower stresses. To reduce the fatigue
failure at attachment site, normally the component needs to be designed from titanium or stainless steel rather than
aluminum.
Titanium
Titanium alloys are very strong, lighter in weight than steel, and corrosion resistant. Titanium is used in some prefabricated orthotic
and prosthetic components when simultaneous strength and light weight are required. In conjunction with light weight composite
shells and plates, metal components of exoskeletons are preferably chosen by titanium alloy to decrease the total weight of device.
Wood
Unique characteristics of wood make it an appropriate material in lower limb prosthetics application. It is lightweight and, strong.
The wooden keel of solid-ankle-cushion-heel (SACH) prosthetic foot is fabricated of maple and encased in a microcellular PUR
shell in shape of normal foot.
Cork is the most common wood used in orthoses. It is exceptionally lightweight and resilient, making it appropriate for shoe
lifts. Sometimes, cork is mixed with rubber or EVA to achieve greater flexibility, cushioning or thermoformability. Except cork,
some other woods might be used for shoe elevation or designing the shoe last.
A last is a mechanical form in shape of a human foot. It is used by shoemakers as a working surface on which the leather forms
and it is made from hardwoods or some high density plastics.
Synthetic Polymers
Polymers are typically classified into three groups: plastics including thermoplastics and thermosets, polyurethanes (PUR) and elas-
tomers. Thermoplastics comprise the majority of available polymers.
Plastics
Development of plastic polymers revolutionized orthotic and prosthetic prescription because these materials offered enhanced
mechanical properties such as formability which introduced them as predominant materials in this technology. The growth of
the plastic industry has been evolving into today’s routine to sophisticated high performance products. There are two major cate-
gories of plastics: thermoplastics and thermosetting plastics. Plastics are usually favored because of their superior appearance,
uniform color, and the ease at which they can be casted, molded or extruded.
Thermoplastics
Thermoplastics are frequently used in O&P. Thermoplastic materials get malleable when heated but will retain the new shape when
cooled. The most useful physical property of a thermoplastic is its glass transition temperature at which it begins to soften. Ther-
moplastics are divided into two groups; low-temperature or high-temperature materials, according to the temperature range at
which they become formable, whereas high-temperature materials require higher temperatures and must be molded over a positive
plaster replica of the patient’s limb. One advantage of thermoplastic materials is that they can be reheated and reshaped, making
possible minor adjustments of an appliance during fittings.
Low-temperature thermoplastics, those moldable in a warm water bath, can be molded directly onto the body. The basic ingre-
dient of many thermoplastics (low-temperature) is polycaprolactone which helps to make the plastic highly conforming. In
contrary, some others include polyisoprene, a synthetic rubber,
Low-temperature thermoplastics such as Orfit are most often reserved for orthotic devices that are designed to provide temporary
low-stress support and protections such as short-time spinal, upper extremity and fracture orthoses (Fig. 7).
Development of low-temperature knitted textile materials can facilitate the fabrication of finger and other small immobilization
orthoses.
The production of most of orthopedic appliances is accomplished by application of high-temperature thermoplastics. Some of
the commonly used materials comprise polyolefins (including polyethylene and polypropylene), acrylic, polycarbonate, acryloni-
trile butadiene styrene (ABS), polyamide, saturated polyester, vinyls and acetals) .In spite of versatility of thermoplastic sheets, poly-
olefins are still considered as most widely used plastics.
As a matter of fact, there is no unique plastic which can meet all the requirements. Therefore, when selecting a sheet of plastic,
several factors should be kept into consideration including desired finished thickness, shrinkage and rigidity.
The characteristics of some of the commonly-used thermoplastics and their typical orthotic and prosthetic application are
summarized in Table 4.
Thermoforming
To fabricate the components of a device such as prosthetic socket and AFO, most of the orthotic and prosthetic laboratories take the
advantage of a common technique called thermoforming (drape forming). In this technique, a heated thermoplastic sheet of plastic
is drawn on a positive mold; the technique is completed once the plastic sheet is vacuumed and conforms to the shape of the mold
(Fig. 8).
Fig. 7 (Top) Low temperature thermoplastics to design different orthoses for upper extremity, (bottom) application of a WHO.
Table 4 Thermoplastics used in orthotic and prosthetic fabrication
Generic name Characteristics Application
Polyolefins • Good toughness and flexibility • Spinal and upper extremity orthosis
Low-density Polyethylene (LDPE) • Arch support
• Flexible prosthetic sockets
Polyolefins • Thermoforming • Spinal orthoses
High-density Polyethylene (HDPE) • Increased rigidity and tensile strength • Lower extremity cuff
• Good fatigue and wear resistance • Resting passive upper limbs orthosis such as
WHOs
Polyolefins • Durable and stiff but more difficult to form • Applications require greater strength
• Decreased impact strength • Upper and lower extremity
• Polypropylene Homopolymer (PP H) • Maximum rigidity among olefins • Spinal orthotics
Polyolefins • Polypropylene/polyethylene copolymer • Upper and lower extremity
• Very good thermoforming • Nonarticulated dynamic AFOS
• Polypropylene Copolymer (PPC) • Rigid yet flexible • Spinal orthotics
• Offers more durability than PP homopolymer
• Good endurance to million cycles of repetitive flexes
Polyethylene (PE) foam • Resilient • Padding and lining straps
• Lightweight • Shock absorbing accommodative FOs
• Available in various durometers • Cosmetic cover in endoskeletal prostheses
• Moldable
Ethylene-vinyl acetate (EVA) • Copolymer of ethylene with 14%–40% vinyl acetate • In foam shape for padding and cushioning in
• Light weight footwear and shoes
• Excellent shock absorbency
• Increased flexibility compared to LDPE
PE foam plus EVA • Denser and stronger than PE foam alone • Semi rigid foot orthosis
• Good shock absorbing capabilities • Posting material
• Moldable
Acrylic • Transparent • Check socket
PMMA (polymethyl methacrylate) • High stiffness • Ocular prosthesis
• Easy to bond to alignment component by using
acrylic resin
Low-temperature thermoplastic • Rubbery thermoplastic • Upper limb orthoses with low force load
Polyisoprene-based (ezoform) • Formable
Low-temperature thermoplastic • A more rigid thermoplastic • Upper limb orthoses
Polycaprolactone–based (Orfit) • Good draping (molding) • Trunk orthoses
Polyamides • Semi rigid plastic • Orthotic joints, which are lighter in weight than
metal ones
• Nylon • Reinforcement fiber
• Aramids (Kevlar)
Polyethylene terephthalate Glycol • In family of saturated (thermoplastic) polyesters • Prosthetic check socket
(PETG) • Transparent • Face mask for sport and burns
• Superior impact strength and resistance over acrylic
thermoplastic
• Appropriate for vacuum forming
PVC (polyvinyl chloride) • Tough • Upper limb orthoses
• Chemical resistant • Spine and cervical orthoses
Polyacetal • High strength and stiffness • Components in orthopedic devices that require
(POM ¼ polyoxymethylene) • Low coefficient of friction a great deal of stiffness such as plastic screws and
nuts
Acrylonitrile butadiene styrene (ABS) • A modified polystyrene with improved impact • Spinal body supports
strength • Seat inserts
• Bondable
Polycarbonate (PC) • Transparent • Check socket
• High durability and impact resistance
Ionomer (surlyn) • Copolymer of ethylene and methacrylic acid • Upper extremity orthosis
• Ionically cross-linked • Spinal orthosis
• Very high tensile strength • Partially flexible inner prosthetic socket
• Transparent • Check sockets
• Tough and durable • Face mask for sport and burns
• Soft • Cranial helmet
• Excellent thermoforming
(Continued)
Table 4 Thermoplastics used in orthotic and prosthetic fabricationdcont'd
Generic name Characteristics Application
Polyvinyl chloride (PVC) • Good stretchability • Cosmetic glove

• Increased perspiration • Top covering material (impregnated with cloth
backing)
Polyvinyl alcohol (PVA) • Soluble in water and alcohol • PVA bags for lamination
Polyether ether ketone (PEEK) • Rod form • Dynamic AFOS
• Tough
• Spring function
Kydex • Composite of acrylic and polyvinyl chloride (PVC) • Supporting material in Cervical collars (neck
• Reinforcing material orthosis)
• Thermoforming • Spinal orthotic such as TLSO body jackets
• Outstanding toughness
Fig. 8 Cranial remodeling orthoses and transparent face masks made by thermoforming techniques.
Thermosetting materials
The cured thermosettings degrade rather than melt upon heating. Thermosetting plastics (Resins) are plastics that are applied over
a positive model in liquid form and then chemically cured to maintain the shape of underlying contour. Thermosets are often
impregnated into various fabrics by a process of lamination. Epoxy resin (polyepoxides), unsaturated polyester resin and acrylic
resins are considered as thermosetting polymers.
Epoxy has the highest performance of all thermosetting plastics as the cured resin represents high strength in tension and
compression.
Acrylic resin is available in different blends each having their own characteristics. Examples include a standard blend of 80%
rigid and 20% flexible resin to be used for vacuum laminations.
The skin color pigment is an active plastics softener, and as a result it should not be mixed more than 2%–3% to the lamination
resin.
Due to the transparency and biocompatibility, acrylic resin also can be used in fabrication of Prosthetic eye (Ocular prosthesis)
as well as finger nail and toe nail in partial hand and foot prostheses.
Polyester resin does not bond well to the fiber. Due to the poor rinsing properties of this resin, it is not recommended for high
performance composite manufacture.
Forearm segment in above elbow prosthesis is usually a prefabricated component which is laminated with polyester resin while
encompassing the steel side bars of a mechanical elbow joint.
Composites
Fiber-reinforced plastics (FRPs) also called composites, have reformed the O&P by producing a material with enhanced strength and
quality yet capable of tolerating compressive and flexural stresses.
The most common fiber reinforcements used in polymer composites are fiberglass, carbon, and Kevlar aramid fibers. In general,
the volume of fibers should be higher than the volume of resin matrix. Carbon fiber reinforced plastics (CFRP) are widely used in
orthoses and prostheses including energy storing keels in prosthetic feet, external supporting frame of hydraulic cylinder in pros-
thetic knee units, pylon tubes in heavy duty cases and orthotic uprights. According to degree of graphitization, this fiber may mani-
fest high strength properties while also being lightweight. Carbon fiber has superior stiffness properties in both compression and
tension but, it has relatively low impact strength. In spite of concern about their biocompatibility, aramid fibers have a very high
tensile strength. Kevlar fibers might be used in some prosthetic feet as a reinforcement structure to protect the PUR foam from
fatigue.
Currently, there are two techniques for processing FRP composites in orthotic and prosthetic laboratories namely hand layup
with vacuum impregnation-consolidation, and heat curing of resin-impregnated fabrics (Pre-preg).
The most common method called vacuum bag lamination includes a hand layup of tubular nylon or cotton stockinette fabrics
and reinforcement fibers in shape of cloth, braid or roving placed on the positive mold. This complex is sealed between two layers of
polyvinyl alcohol (PVA) plastic bags which can accommodate the wet liquid resin to be poured into the enclosure while the applied
vacuum removes the entrapped air from the laminated parts, pressing the resin through the fibers and create a thin-walled structure
(Fig. 9).
Preimpregnated (Pre-preg) materials are composites of one of those three reinforcement fibers as a foundation material impreg-
nated with a predetermined amount of resins, preferably epoxy. Pre-pregs are easier to work with than current wet-lamination tech-
nique while light weight device can attenuate the forces, successfully. These materials are best option for the manufacture of orthoses
in a frame construction such as rigid knee orthoses. As a limitation, they cannot be stored in room or higher temperature for long
time.
Recently, there is an interest to combine the strength and energy storage of carbon fiber with the workability and versatility of the
thermoplastics by extruding the carbon fiber or glass fiber compounds inside the thermoplastics. Nylon, polypropylene and PETG
are the thermoplastics that have been used in large volume. For partial reinforcement in fabrication of orthoses these fiber reinforced
thermoplastic profiles can be formed and bonded to the underlying thermoplastic for enhancement of structure strength.
Expanding foams (foamed plastics)

Both thermoplastic and thermosetting resins can be used to produce a rigid, semi-rigid, or flexible foam structure. Expanding foams
can be used both in orthotic and prosthetic devices, as an interface to protect the skin from the appliance. Bubble gases like
hydrogen or nitrogen can be forced into the plastic solid matrix to create resilient materials. These foams are categorized into
two groups: open and closed-cell. The cellular structure can reduce the shear forces. In open-cell foam, the cells are interlinked while
in closed-cell foam, the cells are completely isolated from each other. Because are liquid-proof, the absorption ratio of body perspi-
ration or odor in closed-cell foams is significantly limited since they are liquid-proof.
These closed-cell polyethylene (PE) foams are available in a wide array of durometer hardness PE foams (e.g., Plastazote, Pe-lite)
can be used in the manufacture of thermoforming soft socket in lower limb prostheses as well as padding in orthoses and cervical
collars.
The base material or top-layer cover of accommodative foot orthoses which are highly prescribed for patients with neuropathies
could be made from materials such as EVA, neoprene rubber, soft PUR foams, and low density PE foams to provide comfort and
shock attenuation.
Sometimes the foamed plastic benefit from some treatments such as a layer of silver bonded to material to possess an antimi-
crobial and antifungal effect. The sandwich foams of PE-EVA with different hardness might be helpful in some patients to absorb the
shock while providing a more resilient combination.
Fig. 9 Materials for vacuum bag lamination.

3D knitted spacer fabrics can be used for padding since they have good compression properties. They might be combined of
polyester and nylon in different combinations. Good air permeability and compression behavior are unique properties propose
these fabrics as good alternatives for padding.
Elastomers
Elastomer comprise a large family of elastic polymers which can snap back to approximately original size and form once the load
causing the deformation is released.
Because of their properties to absorb and dissipate the loads, many of elastomers are used in shock-absorbing shoe inserts.
The classification of four main groups and subgroups of elastomers (e.g., natural and synthetic rubber, PUR, silicone, and ther-
moplastic elastomers (TPE) has been presented in Table 5.
Polyurethane (PUR)
The mixture of polyol (specific alcohol) and isocyanate will result in an instant expansion of a foam or rubber-like product called
PUR which can be found in block or sheet form in versatile stiffness (flexible to rigid).
While soft PUR elastomers are primarily used in body cushioning such as insoles, cosmetic cover in endoskeletal prostheses and
prosthetic liners, rigid PUR foams are especially suitable for load parts of the orthopedic technology range (e.g., length difference
compensating insoles, shank in exoskeletal prosthetic design and carving blocks in milling machines).
PUR elastomers with moderate durometer are generally used to encapsulate the keel of foot unit through a process of injection
molding. Also, outer sole of the shoe and flexural ankle joint in AFO can be made from.
Thermoplastic elastomers (TPE)

TPEs are a class of copolymers which show the advantageous properties of both elasticity and easy processability. The use of TPE in
recent decades has dramatically increased. As an example, TPE gels enriched with vitamins and mineral oil can be ideal option for
use in cushioning and shock absorption products such as prosthetic gel liners, insoles, heel cups and hypertrophic scar management
while they can continuously moisturize the skin.
Rubber
In general, rubber has considerable elasticity, shock absorbency, and toughness. Synthetic rubber has more resistant to corrosion
than natural rubber. Rubber strands may be woven with cotton or other fabrics to create elastic straps. Polychloroprene also called
neoprene is available in various densities, making the low-durometer versions suitable as lining material for orthoses, whereas the
firmer samples are used for sport and support orthoses. The nylon (polyamide)-covered neoprene acts as a shock absorber while
also reducing friction on the foot’s plantar surface.
Silicone
Dimethyl polysiloxanes is the largest group of commercial silicone polymers used in medicine (including prosthetics). Silicone may
form the following polymers: silicone oils (for decreasing the viscosity of silicones), silicone resins (liquid silicone), and silicone
rubber (high consistency solid silicone). Depending on whether the vulcanizing process uses heat or not, silicones are available
as heat vulcanized (HTV) or room temperature vulcanized (RTV). In general, HTV Silicones show higher strength.
Table 5 Classification of elastomers
Name Name in details Comment/application
Natural Rubber Latex Obtained from the tree • Base material of contact
Synthetic Rubber Polyisoprene rubber (IR) Synthetic version of Natural rubber adhesives
Polybutadiene (BR) Mostly used in tire production • Shock attenuation padding
Styrene-butadiene rubber (SBR) Most important sort of synthetic rubber insole
Polychloroprene (CR) also called First synthetic rubber maintain flexibility in • Rubber shoe outsole
neoprene a wide temperature range • Rubber band in some WHOs
and hooks
Polyurethane (PUR) Polyester-urethane Better mechanical properties • Insoles
Polyether-urethane Better cushioning • PUR shoe outsoles
• Flexural ankle joints
• Prosthetic liner
Silicone rubber Dimethyl polysiloxanes • Cosmetic prostheses
• Gel liner
Thermoplastic elastomer Styrene based (TPE-S) • Shoe outsole and footwear
(TPE) Olefin based (TPE-O) • Prosthetic Gel liners
Urethane based (TPE-U)
Fig. 10 (Top and bottom) Silicone hands including artistic designs.
Silicone cushioning and elasticity provide excellent wearing comfort, regardless of whether they are used in a socket for pros-
theses or an orthosis (Fig. 10).
The wide range of medical-grade silicones with different degree of stiffness possesses a variety of options to make appliances
such as:
B End bearing/cushioning pads
B Cosmetic finger and partial hand prosthetics
B Face mask for scar compression treatment
B Cosmetic and functional partial feet
B Cosmetic glove for upper limb prosthesis
B External mammary prostheses
B Maxillofacial prostheses
B Foot orthotics
B Socket liners
To facilitate the donning and doffing of liners, different techniques have been implemented in industry.
Poly-para-xylylene (Parylene) is a polymer used to create an ultra-smooth surface coating with low coefficient of friction. It
bonds to the outer surface of silicone liner at molecular level while this process does not affect the elasticity of substrate silicone.
Fabric to gel bonding is another approach to improve the sliding of the gel surface while the fabric cover is in contact with the
socket.
Adhesives
The adhesives used for orthopedics applications must fulfill a maximum amount of adhesion power, decrease curing speed and be
compatibility with a greater range of materials for surface bonding. Examples of the most common glues used in orthopedic and
footwear industry include one-part, rubber-based contact adhesive (e.g., polychloroprene, styrene-butadiene rubber) and PUR-
based contact adhesive. These materials are mostly air drying as the solvent will be evaporated to provide excellent final bond
strength.
Surface Finishing
Surface coating will improve the acceptance of orthoses and prostheses to the patient. Water transfer printing (hydrographics) and
thermopapers are two techniques to match the device with patient’s expectations.
Water transfer painting is a coating technology to print and decorate any design on orthopedic devices by using a printed PVA
hydrographic film which is placed on the surface of water. Due to the surface tension of water, floating ink pattern will curve and
bond around the component.
Colorful transfer papers are widely used to cover the thermoplastic material with a specific pattern. Usually an ink-jet printer is
used to print an image or pattern on the paper. Then a heat press can transfer image onto the plastic sheets. The final product might
be more appealing to the patient and especially in case of pediatric devices, the compliance might be improved.
3D Printing
3D printing, as a process of creating solid objects from digital files has become the medium of new technological revolution in the
medical science, including O&P. Due to fabrication ability of customized sophisticated devices, 3D printing has the potential to
facilitate the design of the orthopedic devices. At the moment this technology is being implemented to fabricate the prosthetic
sockets, hand prostheses, AFOs and spinal orthoses.
There is a wide variety of material types that are supplied in different states (powder or filament forms) to address the 3D
printing technologies including the selective laser sintering (SLS) and fused deposition modeling (FDM) processes. ABS, nylon,
and composite form of materials with improved mechanical stiffness such as blend of carbon fiber and polylactic acid (PLA),
a biodegradable thermoplastic polyester derived from renewable resources, carbon fiber filled PETG and glass filled nylon are
some examples of those materials nowadays used to print the orthopedic devices.
There is a potential to use the metal powder of Stainless steel and Titanium for 3D printing of Orthotic and prosthetic compo-
nents, as well.
Summary and Possible Future Direction
There is no doubt that O&P field has developed dramatically and technology advancement has offered outstanding improvement in
application of materials.
A continually increasing variety of new materials is being used for orthotic and prosthetic fabrication. Using new materials have
largely supplanted the traditional methods using leather and steel. Application of Thermoplastics, composites and improved metal
alloys in orthopedic devices has offered progress in terms of weight, strength and energy return to customize for individual patient.
Materials with better physical properties will be emerging from plastic industry. We are passing from PP, PE and copolymers, wet
lamination of sockets and pre-preg sheets to now extruding the plastic with carbon fiber in it and in this way, technology is going to
change the field.
Next generation of composites might be more evolutionary such as placement of piezoelectric fibers in laminated layers of appli-
ance. Since the piezo crystals can generate electricity when subjected to physical stress, there would be a potential to implement
them as a mechanism for battery recharging during walking with prostheses or a method to change the stiffness of a component
when electrified.
Elastic actuators such as pneumatic artificial muscle (PAM) are used in the field of exoskeletons due to their ability to generate
linear forces and motions with a simple mechanism, while remaining lightweight. In most cases, these actuators are composed of
elastomeric cylindrical bladders constrained by flexible but inextensible mechanisms such as meshes and embedded fibers. In close
future, artificial muscles might be made of fabrics to be integrated into clothing to aid mobility. By using fabrics and textiles, soft
wearable exosuits will probably provide a more comfortable interface without any mechanical constrain on wearer’s joints as the
hard exoskeletal joints do.
Growing experience with osseous integrated prostheses suggest that some patients may achieve greater comfort and function
with direct attachment of prosthetic adaptor to implanted metal shaft that protrudes from the limb and by doing so, application
of prosthetic sockets may gradually decrease. As a result, in future, the investment might lead to further development of active pros-
thetic components with new materials and enhancement of control systems including neural control.
3D printing revolutionizes the fabrication techniques of a product and moves the industry forward by making the orthopedic
devices more accurate, innovative and improved appearance.
There is a very good potential for the application of 3D printing in fabrication of different orthotic and prosthetic components as
well as exoskeletons.
The concept of natural fibers is being tested to low-cost orthotic and prosthetic devices. Application of composite materials for
3D printing can improve the strength of the final product significantly without affecting the flexibility. Development of recycling
and long-lasting materials as well as decreasing the total time of printing will further enhance the feasibility of this technology
to serve in orthopedic devices.
In summary, the final choice for material selection will rely on local availability of materials, technical expertise and preference of
orthotist/prosthetist, expected function and patient’s previous experience; However the interplay of materials science and processing
techniques will reveal new insights, and therefore consistent development and enhancement of materials guarantees the achieve-
ments of dreams and forthcoming expectations in O&P industry.
Further Reading
Coppard, B. M., & Lohman, H. (2014). Introduction to orthotics: A clinical reasoning and problem-solving approach. ElsevierdHealth Sciences Division.
Faustini, M. C., Neptune, R. R., Crawford, R. H., & Stanhope, S. J. (2008). Manufacture of passive dynamic ankle–foot orthoses using selective laser sintering. IEEE Transactions on
Biomedical Engineering, 55, 2.
Herr, H. (2009). Exoskeletons and orthoses: classification, design challenges and future directions. Journal of Neuroengineering and Rehabilitation, 6, 21.
Hsu, J. D., Michael, J., & Fisk, J. (2008). AAOS atlas of orthoses and assistive devices E-book. Elsevier Health Sciences.
Jacobs, M. L. A., & Austin, N. M. (2013). Orthotic intervention for the hand and upper extremity: Splinting principles and process, 2nd edn. Lippincott Williams & Wilkins.
Krajbich, J. I., Pinzur, M. S., Potter, B. K., & Stevens, P. M. (2016). Atlas of amputations and limb deficiencies (4th edn.). American Academy of Orthopaedic Surgeons.
Lusardi, M. M., Jorge, M., & Nielsen, C. C. (2013). Orthotics and prosthetics in rehabilitationdE-book. Elsevier Health Sciences.
Walbran, M., Turner, K., & McDaid, A. J. (2016). Customized 3D printed ankle-foot orthosis with adaptable carbon fibre composite spring joint. Cogent Engineering, 3, 1227022.
Wise, D. L., Trantolo, D. J., Altobelli, D. E., Yaszemski, M. J., & Gresser, J. D. (2013). Human biomaterials applications. New York: Humana Press.
List of Relevant Websites
http://enablingthefuture.org/d“Enabling the future”.

https://www.orfit.com/d“Orfit”.
https://www.ossur.com/d“Ossur”.
http://www.ottobock.com/en/d“Ottobock”.
https://wyss.harvard.edu/technology/soft-exosuit/d“Wyss Institute”.
Microfluidics for Biomedical Applications
Shiyu Cheng*, Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of
Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, Beijing,
P. R. China
Jinqi Deng*, Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of
P. R. China; and Sino-Danish College, University of Chinese Academy of Sciences, Beijing, P. R. China
Wenfu Zheng, Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of
P. R. China
Xingyu Jiang, Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of
P. R. China; and Sino-Danish College, University of Chinese Academy of Sciences, Beijing, P. R. China
Introduction 369
Microfluidics for Manipulating Cells 369
Microfluidics for Cells Adhesion on Surfaces 369
Adhesion of single type of cells 369
Adhesion of multiple types of cells 369
Dynamic control of cell adhesion 369
Microfluidics for Cells Migration on Surfaces 369
Topographic properties 370
Shape of cells 371
Cell–cell interactions 372
Wound healing 372
Microfluidics for 3D Patterning 372
Spatial control 372
Soluble molecular gradients 375
Microfluidic simulation of mechanical stimuli 375
Microfluidic-Based Biochemical Analysis 375
Nucleic Acid Detection 376
Protein Analysis 378
Barcode-Based Multiplex Analysis 379
Cell Analysis 381
Further Reading 383
Glossary
Antibodies Proteins that are produced by plasma cells and can bind tightly to their targets, which are called antigens.
Cell adhesion Cells interact and attach to a surface, substrate, or another cell by interactions between molecules of cell surfaces.
Cell migration Movement of cells in particular directions to specific locations in response to specific external signals, including
chemical and mechanical signals.
Cell patterning A process to position cells on a substrate with defined spatial selection and in turn to keep stable cell activities
on the substrate.
Circulating tumor cells Tumor cells that shed from primary lesions and circulate in the bloodstream. The existence of
circulating tumor cells can cause tumor recurrence and cancer metastasis.
Loop-mediated isothermal amplification An isothermal method for nucleic acids amplification.
Microfluidics Precise control and manipulation of fluids that are geometrically constrained to a sub-millimeter scale with
specific nano-/micro-structures.
*
These authors contribute equally to this work.

Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Microfluidics for Biomedical Applications 369
Introduction
Microfluidics typically refers to the precise control of minute amounts of fluids with micro-scale structures. Soft lithographic tech-
niques, based on self-assembly and replica molding, enable the micro-/nano-fabrication and advance the field of microfluidics in
a convenient, effective, and low-cost manner. Microcontact printing (mCP) is one of the key soft lithographic techniques, a stamp
with patterned relief structures on its surface could generate patterns with feature sizes ranging from 30 nm to 100 mm. Based on soft
lithography, several representative advances emerged and have become useful for biomedical applications.
In this article, we will introduce recent developments in microfluidics for manipulating cells and microfluidic-based biochemical
analysis. We will focus on microfluidic interfacial methods and their extensive applications in fundamental biological research.
Microfluidics for Manipulating Cells
Precise control of cell behaviors has been increasingly useful with the development of cell biology and regenerative medicine. The
polydimethylsiloxane (PDMS)-based microfluidic chips, a transparent soft material with good oxygen permeability and high level
of compatibility, could enable suitable micro-environment for cell culture and real-time observation, thus comprising a great tool
for manipulating cells and studying cell-cell interactions.
Microfluidics for Cells Adhesion on Surfaces

Most cells require adhesion to solid substrates to carry out biological activities. Mammalian cells could obtain good attachment on
surfaces only if suitable extracellular matrix (ECM) proteins are present. ECM, populated by extensive proteins and other molecules,
is essential for the regulation of cellular and organismal physiology. It provides a variety of physical and chemical cues to regulate
cell behaviors. In particular, physical cues such as topography and stiffness provide mechanical support to cells while chemical cues
such as cytokines, ionic strength, and pH values provide chemical stimulations for cells. By employing microfluidic chips, it is
straightforward to realize the specific modification of the surface and precisely pattern cells in microchannels with micrometer preci-
sion. We will introduce methods for controlling cell adhesion in a confined area, releasing cells from the surface, and dynamic
control of cell adhesion.
Adhesion of single type of cells

We could control cell adhesion in a specific area by exerting self-assembled monolayers (SAMs) and electrochemical desorption.
SAMs are one of the most intensively studied methods to modify the surface. SAMs allow us to control the properties of a surface
on the molecular scale with the adsorption of u-substituted alkanethiols on the surface of gold (Au) and palladium (Pd). SAMs can
be easily prepared by immersion of a substrate in the solution containing a ligand (Y(CH2)nX) reactive toward the surface, or by
exposure of the substrate to the vapor of a reactive species. The thickness of a SAM can be controlled by changing the number
(n) of methylene groups in the alkyl chain. EG3-terminated SAMs could resist the absorption of proteins, and thus resist cells attach-
ment and spreading. We confined single human umbilical artery endothelial cells to different micropatterns with SAMs on Au and
Pd (Fig. 1A). We could also easily remove the confinement by electrochemical desorption of the (EG)3-terminated SAM. We
confined bovine capillary endothelial (BCE) cells into patterns by using mCP and thiols-HS(CH2)11(OCH2OCH2)3OH (C11EG3)
and HS(CH2)17CH3(C18). By exerting a cathodic voltage pulse ( 1.2 V for 30 s), BCE cells can attach to, and spread across previ-
ously protein-inert areas and release patterned cells from the constraints of these patterns (Fig. 1B).
Adhesion of multiple types of cells

Situations would be more sophisticated for real tissues, which are usually composed of more than one type of cells. Combining
SAMs and microfluidic channels, we could easily confine two or more types of cells to specific locations on surfaces without physical
constraints and we could also control the motility of these different types of cells. We confined NIH 3T3 cells and Hela cells in the
middle and two sides of stripes (Fig. 1C). Cells moved freely when exerting a cathodic potential of 1.2 V for 30 s (Fig. 1C). This
approach is extensively applicable for studying fundamental biomedical problems based on cell–cell interactions.
Dynamic control of cell adhesion

Dynamic control of cell adhesion on surfaces allows us to control the mobility of a single cell, or a group of cells at will. We realized
reversible cell adhesion using azobenzene-embedded self-assembled monolayers. The azobenzene moiety can be converted photo-
chemically between E and Z configuration to either present or mask RGD ligand and hence modulate cell adhesion (Fig. 1D). The
two states of the surface are reversible at different wavelengths of light by the mercury lamp of a standard fluorescence microscope.
Microfluidics for Cells Migration on Surfaces

Cell migration plays a fundamental role in many physiological activities, such as embryogenesis, morphogenesis, wound healing,
and disease progression. The migration of mammalian cells typically includes several steps: (1) morphological polarization; (2)
extension toward the direction of motility; (3) attachments between the leading membranes and the substrates; (4) movement
370 Biomaterials: Biomaterial Applications and Advanced Medical Technologies j Microfluidics for Biomedical Applications
Fig. 1 Microfluidics/surface engineering for cells adhesion. (A) Single human umbilical artery endothelial cells with different micropatterns confined
by SAMs on Au (left) and Pd (right). Fibronectin, actin, and the nucleus were stained by anti-fibronectin (green), phalloidin-Texas red (red), and DAPI
(blue), respectively. (B) BCE cells were allowed to attach to a surface via SAMs. Application of a cathodic voltage pulse released the cells from the
microislands. The numbers indicate the time elapsed (in minutes) after the voltage pulse. (C) Top: Phase-contrast and fluorescence micrographs of
NIH 3T3 cells and Hela cells patterned on a substrate modified with EG6 by using selective desorption of SAMs. Hela and NIH 3T3 were stained with
celltrace calcein green AM and celltrace calcein redorange AM, respectively. Bottom: Application of a cathodic potential on the substrates allowed all
cells to move freely on the surface. The numbers indicate the time (in hours) after application of the pulse. (D) Upper panel: The azobenzene moiety
can be converted photochemically between the E and Z configurations to either present or mask the RGD ligand. Lower panel: Cells adhered onto
SAMs with the azobenzene group in the E configuration. Few cells adhered to the same SAMs with azobenzene in the Z configuration. Cells adhered
to the SAMs again when the conformation of azobenzene changed from Z to E. Scale bars are 200 mm. (A) Jiang, X., Bruzewicz, D. A., Thant, M. M.,
and Whitesides, G. M. (2004). Palladium as a substrate for self-assembled monolayers used in biotechnology. Analytical Chemistry 76, 6116–6121,
with permission from American Chemical Society. (B) Jiang, X., Ferrigno, R., Mrksich, M., Whitesides, G. M. (2003). Electrochemical desorption of
self-assembled monolayers noninvasively releases patterned cells from geometrical confinements. Journal of American Chemical Society 125, 2366–
2367, with permission from American Chemical Society. (C) Li, Y., Yuan, B., Ji, H., et al. (2007). A method for patterning multiple types of cells by
using electrochemical desorption of self-assembled monolayers within microfluidic channels. Angewandte Chemie International Edition 46, 1094–1096,
with permission from Wiley-VCH. (D) Liu, D., Xie, Y., Shao, H., Jiang, X. (2009). Using azobenzene-embedded self-assembled monolayers to photo-
chemically control cell adhesion reversibly. Angewandte Chemie International Edition 48, 4406–4408, with permission from Wiley-VCH.
of the bulk of cell body; (5) release the attachment at the sharp end. These processes are influenced by substrate characteristics such
as topographic properties, the shape of the cells, and cell–cell interactions.
Topographic properties
Most types of cells can polarize and move directionally with topographic stimulus. Researchers studied the role of groove/elevation
(ridge) dimensions at the micrometer scale on fibroblast cell migration by correlating cell shape, migration angle, cell orientation
and velocity with these dimensions. They found that surface structures significantly influenced migration direction, cell orientation
and mean velocity, as well as migration speed in the directions parallel and perpendicular to the grooves/elevations in a surface
structure dependent way. Cell migration velocity parallel and perpendicular to the structures was significantly affected by the geom-
etries and dimensions of the substrates. To study influences of the topographic properties, we fabricated microgroove and
microgroove-composed structures by applying electrospun (ES) fibers as the template and replica molding the fibers with
PDMS. We combined microfluidics with the replica-molded substrate from ES fibers to guide neurites and control the morphology
of neurons (Fig. 2A).
Shape of cells
We identified that apart from the environmental stimulus, the shape of a cell could also determine the direction of mobility. We
designed a teardrop-like asymmetric pattern and restricted cells by employing SAMs, then released the constraint on the shape
and assessed the direction of motility for individual cells. The cells tend to move toward blunt ends after release from the defined
area (Fig. 2B).
Fig. 2 Microfluidics/surface engineering for cells migration. (A) Integration of microfluidic channels with replica mold of aligned ES fibers for
guiding neurites. The green fluorescence was neuron-specific marker Tuj1. (B) Left: Migration of a typical mammalian cell on a flat surface. Right:
Time-lapse images (in minutes) show the motility of an initially polarized 3T3 fibroblast/COS-7 after the constraint is released. (C) Left: The strategy
for patterning multiple types of cells on the same substrate that allows three types of naturally occurring cell–cell interactions. Right: time-lapse
phase fluorescence micrographs for the three types of cell–cell interactions between 3T6 and NIH3T3 cells. (D) Patterning different types of cells in
a matrix with (left) or without (right) wound at the “wound edge” according to different properties of the surface of PDMS membrane. (E) Micro-
fluidic chip for investigating the neuron-cancer cell interaction in vitro. Neurons, cancer cells and nuclei are stained with Tuj1 (green), panCK (red)
and DAPI (blue), respectively. (A) Liu, Y., Sun, Y., Yan, H., et al. (2012). Electrospun fiber template for replica molding of microtopographical neural
growth guidance. Small 5, 676–681, with permission from Wiley-VCH. (B) Jiang, X., Bruzewicz, D. A., Wong, A. P., Piel M., and George M. White-
sides G. M. (2005). Directing cell migration with asymmetric micropatterns. Proceedings of the National Academy of Sciences USA 102, 975–978, with
permission from National Academy of Sciences. (C) Chen, Z., Li, Y., Liu, W., et al. (2009). Patterning mammalian cells for modeling three types of
naturally occurring cell-cell interactions. Angewandte Chemie International Edition 48, 8303–8305, with permission from Wiley-VCH. (D) Yuan, B., Li,
Y., Wang, D., et al. (2010). A general approach for patterning multiple types of cells using holey PDMS membranes and microfluidic channels.
Advanced Functional Materials 20, 3715–3720, with permission from Wiley-VCH. (E) Lei, Y., Li, J., Wang, N., et al. (2016). An on-chip model for
investigating the interaction between neurons and cancer cells. Integrative Biology 8, 359–367, with permission from Royal Society of Chemistry.
Cell–cell interactions
Apart from the physical and chemical cues, cell–cell interactions also influence cell behavior. Microfluidic system is a great tool for
simulation of cell–cells interaction in vitro by selectively modifying the surface on the same substrates in micrometer-scale preci-
sion. In vivo cell interactions between different type of cells can be divided into three types: (1) both types of cells are immobilized
and confined to isolated areas, such as epithelial cells and fibroblasts during ovarian development; (2) one type is immobile and
another moves freely, for example, glial cells and neurons during the development of nervous system; (3) both types of cells could
move freely, for instance, hepatocytes and fibroblasts in the liver. We used an alkanethiol and a kind of ECM fibronectin to resist and
promote cells adhesion, respectively, thus created areas with different surface properties. We transported different types of cells to
designed locations on the same surface by employing microchannels and realized patterning multiple types of cells on the same
substrate to simulate three types of cell–cell interactions (Fig. 2C).
Patterning different types of cells on diverse substrates is crucial for studies of many processes in vitro such as cancer formation,
angiogenesis, and metastasis. We circumvented the substrates limitations of previous patterning techniques by employing a thin
PDMS membranes as the physical barrier to achieve desired surface properties and to deliver cells. We patterned three types of cells:
NIH 3T3 cells, Mardin Darby canine kidney (MDCK) cells, and JEC cells on substrates with micro-scaled grooves (Fig. 2D). After
cells adhesion, we removed the microfluidic channels and cells were free to migrate under the influence of each other and eventually
contacted each other. We patterned MDCK and NIH 3T3 cells onto PDMS substrates with microgrooves. Although MDCK cells
migrate much more slowly than 3T3 on flat substrate, the velocity of migration of MDCK parallel to the grooves is higher than
that of 3T3 perpendicular to the grooves. This experiment shows that both cell–cell and cell–substrate interactions simultaneously
influent cell group behaviors.
For cancer development, we designed a microfluidic compartmentalized device to simulate the interaction between neurons and
cancer cells (Fig. 2E) and demonstrated that nerves provided biophysical support for cancer cells and guidance for their directional
migration, which was consistent with clinical metastatic behaviors of multiple types of cancers.
Wound healing
Wound healing is a complex process, including hemostasis, inflammation, proliferation, and remodeling, which is involved in
multicellular activities. Microfluidics is an important tool to study these activities in vitro. For wound healing, we established
a microchip model comprising three parallel microfluidic channels for co-culture and wounding assay to study epithelial collective
migration. To mimic the scenario of wound tissue, we filled distilled water to the middle channel, causing wounded and normal
epithelial cells co-exist on the same substrate. We co-cultured MDCK and NIH 3T3 cells and confirmed the lysed epithelial cells and
fibroblasts could enhance epithelial collective migration without the influence of free physical space (Fig. 3A). The microfluidic chip
also allows us to in vitro screen wound dressing candidates that can minimize the use of animals for developing better method for
wound care. We also employed a cell-on-a-chip model to simulate the cutaneous wound and to in vitro screen the performances of
several ES fibrous wound dressings in enhancing wound healing. NIH-3T3 cells and MDCK cells were patterned on the Petri dish
using the microchip and then covered by the PCL/gelatin ES mats and bacterial cellulose (BC) mats (Fig. 3B). After peeling off the
covered mats, we could observe the migration of cell patterns and measure the distance of cell patterns to evaluate the effect of
different wound dressing on cell migration.
Microfluidics for 3D Patterning

Patterning cells on 2D substrates is significant in fundamental cell biology research, but is limited in mimicking the physiological
structure of tissues in vivo. 3D cell culture is rapidly gaining its popularity since embedment of cells in a 3D extracellular matrix is
associated with more relevant physiological behaviors. Microfluidics allow special control over fluids in micrometer-scale channels
and hence extend the physiological relevance of 3D culture models. By employing microfluidic techniques in 3D cell culture, we
could co-culture cells in a spatially controlled manner, control over signaling gradients, and mimic the microenvironments of in vivo
tissues.
Spatial control
Spatial control, as the basis of microfluidic 3D cell culture, allows patterning of cells and extracellular microenvironment. A recent
trend is to use hydrogels to offer cells more physiologically relevant 3D matrix. Hydrogels enable cells cluster together without need
for surface adhesion. If confined in the appropriate 3D microarchitecture, cells have the intrinsic potential to form aligned tissues
in vivo will self-organize into functional tissues in vitro.
Microvascular networks support metallic activity and define microenvironment conditions within tissues in health and
pathology. Recapitulation of functional microvascular structure in vitro could provide a platform for the study of complex vascular
phenomena, including angiogenesis and thrombosis. We engineer 3D vascular network in hydrogel by utilizing fibrillogenesis of
collagen and a liquid mold (Fig. 4A). The well controlled vascular network exhibited both mechanical stability for perfusing solu-
tions and biocompatibility for cell adhesion and coverage. This approach could be used for modeling of mass transfer-involved
physiology in vasculature-rich tissues and organs for regeneration and drug screening.
Blood vessel, composed of intima, media, and adventitia, is a typical complex 3D structure. By using a stress-induced rolling
membrane (SIRM) technique, we could transform simple patterns on 2D membranes into complex patterns in 3D tubes. These
tubes could mimic blood vessels in which different types of cells constitute different layers of the tubular wall, and cells on certain
Fig. 3 Microfluidics for wound healing. (A) Left: The schematic illustration of a microchip model for co-culture and selective wounding. Right:
MDCK cells alone or co-cultured with NIH 3T3 were patterned on a surface. MDCK cells in the middle channel were selectively lysed by distilled
water. (B) Left: The schematic illustration of cutaneous wound model in a cell-on-a-chip device. Cells were patterned on the bottom of a Petri dish
using the microchip. Right: NIH-3T3 alone, MDCK cells alone, co-cultured NIH-3T3 and MDCK cells were delivered to two sides to study the effects
of the wound dressing. (A) Xie, Y., Zhang, W., Wang, L., et al. (2011). A microchip-based model wound with multiple types of cells. Lab Chip 11,
2819–2822, with permission from Royal Society of Chemistry. (B) Li, Y., Wang, S., Huang, R., et al. (2015). Evaluation of the effect of the structure
of bacterial cellulose on full thickness skin wound repair on a microfluidic chip, Biomacromolecules 16, 780–789, with permission from American
Chemical Society.
Fig. 4 Microfluidics for 3D patterning. (A) Left: Schematic illustration of engineering of the vascular network in hydrogel. Middle: The vascular
network in serpentine and tree-like shapes. Right (top): The monolayer of HUVECs on the wall of branched hydrogel microchannels, stained by
Calcein-AM. Right (bottom): The left three images show one microchannel at different depths. The right image shows the cross-section. (B) Left:
Schematic illustration of a SIRM and tubes with multiple types of cells. Right: Three types of cells on a SIRM before and after rolling. Red, endothe-
lial cells; green, smooth muscle cells; blue, fibroblasts. (C) Schematic illustration of fabrication of the cell-laden multilayered PCL–PLGA tubes and
morphology changes during long-term incubation. (D) 3D neuronal networks on assembled microspheres. The left three are SEM images of neurons
on borosilicate microspheres. Middle: Spatial distribution of neurons. Different colors represent the normalized height to the substrate. Right: Polarity
of axons (smi312, arrows) and somatodendrites (MAP2, arrow heads). (A) Mu, X., Zheng, W., Xiao, L., Zhang, W., and Jiang, X. (2013). Engineering
a 3D vascular network in hydrogel for mimicking a nephron. Lab Chip 13, 1612–1618, with permission from Royal Society of Chemistry. (B) Yuan,
B., Jin, Y., Sun, Y., et al. (2012). A strategy for depositing different types of cells in three dimensions to mimic tubular structures in tissues.
Advanced Materials 24, 890–896, with permission from Wiley-VCH. (C) Cheng, S., Jin, Y., Wang, N., et al. (2017). Self-adjusting, polymeric multilay-
ered roll that can keep the shapes of the blood vessel scaffolds during biodegradation. Advanced Materials 29, 1700171, with permission from Wiley-
VCH. (D) Huang, Z., Sun, Y., Liu, W., et al. (2014). Assembly of functional three-dimensional neuronal networks on a microchip. Small 10,
2530–2536, with permission from Wiley-VCH.
layers align either longitudinally or circumferentially around the tube, mimicking their natural arrangement in vivo (Fig. 4B). Based
on SIRM technique, we designed structures, such as tubes with wrinkled walls, tubes-in-a-tube structures and spirals that are difficult
to fabricate with existing approaches. We also fabricated SIRM using non-elastic materials whereas reported structures of this type
usually require elastic materials. Based on the SIRM technique and electrospinning technique, we used PCL film as inner layer and
PLGA film as outer layers to form multicell-laden tubular structure (in vitro) or multilayered, acellular tubular structure (in vivo)
(Fig. 4C). The inner (PCL) layer of the tube can expand whereas the outer (PLGA) layers will shrink to maintain the stability of the
shape and the inner space of the tubular shape both in vitro and in vivo over months. This approach can be generally useful for
making scaffolds that require the maintenance of a defined shape, based on FDA-approved materials.
To develop 3D neuronal networks, we used microchamber-assisted assembly of borosilicate glass microspheres to fabricate scaf-
folds to support neurons (Fig. 4D). These neuron-carrying microspheric building blocks have many advantages, including a rela-
tively large surface to volume ratio, a highly ordered manner with the geometrical confinement of microchambers, sufficient
nutritional exchange through the spaces between the building blocks, good long-term stability of the 3D structure. By contrast,
the polymeric/hydrogel systems tend to deform and collapse.
Soluble molecular gradients

Soluble molecular gradients exist in various biological activities in vivo, such as angiogenesis, invasion and migration. The gradients
can be precisely controlled since microfluidics enables spatial control over fluids by altering the channel geometry and flow rates.
We fabricated gradients that range from pure laminin to pure bovine serum albumin (BSA) by using laminar flows in microchan-
nels. We cultivated rat hippocampal neurons on these substrate-bound gradients and found that axon specification is oriented in the
direction of increasing surface density of laminin. To generate temporally and spatially soluble gradients with more complex geom-
etries, researchers patterned ported microchannels within a 3D ECM and examined how geometry of endothelial vasculature alters
the special patterning of diffusive gradients and in turn influences the invasion of endothelial cells during angiogenic sprouting.
Microfluidic simulation of mechanical stimuli

Mechanical stimuli play important roles in cell regulate microenvironment of cells and development of many tissues. It is straight-
forward to establish dynamic microenvironment to mimic the in vivo condition of cells by employing microfluidic chips.
To realize real-time imaging of molecular dynamics of live cells, we fabricated a device for stretching cells adhering to elastic
membranes in equiaxial or uniaxial mode and obtained high-resolution images of stress fibers during the entire process of the stress
(Fig. 5A). We observed that several adjacent stress fibers reassembled into a single one after stretching, which indicated that mechan-
ical stretching played important roles in the rearrangement of actin filaments. To clarify specific mechanical properties of different
cells and tissues, we built an in vitro model to mimic the cellular microenvironment by combining both mechanical stretch and
geometrical control (Fig. 5B). We found that mechanical stretch determined the optimal geometry of myoblast C2C12 cells,
whereas vascular endothelial cells and fibroblasts had no such dependency.
In the cardiovascular system, endothelial cells (ECs) are constantly subjected to haemodynamic forces in the form of fluid shear
stress (FSS) and cyclic stretch (CS) resulting from blood flow and blood pressure. The two mechanical stimulations on ECs are
important for the maintenance of the physiological condition of the cardiovascular system and the development of cardiovascular
and cerebral diseases. To study the blood vessel biomechanics, we developed a microfluidic flow-stretch chip, which integrated FSS
and CS (Fig. 5C). By imposing FSS-only, CS-only, and FSS þ CS stimulation on rat mesenchymal stem cells and human umbilical
vein endothelial cells, we found that the alignment of the cellular stress fibers varied with cell type and the type of stimulation. This
flow-stretch chip is an ideal model for simulating the haemodynamic microenvironment.
The extravasation of tumor cells is critical in tumor metastasis. To study the mechanism underlying tumor cell extravasation, we
fabricated a microfluidic chip to simulate both mechanical and biochemical environments of human vascular systems and analyze
their synergistic effects on tumor extravasation (Fig. 5D). The Hela cells exhibited highest viability and adhesion activity in the envi-
ronment of capillary.
The arterial microenvironment plays a vital role in the pathology of atherosclerosis (AS). To address the interplay between arte-
rial microenvironment and atherogenesis, we employed a microfluidic AS model to recapture the atherogenic responses of endo-
thelial cells in ways that Petri dish could not (Fig. 5E). We discovered significant cytotoxicity of a clinical anti-atherosclerotic drug
probucol on the model, which does not appear on a Petri dish but is supported by previous clinical evidence.
In this part, we introduced recent advances of microfluidics for the manipulation of cell adhesion, migration, and surface engi-
neering. We also focused on microfluidic chip models for cell–cell interactions and wound healing. Apart from that, we discussed
microfluidic models for 3D patterning, which could mimic the complex in vivo microenvironments and precisely image dynamic
behaviors of cells. The microfluidics allows us to integrate multiple cell types, ECM, and various physical and chemical cues to
address basic problems in cell biology and provide robust platforms for fundamental biological research.
Microfluidic-Based Biochemical Analysis
Biochemical analysis is important for diagnosis and treatment of diseases. Many different methods enable biochemical analysis.
However, most of these methods rely on expensive equipment, trained technicians and complex operations, making them unsuit-
able for resource-limited areas. Microfluidic platform is a good choice for biochemical analysis as most microfluidic devices are
inexpensive, portable and easy to operate. In this part, we will show several typical methods for biochemical analysis based on
microfluidics, including nucleic acid detection, protein analysis and cell analysis.
Nucleic Acid Detection

Nucleic acids are the carriers of genetic information and exist in nearly all organisms. The detection of nucleic acid is thus quite
important for disease diagnosis and treatment. In some cases, the nucleic acid analysis can provide more accurate results than
the protein analysis since the genetic information of organisms is more stable than proteins, whose expression can be affected
by the environment. Besides, nucleic acid detection can be quite highly sensitive because nucleic acids can be amplified easily
by using different technologies, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP).
Fig. 5 Microfluidics for simulation. (A) Left: Design of the device and stretch modes: equiaxial stretch; uniaxial stretch. Black arrow heads represent
the force applied on elastic membranes. Right: Stress fibers of live NIH 3T3 fibroblast cells, transfected with EGFP-actin. Cells were stretched to
different extents in the uniaxial mode 16%; 26%; 32%, and relaxed. (B) Left: Top and side views of the stretching device. Right: Single C2C12 cells
were successfully confined in different shapes (blue, nuclear stained by DAPI; red, F-actin stained by Rhodamine Phalloidin). (C) Left: Schematic
illustration of the microfluidic flow-stretch chip. Right: The stress fibers tend to align in the direction parallel to that of the FSS, CS and the resultant
force of the FSS and CS. (D) Left: Schematic illustration of the vascular model chip. Middle: The adhesion of the tumor cells (HeLa, green) on the
ECs monolayer (HUVEC, orange) under different mechanical microenvironments of main artery, medium-sized artery, and capillary respectively. Right:
The adhesion of HeLa cells on the wall of the simulated major artery, medium-sized artery, and capillary respectively. (E) Left: Schematic illustration
of the early-stage AS model. Right: The morphology change of HUVECs on the Petri dish and the AS model before and after probucol treatment. The
white arrowhead points to contracted cells, the white arrow points to space between contracted cells. (A) Wang, D., Xie, Y., Yuan, B., et al. (2010). A
stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire
process of the stretch. Integrative Biology 2, 288–293, with permission from Royal Society of Chemistry. (B) Wang, D., Zheng, W., Xie, Y., et al.
(2015). Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment. Scientific Reports 4, 6106, with permis-
sion from Springer Nature. (C) Zheng, W., Jiang, B., Wang, D., et al. (2012). A microfluidic flow-stretch chip for investigating blood vessel biome-
chanics. Lab Chip 12, 3441–3450, with permission from Royal Society of Chemistry. (D) Huang, R., Zheng, W., Liu, W., et al. (2016). Investigation of
tumor cell behaviors on a vascular microenvironment-mimicking microfluidic chip. Scientific Reports, 5, 17,768, with permission from Springer
Nature. (E) Zheng, W., Huang, R., Jiang, B., et al. (2016). An early-stage atherosclerosis research model based on microfluidics. Small 12, 2022–
2034, with permission from Wiley-VCH.
Fig. 5 (continued).
Combining the amplification methods with microfluidic chips, the nucleic acids can be detected accurately with small amount of
samples.
PCR is a non-isothermal method for DNA amplification. A typical cycle of the PCR process includes three steps: denaturing (90–
95 C), annealing (60–65 C) and extension (70–75 C). So for a PCR-based microfluidic chip, the temperature should be controlled
carefully, which makes the microchip more complex. Researchers have made more efforts on isothermal amplifications, such as
LAMP.
LAMP is an isothermal nucleic acid amplification method. Besides DNA polymerase and deoxy-ribonucleoside triphosphates
(dNTPs), six different primers are also required for LAMP. Unlike PCR, whose products are DNA duplex exactly the same as their
parental chain, the products of LAMP have a loop structure. Compared with other isothermal amplification methods, LAMP has
high amplification efficiency and selectivity.
We integrated the LAMP on an eight-channel microchip for the detection of Pseudorabies virus (PRV) successfully (Fig. 6A).
After the injection of sample and the reaction mixture of LAMP, the microchannels were sealed with uncured PDMS to form isolated
microchambers. The microfluidic chip was incubated at 63 C for 1 h for the LAMP process. After amplification, the results could be
analyzed either by the naked eyes or by optical absorbance. The total process could be accomplished within 2 h, and 10 fg mL 1
DNA could be detected successfully. We designed an octopus-like multiplex microfluidic loop-mediated isothermal amplification
(mmLAMP) assay for the multiplex gene detection (Fig. 6B). With this mmLAMP, we could identify three human influenza A sub-
strains simultaneously, and the limit of detection (LOD) was less than 10 copies mL 1 in 2 mL quantity of sample.
In order to reduce the complexity of device fabrication, we used microcapillary to replace PDMS chip for LAMP process. We re-
ported a microcapillary-based LAMP for the detection of two RNA sequences of human immunodeficiency virus (HIV) (Fig. 6C).
The samples and reagents were introduced into the microcapillaries, and microcapillaries are sealed with epoxy glue. We put the
microcapillaries in two pieces of pocket warmers for 45 min for amplification. After that, fluorescence signal could be caught under
excitation. This device was totally equipment-free and easy to operate, and it was quite convenient for the areas with limited
resources.
To simplify the operation of nucleic acid extraction, we designed an integrated microcapillary for sample-to-answer nucleic acid
detection (Fig. 6D). In this device, a Flinders Technology Associates (FTA) membrane was inserted into the microcapillary for the
nucleic acid extraction and the relevant reagents were preloaded in the microcapillary for the LAMP process. When we carried out
the detection, we introduced the sample into the microcapillary through negative pressure and the nucleic acids were extracted when
the sample flow through the FTA membrane. We added a positive pressure at the other end of the microcapillary to push the
washing reagents and LAMP mix flow through the FTA membrane for the LAMP process. During the process, the two ends of
the microcapillary were sealed with the epoxy glue to avoid contamination. After amplification, the fluorescence signal could be
captured using a hand-held flashlight. We also reported a micro-pipette tip-based nucleic acid test (MTNT) (Fig. 6E). Combined
Fig. 6 Microfluidic-based nucleic acid detection. (A) Photograph (left) and schematic drawing (right) of the eight-channel microfluidic chip for
LAMP. (B) Photograph of the octopus-like multiplex microfluidic LAMP system. (C) Microcapillary-based loop-mediated isothermal amplification for
nucleic acids detection. The samples and reagents were introduced into the microcapillaries and the microcapillaries were sealed and put in two
pieces of pocket warmers for 45 min for amplification. Fluorescence signals indicated results. (D) Integrated microcapillary for nucleic acids detec-
tion. The FTA card and relevant reagents were preloaded into microcapillaries. The samples were introduced into microcapillaries through negative
force for nucleic acid extraction. The other reagents were pushed to the FTA card through positive pressure. After amplification, the fluorescence
signal could be caught be naked eyes. (E) Schematic illustration of the high throughput micro-pipette tip-based nucleic acid test for sample-to-
answer analysis. (A) Reproduced from Fang, X., Liu, Y., Kong, J. and Jiang, X. (2010). Loop-mediated isothermal amplification integrated on micro-
fluidic chips for point-of-care quantitative detection of pathogens. Analytical Chemistry 82, 3002–3006, with permission from American Chemical
Society. (B) Reproduced from Fang, X., Chen, H., Yu, S., Jiang, X. and Kong, J. (2011). Predicting viruses accurately by a multiplex microfluidic
loop-mediated isothermal amplification chip. Analytical Chemistry, 83, 690–695, with permission from American Chemical Society. (C) Reproduced
from Zhang, Y., Zhang, L., and Sun, J. et al. (2014). Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated
isothermal amplification. Analytical Chemistry 86, 7057–7062, with permission from American Chemical Society. (D) Reproduced from Zhang, L.,
Zhang, Y., and Wang, C. et al. (2014). Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Analyt-
ical Chemistry 86, 10,461–10,466, with permission from American Chemical Society. (E) Reproduced from Lu, W., Wang, J., and Wu, Q. et al.
(2016). High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips. Biosensors and Bio-
electronics 75, 38–33, with permission from Elsevier.
with a multichannel pipette, high throughput nucleic acid test could be achieved, and two copies of DNA could be detected success-
fully. Besides, this MTNT could be also used for different crude samples, including bacteria, cells and even solid plants.
Protein Analysis
Many proteins are biomarkers of diseases or physical conditions. So detecting proteins with fast and accurate methods is quite
meaningful for clinical use, as well as fundamental research. One of the biggest challenges for protein detection is that proteins
cannot amplify, unlike nucleic acids. So the methods for proteins detection should be highly sensitive and highly specific. The most
commonly used methods for protein detection are antibody-based immunoassays.
Antibodies are immunoglobulin that can recognize and bind tightly to their targets, which are known as antigens. Antibody-
based protein detection has both excellent sensitivity and selectivity because of the great affinities between either antibodies and
antibodies or antigens and antibodies. Enzyme-linked immunosorbent assays (ELISA) are the most commonly used method for
protein detection. There are several different kinds of ELISA, including direct ELISA, indirect ELISA, sandwich ELISA and competitive
ELISA.
Although there are different protocols according to different ELISA types, they all need the solid substrate to immobilize the
antigens or antibodies. So the substrate with higher immobilization efficiency is beneficial for detection. Many efforts have been
put on this field, including searching for better substrate, modifying the surface of the substrate, and changing the morphology
of the substrate. We compared electrospun polycarbonate (ESPC) membranes and track-etched polycarbonate (TEPC) membranes
as substrates for microfluidic immunoassays (Fig. 7A). Due to the larger specific surface area, the immunoassay using ESPC
exhibited higher sensitivity for HIV detection.
Blocking is the second step of conventional ELISA. We reported a microfluidic assay without blocking (MAWB) for HIV detection
(Fig. 7B). For the MAWB method, we incubated antigens in the microchannels to form protein stripes on the PDMS slab. Then the
microchannels as well as PDMS slab were washed with 0.05% PBST (phosphate buffered saline with 0.05% of Tween-20). The
microchannels were peeled off and the PDMS slab was rinsed with water and dried in the air. This process helped to block the acti-
vated sites with Teewn-20 quickly. Another microchip with parallel microchannels was utilized to combine with the PDMS slab
with the channels vertical to the stripes of antigens. The samples and the labeled secondary antibodies were introduced into the
microchannels for the immune-reaction. The LOD of this method was comparable to the conventional ELISA but the total assay
time was shortened about 25%.
To increase the detection sensitivity, many nanomaterials have been used as alternatives of enzyme for the signal readout. We
reported a nano- and micro-integrated protein chip (NMIPC) that uses water-soluble CdTe/CdS core-shell quantum dots (QDs) as
signal probes (Fig. 7C). We used this NIMPC to detect carcinoembryonic antigen (CEA). The dynamic range covered six orders of
magnitude, and the LOD was 500 fmol L 1, much lower than the conventional fluorophore-based methods. We also reported
a label free platform for protein analysis (Fig. 7D). We embedded gold nanoparticles (AuNPs) on a glass substrate through micro-
wave plasma methods to form Au substrate. The Au substrate was blocked with poly (ethylene glycol) (PEG) and functionalized
with biotin moiety. Streptavidin and glutaraldehyde were used to capture the antigens on the surface of the substrate. After the
capture of antigens, the interaction between AuNPs was affected and the local surface plasmon resonance (LSPR) peak of AuNPs
shifted. There was a positive correlation between the antigen concentration and peak shift. With a portable optical bench, the
peak shift could be measured by the UV–Vis spectrophotometer easily and the concentration of targets could be determined.
Conventional ELISA process needs several hours and researchers have made great efforts to shorten the reaction period. We
described a vacuum-accelerated microfluidic immunoassay that can shorten the reaction time significantly (Fig. 7E). This device
was consisted with two PDMS layers and a polycarbonate filter membrane that was sandwiched between the two layers. We intro-
duced the antigen solution in the top layers and applied a vacuum to the whole device. Because of the pressure difference, the solu-
tion flowed to the bottom layer, and the antigens were immobilized on the filter membrane. We introduced the labeled antibodies
to the bottom layer and applied a vacuum again for the immune reaction. We used this device to detect Sudan Red, an illegal food
additive. The total time required was less than 15 min and a LOD of 1 ng mL 1 was achieved.
Multiple protein detection is quite meaningful for biological applications. Western blot is a commonly used method for protein
detection. But normally just one kind of protein can be detected at one time. We reported a microfluidic-based Western blot
(Fig. 7F). With this microfluidic-based method, at least 10 proteins could be detected simultaneously, and the amount of antibody
needed was just 1% compared with the conventional Western blot.
Barcode-Based Multiplex Analysis

Recently, advanced microfabrication techniques make it available to detect multiplex samples or multiplex targets simultaneously,
but the data acquisition and analysis process still limit the throughput of these microdevices. We reported several barcode-related
devices for high-throughput multiplex analysis.
One of the limitations of data analysis is the lack of “function patterns,” so the region of interest (ROI) needs to be defined
manually. We designed a 33-channel microfluidic chip for multiplex analysis (Fig. 8A). A piece of microchannels was assembled
on a PDMS slab for the antigen immobilization. After immobilization, that piece of microchannels was peeled off and a second
one was assembled on the PDMS slab, perpendicular to the previous one, so that a 33 33 matrix was generated, just like
a two-dimensional barcode. During the process of immune reaction, we introduced positive control at the edges of the matrix,
which could act as “function pattern.” Within a computer program, the ROI could be performed automatically. The total time
required for the data acquisition and analysis was 10 s, more than 500 times faster than the conventional process.
The barcode readers and smart phones can make the process of data analysis easier. We designed a one-dimensional barcode-
based microchip that used the microchannels with different widths to imitate the common one-dimensional barcode (Fig. 8B). The
applicability of this design was demonstrated by the simultaneous detection of three HIV-related proteins and seven pathogen-
specific oligonucleotides. Recently, we reported a barcoded paper-based assay (BPA) for multiplex analysis (Fig. 8C). For this
BPA system, the constant regions which were not involved in the immune reaction were printed by the inkjet printer, and the
Fig. 7 Microfluidic-based protein analysis. (A) Top: A microfluidic device for bioassays with the ESPC membrane as solid substrate. Bottom: Sche-
matic illustration of the process of the immunoassay. (B) Schematic illustration of the microfluidic assay without blocking. Step-1: antigens immobili-
zation. Step-2: plasma introduction for immune reaction. Step-3: secondary antibodies introduction for signal readout. (C) Schematic illustration of
nano- and micro-integrated protein chip with water-soluble QDs as signal probe. (D) Schematic illustration of the label-free microchip for protein
analysis. (E) Schematic illustration of the vacuum-accelerated microfluidic immunoassay. (F) Schematic illustration of microfluidic-based Western
Blotting. Left: Proteins were transferred to polyvinylidene fluoride (PVDF) membrane by electroblotting and form protein bands. Right: Antibodies
were introduced into the microchannels that are perpendicular to the protein bands. (A) Reproduced from Yang, D., Niu, X., and Liu, Y., et al. (2008).
Electrospun nanofibrous membranes: A novel solid substrate for microfluidic immunoassays for HIV. Advanced Materials 20, 4770–4775, with
permission from Weily-VCH. (B) Reproduced from Song, L., Zhang, Y., and Wang, W. et al. (2012). Microfluidic assay without blocking for rapid HIV
screening and confirmation. Biomedical Microdevices 14, 631–640, with permission from Springer. (C) Reproduced from Yan, J., Hu, M., and Li, D.
et al. (2008). A nano- and micro- integrated protein chip based on quantum dot probes and a microfluidic network. Nano Research 1, 490–496, with
permission from Springer. (D) Reproduced from Zhang, Y., Tang, Y. and Hsieh, Y. et al. (2012). Towards a high-throughput label-free detection
system combining localized-surface plasmon resonance and microfluidics. Lab on a Chip 12, 3012–3015, with permission from Royal Society of
Chemistry. (E) Reproduced from Liu, Y., Yu, J. and Du, M. et al. (2012). Accelerating microfluidic immunoassays on filter membranes by applying
vacuum. Biomedical Microdevices 14, 17–23, with permission from Springer. (F) Reproduced from Pan, W., Chen, W. and Jiang, X. (2010). Micro-
fluidic western blot. Analytical Chemistry 82, 3974–3976, with permission from American Chemical Society.
Fig. 8 Barcode-based multiplex analysis. (A) The layout of the barcode-based two-dimensional microfluidic chip. (B) A barcoded microchip for
multiplex biomolecules analysis using barcode readers or smart phones. (C) Schematic illustration of the barcoded paper-based assay for multiplex
proteins and nucleic acids detection. (A) Reproduced from Zhang, Y., Qiao, L., and Ren Y. et al. (2013). Two-dimensional barcode-inspired automatic
analysis for arrayed microfluidic immunoassays. Biomicrofluidics 7, 034110, with permission from American Institute of Physics. (B) Reproduced
from Zhang, Y., Sun, J., and Zou,Y. et al. (2015). Barcoded microchips for biomolecular assays. Analytical Chemistry 87, 900–906, with permission
from American Chemical Society. (C) Reproduced from Yang, M., Zhang, W., Zheng, W, Cao, F. and Jiang, X. (2017). Inkjet-printed barcodes for
a rapid and multiplexed paper-based assay compatible with mobile devices. Lab on a Chip 17, 3874–3882, with permission from Royal Society of
Chemistry.
variable regions which have capture probes (CPs) were dispensed by the XYZ dispensing platform. We also designed a new group of
barcodes and programmed a new application (APP) for the readout of the barcodes. This BPA could be used to detect both proteins
and nucleic acids. Using this assay, the whole process of reaction and results analysis could be completed within 10 min.
Cell Analysis
Cells are the basic units of organisms for life activities. Isolation and analysis of certain cells are quite powerful methods for biomed-
ical research and applications. However, conventional approaches cannot manipulate cells precisely as the size of cells is quite
small, just around 10 mm. In this field, microfluidic methods have excellent performance as the micro-fabricated structures allow
better spatial and temporal control of biological samples.
Circulating tumor cells (CTCs) are tumor cells that shed from primary lesions and circulate in the bloodstream. CTCs are the
main reason for tumor recurrence and cancer metastasis. And CTCs detection can be employed to cancer diagnosis and therapy
monitoring. The main challenge for CTCs detection is that the abundance of CTCs in the bloodstream is very low, compared
with other blood cells. Many researchers have made efforts to develop effective methods to isolate CTCs with high efficiency, either
by physical force difference or by the affinity difference.
We designed a label-free cell sorter for CTCs isolation (Fig. 9A). The cell sorter was composed of a double-spiral microchannel
with one inlet and three outlets. When the blood sample flowed through the microchannel, different cells could be isolated due to
Fig. 9 Microfluidic-based cell analysis. (A) Top: Schematic illustration of the double-spiral microfluidic cell sorter for particle/cell separation.
Bottom: Schematic illustration of the two counter-rotating Dean vortices forming in the top and bottom halves of the microchannel. The height of the
microchannal was 50 mm. The CTCs flowed out from the middle outlet. (B) Schematic illustration of the microfluidic cell sorter with double-spiral
microchannel for cell separation. The height was 85 mm. The large CTCs flowed out through the inner outlet. (C) Schematic illustration of the inte-
grated microfluidic cell sorter for size-based CTCs isolation and enrichment. The height of the microchannel was 40 mm. After isolation, small lysed
red blood cells (RBCs) and white blood cells (WBCs) were removed from the side outlets, while the large CTCs were deflected into the middle outlet
and enriched by the membrane filter for further nucleic acid analysis. (A) Reproduced from Sun, J., Li, M. and Liu, C. et al. (2012). Double spiral
microchannel for label-free tumor cell separation and enrichment. Lab on a Chip 12, 3652–3660, with permission from Royal Society of Chemistry.
(B) Reproduced from Sun, J., Liu, C. and Li, M. et al. (2013). Size-based hydrodynamic rare tumor cell separation in curved microfluidic channels.
Biomicrofluidics 7, 011802, with permission from American Institute of Physics. (C) Reproduced from Wang, J., Lu, W. and Tang, C. et al. (2015).
Label-free isolation and mRNA detection of circulating tumor cells from patients with metastatic lung cancer for disease diagnosis and monitoring
therapeutic efficacy. Analytical Chemistry 87, 11,893–11,900, with permission from American Chemical Society.
their size difference. After separation, the small blood cells flowed out from the inner outlet, while the large CTCs flowed out from
the middle outlet. The recovery rate of tumor cells could be 88.5%. When the height of microchannel increased from 50 to 85 mm,
the small cells flowed out from the middle outlet, and the large cells flowed out from the inner outlet (Fig. 9B). Spiked HeLa cells
could be separated successfully from the 20 diluted blood sample using this cell sorter.
For the further analysis of CTCs, we integrated an 8 mm filter on the outlet for cell enrichment (Fig. 9C). After separation, the
CTCs were enriched by the filter. The capture efficiency of this cell sorter was determined to be 74.5 6.1%, and the enrichment
factor reached 3.75 106. We used LAMP for CTCs analysis with CK-19 mRNA as target nucleic acid. The results showed correlation
with the assessment gotten from X-ray computed tomography (CT). Compared with FDA-approval CellSearch system, this method
showed higher detection efficiency, especially for the epithelial mesenchymal transition (EMT) phenotype.
Microfluidic-based method has been widely used in biochemical analysis, both on molecule level and cell level. Compared with
conventional methods, microfluidic-based methods are time-saving, cost-saving and labor-saving. Besides, microfluidic-based
devices have been broadly used in point-of-care testing area, as it is convenient to make integrated platform by using microfluidic-
based devices.
Further Reading
Chen, Z., Li, Y., Liu, W., et al. (2009). Patterning mammalian cells for modeling three types of naturally occurring cell-cell interactions. Angewandte Chemie International Edition,
48, 8303–8305.
Chen, Y., Xianyu, Y., & Jiang, X. (2017). Surface modification of gold nanoparticles with small molecules for biochemical analysis. Accounts of Chemical Research, 50, 310–319.
Cheng, S., Jin, Y., Wang, N., et al. (2017). Self-adjusting, polymeric multilayered roll that can keep the shapes of the blood vessel scaffolds during biodegradation. Advanced
Materials, 29, 1700171.
Fang, X., Chen, H., Yu, S., Jiang, X., & Kong, J. (2011). Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip. Analytical Chemistry,
83, 690–695.
Gong, P., Zheng, W., Huang, Z., et al. (2013). A strategy for the construction of controlled, three-dimensional, multilayered, tissue-like structures. Advanced Functional Materials,
23, 42–46.
Huang, Z., & Jiang, X. (2013). Micro/nano-scale materials and structures for constructing neuronal networks and addressing neurons. Journal of Materials Chemistry C, 1,
7652–7662.
Jiang, X., Ferrigno, R., Mrksich, M., & Whitesides, G. M. (2003). Electrochemical desorption of self-assembled monolayers noninvasively releases patterned cells from geometrical
confinements. Journal of the American Chemical Society, 125, 2366–2367.
Jiang, X., Bruzewicz, D. A., Wong, A. P., Piel, M., George, M., & Whitesides, G. M. (2005). Directing cell migration with asymmetric micropatterns. Proceedings of the National
Academy of Sciences of the United States of America, 102, 975–978.
Jiang, B., Zheng, W., Zhang, W., & Jiang, X. (2014). Organs on microfluidic chips: A mini review. SCIENCE CHINA Chemistry, 57, 356–364.
Li, X. J., & Zhou, Y. (2013). Microfluidic devices for biomedical applications. The United Kingdom: Woodhead Publishing Ltd.
Li, Y., Yuan, B., Ji, H., et al. (2007). A method for patterning multiple types of cells by using electrochemical desorption of self-assembled monolayers within microfluidic channels.
Angewandte Chemie International Edition, 46, 1094–1096.
Liu, D., Xie, Y., Shao, H., & Jiang, X. (2009). Using azobenzene-embedded self-assembled monolayers to photochemically control cell adhesion reversibly. Angewandte Chemie
International Edition, 48, 4406–4408.
Mu, X., Zheng, W., Sun, J., Zhang, W., & Jiang, X. (2012). Microfluidics for manipulating cells. Small, 9, 9–21.
Sun, J., Li, M., Liu, C., et al. (2012). Double spiral microchannel for label-free tumor cell separation and enrichment. Lab on a Chip, 12, 3652–3660.
Sun, J., Xianyu, Y., & Jiang, X. (2014). Point-of-care biochemical assays using gold. nanoparticle-implemented microfluidics. Chemical Society Reviews, 43, 6239–6253.
Wang, J., Lu, W., Tang, C., et al. (2015). Label-free isolation and mRNA detection of circulating tumor cells from patients with metastatic lung cancer for disease diagnosis and
monitoring therapeutic efficacy. Analytical Chemistry, 87, 11893–11900.
Wu, J., He, Z., Chen, Q., & Lin, J. (2016). Biochemical analysis on microfluidic chips. Trends in Analytical Chemistry, 80, 213–231.
Yang, D., Niu, X., Liu, Y., et al. (2008). Electrospun nanofibrous membranes: A novel solid substrate for microfluidic immunoassays for HIV. Advanced Materials, 20, 4770–4775.
Yang, M., Zhang, W., Zheng, W., Cao, F., & Jiang, X. (2017). Inkjet-printed barcodes for a rapid and multiplexed paper-based assay compatible with mobile devices. Lab on a Chip.
https://doi.org/10.1039/c7lc00780a.
Yuan, B., Li, Y., Wang, D., et al. (2010). A general approach for patterning multiple types of cells using holey PDMS membranes and microfluidic channels. Advanced Functional
Materials, 20, 3715–3720.
Yuan, B., Jin, Y., Sun, Y., et al. (2012). A strategy for depositing different types of cells in three dimensions to mimic tubular structures in tissues. Advanced Materials, 24,
890–896.
Zhang, Y., & Jiang, X. (2013). Microfluidic tools for DNA analysis. In C. Fan (Ed.), DNA nanotechnology (pp. 113–153). Heidelberg: Springer.
Zhang, L., Zhang, Y., Wang, C., et al. (2014). Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Analytical Chemistry, 86,
10461–10466.
Zhang, Y., Zhang, L., Sun, J., et al. (2014). Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification. Analytical
Chemistry, 86, 7057–7062.
Zhang, Y., Sun, J., Zou, Y., et al. (2015). Barcoded microchips for biomolecular assays. Analytical Chemistry, 87, 900–906.
Zheng, W., & Jiang, X. (2014). Precise manipulation of cell behaviors on surfaces for construction of tissue/organs. Colloids and Surfaces B: Biointerfaces, 124, 97–110.
Organs-on-Chips
Yunki Lee, Song Ih Ahn, and YongTae Kim, George W. Woodruff School of Mechanical Engineering, Wallace H. Coulter Department
of Biomedical Engineering, Institute for Electronics and Nanotechnology, Parker H. Petit Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, Atlanta, GA, United States
Introduction 384
Vascular System 385
BraindThe Blood–Brain Barrier 385
Heart 386
Liver 387
Gut (Intestine) 388
Muscle 389
Lung 390
Kidney 391
Current Challenges and Future Prospect 392
Further Reading 392
Glossary
Clinical translation Clinical translation involves the application of discoveries made in the laboratory to diagnostic tools,
medicines, procedures, policies and education, in order to improve the health of individuals and the community.
Microfluidics The technology for the manipulation of nanoscale amounts of fluids in microscale fluidic channels for a wide
range of applications that include chemical synthesis, and biological analysis and engineering.
Organs-on-chips A microscale device that mimics biological systems and is used to probe complex human problems.
Introduction
The drug development has been strained by lengthy time lines and poor predictive power of preclinical studies, leading to
increased development costs. It is estimated that only one in nine drug candidates that enter clinical trials can reach the
market and that two thirds of the development costs are spent in the clinical testing stage. The low success rate and the
high expense in the later development stage highlight the importance of earlier stage predictive power using more versatile
and rapid preclinical models that more accurately predict drug safety and efficacy. However, current in vivo studies using
animal models may bear little relation to whole human physiology, and conventional in vitro models are based on two-
dimensional (2D) culture of transformed cell lines. Recent advanced three-dimensional (3D) cell culture models using custom
hydrogels remain to pose technical challenges in recapitulating physiologically relevant microenvironments of human organs
or tissues.
Meanwhile, microfluidic systems have shown great potential to mimic the physiology and functionality of an organ or tissue
interface. These microsystems are engineered with 3D cell culture and controlled mechanical and chemical cues to investigate
cellular dynamics and molecular pathways, which are extremely difficult to observe using current in vivo models. This so-called
“organs-on-chips” technology aims to recapitulate the chemical and mechanical microenvironment within human organs. The
underlying microtechnology of organs-on-chips overcomes several practical limitations including the large number of cells required
to build organ structure and function, the large volume of drug and other reagents needed to study, and the low throughput of
existing studies needed to drive the economics of the preclinical drug evaluation process. Recent years have reported new
approaches for organs-on-chips that reconstitute the structure and function of human organs and their interactions. Successful
development of organs-on-chips will enable scientists to predict more accurately the efficacy of therapeutic drug candidates, revo-
lutionizing drug development, disease modeling, and personalized medicine.
This article provides the fundamental understanding of the structure and function of key human organs, describes the cellular
features and microenvironments, and highlights current technologies to microengineer the key functional units in organs-on-chips
systems. It also includes brief discussion on current challenges and future prospects for these technologies to decode its potential to
practice.

Biomaterials: Biomaterial Applications and advanced Medical Technologies j Organs-on-Chips 385
Vascular System
The vascular system or the circulatory system carries blood and lymph throughout the body. The blood vessels like arteries and veins
carry blood throughout the body, transporting oxygen and nutrients to the pheripheral tissues and removing the wastes from target
tissues. The lymph vessels filter and drain lymph from the body to maintain the fluid environment. The dysfunction of the vascular
system therefore causes critical pathologies including cardiovascular diseases (e.g., hypertension, atherosclerosis, and restenosis),
tumor angiogenesis, and cancer metastasis.
The endothelium lining the inner layers of blood vessels in the cardiovascular system experiences pulsatile flow-induced wall
shear stress and transmural pressure, and pericytes and smooth muscle cells wrapping the outer walls of blood vessels contribute
to the stabilization of the vascular system (Fig. 1A). Intercellular communication between these cells is regulated by signaling path-
ways such as through gap junctions that allow the exchange of metabolites, ions, and other essential molecules. Direct interaction
between endothelial cells and smooth muscle cells faciliates the synchronization of their behaviors along the vascular wall.
The microengineered vascular system represents an undisputed advance in developing physiologically relevant vasculature
models. The microfluidic device integrated with micropost structures allows for the investigation of microvessel formation, organi-
zation, and function under highly controlled conditions including biochemical gradients and flow-mediated dynamics. One repre-
sentative approach is to replicate vasculogenic formation and angiogenic sprouting morphogenesis at the intraluminal side of the
microvascular network (Fig. 1B). The extracellular matrix-mimicking hydrogels are being developed to provide more physiologi-
cally relevant 3D microenvironment to vascular endothelial cells, pericytes, and smooth muscle cells. Recent advances in 3D bio-
printing technology allow high-precision construction of microarchitechture with hydrogel bioinks containing vascular cells and
extracellular matrix components. This recapitulation of the complex microarchitechture of a human vascularized tissue combined
with precisely controlled flow conditions with physiological context enables a perfusable capillary network within 3D extracelluar
matrix and maintains the endothelial barrier function.
Despite progress in engineering vascular systems, reproduction of the key biophysical and biochemical features of the basement
membrane and interstitial matrix in the microengineered vascular systems with the desired luminal structure and size requires more
physiologically relevant biomaterials and advanced microfabrication approaches. Co-culture with stromal cells and immune cells in
the developed vascular models is being studied with better maintenance of phenotypic cell stability and intercommunication.
Modeling of complicated vascular functions including regulation of blood pressure, vasoactivity, hemostatic balance, permeability,
and immunity and complex pathological conditions including thrombus formation and ischemia/reperfusion injury remains
challenging.
BraindThe Blood–Brain Barrier
The brain is a highly specialized organ serving as the centeral nervous system that controls dynamic and coordinated responses to
the environmental changes in other organs. These highly coordinated biological activities are maintained by the blood–brain
barrier, a selectively permeable structure that restricts the passage of nonlipophilic species or large substances (> 400 Da). Dysfunc-
tion of the blood-brain barrier leads to disruption of brain homeostasis, ultimately causing a variety of neurological disorders
including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis.
The blood-brain barrier is a highly organized structure that consists of specialized endothelial cells that line the blood vessels
with the basal lamina, pericytes that wrap around these vessels, and end-feet of astrocytes extending to the vesselsdcollectively
referred to as the blood–brain barrier contributing to the brain homestasis (Fig. 2A). The crosstalks between these cells maintain
the integrity of the blood-brain barrier. Pericytes and astrocytes act on the specialized endothelial cells to maintain an extensive
Fig. 1 (A) Schematic illustration of vascular system. (B) Microengineered vascular system to investigate microvascular network organization by
mechanical and biochemical stimuli. Reproduced from Kim, S., Chung, M., Ahn, J. et al. (2016). Interstitial flow regulates the angiogenic response
and phenotype of endothelial cells in a 3D culture model. Lab on a Chip 16(21), 4189–4199, with permission from Royal Society of Chemistry.
386 Biomaterials: Biomaterial Applications and advanced Medical Technologies j Organs-on-Chips
Fig. 2 (A) Schematic illustration of neurovascular functional unit consisting of neurons, blood–brain barrier including endothelial cells, pericytes,
astrocytes and glial cells. (B) Engineered neurovascular unit on a chip. (C) 3D human blood-brain barrier on a chip. (A and B) Reproduced from
Alcendor, D. J., Block, F. E., Cliffel, D. E. et al. (2013). Neurovascular unit on a chip: Implications for translational applications. Stem Cell Research &
Therapy 4(Suppl 1), S18, with permission from BioMed Central Ltd. Reproduced with permission from Herland, A.,van der Meer, A. D., FitzGerald, E.
A. et al. (2016). Distinct contributions of astrocytes and pericytes to neuroinflammation identified in a 3D human blood-brain barrier on a chip. PLOS
One 11(3), e0150360.
network of tight junctions, promote the expression of specialized transporters, and limit the rate of transcellular pinocytosis. In
addition, the extracellular matrix as well as the basement membrane of the endothelium plays a key role in the brain microenvi-
ronment homeostasis. While the basement membrane is predominantly made up of collagen, laminin, and fibronectin, the neural
extracellular matrix comprises a dense network of proteoglycans, hyaluronan, tenascins, and small amounts of fibrous proteins and
adhesive glycoproteins.
In vitro systems of the blood–brain barrier that incorporate all those key components with its capability for spatiotemporal regu-
lation of the microenvironment may provide a promising tool for prescreening and optimization of new drugs for neurological
diseases prior to animal and clinical trials. However, due to the complexity, no existing in vitro models have closely recapitulated
the blood–brain barrier with physiological context yet. The current state-of-the art model is a 3D multicompartment microfluidic
device that includes the central nervous system and cerebral spinal fluid, which is used to investigate the role of both the blood–
brain barrier and the blood-cerebral spinal fluid barrier in modulating chemical body–brain interactions, and to characterize the
interactions of pericytes, astrocytes, microglia, and neuronal and endothelial cells in the brain and its barriers (Fig. 2B). The critical
aspect of the in vitro blood–brain barrier model development is to mimic a 3D microenvironment of a brain tissue. Several multi-
component hydrogel scaffolds are widely utilized with manipulation of physicochemical properties such as matrix stiffness and
surface chemistry. The in vitro 3D models with an endothelial lumen structure have been primarily developed in a collagen type
I gel where the endothelial cells arranged in a cylindrical monolayer inside the microfludic platforms construct similar geometry
to brain microvessel (Fig. 2C).
No physiologically relevant in vitro neurovascular unit systems including the blood–brain barrier and 3D glial and neuronal
networks exist due to the complexity. Mimicking brain microenvironments for better maintenance of brain cell phenotypic stability
remains challenging. In addition to the recent focus on the development of blood–brain barrier models, 3D neural cultures
designed to manipulate the complex neuronal networks, long processes, and synapses are required for better modeling neurological
diseases, as well as drug screening.
Heart
The heart is a muscular organ that pumps blood through the circulatory system. The heart wall consists of three cellular layers: the
outer epicardium, middle myocardium, and inner endocardium. The epicardium, a thin membrane that covers the myocardium,
functions as a source for multipotent progenitor cells, and regulates cardiomyocyte proliferation, differentiation, and regeneration.
The myocardium, a layer located between the epicardium and the endocardium, comprises the primary contractile unit of the heart
(Fig. 3A).
A cardiomyocyte is the primary cell type in the myocardium that terminally differentiate into a muscular cell, which accounts for
the transmission of electrical signals (e.g., action potential) through the gap junctions, coordinating the contractile activity of the
muscle. These physiological functions of the cardiac tissue are maintained with other important integrated components: fibroblasts,
extracellular matrix, and vascular systems. The components for the extracellular matrix in the heart tissue include collagen (type I
and III), proteoglycans, fibronectin, and basement membrane proteins. The cell–cell interactions have a significant impact on the
tissue responses via the secretion of a combination of different factors (e.g., growth factor, cytokine) that modulate the molecular
composition of the cardiac tissue microenvironment. Such an organized orchestra of cells and tissue factors regulates, stabilizes, and
reinforces the cardiac phenotype. It is the concerted interactions between the cells, extracellumar matrix, and signaling molecules all
together that contribute to the development and maintenance of the functions of the heart.
Fig. 3 (A) Schematic illustration of anatomic structure and organization of cardiac-muscle tissue. (B) Engineered heart-on-a-chip device based on
muscular thin film technique. Reproduced from Agarwal, A., Goss, J. A., Cho, A. et al. (2013). Microfluidic heart on a chip for higher throughput
pharmacological studies. Lab on a Chip 13(18), 3599–3608, with permission from Royal Society of Chemistry.
To study cardiac physiology, pathology, and pharmacology, engineered heart-on-a-chip platforms have to provide precise
control over these essential factors; for example, combination of topographical and electrical cues to engineer the cardiac tissue
with matured neonatal rat cardiomyocytes in microscale devices allows for increased cellular maturation and elongation with
gap junction formation. The current state-of-the-art of heart-on-a-chip is to use a muscular thin film technique to recapitulate
the structure and function of the myocardium (Fig. 3B). This system enables the real-time measurement of the cardiac muscle tissue
contraction in response to pathophysiological conditions combining mechanical, electrical, and chemical stimuli. The advance of
this technology has demonstrated not only microcontact printing of fibronectin on the thin flexible PDMS films to electrically stim-
ulate anisotropically organized cardiomyocytes, but also, multiple engineered cardiac tissues on a chip for drug testing applications
in a robust and high-throughput manner using 3D printing technology.
While most heart-on-a-chip platforms use animal-derived primary cells, more focus on integration of human origin and human-
derived iPSCs with these models will be required for better mimicking human physiological response. In addition, development of
3D engineered myocardium microenvironment and integration of these platforms with vasculature remains challenging. Engi-
neered cardiac tissue constructs that include both physiologically relevant mechanical and electrical properties and provide real-
time measurement of contractility and action potential signals will impact the field for the applications of in vitro drug testing,
disease modeling, and biological mechanistic studies.
Liver
The liver, the largest organ in the body, performs essential metabolic functions that include synthesis of proteins (e.g., albumin),
detoxification of endogenous and exogenous substances ingested through the gastrointestinal tract, and production of biochemicals
necessary for digestion. The internal structure of the liver has approximately 100,000 small hexagonal functional units known as
lobules, where the oxygen consumption creates the functional heterogeneity between periportal and perivenous zones (i.e., liver
zonation) during methabolism and detoxification process.
The hepatocyte, a majority of cells in the liver that constitute 70%–85% of the liver’s mass, performs primary functions including
metabolism, detoxification, storage, digestion, and bile production. The walls of hepatic sinusoid are lined by three different cell
types: sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells (Fig. 4A). The liver sinusoids are lined with sinusoidal
endothelial cells, separating hepatocytes from flowing blood. A thin extracellular matrix in the space of Disse between hepatocytes
and endothelial cell layer is composed of fibronectin, collagen, laminin, and other basement membrane proteins, and retains
important substances including glycosoaminoglycans, growth factors (e.g., basic fibroblast growth factor, hepatic growth factor,
and vascular endothelial growth factor) and cytokines. Kupffer cells preferentially reside in the sinusoid of the periportal zone,
and form the macrophase population. Quiescent stellate cells located in the space of Disse store vitamin A, and regulate regener-
ation, vascular remodeling, differentiation, and inflammation.
To mimic the liver microenvironment, long-term maintenance of hepatocyte viability and functionality is required for the
metabolism and liver toxicity studies. Incorporation of a perfusable flow provides continuous nutrient/oxygen supply and physi-
ological shear stress required to recapitulate liver tissue microenvironment. These approaches are enhanced with efforts to simulate
the physiologically relevant morphology of the liver functional units; endothelial barrier of liver sinusoid and hepatic lobule struc-
ture, highlighting the importance of structure-function relationship of these modeling approaches and combining physiological
cues such as dynamic flow and vascularization within the liver-like structure (Fig. 4B). To predict drug and biothreat agent toxicity
under physiological conditions in real-time instead of conventional end-point assays, microengineered liver-on-a-chip platforms
have to integrate electrochemical measurement technology. Incorporation of oxygen sensors in a liver-on-a-chip bioreactor device
allows for the detection of minute changes of oxygen concentrations across the tissue and the real-time tracking of the dynamics of
Fig. 4 (A) Schematic illustration of the basic liver tissue unit, hepatic lobule and sinusoid. Microengineered (B) in vitro lobule-mimetic and (C) liver
sinusoid-like bioreactor models consisting of hepatocytes and endothelial cells. Reproduced from Ho, C. T., Lin, R. Z., Chen, R. J. et al. (2013).
Liver-cell patterning lab chip: Mimicking the morphology of liver lobule tssue. Lab on a Chip 13(18), 3578–3587; Domansky, K., Inman, W., Serdy, J.
et al. (2010). Perfused multiwell plate for 3D liver tissue engineering. Lab on a Chip 10(1), 51–58, with permission from Royal Society of Chemistry.
metabolic adaptation to mitochondrial dysfunction (Fig. 4C). In addition, combining this monitoring technology with 3D bio-
printing platforms, researchers have encapsulated hepatic spheroids in hydrogels printed directly into the microfluidic bioreactor
device, allowing for the evaluation of drug-induced toxicity through dynamic monitoring of metabolite production.
Current challenges include the development and long-term validation of reliable 3D disease models that mimic vascularization
and dynamic flow in the liver and the availiability of human cell sources such as iPSC-derived human liver cells, which may extend
the potential for the personalized drug screening applications.
Gut (Intestine)
As a vital organ in the gastrointestinal tract of digestive system, the intestine operates several essential functions such as digestion,
nutrient transport into blood circulation, metabolism, and elimination of toxic substances. The intestinal wall composed of various
differentiated cell types with a tight junction and covered by a mucus layer forms a highly selective barrier whose physiological func-
tions are closely associated with the peristalsis, villi of the mucus layer, and microbiome activity (Fig. 5A). The intestinal epithelium
regulates these fundamental immune-regulartory functions through the secretion of mucins and antimicrobial peptide while gut
microbiome prevents infection and controls immune system through the balanced host-gut microbiome crosstalk. Infections,
inflammation, and drug toxicity can cause epithelial dysfunction and gut microbiome imbalance, such as inflammatory bowel
disease.
Fig. 5 (A) Schematic illustration of native intestinal wall with villi structure and tissue interface with blood vessel. (B) Design of human gut-on-a-
chip microdevice composed of the gut lumen and blood compartment. Cyclic suction to both side channels exerts peristalsis-like motion, ultimately
producing relevant host-microbe interaction. Reproduced from Kim, H. J., Huh, D., Hamilton, G. et al. (2012). Human gut-on-a-chip inhabited by
microbial flora that experiences intestinal peristalsis-like motions and flow. Lab on a Chip 12(12), 2165–2174, with permission from Royal Society of
Chemistry.
Most substances and drugs absorbed through the intestine into the blood stream diffuse across the mucus layer, the epithelial
cell layer of the intestine wall, basement membrane, and the endothelial cell layer lining the capillaries. The intestinal epithelium is
composed of different types of epithelial cells mainly including enterocytes and goblet cells. The enterocytes covered with surface-
enhancing microvilli produce various catabolic enzymes on their exterior luminar surface for the absorption and methabolism func-
tions. In most areas of the intestine, the goblet cells secrete the mucus layer which protects the epithelial layer from the luminal
contents, and play critical roles in regulating innate immune responses and homeostasis. The basement membrane, consistsing
of laminin, collagen type IV, and proteoglycans, supports the intestinal epithelium for tissue development, function, and repair.
The current state-of-the art model of gut-on-a-chip is based on double-layered perfusable channels separated by an extracellular
matrix-coated porous flexible membrane in order to simulate the intestine lumen and the blood compartment (Fig. 5B). The intes-
tinal epithelial cells form a high integrity epithelium-barrier on one side of membrane with contiuous fluid shear stress while the
cyclic mechanical deformation triggered by stretching and relaxing the porous membrane mimics physiological peristaltic motions.
This mechanically active platform enables the cell polarization and differentiation into a columnar epithelium and the subsequent
intestinal villi formation. When co-cultured with a normal intestinal microbe on the luminal surface, the intestinal microenviron-
ment engineered to mimic peristalsis-like motion and fluid flow improves the intestinal barrier function. This physiological organ-
level function reconstitution has not been possible in 2D static culture system due to contamination and overgrowth of the microbe.
This platform is being further adapted to study inflammation response at tissue and organ levels, and antibiotic and probiotic
therapeutic effects toward injured intestinal epithelium, thereby verifying host-microbiome crosstalks in intestinal pathophysiology
and dissect disease mechanisms. These in vitro models provide new insights of the interplay between multiple different types of cells
and tissues (e.g., intestinal epithelial/endothelial cells, immune cells, commensal microbe and pathogenic bacterial), molecular
components, and physical cues in an organ-relevant microenvironment. Some other recent modeling approaches include host-
microbe interface model under aerobic-anaerobic conditions that highlight the significant technical advance in manipulating meta-
bolically mismatched microenvironments. Current challenge in this technology is to obtain healthy primary human cells for
improved extrapolation of results to humans and to contain a host of “non-GI” cells including mast cells which play a key part
in the barrier integrity and physiology of the intestinal epithelium.
Muscle
Skeletal muscle tissue accounts for 30%–40% of total body weight, and is responsible for gross movements. Skeletal muscle consists
of myofilaments, sarcomeres, myofibrils and muscle fiber. The sarcomeres, the muscle contractile units, result from the assembly of
myosin and actin myofilaments. The repetition of sarcomeres generates myofibrils, which assemble into myofibers, long cylindrical
multinucleated muscle cells. Muscle fibers are the basic unit of muscles and organize into fascicles, which are surrounded by
a network of blood vessels and nerve fibers (Fig. 6A). Pathological conditions include muscular genetic disease such as Duchenne
Fig. 6 (A) Anatomic structure of organized skeletal muscle fiber surrounded by capillary blood vessels and neuronal network. (B) In vitro model
of neuromuscular junction cocultured with motoneuron cell bodies and muscle bundles in the microfluidic device. (i–ii) Representative images of
neuron-muscle coculture and neurite extension. Axons and myocytes are immunoreactive for NFM and desmin, respectively. (iii) Visualization of
fibroblast (green), motor axon (red) and myotubes (blue) in the distal chamber. Reproduced from Southam, K. A., King, A. E., Blizzard, C. A. et al.
(2013). Microfluidic primary culture model of the lower motor neuron-neuromuscular junction circuit. Journal of Neuroscience Methods 218(2), 164–
169, with permission from Elsevier.
muscular dystrophy and neuromuscular degenerative disease such as amyotrophic lateral sclerosis. In case of the mutation in the
dystrophin gene, the abnormal transmission of contraction alters the muscle contractility, leading to muscle atrophy.
The microenvironment of the skeletal muscle is composed of myofiber, extracellular matrix, and cellular components including
muscle satellite cells, fibroadipogenic progenitors, and endothelial cells in the microvalsculature and neuronal network. The satellite
cells, which make up approximately 2%–10% of the total number of myonuclei, are quiescent in physiological condition, but are
activated for proliferation and differentiation to form new myofibers consisting of multinucleated myotubes in response to
mechanical and chemical stimuli resulting from injurious events. These myofibers are covered by a thin layer of connective tissues
mostly composed of laminin and collagen type IV. The cells in the muscle microenvironment are anatomically adjacent to each
other, and their behaviors are strictly regulated through the dynamic interplay with vasculature. Vascular inflow into muscle takes
place through the arteries that are distributed along muscle fibers. Vessel branching occurs obliquely or perpendicularly to the main
vessels thereby surrounding muscle fibers, and allow for their perfusion. The contractitility of aligned muscle fibers is modulated by
electrical stimulation. Motoneurons located in the spinal cord innervate skeletal myofibers by forming neuromuscular junctions
and trigger muscle contraction by releasing transmitter acetylcholine.
Generation of functional muscle fibers requires fine control of topographical (e.g., surface micropatterning/microgrooves),
biochemical (e.g., soluble factors and matrix composistion), mechanical (e.g., cyclic strain and matrix stiffness), and electrical
ques in the microdevices. Incorporation of these stimulations increases cellular actitivites including myogenic differentiation
and myotube formation, maturation and alignment, thus effectively improving the contractile behavior of engineered muscle tissue
units. Another important characteristic in the muscle microenvironment, the neuromuscular junctions are constructed to investigate
cell–cell interaction between motor neurons and muscle cells, which is crucial to understand mechanisms leading to muscle degen-
eration or diseases (Fig. 6B). Precise spatiotemporal control of local chemical environment allows for investigation of the effect of
chemoattactant gradients or neuron-secreted factors on myotube functions in co-culture condition. Incorporation of compartmen-
talization system with microgrooves allows axonal outgrowth from motoneuron cell bodies and formation of neuromuscular junc-
tions with muscle cells. More recent approaches mimic more realistic morphology, and increase physiological relevance with 3D
functional neuromuscular junction platforms engineered by coculturing with myoblast-derived 3D muscle strips and motor
neurons within a hydrogel matrix. These 3D muscle tissue models compartmentalize muscle fibers and motor meurons, mimicking
the geometry of the spinal cord-limb physical separation and facilitating real-time monitor axonal outgrowth and muscle innerva-
tion. The combination strategy of microfluidic and optogenetic technologies enables development of highly controllable and phys-
iologically relevant tissue model for motor units and ultimately for drug screening assay applications.
The major challenges lie in the manipulation of cues that induce differentiation toward contractile lineage, the understanding of
how contraction is impaired in the muscular disorders and how to rescue it, and the engineering of de novo surrogates for tissue
repair or nonmedical applications. Current models still lack important features that would enable them to fully characterize skeletal
muscle pathologies. To better characterize the response of pathological muscle models, the readout of important parameters such
as muscle contractility, indicative of muscle health, needs to be added to the systems on the basis of similar apparatuses reported for
muscle models. Lastly, novel findings indicate how the pathological muscular endothelium is an important element to be consid-
ered, although vascularized pathological muscle model is not currently available.
Lung
The lung is a primary organ of the respiratory system that performs gas (oxygen–carbon dioxide) exchange through passive diffusion
in the basic functional unit, alveoli, which are hollow cavities surrounded by numerous pulmonary capillaries. The alveolar wall is
a physical barrier that is composed of two cell layers of the alveolar epithelium and the vascular endothelium, and that contributes
to the regulation of lung tissue homestasis (Fig. 7A). The alveolar epithelial cells are exposed to mechanical stresses that have critical
roles in the regulation of the key pulmonary functions, including their response to environment-induced damage by releasing bio-
logically active factors.
The defense mechanisms of lung tissue against various environmental insults (e.g., pathogens and tissue damaging agents)
include secreting antimicrobial peptides and inflammatory mediators, and initiating immune cell recruitment to the damaged or
infected site. Through the epithelial-endothelial crosstalk, various mediators including chemokines, cytokines, growth factors
and other small molecules such as nitric oxide or reactive oxygen species are released to recruit innate and adaptive immune cells.
The infection and chronic inflammation are responsible for most lung diseases, particularly chronic obstructive pulmonary diseases
including chronic bronchitis, emphysema, and asthma. The extracellular matrix of the pulmonary alveoli is composed of collagen
(type I and III), elastin, glycosaminoglycans and proteoglycans. In the lung tissue, the pulmonary extracellular matrix determines
lung-tissue strucuture, and provides mechanical stability, elastic recoil and maintenance of normal interstitial fluid dynamics, which
are critical for physiological lung function. Moreover, biochemical/mechanical signals initiated by the extracellular matrix regulate
cellular behavior, and play key roles in development, remodeling and homeostasis of the lung tissue.
To replicate the strcutrual and functional complexity of the alveolar-capillary interface in the lung, the state-of-the art lung-on-a-
chip system leverages compartmentalized microfluidic chambers separated by a porous membrane to create the microarchitecture
and dynamic microenvironment of the alceolar-capillary unit (Fig. 7B). The epithelial and endothelial cells are co-cultured on the
opposite sides of extracellular matrix-coated membrane, and are respectively exposed to air and flowing media, ultimately resulting
in air–liquid barrier formation in microdevices. The cyclic stretching implemented in the system allows for the simulation of the
Fig. 7 (A) Schematic illustration of the lung functional unit. (B) (i) Cyclic stretchable lung-on-a-chip microdevice consisting of epithelial and endo-
thelial cell layers for reproducing the alveolar–capillary barrier in a microfluidic device. (ii) A tissue–tissue interface consisting of a single layer of the
alveolar epithelium closely apposed to a monolayer of the microvascular endothelium by long-term microfluidic co-culture. Reproduced from Huh, D.,
Kim, H. J., Fraser, J. P. et al. (2013). Microfabrication of human organs-on-chips. Nature Protocols 8(11), 2135–2157, with permission from Nature
Publishing Group.
lung physiological breathing motion. This mechanically active microdevice reproduces organ-level responses to bacteria and proin-
flammatory cytokines as well as nanoparticles, which provides more realistic in vitro models to study pathogen-triggered patholog-
ical mechanism and body’s response to drugs. This platform is continuously used to simulate human lung diseases such as
pulmonary inflammation by perfusing proinflammatory factors. Chronic obstructive pulmonary diseases are also modeled by
lining the airway with epithelial cells and stimulating with viral mimic polyinosinic–polycytidylic acid or lipopolysaccharide
endotoxin.
Current challenge in the microengineered lung-on-a-chip models include the applications of human primary cells or specific
patient-derived cells, which would enable creating more relevant and complex hallmarks of human lung diseases. In addition,
more cell types such as pneumocyte II or macrophage immune cells would be integrated in the well-estabilised model to assess
some mechanisms in certain aspects of lung response to inflammation and infection.
Kidney
The kidney is a vital organ that maintains hemeostasis in the body and controls blood pressue through blood filtration, waste excre-
tion, and essential substance reabsorption. It also plays a primary role in the elimination of toxins and their metabolites, therefore is
susceptible to injury during the toxin removal process. The kidney tissue consists of an inner medulla, an outer cortex and millions
of nephrons, the basic structural and functional unit of the kidney. The nephron is composed of two main structures: the glomerulus
that is responsible for renal ultrafiltration and the renal tubule for reabsorption of useful substances such as water, glucose, and
amino acid (Fig. 8A).
The glomerular filtration barrier consists of endothelial cells, podocytes, mesangial cells, and epithelial cells. The endothelial and
epithelial monolayers share the extracellular matrix known as the glomerular basement membrane where these three distinctive
layers form the glomerular filtration barrier. The filtration and excretion mechanism include size, charge, and shape selectivity at
the glomerular barrier that has size-selective slit nanopores in foot processes and surface negative charge of glomerular endothe-
lium. In contrast, the renal tubular reabsorption is the process where the removed water and solutes from the glomerular capillaries
transport into the blood circulatory system to maintain homeostasis, which mostly occurs in the proximal tubule by osmotic pres-
sure and active transport of the tubular epithelial cells. Impairment of the renal filtration by disrupted epithelial cell junctions causes
failure of homeostasis, leading to kidney diseases. Acute renal injury is caused by decreased blood flow to the kidneys or injury from
drugs and viral infection. Chronic renal failure includes inflammation such as glomerulonephritis or pyelonephritis, polycystic
kidney disease, renal fibrosis and kidney stone.
The kidney-on-a-chip platforms recapitulate tissue- and organ-level physiology of the nephron functional unit, focusing on the
renal tubule and capillary microenvironment of the kidney-specific functional unit on a chip. The state-of-art proximal tubule
model is a multilayer microfluidic device system consisting of luminal flow channel and interstitial space compartmented by
a porous membrane. The integration of relevant fluidic shear stress and transepithelial osmotic gradients increases the physiological
relevance, demonstrating polarization and cytoskeletal reorganization of the renal tubular epithelial cells. This approach is
enhanced to construct the kidney glomerulus as well as disease model. The diabetic nephropathy disease model of the glomerular
endothelial barrier was estabilished in the compartmentalized microfluidic device consisting of tissue-specific cellular components
and 3D extracellular matrix (Fig. 8B). Under fluid flow condition, 3D hydrogel membrane was lined by endothelial cells and podo-
cytes, and served as the glomerular filtration barrier, showing selective permeability for large proteins. This device was also
employed to verify high glucose-induced pathological responses where hyperglycermia proved to cause glomerular dysfunction
Fig. 8 (A) Schematic illustration of nephron functional unit (left) and glomerular structure (right) in kidney tissue. (B) In vitro glomerular model
consisting of glomerular endothelial cells, podocytes and hydrogel membrane. This design mimics the glomerular filteration barrier. Reproduced from
Wang, L., Tao, T., Su, W. et al. (2017). A disease model of diabetic nephropathy in a glomerulus-on-a-chip microdevice. Lab on a Chip 17(10), 1749–
1760, with permission from Royal Society of Chemistry.
with increased barrier permeability. In addition, to better recapitulate in vivo microenvironment, with combining bioprinting tech-
nique, perfusable 3D human renal proximal tubule was created on a chip. This engineered 3D tubule model promoted tissue-like
epithelium formation with enhanced epithelial morphology and functional properties, compared to the 2D system.
Most work leverages double-layered microfluidic compartmentalization to develop the renal tubular microenvironment of the
nephron. More studies are now focusing on mimicking a glomerular filtration barrier structure and function, which will contribute
to the modeling of many renal diseases that are related to impaired activity of glomerulus. Glomerular dysfunction leads to the
alteration of podocyte foot processes, which is responsible for inefficient blood ultrafiltration and ultimately proteinuria and other
pathological diseases. Introduction of primary human cells into this microengineered nephron functional unit would provide more
realistic physiological and pathological in vitro models for studying drug therapeutic efficacy and toxicology to kidney tissues.
Current Challenges and Future Prospect
The overall challenge of this field is to ultimately scale up these microengineered organ constructs to the volume required for phar-
maceutical applications and to achieve reliable high-throughput systems for efficient drug screening. More practically, the central
question lies in how to create the simplest yet physiologically relevant (and also useful) models that reproduce the critical functions
of interest organs with reliable human primary cell sources.
To further recapitulate the structure and function of human organs, the current organs-on-chips technologies need to maintain
a healthy physiological state where nutrients and waste products do not reach unacceptably low or high levels and establish preci-
sion fluid flow on organ model platforms for controlling drug, nutrient, and metabolite concentrations and applying fluid shear to
cultured cell populations. Moreover, automation technologies should be incorporated for monitoring cell culture conditions in
multiwell plates as well as real-time assessment of morphology, cell growth, and protein expression. Real-time measurement of
basic cell culture parameters such as oxygen, temperature, pH, and molecular measurements will further expand this innovative
technologies for the rapid translation of drug candidates to the clinic. Lastly, due to the complexity of each organ-on-a-chip system,
multiorgan system integration and scaling remain challenging in the context of the application to drug screening.
Successful development of organs-on-chips with these advances will be able to improve the cost effectiveness and predictive
power of preclinical studies assessing the safety and efficacy of compounds (e.g., absorption, distribution, metabolism, and excre-
tion), and eliminate ineffective drug candidates as early as possible. Furthermore, this innovative approach may allow us to be able
to respond to newly evolving demands including personalized or precision medicine, rising rates for many chronic diseases, and
threats from emerging infectious diseases.
Further Reading
Benam, K. H., Villenave, R., Lucchesi, C., et al. (2016). Small airway-on-a-Chip enables analysis of human lung inflammation and drug responses in vitro. Nature Methods, 13(2),
151–157.
Bersini, S., Arrigoni, C., Lopa, S., et al. (2016). Engineered miniaturized models of musculoskeletal diseases. Drug Discovery Today, 21(9), 1429–1436.
Huh, D., Matthews, B. D., Mammoto, A., et al. (2010). Reconstituting organ-level lung functions on a chip. Science, 328(5986), 1662–1668.
Khetani, S. R., & Bhatia, S. N. (2008). Microscale culture of human liver cells for drug development. Nature Biotechnology, 26(1), 120–126.
Kim, H. J., Li, H., Collins, J. J., et al. (2016). Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.
Proceedings of the National Academy of Sciences of the United States of America, 113(1), E7–E15.
Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A., & Lewis, J. A. (2016). Three-dimensional bioprinting of thick vascularized tissues. Proceedings of the National Academy of
Sciences of the United States of America, 113(12), 3179–3184.
Lind, J. U., Busbee, T. A., Valentine, A. D., et al. (2017). Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing. Nature Materials, 16(3),
303–308.
McCain, M. L., Sheehy, S. P., Grosberg, A., Goss, J. A., & Parker, K. K. (2013). Recapitulating maladaptive, multiscale remodeling of failing myocardium on a Chip. Proceedings of
the National Academy of Sciences of the United States of America, 110(24), 9770–9775.
Park, G. S., Park, M. H., Shin, W., et al. (2017). Emulating host-microbiome ecosystem of human gastrointestinal tract in vitro. Stem Cell Reviews and Reports, 13(3), 321–334.
Uzel, S. G., Platt, R. J., Subramanian, V., et al. (2016). Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units. Science
Advances, 2(8), e1501429.
Wilmer, M. J., Ng, C. P., Lanz, H. L., et al. (2016). Kidney-on-a-Chip Technology for drug-induced nephrotoxicity screening. Trends in Biotechnology, 34(2), 156–170.
Wong, K. H., Chan, J. M., Kamm, R. D., & Tien, J. (2012). Microfluidic models of vascular functions. Annual Review of Biomedical Engineering, 14, 205–230.
Yi, Y., Park, J., Lim, J., Lee, C. J., & Lee, S. H. (2015). Central nervous system and its disease models on a chip. Trends in Biotechnology, 33(12), 762–776.
Yoon No, D., Lee, K. H., Lee, J., & Lee, S. H. (2015). 3D liver models on a microplatform: Well-defined culture, engineering of liver tissue and liver-on-a-chip. Lab on a Chip,
15(19), 3822–3837.
Shape-Memory Polymer Medical Devices
Muhammad Y Razzaq, Markus Reinthaler, Mark Schröder, Christian Wischke, and Andreas Lendlein, Institute of Biomaterial
Science and Berlin Brandenburg Center for Regenerative Therapies, Teltow, Germany
Medical Devices 395

Characteristics and General Requirements 395
Upcoming Clinical Demands for Smart Medical Devices 395
Shape-Memory Polymers 395
Working Principle 395
Polymer Network Structure 396
Molecular Switches Respond to External Stimulation 396
Programming Processes Are Mandatory for the SME 397
From Dual-Shape Effects to Complex Shape Switching 397
Quantitative Analysis of the SME 398
Tailoring Shape-Memory Polymers for Use in Medical Devices 398
Switching Temperature 398
Cell and Tissue Compatibility 399
Degradation Rates 399
Sterilization 400
Medical Devices Based on Shape-Memory Polymers 400
Overview 400
Surgical Devices 400
Sutures 400
Surgical fasteners 401
Vascular Devices 401
Intravascular stents 401
Clot removal 402
Aneurysm therapy 402
Orthopedic Devices 404
Filling of bone cavities 404
Drug Delivery Devices 404
Miniaturization of SMP 404
Outlook 405
Further Reading 405
Glossary
Medical device An instrument, apparatus, implant, in vitro reagent, or similar or related article that is used to diagnose,
prevent, or treat disease or other conditions, and does not achieve its purposes in or on the human body primarily by
pharmacological, immunological, or metabolic meansdbut typically by physical principles.
Minimally invasive intervention Surgical technique that limits the size of incisions needed and so lessens wound healing time,
associated pain and risk of infection.
Polymer network A structure, in which all polymer chains are interconnected by crosslinks to form a single macroscopic entity.
Programming An external mechanical manipulation of the SMP to achieve a temporarily fixed, second shape.
Shape-memory polymer (SMP) A stimuli-sensitive polymeric material that has the ability to return from a deformed state
(temporary shape) to its original (permanent) shape by exposure to an external stimulus (trigger), such as heat, light etc.
Stent Biomaterial-based tubulous construct inserted into the lumen of an anatomic vessel or duct to keep the passage way
open.
Nomenclature
PCLDMA Poly(ε-caprolactone)dimethacrylate

Biomaterials: Biomaterial applications and advanced medical technologies j Shape-Memory Polymer Medical Devices 395
Rf Shape fixity ratio

Rr Shape recovery ratio
SME Shape-memory effect
SMP Shape-memory polymer
Tg Glass transition temperature
Tm Melting transition temperature
Tsw Switching temperature
Ttrans Thermal transition temperature
Ts,max Temperature, at which the maximum stress is generated
Medical Devices
Characteristics and General Requirements
Medical devices are systems dedicated to diagnose, prevent, or treat diseases or other conditions. Examples include man-made
implants, which are employed to replace a missing biological structure, support a damaged biological structure, or enhance an exist-
ing biological structure, for example, by physical principles. These devices are usually implanted by surgical procedures. As they are
directly exposed to tissue, they should be nontoxic, nonimmunogenic and sterile.
From a historical point of view, dental implants are important medical devices, as this technology has been used and developed
since the earliest civilizations. For example, ancient Egypts used ivory to replace single teeth, whereas metals and more recently poly-
mers are the present materials of choice.
Beside mechanical properties, as illustrated by dental devices, modern implants also need to exhibit additional features, which
are highly dependent on the application area of a device. For example, hemocompatibility and low thrombogenicity are essential
characteristics for devices, which are applied in the vascular system and therefore directly interact with the blood stream. Stents,
artificial heart valves as well as extracorporal circulatory systems may be mentioned in this context.
A group of medical devices with increasing relevance are temporary implants, which degrade after a certain period. In this way,
they are able to support organ functions just as long as necessary without leaving behind foreign materials. This concept may avoid
long-term side effects like chronic inflammation (foreign body response). Furthermore, the diffusion characteristics or the erosion
behavior of polymeric biomaterials were prerequisites for their application as matrix materials in controlled drug-release systems.
Importantly, the degradation products need to be either metabolized or excreted and should, again, be nontoxic.
Upcoming Clinical Demands for Smart Medical Devices

Over the past few years, minimally invasive interventions have increasingly replaced standard surgical procedures. This trend
continues and imposes specific requirements on materials and upcoming technologies. One of the key questions arising is how
to adapt bulky devices in order to precisely deliver them to certain areas inside the body through small incisions. Furthermore,
a general problem in device based procedures is the high anatomic variability of every individual patient, raising the need for
“personalized designs.” For example, heart valves and valve dysfunctions exist in numerous anatomic variabilities and the optimal
percutaneous treatment will require individually adapted devices.
Here, materials that can change their shape in a predefined way on demand could potentially play an important role. An implant
could be inserted into the body through a small incision in a temporary stabilized compressed or elongated shape. As soon as the
implant is placed in the body, it is heated to body temperature, which may trigger its change into the desired bulky application-
relevant shape. Furthermore, the type of shape switch might be adjusted to the individual needs of the patient. It may also be of
interest to combine the shape switch with other functions, such as degradability or drug release.
In this article, we discuss shape-memory polymers (SMPs) as stimuli-sensitive matrix materials that allow spatially directed
shape switches and their possible applications in medical devices. This includes an overview of fields of applications including
general surgery, the cardiovascular system or applications in orthopedic surgery. Furthermore, the general principle of the thermally
induced SME is described, and selected examples for biomaterials with SME are introduced.
Shape-Memory Polymers
Working Principle
The shape-memory effect (SME) of a polymer describes the capacity to switch from a temporary shape to a permanent shape. In
order to allow a SME in polymers, a number of preconditions need to be fulfilled: the material needs to exhibit a polymer network
structure, suitable elasticity, a process to transfer the material to the temporary shape by elastic deformation, and molecular switches
with the ability to form reversible crosslinks for fixation of the temporary shape. The permanent shape of the sample is determined
396 Biomaterials: Biomaterial applications and advanced medical technologies j Shape-Memory Polymer Medical Devices
Fig. 1 Principle of the thermally induced SME in polymers. (A) Schematic demonstration of a thermomechanical cycle of programming a SMP and
its shape recovery. (B) Photo series demonstrating the macroscopic SME for a poly(3 -caprolactone) based shape-memory polymer network with
a Ttrans ¼ 51 C (length of sample is 6 cm).
by the polymer network structure, while the temporary shape is achieved by a programming process that may involve stretching,
compression, bending and/or twisting deformations. Recovery to the original shape is carried out by application of an external stim-
ulus such as heat, light or moisture to remove the temporary crosslinks. A schematic demonstration of the programming and
recovery of a thermally induced SMP is provided in Fig. 1A. The process of programming and recovery to the original shape can
be repeated several times depending on the application.
Polymer Network Structure

SMPs exhibit a network architecture consisting of netpoints, chain segments in between the netpoints, and molecular switches. The
netpoints can be either of physical or chemical nature and determine the permanent shape to the polymer network.
Multiblock copolymers, whose morphology consists of at least two types of phase-segregated domains, are examples for phys-
ically crosslinked polymers. Here, the domains providing the highest thermal transition temperature act as netpoints. The domains
associated with the second highest thermal transition can serve as the molecular switch. Such materials are examples of temperature-
induced SMPs.
Shape-memory polymer networks with covalent netpoints can be obtained either by in situ crosslinking during polymer
synthesis or by postpolymerization, which includes radiation or chemical methods. Due to the covalent nature of netpoints, cova-
lent networks are often capable of a more quantitative switching toward the original shape. Besides temperature-sensitive SMPs with
covalent network structure, also other types of molecular switches can be introduced.
Molecular Switches Respond to External Stimulation

In principle, molecular switches can be selected from a variety of chemical components as long as they are able to form crosslinks
that are reversible in nature. The most frequently reported SMPs are thermo-sensitive and can be activated by heat as an external
stimulus. Here, the temporary physical crosslinks base on domains, which are formed from switching segments and are associated
with distinct thermal transition temperatures Ttrans (e.g., melting transition Tm or glass transition Tg) (Fig. 2). The fixation of
a temporary shape can in this case be achieved by vitrification or crystallization of switching domains upon cooling, while shape
recovery can subsequently be induced by heating. The macroscopic shape recovery of a SMP with crystallizable switching domain is
shown in Fig. 1B. Heat can be applied directly, but also indirectly in a non-contact mode. Indirect heating can be realized by incor-
porated dyes or magnetic nanoparticles, which can transform energy provided by light or alternating magnetic fields, respectively,
into heat locally.
Other types of molecular switches base on reversible covalent bonds, which are formed between two functional groups incor-
porated in the polymer network at a given condition, and can be cleaved at a different condition. Examples include the light-
induced SME, which can be controlled independently from heat and is obtained by incorporating light-sensitive reversibly reacting
(A) (B)
Fig. 2 Schematic representation of molecular architecture of covalently crosslinked SMPs (black lines: switching segments, red circles: netpoints)
with (A) amorphous and (B) crystallizable switching segments.
molecular switches into the polymer networks. Another example is SME induced by ultrasound cavitation, which is based on revers-
ible dissociation of metal–ligand coordination bonds under the effect of ultrasonic waves. Furthermore, redox active moieties can
also be utilized to create reversible covalent bonds in SMPs. For instance, cellulose derivatives having a crosslinkable mercapto
group enabled a redox sensitive SME. However, the shape-recovery ratio (Rr), a value that would be 100% for full recovery to
the original shape, decreased with the repetition of the redox treatment because side reactions reduced the number of available mer-
capto groups.
Programming Processes Are Mandatory for the SME

Along with the polymer network structure, a suitable elasticity is required for SMPs as mentioned above. This elasticity is essential to
allow the deformation of the polymer during a programming step and, importantly, to drive the shape recovery to the permanent
shape. Based on their mandatory role to realize a SME, the programming technologies are also called shape-memory creation proce-
dures (SMCP).
In case of temperature-sensitive SMP, the programming of the polymer network is carried out by the action of an external stress
applied at a temperature T > Ttrans (Fig. 1). At this temperature, the chain segments are flexible and can be deformed to a less coiled
conformation, resulting in a loss of entropy. The overall deformation capability can be adjusted by the length and flexibility of the
polymer chain segments, from which the network has been constructed. Cooling the sample to T < Ttrans causes the crystallization
or the vitrification of the switching segments, preventing the immediate recoiling of the polymer chains and thus fixing the tempo-
rary shape of the polymer sample. The macroscopic recovery to the permanent shape, in this case induced by heating to T > Ttrans, is
enabled by entropy elasticity of the switching segments that move toward an entropically favored conformation.
From Dual-Shape Effects to Complex Shape Switching

By introducing a second type of switching segments into the polymer network, the capability of memorizing two distinguishable
shapes can be implemented, thus extending the dual-shape capability to a triple-shape effect. Such a triple-shape polymer network
consists of at least two segregated domains with two transition temperatures Ttrans,A and Ttrans,B and can change on demand from
a first shape (A) to a second shape (B) and from there to a third shape (C) when stimulated by two subsequent temperature
increases. An example of such a multiphase polymer network is an AB copolymer network based on crystallizable poly(ε-caprolac-
tone) (PCL, Ttrans,A ¼ Tm,PCL z 50 C) and amorphous poly(cyclohexyl methacrylate) (PCHMA, Ttrans,B ¼ Tg,PCHMA z 140 C)
segments.
By adding further distinct phase transitions in a polymer or by using a polymer with a broad thermal transition associated with
the switching domain, the number of temporary shapes can be increased. In this way, a multishape effect (MSE) can be enabled in
polymers. These polymers are capable to memorize and recover more than two predefined shapes. Such an effect has been realized
in covalently crosslinked copolymer networks, interpenetrating polymer networks, and in multimaterial polymer systems. However,
the dual-shape and MSEs are categorized as “one-way” phenomena as they are not reversible and an external manipulation is always
required to again program the samples.
A reversible actuation can be observed in polymer networks containing at least one crystalline switching domain. Here, a constant
stress (sc) has to be maintained on the sample at all times, which induces crystallization along the direction of the load. On the
molecular level such a reversible SME (rSME) is based on the crystallization-induced elongation (CIE) of preoriented switching
chain segments, which occurs during cooling to temperatures Tlow < Tm under a specific sc, while heating to temperatures above
Tm results in melt-induced contraction (MIC).
By using a polymer network with two crystallizable switching domains, an rSME has been demonstrated, which is possible under
stress-free conditions. This actuation effect requires a modified thermomechanical programming procedure and is named reversible
bidirectional SME (rbSME).
In principle, a SME is not limited to large samples but also works for microstructures, which are useful for miniaturized biomed-
ical devices and microfluidic systems. A variety of shape-memory micro-objects has been explored and their one-way and rSMEs are
investigated. However, specific thermomechanical programming and characterization procedures at microscale are mandatory to
observe the SME at micro-level.
Quantitative Analysis of the SME

The thermally induced SME of macroscopic samples can be quantified in cyclic, thermomechanical tests that consist of the program-
ming as well as the recovery step. A variety of test protocols has been developed, which are based on recovery under stress-free or
strain-controlled conditions. They allow determining the switching temperature (Tsw) of thermo-sensitive SMPs, at which the
maximum recovery takes place during stress-free recovery conditions, or the temperature, at which the maximum stress is generated
(Ts,max) during strain-controlled recovery process. Other important shape-memory properties include the extent, to which the
temporary shape can be fixed (shape fixity ratio Rf) and the permanent shape is recovered (shape recovery ratio Rr). These
shape-memory characteristics strongly depend on the polymer’s structural design as well as on the parameters (e.g., heating or cool-
ing rates or the type of mechanical deformation) applied during the SMCP.
Tailoring Shape-Memory Polymers for Use in Medical Devices

Switching Temperature
Heat can be considered as the most commonly evaluated switching mechanism in the body, which, however, must be carried out at
a physiological temperature at or slightly above 37 C. For SMPs with a Tsw lower than body temperature, a premature recovery
before implantation can take place, whereas hyperthermia to induce SMPs with Tsw well above 37 C may cause cell and tissue
damage. General principles to tailor Tsw include modification of the respective Ttrans by changing the segment length between net-
points and / or by copolymerizing different types of monomers. Some of the common monomers used for the synthesis of degrad-
able SMPs with tailorable Tsw are provided in Fig. 3A. Copolymerization of these cyclic monomers has also enabled controlling the
hydrolytic degradation rate of the resulting polymers.
(A) (B)
Glycolide oligo[(rac-lactide)-co-
glycolide]tetrol
L,L-Dilactide D,D-Dilactide Diglycolide
I-[OH]4 Caprolactone oligo[(rac-lactide)-co-
(ε -caprolactone)]tetrol
rac-Dilactide
p-Dioxanone ε -Caprolactone Morpholino-2,5-dione
pentaerythriteI = p-Dioxanone oligo[(rac-lactide)-co-
(p-dioxanone)]tetrol
(D)
(C)
MW Loss
150
125
100
Time
ε (%)
75 Alternating PLGA Homogeneous

Hydrolysis
50
MW Loss
25
0
0 10 20 30 40 50 60 70
T (°C) Time
Heterogeneous
Random PLGA
Hydrolysis
Fig. 3 (A) Common cyclic monomers used for synthesis of biodegradable SMPs with different Tsw, (B) synthesis of star-shaped rac-dilactide-based
macrotetrols used for the synthesis of polyesterurethanes with different Tsw, (C) strain recovery process of different copolyesterurethanes with Tsw
ranging from 14 C to 56 C (Tsw ¼ inflection point of recovery curve), (D) effect of monomer sequence on degradation behavior of PLGA copolymer.
Reprinted with permission from Biomacromolecules 2009, 10, 975–982, Copyright 2009, American Chemical Society and J. Am. Chem. Soc., 2011,
133 (18), 6910–6913, Copyright 2011, American Chemical Society.
The Tm of semicrystalline polymer networks prepared by photo-crosslinking of oligo(3 -caprolactone) dimethacrylate could be
varied in a range from 30 C to 50 C depending on the molecular weights of the oligomeric precursor. Similarly, the terpolymer
poly(L-lactide-ran-glycolide-ran-trimethylene carbonate) presented Tgs in the range of 38 C–42 C, which could be tailored by using
different weight contents of L-lactide. Furthermore, the Tgs of amorphous polymer networks from star-shaped rac-dilactide-based
macrotetrols and a diisocyanate can be controlled systematically by incorporation of p-dioxanone, diglycolide, or ε-caprolactone as
comonomer (Fig. 3B and C). The resulting Tsw could be adjusted between 14 C and 56 C by selection of comonomer type and ratio
without affecting the advantageous elastic properties of the polymer networks.
Cell and Tissue Compatibility

SMPs designed for biomedical applications are expected to be nontoxic. Therefore, systematic studies both in vitro and in vivo are
required to better address the concern of biocompatibility. The use of natural polymers such as polypeptides or polysaccharides is
an important approach to design biocompatible SMPs. Another strategy is to use synthetic macromolecules built from compounds
naturally occurring in the human body to minimize potential toxic side effects. For example, polyesters can be made based on mole-
cules abundant in human metabolism, like glycerol and lactic, citric or sebacic acids. To create such shape-memory polyesters, poly-
condensation of polyols and diacids endogenous to human metabolism is carried out. A variety of biocompatible polymers based
on poly(glycerol-sebacate), poly(glycerol-dodecanoate), and polydiol citrate (using 1,8-octanediol, 1,12-dodecanediol, and 1,16-
hexadecanediol together with citric acid) have been synthesized and their shape-memory performance was investigated. Further-
more, a bio-based poly(propylene sebacate) was synthesized from 1,3-propanediol, sebacic acid, and itaconic acid produced via
fermentation or extraction. The Tsw could be tuned in a range from 12 C to 54 C by a copolymerization with diethylene glycol
to tailor the chain flexibility. In vitro fibroblast response demonstrated that these polymers are potentially cell compatible and
are promising candidates for medical devices.
Along with biopolymers, the compatibility of commercially available polyurethane based SMPs (Mitsubishi, Japan) is investi-
gated. The absence of both cytotoxic effects on L929 fibroblast cultures and mutagenicity with strains of Salmonella typhimurium were
first relevant results to allow further development of these materials to be used for endovascular interventions.
Also, the behavior of L929 mouse fibroblasts and other cells from human and rat in contact with biodegradable oligo(ε-capro-
lactone)dimethacrylate (OCLD) based SMP networks was investigated. The differentiation capacity of mesenchymal stem cells into
osteoblasts and adipocytes was supported by the polymer network. The shape-memory effect did not affect the majority of the
adherent cells.
While in vitro studies may give a first hint on cellular response for selected types of cells, often based on immortalized cell lines,
the exploration of in vivo behavior of implanted materials provides insights in complex cellular responses of multiple involved cell
types. As the place of application (e.g., subcutaneous or intravascular), the disease and surgery-associated trauma (e.g., bone frac-
tures or intervention-induced tissue response based on surgical skills), the type and properties of SMP material including potential
impurities (e.g., chemical composition, surface properties, sterility, degradation products), the mode of action of the SMP device
(e.g., pressure on adjacent tissues) and individual factors associated to the treated person/animal (e.g., immunological state) all
can affect in vivo response, a generalized statement on histocompatibility of SMP cannot be given. However, several preclinical
studies demonstrated a promising in vivo behavior. For instance, tissue integration and angiogenesis was observed in rats along
with slow degradation of multiblock copolymers composed of oligo(p-dioxanone)- and oligo(ε-caprolactone)-segments. Photo-
crosslinked SMP networks from poly(ε-caprolactone)-co-(a-allyl carboxylate ε-caprolactone) showed no inflammatory response
when implanted adjacent to femoral artery ligations in mice, a model for ischemia. Polymer networks from poly(D,L-lactide) con-
taining polyhedral oligomeric silsesquioxane (POSS) netpoints demonstrated that different phases of generally mild inflammatory
responses can be distinguished after subcutaneous implantation in rats before and after the onset of material degradation. The asso-
ciated formation of fibrous capsules around the implant depict the cellular response to the foreign body, which is characteristic to
most implants and may preferentially be resorbed with time. In specific applications, however, fibrous tissue formation in response
to the implant may be desired to permanently alter the implantation site, as long as this response is well controlled, aseptic, and
local. A low level chronic inflammatory reaction to a spiral shaped SMP occluder from poly(DL-lactic acid)-based poly(urethane
urea) inserted for contraception in rabbit fallopian tubes was reported to result in a permanent tubal occlusion due to an almost
complete replacement of mucosa and muscle by fibrous tissue. For occlusion of aneurisms with polyurethane SMP foams (see
“Aneurism Therapy” section), the desired ingrowth of connective tissue with only a mild inflammatory reaction was confirmed.
Degradation Rates
The degradability of SMP can be achieved by introducing hydrolyzable bonds in the polymer chains, which can be cleaved under
physiological conditions. Polymeric materials may be degraded via chemical and enzymatic oxidation upon exposure to body fluids
and tissues, via hydrolysis catalyzed by acids, bases, or salts, and via enzyme-catalyzed hydrolysis reactions. The degradation rates of
the SMPs can be tailored by copolymerization and crosslinking procedures. For example, the hydrolytic degradation rate of SMP
based on oligo[(ε-hydroxycaproate)-co-glycolide] dimethacrylates and butyl acrylate can be tailored by adjusting the weight content
of glycolide units and n-butyl acrylate weight content during the crosslinking reaction. The degradation rate could be accelerated by
increasing the glycolide content and reduced by increasing amounts of n-butyl acrylate units. The enzymatic degradation of cross-
linked poly(ε-caprolactone)-based SMP can also be controlled by adding pendant coumarin groups. Here, the coumarin groups
hindered the excess of enzyme to the degradable polymer chains resulting in a decreased degradation rate. It should be noted that
also the order of how monomers are build in the polymer chain during copolymerization affects the hydrolytic degradation of the
polymers. A dramatically different hydrolysis rate of alternating PLGA and random sequence PLGA was observed (Fig. 3D). Along
with chemical composition, other factors such as water diffusion, monomer solubility and diffusion, material homogeneity, pro-
cessing technique, and device geometry and size play a role to control the degradability. Furthermore, polymer blending and surface
modification can be used to adjust the degradation of the SMPs.
Sterilization
Sterility is a mandatory feature of SMP medical devices for implantation. However, the sterilization method can influence the poly-
mer structure, SME functionality, biocompatibility and thus performance of a device. For temperature-induced SMP, the steriliza-
tion technique should ideally be operated below Ttrans, which typically excludes heat (160 C) or steam sterilization (121 C) from
the list of possible methods at least for thermoplastic or programmed SMP samples. Thus, low-temperature sterilization (LTS)
methods based on g- or e-beam-irradiation, hydrogen peroxide vapor, low-temperature gas plasma and ethylene oxide (EO)
may be preferred. Among them, a careful selection is needed as also these techniques may induce changes in polymer structure
under certain conditions. For instance, hydrogen peroxide vapor or EO may react with certain chemical groups of the SMP, while
g-, e-beam, and plasma sterilization may be associated with changes in the molecular structure of the polymer. Recently,
low-temperature plasma (LTP) sterilization was used for SMP polyurethane foams and the effects of sterilization on the chemo-
physical, thermomechanical, and shape-memory performance of polyurethane were investigated. Plasma sterilization (Sterrad-
100St, H2O2, 52 min) did not influence the SME of PU foams, but an increase in the open porosity was observed. Furthermore,
plasma treatment had no significant effect on the material’s cytotoxicity on L929 cells in vitro. In another study, six sterilization
techniques (LTP, steam, ethylene oxide, e-beam radiation, gamma radiation, and nitrogen dioxide) were evaluated for acrylate
based SMP networks from poly(ethylene glycol) dimethacrylate copolymerized with tert-butyl acrylate or methyl methacrylate.
The samples were shown to be cytotoxic after LTP sterilization as the surface was oxidized, which negatively affected the biocom-
patibility. Gamma radiation decreased the Tg and significantly increased the rubbery modulus. The other sterilization methods did
not significantly alter the thermomechanical properties. The sterilized SMPs exhibited a full shape recovery under free-strain condi-
tions even after a storage of 1 year at 20 C. The Tsw increased by up to 9 C and the speed of the strain recovery by up to 9% due to
physical aging. Storage and aging of SMPs would allow for faster activation in vivo if the increase in Tsw did not negatively affect
recovery.
Medical Devices Based on Shape-Memory Polymers

Overview
Owing to a combination of advantageous features such as lightness, low cost, easy processability, high recoverable strains, tailored
thermomechanical properties, and shape-recovery temperatures as well as possibly in vivo degradation, SMP could be beneficially
used in the fabrication of various medical devices, particular for minimally invasive applications. However, the promise of SMPs in
biomedical applications has yet to be fully realized and so far is restricted to conceptional studies in some clinical fields. Potential
medical applications of SMPs are provided in Fig. 4. In this section, selected SMP-based medical devices are discussed, with special
emphasis on those works where evidence of functionality either in vitro or in vivo has been obtained.
Surgical Devices
Sutures
One of the cornerstones in conventional surgery is to create stable tissue connections or to secure implants, which has traditionally
been performed by surgical sutures. In order to safely adapt tissue margins, the suture has to be tied at an extent providing sufficient
stress on the wound lips. Both, inadequately high or low suturing forces may result in poor healing.
Especially in minimally invasive (keyhole) surgeries, stitching may become a major limitation due to lack of space. Self-
tightening sutures may therefore be of particular relevance in these types of operations. The first biodegradable self-tightening suture
was based on a multiblock copolymer synthesized from oligo(ε-caprolactone)diol and crystallizable oligo(p-dioxanone)diols. The
suture was first stretched (about 200%) to achieve a temporary shape. Modification of the stretching conditions or the molecular
structures of the multiblock copolymer were used to adapt the exerted tension of the SMP suture.
Series of experiments were conducted to demonstrate the feasibility of these shape-memory sutures. Once the suture was applied
for example to close an incision, the SME capability of the polymer was activated by the body temperature, tying together the wound
margins. As the adopted polymer may also be degradable, there may be no need to remove the suture after the incision has healed.
The sutures proved to be cell compatible when incubated with 3T3 fibroblasts, umbilical endothelial cells and human fibroblasts.
For the selected programming conditions, shape-memory based contraction of the polymer suture was 25%, when tested in 0.9%
(w/v) saline, blood or air at 38 C–45 C.
Sutures
Surgical fastener
Stents for intraluminal prothesis
Soft tissue fixation to bones
General
surgical
Archwires uses Aneurysm closure
Orthopedics
& Ortho- Intravascular
dontics
Glaucoma stents applications
Ocular
Clot removal
Thermally-induced devices
SMP material
Heat packs External
platforms
Brain
Microgripper &
Neuronal electrode retrieval devices
Urogenital Percutaneous
tract vascular access
products
Retrieval devices
Fertility control plug Dialysis needle
Stents for Catheter for

intraluminal prothesis Anchoring apheresis
cannula
Fig. 4 Potential medical applications of shape-memory polymers. Reprinted from Comprehensive Biomaterials 2011, 479–496, Copyright 2011, with
permission from Elsevier.
Surgical fasteners
Another application of SMP has recently been suggested for the fixation of medical devices during surgery. The concept involves
needles of temporary straight shape that are pierced through the implanted device, such as a hernia mesh, and the respective subja-
cent tissue. Upon heating to body temperature, both ends of the needle recover to a helical shape, thus fixing the implant in the
desired place.
The temperature-induced self-tightening of a bioabsorbable PLGA based subcuticular staple has been demonstrated. The use of
these fasteners reduces the need for staples of different sizes and provides gentle force to close the wound. Furthermore, cannulas
containing SMP-based anchoring structures are proposed for minimally invasive procedures. The cannulas are utilized to provide an
access port for surgical procedures. The proposed cannula include an elongated longitudinal shaft having a distal opening, a prox-
imal opening for receipt of the operating instruments, an anchoring member including a SMP on the outer surface of the elongated
longitudinal shaft, and a sliding tube mounted on the outer surface of the cannula. After inserting the cannula, it can be prevented
from migrating or slipping out through the incision using the SMP-based anchoring structures on the distal portion of the cannula.
Vascular Devices
The use of medical devices inside of blood vessels is necessary in highly critical medical conditions such as vessel occlusion. High
precision of device insertion to the site of placement, control of device expansion and/or fixation in the tissue, and long-term perfor-
mance are typical key requirements of vascular devices, which make these applications highly relevant for SMP-based implants but
at the same time also more challenging than some other applications introduced above.
Intravascular stents
Stents are tubular structures, designed to re-open a stenosed/occluded vessel and therefore to establish adequate blood flow as
shown in Fig. 5. As demonstrated in coronary interventions, supporting the vessel wall by implanting these devices has become
one of the crucial steps to ensure durability of the treatment as they prevent elastic recoil. Traditionally, metallic stents were
mounted on a balloon catheter and delivered from a peripheral access. Beside balloon-expandable stent technologies, self-
expandable designs have been developed, which are typically used to treat peripheral vascular disease. Balloon-expandable metal
stents are commonly made of stainless steel, cobalt or titanium, whereas nitinol-alloys are mainly used in self-expandable stents.
Vessel
Stent
Plaque
Fig. 5 Reopening of a stenosed vessel by using a stent.
Polymer based scaffolds, which offer the option of biodegradability, have recently been introduced in the interventional treat-
ment of coronary artery disease. The idea behind this technology is to support the vessel wall just as long as necessary and therefore
avoid long-term complications like late stent thrombosis. This problem has especially been observed in drug eluting stents. In this
context, degradation time seems to be an important parameter and according to current opinion should be a period of 6–9 months.
Recently, the ABSORB (Abbott vascular) and the DESolve (Elixir Medical corp.) devices based on degradable polymers have been
granted CE marks and are commercially available. However, so far metallic stents are still the gold standard in cardiovascular medi-
cine and the field of polymer stents is still under development.
SMP-based stent technologies have not been in clinical use so far, however due to a strong clinical trend toward minimally inva-
sive techniques, SMP-based stent technologies have been continuously explored. These stents can be preprogrammed to a compact
shape, which on the one hand may be activated at body temperature, resulting in natural deployment without need for auxiliary
devices. Proposed stent designs include spiral shaped structures based on copolymer networks of star-shaped poly(ε-caprolactone)
(acting as switching segment) and polyester poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) (acting as hard
segment). These spirals would partially uncoil upon shape recovery, a process resulting in a twisting of each end of the spiral
and possibly a reduction in device length. In another study, either solid or faster expanding, perforated tubular stents based on
tert-butyl acrylate and poly(ethylene glycol)dimethacrylate (PEGDMA) crosslinkers were programmed to a small temporary shape
by furling and rolling, which would have to be reversed at the site of placement. In both cases, practical concerns in the precision of
placement would need to be addressed. More advanced concepts have considered a highly perforated tubular design with small
struts, which should be programmed in situ by aid of balloon catheters. This approach would allow stent removal by diameter
reduction via the shape-memory effect. In addition, SMPU based neurovascular stent prototypes were fabricated. These tubular
devices were laser etched for more flexible stents that also could navigate through small tortuous vesselsdhowever, it has been
revealed that resistance to withstand the critical collapse pressure in the vessel at elevated body temperature may not be given in
all cases.
Clot removal
Micro-devices for mechanical clot removal have recently attracted significant attention in minimally invasive surgery as an alterna-
tive to thrombolytic agents, as these drugs are associated with bleeding complications. First experimental devices have already been
used and offer a transcatheter approach, guided from a peripheral access. After crossing the clot, the SMP-based device is activated by
an external stimulus and converted in a grabber-like shape, which allows extraction of the embolus. For example, a corkscrew
temporary shape had been proved to be effective under physiological flow and pressure conditions (Fig. 6). Controllable shape
switching can be achieved by indirect heating using a light-absorbing dye incorporated into the SMP or by electro resistive heating
of hybrid devices with a nitinol core covered with a polymeric shell. This hybrid device enabled a higher recovery and therefore
retraction force in the blood flow. The functionality was demonstrated in vitro and in vivo in a water-filled silicone neurovascular
model and a rabbit carotid occlusion model, respectively.
Aneurysm therapy
An aneurysm is a localized, blood-filled balloon-like bulge in the wall of a blood vessel. Especially in the brain, aneurysms can cause
symptoms by compressing adjacent structures or can rupture, which is a life threatening complication. Based on the aneurysm
anatomy and the clinical circumstances, endovascular or surgical treatment-strategies are available. Both treatment modalities
attempt to disconnect the aneurysm from the blood stream either by clipping, coiling, or stenting. The idea of coiling is to insert
a filling material into the vascular bulge (usually platinum coils), inducing thrombosis and therefore blocking off the aneurysm
Microcatheter
Clot
Straightened
Helical coil
coil
1. Blood vessel 2. Insertion of a 3. Coil returning back 4. Ensnaring of the 5. Cleared blood
with a clot microcatheter with to its original helical clot vessel
straightened coil shape by laser heating
to the site of clot
Fig. 6 Procedure of mechanical removal of embolus using a SMP-based clot removal device.
cavity. In 2013, 28,000 people in Western Europe received a coil based minimally invasive aneurysm treatment (Clin. Neuroradiol.
2015, 25 (2), 317–324). However, coils are prone to incomplete cavity filling or to rupture the aneurysm wall by coil puncture.
The utilization of SMPU spherical foams as filling materials has shown to be a good alternative to metallic coils. These devices are
softer compared to metallic coils, reducing the risk of rupture, and may also improve the closure performance due to enhanced
alignment to irregular aneurysm shapes. From a procedure point of view, the device can be programmed to its temporary shape
for delivery by crimping onto the catheter. The device should revert back into its permanent shape inside the aneurysm when
exposed to the body temperature (Fig. 7). Furthermore, by tuning the pore size of a SMP foam by the composition and synthesis
condition, cellular infiltration can be controlled to support the blood clotting and anchoring of the clot in the aneurysm. An impor-
tant requirement is radio-opacity of devices as the procedure is fluoroscopy guided, for which tungsten particulate filler were incor-
porated into the SMP. In vitro models of shape-memory polymers for embolization of aneurisms were explored to predict the
Catheter
Aneurysm
Stent
compressed
expanded
SMP-Foam
SMP-Foam
1. Vessel with 2. Stenting and 3. Foam expansion 4. Remainingfilled

Aneurysm delivery of the aneurysm
compressed Foam
Fig. 7 Procedure of aneurysm treatment by using a SMP-based occluder device and additional stent.
stresses caused by the SMP, thermal and fluid dynamic changes, and changes in vascular dynamics. Also in vivo studies have been
conducted with SMP foams versus metal coils in a vein pouch aneurism model, which provides similar aneurism sizes to those
found in the human intracranial vasculature. These studies suggested a much higher occlusion of aneurism volume by SMP foams
compared to bare metal coils when examined 180 days after implantation. While significant research has gone into this application
of SMPs, FDA clearance for this application is still required.
In addition to neurovascular applications, SMP occluder systems including foams as well as fibrous SMP coils are explored
in vivo in peripheral vasculature to treat, for example, aneurisms or hemorrhage, or to block the blood supply to tumors.
Orthopedic Devices
Filling of bone cavities
Bone tissue typically allows spontaneous healing of defects up to a critical size. Larger defects above the critical size are currently
preferentially treated by using bone autografts from sources like the iliac crest or the ribs (gold standard), which are not always avail-
able and/or bring additional burden to patients during harvesting. An unmet need are devices that would enable the simple access
and optimal adaption to irregular shapes of defect cavities as well as eventually the regeneration of bone.
Other than synthetic grafts that have to be tailored manually to the defect for the respective patient, SMP foams can be fixed in
a compressed temporary shape, which allows simple insertion of the implant to the defect. By shape recovery, the foam expands
until it fits the cavity boundaries and remains anchored by applying its recovery force to the cavity boundaries during constraint
recovery. This anchoring method works like a press-fit fixation.
Often, materials with a temperature-induced shape recovery, in some cases at critically high temperatures, have been proposed.
An alternative may be materials with a water-induced shape-memory effect, which can be realized by water-induced plasticization of
glassy switching domains. An example can be found in gelatin-based porous hydrogels stabilized via covalent crosslinks. When
water was added to compressed dry-state samples, the original shape was recovered (Rr of 88% 5% to 95% 5%) along with
a reduction of the Tg of the switching domains from 50 C–60 C to 0 C. The inherent cell-binding motives of gelatin-based material
and the merging of smaller pores to larger sized cavities during degradation were beneficial as concluded from cell proliferation,
support of cell differentiation, and a purely material-induced regeneration in different animal models with outcomes comparable
to less easily accessible cancellous bone graft.
Beyond filling of bone defects, orthopedic applications of SMP expand also to the fixation of tendon and ligaments. For a bio-
stable polyether ether ketone SMP medical device, which is designed for nonrotational insertion in (drilled) bone cavities and
prevents rotational force on the tissue to be fixated, an FDA clearance has been granted.
Drug Delivery Devices

In recent years, degradable SMPs have been equipped with drug-release functionality by drug incorporation, which leads to multi-
functional SMPs. A detailed analysis including examinations of the kinetics of drug incorporation and in vitro release as well as
tissue compatibility, in vivo release and degradation are pivotal for these devices. Furthermore, the incorporation of drugs may
result in a disturbance of polymer morphology or thermomechanical properties, and thus may lead to an impaired SME of a temper-
ature-induced SMP. Therefore, investigations concerning the interplay between drug payload, SME and polymer degradation are
required in order to obtain tailored properties of a device for a specific application. In recent years, a variety of SMP-based materials
for drug delivery systems has been proposed.
Degradable SMP networks prepared by UV-curing of oligo[(ε-caprolactone)-co-glycolide]-dimethacrylates precursors were
loaded with ethacridine lactate or enoxacin as examples of hydrophilic and hydrophobic test drugs, respectively, by swelling
and by incorporation during network synthesis. The investigation of the SME and drug delivery for this set of materials showed
that the shape-memory functionality at relevant temperatures (28 C–42 C) was maintained after drug incorporation and the
diffusion-controlled release was timely independent of the polymer degradation. Similar systems were further proposed as implant-
able devices with body temperature-induced shape switch to avoid implant migration by anchoring in the tissue, enabling local or
systemic drug delivery. Further covalent polymer network systems were shown to have controlled drug-release capacity and, in
animal studies, slowly degraded over the period of weeks when implanted in rats. Although there are not yet massive publications
in this field, drug loaded SMPs hold substantial promise for applications in the field of controlled drug release.
Miniaturization of SMP
SMPs are promising candidates to generate switchable micro-objects or miniaturized biomedical devices such as drug delivery
systems. Recently, shape-memory particles composed of biodegradable multiblock copolymers of poly(u-pentadecalactone) and
poly(ε-caprolactone) have demonstrated the ability to be switched from prolate ellipsoids to spheroid. Recently, SMP microparti-
cles based on poly(ε-caprolactone) and poly(ethylene glycol) have shown a reversible switch from spherical to ellipsoidal shape by
cyclic heating and cooling between 0 C and 43 C. In another study, a soft lithographic technique is used to fabricate SMP micro
cuboids. Resulting microcuboids enabled a nano (surface roughness) and micro-level (cuboidal geometry) shape recovery process
after programming by different compression ratios.
Miniaturization of SMP-based features has also been used to design responsive micro-structured surfaces with switchable mate-
rial properties such as wetting, adhesion and friction. For example, micro-tipped SMP surfaces were used to fabricate reversible dry
adhesives. The micro-tips in the deformed state achieved a tight contact and bonding with the substrate. Upon heating, the micro-
tips returned to their protruded state, causing de-bonding. In another example, surface wetting was controlled by using micro-pillars
developed on the surface of SMP. The deformed and original/recovered SMP pillar array displayed distinct wettability, which was
characterized by the static/dynamic water contact angles and the droplet sliding angle. Furthermore, as cell growth is considered to
be sensitive to the topographic features of a substrate, different cell behaviors were observed in response to changes of the surface
micro-structure changes in SMPs.
Outlook
In summary, the unique capability of shape-memory polymers to undergo predefined shape switches between two or more desired
shapes as well as the recent demonstration of reversible bidirectional actuation by SMP materials underline the high potential of
SMP in biomedical applications. This is reflected also in the high attention that SMP experience in academic research. Some first
SMP-based medical devices have progressed far towards clinical application including successful examination in approval processes.
These systems are pioneers for other SMP-based medical devices that will follow.
Further Reading
Behl, M., Kratz, K., Noechel, U., Sauter, T., & Lendlein, A. (2013). Temperature-memory polymer actuators. Proceedings of the National Academy of Sciences of the United States
of America, 110(31), 12555–12559.
Chen, M. C., Tsai, H. W., Chang, Y., Lai, W. Y., Mi, F. L., Liu, C. T., Wong, H. S., & Sung, H. W. (2007). Rapidly self-expandable polymeric stents with a shape-memory property.
Biomacromolecules, 8(9), 2774–2780.
Filion, T. M., Xu, J., Prasad, M. L., & Song, J. (2011). In vivo tissue responses to thermal-responsive shape memory polymer nanocomposites. Biomaterials, 32(4), 985–991.
Hardy, J. G., Palma, M., Wind, S. J., & Biggs, M. J. (2016). Responsive biomaterials: Advances in materials based on shape-memory polymers. Advanced Materials, 28,
5717–5724.
Horn, J., Hwang, W., Jessen, S. L., Keller, B. K., Miller, M. W., Tuzun, E., Hartman, J., Clubb, F. J., & Maitland, D. J. (2017). Comparison of shape memory polymer foam versus
bare metal coil treatments in an in vivo porcine sidewall aneurysm model. Journal of Biomedical Materials Research Part B Applied Biomaterials, 105(7), 1892–1905.
Jung, F., Wischke, C., & Lendlein, A. (2010). Degradable, multifunctional cardiovascular implants: Challenges and hurdles. MRS Bulletin, 35, 607–613.
Lendlein, A., & Langer, R. (2002). Biodegradable, elastic shape-memory polymers for potential biomedical applications. Science, 296, 1673–1676.
Lendlein, A., Behl, M., Hiebl, B., & Wischke, C. (2010). Shape-memory polymers as a technology platform for biomedical applications. Expert Review of Medical Devices, 7,
357–379.
Neffe, A. T., Pierce, B. F., Tronci, G., Ma, N., Pittermann, E., Gebauer, T., Frank, O., Schossig, M., Xu, X., Willie, B. M., Forner, M., Ellinghaus, A., Lienau, J., Duda, G. N., &
Lendlein, A. (2015). One step creation of multifunctional 3D architectured hydrogels inducing bone regeneration. Advanced Materials, 27(10), 1738–1744.
Rodriguez, J. N., Clubb, F. J., Wilson, T. S., Miller, M. W., Fossum, T. W., Hartman, J., Tuzun, E., Singhal, P., & Maitland, D. J. (2014). In vivo response to an implanted shape
memory polyurethane foam in a porcine aneurysm model. Journal of Biomedial Materials Research Part A, 102(5), 1231–1242.
Serrano, M. C., & Ameer, G. A. (2012). Recent insights into the biomedical applications of shape-memory polymers. Macromolecular Bioscience, 12, 1156–1171.
Small, W., Singhal, P., Wilson, T. S., & Maitland, D. J. (2010). Biomedical applications of thermally activated shape memory polymers. Journal of Materials Chemistry, 20,
3356–3366.
Wischke, C., Neffe, A. T., & Lendlein, A. (2010). Controlled drug release from biodegradable shape-memory polymers. Advances in Polymer Science, 226, 177–205.
Wischke, C., Schossig, M., & Lendlein, A. (2014). Shape-memory effect of micro-/nanoparticles from thermoplastic multiblock copolymers. Small, 10(1), 83–87.
Yakacki, C. M., Shandas, R., Lanning, C., Rech, B., Eckstein, A., & Gall, K. (2007). Unconstrained recovery characterization of shape-memory polymer networks for cardiovascular
applications. Biomaterials, 28, 2255–2263.
REGENERATIVE ENGINEERING
Adult Bone Marrow-Derived Stem Cells: Immunomodulation in the Context

of Disease and Injury
AE Ting and SA Busch, Athersys, Inc., Cleveland, OH, USA
Introduction 406
Immunomodulatory Properties of BMSCs 407
Graft-versus-Host Disease (GvHD) 408
Type 1 Diabetes 409
Spinal Cord Injury 410
Multiple Sclerosis 410
Stroke 411
Potential Safety Issues Associated with BMSCs 411
References 412
Glossary
Allogeneic from an organism of the same species but dissimilar in genotype.
Angiogenesis formation of new blood vessels.
Autologous from the same organism.
Cytokine small cell-signaling molecules important in cell–cell communication and immune modulation.
Immunomodulatory the ability to suppress or enhance an immune system response.
Xenogeneic cells originate from a donor of a different species than the recipient.
Introduction
Adult bone marrow-derived stem cells, or BMSCs, are a heterogeneous population of multipotent progenitor cells. BMSCs are
adherent and form colonies when grown in culture and are capable of multilineage differentiation as they have the capacity to
produce multiple tissue types within the mesenchymal lineage including bone, adipose, and cartilage. These cells are positive for
the cell surface markers CD73, CD90, and CD105, but negative for CD11b, CD45, CD79a, and HLA-DR; however, the phenotype
of these cells is dependent on passage, cell density, and culture media (Ankrum and Karp, 2010). Under the umbrella of BMSCs are
multiple characterized cell types including mesenchymal stem cells, marrow isolated adult multilineage inducible cells, and multi-
potent adult progenitor cells (Mays et al., 2007; Le Blanc, 2002). While these cells were first isolated from bone marrow, they have
since been discovered in fat and several other adult tissues, and it has been determined that the source also dictates their phenotype
and differentiation capacity.
BMSCs were initially considered for therapeutic applications based on their multilineage differentiation capacity and their poten-
tial to replace tissue, but it was later determined that these cells primarily exert their therapeutic effects through the secretion of para-
crine factors that can significantly modulate both the immune response and inflammatory processes in the context of disease and
injury. Furthermore, BMSCs home to sites of injury using the CXCR4-SDF1 chemotactic axis and the secretion of stromal cell-
derived factor 1 (SDF1) by BMSCs can lead to the recruitment of endogenous stem cells to aid in repair (Sordi, 2009). The shift
in scientific thought from considering BMSCs as replacement cells that would differentiate and regenerate injured tissue to the current
hypothesis that BMSCs exert benefit through the production of trophic factors and modulation of inflammation has also led
researchers to change their therapeutic approach from local to systemic administration. The ability to isolate, culture, expand, and
characterize the properties of BMSC have led to an ever-increasing exploration of the clinical utility of BMSCs. Currently, more
than 100 clinical trials are being conducted using BMSCs to treat a variety of diseases including: graft-versus-host disease (GvHD),
acute myocardial infarction, diabetes, multiple sclerosis (MS), spinal cord injury (SCI), and stroke (Ankrum and Karp, 2010).

Regenerative Engineering j Adult Bone Marrow-Derived Stem Cells 407
Immunomodulatory Properties of BMSCs
As a biological therapeutic, BMSCs have a number of important properties. First, the cells do not require immunosuppressive drugs
for host acceptance (as is required for a bone marrow or hematopoietic stem cell transplant) and second, the cells actually suppress
the host T-cell response. Unlike most cells, BMSCs do not elicit an allogeneic response when presented to T cells, in vivo or in vitro (Di
Nicola et al., 2002; Bartholomew et al., 2002; Liechty et al., 2000). Major histocompatibility complex (MHC) molecules are
expressed on all mature cells and used to distinguish self from nonself. While BMSCs express low to medium levels of MHC class
I and escape lysis by natural killer (NK) cells (Rasmusson et al., 2003), there is no detectable MHC class II present to activate T cells.
Furthermore, there is no expression of the costimulatory molecules CD40, CD80, and CD86 that are required for T-cell activation
(Pittenger et al., 1999). Therefore, in contrast to other forms of cell transplantation (e.g., hematopoietic stem cell transplant), no
immunosuppressive agents are required when using allogenic BMSCs.
In addition to being able to avoid recognition by the immune system, BMSCs are also able to suppress the allogeneic T-cell
response. This has been demonstrated to be a dose-dependent response and is observed both in vitro and in vivo and shown to occur
for both naive and memory T cells (Di Nicola et al., 2002; Dazzi et al., 2002). A number of studies have been performed to examine
the mechanism by which BMSCs suppress T-cell function (Table 1). While the mechanisms underlying the immunosuppressive
effects are not clearly understood, there are several hypotheses that have been explored. Because the majority of these effects do
not require cell–cell contact (Yagi et al., 2010), these hypotheses have focused on the inhibition of T-cell proliferation by secreted
factors such as hepatocyte growth factor, transforming growth factor beta (TGF-beta) 1, prostaglandin E2 (PGE2), IL-2, IL-10, indo-
leamine 2,3 deoxygenase, iNOS, soluble HLA-G, and soluble IL-1 receptor. While the precise mechanism has not been determined,
a number of factors have been identified while others are more controversial. What is clear is that BMSCs secrete a variety of factors
that can modulate the immune system and that there are probably undiscovered additional factors involved.
Importantly, it also appears that BMSCs require interaction with T cells in order to generate immunosuppressive trophic factors,
in a process called licensing. When conditioned media from naive BMSCs alone is tested, no inhibition of T-cell activation is
observed. It is only after exposure of BMSCs to T cells that the BMSC-conditioned media is found to inhibit T-cell activation.
Thus, the coincubation of BMSCs with T cells is a requirement for the production of inhibitory factors. The secretion of the inflam-
matory cytokine interferon gamma (IFNg) by T cells has been implicated as one of the major factors required for licensing. When
BMSCs are stimulated with IFNg, the conditioned media from these BMSC is then capable of inhibiting T-cell activation (Ren et al.,
2008; Polchert et al., 2008). Other cytokines that have been suggested to be involved in licensing include tumor necrosis factor
alpha (TNFa) and IL-1b.
BMSCs have also been observed to have effects on other cells from both the innate and adaptive immune system including
helper T cells, regulatory T cells (Tregs), macrophages, antigen-presenting cells (APC), NK cells, and B cells (Figure 1). When BMSCs
are incubated with monocytes, they inhibit their differentiation into dendritic cells and thus reduce the ability to create APC. BMSCs
can alter the cytokine profile of macrophages, DCs, naive and effector T cells (TH1 and TH2), and NK cells from a proinflammatory
to antiinflammatory state. This has been observed primarily by the types of cytokines secreted by the cells, either proinflammatory
(i.e., IFNg, TNFa, IL-1b) or antiinflammatory (i.e., IL-4, IL-10) (Le Blanc and Ringden, 2007; English et al., 2010; Tolar et al., 2010).
BMSCs decrease dendritic cell production of TNFa and increase production of IL-10. Similarly, helper T cells decrease IFNg and
increase IL-4 production (Aggarwal and Pittenger, 2005). BMSC not only drive immune cells toward an antiinflammatory pheno-
type, but also increase the presence of Tregs that are important for regulating the tolerance of the host immune system and may be
involved in reducing the alloreactive T-cell response in the presence of BMSC. The production of Tregs requires cell–cell contact with
Table 1 Potential mechanisms of allogeneic T-cell inhibition
Factor Mechanism Evidence
Prostaglandin E2 (PGE2) Secretion of PGE2, a known immune regulator, PGE2 inhibitors reduce the ability of MSCs to inhibit
by MSCs is upregulated in the presence of T cells T-cell proliferation
Indoleamine 2,3 IFNg stimulated MSCs express IDO, an enzyme An antagonist of IDO, 1-methyl-L-tryptophan blocks
deoxygenase (IDO) that breaks down tryptophan into kynurenine, the effects of MSCs on T-cell proliferation
which has been demonstrated to block
T-cell proliferation
Hepatocyte growth factor (HGF), Cytokines secreted by MSCs are known to Antibodies against HGF and TGF-beta block MSCs
transforming growth factor inhibit T cells ability to inhibit T-cell proliferation
beta (TGF-beta)
Nitric oxide (NO) MSCs produce NO in the presence of T cells and iNOS deficient MSCs, which cannot express NO, are
NO suppresses Stat-5 phosphorylation, which is unable to inhibit T-cell proliferation and unable to
required for T-cell proliferation provide benefit in a mouse GvHD model
HLA-G HLA-G is a nonclassical HLA Class I molecule that Antibodies against HLA-G block the ability of MSC to
inhibits T-cell proliferation and is secreted by inhibit T-cell proliferation
MSCs in an IL-10 dependent manner
MSC, mesenchymal stem cells; GvHD, graft-versus-host disease.

408 Regenerative Engineering j Adult Bone Marrow-Derived Stem Cells
Figure 1 Diagrammatic summary of the major CD4þ and CD8þ T-cell effector subtypes (as derived from naïve CD4þ and CD8þ T cells), the re-
ported effects of adult bone marrow-derived stem cells (BMSCs) on these effectors, and some clinically important disease associations for each. Bidi-
rectional arrows indicate reported interconversion (plasticity) between Th1/Th17 phenotypes and Th17/iTreg phenotypes that may be of relevance to
BMSC immune modulatory effects. CTL, cytotoxic T lymphocyte; DC, dendritic cell; DTH, delayed-type hypersensitivity; FOXP3, forkhead box P3 tran-
scription factor; GvHD, graft-versus-host disease; IFNg, interferon-gamma; IL, interleukin; iTreg, induced regulatory T cell; nTreg, natural regulatory
T cell; Th1, T helper type 1 cell; Th2, T helper type 2 cell; Th17, T helper type 17 cell; Treg, regulatory T cell. Duffy, M.M., Ritter, T., Ceredig, R.,
Griffin, M.D., 2011. Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res. Ther. 2, 34.
BMSC and PGE2, and TGF-beta that induces FoxP3, a transcription factor critical for the differentiation of Tregs (English et al.,
2009). Although this process is not nearly as well characterized, BMSCs have also been demonstrated to have effects on B-cell prolif-
eration upon stimulation (Asari et al., 2009). In vivo studies have demonstrated that BMSCs can reduce antibody production in
a heart transplant model (Ge et al., 2009).
Graft-versus-Host Disease (GvHD)
Bone marrow transplants or hematopoietic stem cell transplantations (HSCT) have been used for over 40 years to treat hematolog-
ical malignancies. One of the major complications that can occur with an allogeneic HSCT is GvHD, a severe inflammatory condi-
tion that occurs when the donor allogeneic T cells are activated by the host APC resulting in further stimulation of cellular and
proinflammatory responses that result in tissue injury (Krensky et al., 1990). While GvHD is a multiorgan disease, the most clin-
ically relevant organ damage occurs in the epithelial cell layer of the skin, gastrointestinal tract, and liver.
Based on in vitro studies that demonstrated the immunomodulatory properties of BMSC, a number of preclinical and clinical
studies were performed to determine if BMSCs could be used to treat GvHD (Tolar et al., 2011). The first clinical study was per-
formed by Le Blanc and colleagues in a child suffering from GvHD who was treated with BMSCs isolated from the mother (Le Blanc
et al., 2004). Subsequently, both the gut and liver GvHD were reversed after two infusions of BMSC. Based on this initial finding,
eight additional patients with steroid-refractory GvHD were treated with BMSC, of which six patients had complete resolution of
their GvHD (Ringden et al., 2007). The significant response observed in these patients led to a number of clinical trials using HLA-
identical allograft donors, haploidentical donors, or unrelated HLA-mismatched donors. One of the first studies ever done was to
give patients BMSCs from either HLA-identical haploidentical or third-party donors. Of the 55 patients in this study (Le Blanc et al.,
2008), 27 exhibited a complete response with 24 of these receiving third-party BMSC. These results demonstrated the utility of using
allogeneic BMSCs to treat GvHD.
The ability to use allogeneic or third-party BMSCs provides many benefits. First, allogeneic BMSCs can be generated in advance
from healthy donors, thus providing a more economical and uniform source of donor cells. Furthermore, allogeneic BMSCs are
available on demand for the treatment. In fact, an allogeneic BMSC product has recently been approved for commercial use in Can-
ada and New Zealand for the treatment of steroid-refractory GvHD in pediatric patients (www.osiris.com). In addition to the treat-
ment of GvHD, there has been a recent study examining the potential use of BMSC as a prophylactic treatment for GvHD. In a Phase
I clinical trial, BMSCs were administered to individuals undergoing allogeneic HSCT for the treatment of leukemia and related
conditions, the BMSCs were well tolerated in both the single infusion and repeat infusion arms and the data also suggested that
the therapy may provide benefit to recipients of allogeneic HSCT, by reducing the incidence and severity of GvHD as compared
to historical clinical experience (Maziarz et al., 2012).
Type 1 Diabetes
Type 1 diabetes is a T-cell-mediated, autoimmune disorder that leads to destruction of pancreatic b islet cells, and is characterized by
the presence of antiislet cell antibodies and severe insulitis (Vija et al., 2009). BMSCs have been shown to suppress autoreactive T-
cell responses in models of autoimmunity, making them an ideal candidate for use in Type 1 diabetes mellitus (T1DM). The efficacy
of BMSCs in the treatment of T1DM has been tested or proposed at several time points of intervention: (1) as a preventative treat-
ment or as a means to delay the full development of the disease, (2) as a therapy to treat complications arising from T1DM, (3) as a
supportive treatment during the isolation of donor pancreatic islet cells for an islet transplant, (4) as an adjunct therapy with islet
transplantation, and (5) as a rescue therapy upon onset of islet graft failure. Several studies have attempted to drive BMSCs to differ-
entiate into insulin-secreting cells, with mixed results, but the field remains focused on the use of BMSCs as immunomodulators in
the context of diabetes.
BMSCs have been shown to significantly delay disease onset in diabetic mouse models (Busch et al., 2011a). In a streptozotocin-
induced model of diabetes, green fluorescent protein (GFP) donor BMSCs significantly reduced blood glucose levels as compared to
controls. Transplantation of bone marrow resulted in an increase in insulin positive islets, but no GFPþ/insulinþ cells were
observed, suggesting that the results were due to proliferation of host islet cells. It has also been demonstrated that upon islet trans-
plantation, recipient islet precursor cells do not divide and contribute to graft function, so understanding the BMSCs-induced stim-
ulation of host cell division or insulin production may be particularly important. These studies have led to the working hypothesis
that BMSCs exert their beneficial effects through indirect mechanisms such as protection of remaining b-cells or stimulation of
endogenous b-cell replacement.
The early success of BMSCs in delaying the onset of diabetes led to studies examining the ability of BMSCs to prevent initial islet
loss and promote engraftment of islets after pancreatic islet transplantation. It is thought that an islet transplant has the potential to
cure Type 1 diabetes; however, there are currently major limitations to widespread implementation, including loss of large numbers
of islets in the immediate setting posttransplant, lack of revascularization, and transplant rejection (Korsgren et al., 2005). The effect
of multiple doses of BMSCs, in combination with immunosuppressive therapy, on islet graft rejection has been examined in dia-
betic rats (Solari et al., 2009). The results demonstrated that both intraportal and IV-administered BMSCs prolonged graft function
through prevention of acute rejection in a dose-dependent fashion, and no difference was seen between syngeneic or allogeneic
BMSCs. The ability of BMSC transplants to prevent rejection was similar to immunosuppressive therapy, but the combination of
BMSC and immunosuppressive therapy together were not more efficacious.
Islet grafts can deteriorate over time due to chronic allograft rejection, local islet toxicity as a result of the immuno-suppressive
regimen, recurrent autoimmunity, and/or failure of islet regeneration (Ding et al., 2010). Drugs used to prevent islet allograft loss
adversely effect b-cell function and glycemic control. BMSC therapy may have the potential to eliminate maintenance of some
systemic immunosuppressive drug therapies, thus relieving negative effects of these drugs on the graft itself in addition to removing
the risks of continued immunosuppression. The failure of the islet graft and the loss of insulin independence can involve rejection
due to the activation of alloreactive T cells and a reoccurrence of the original autoimmune disease. The breakdown of immunologic
tolerance may result in a cross-reactive memory response against the transplanted islets, resulting in loss of b-cell mass. Leukopenia,
a decrease in the number of white blood cells in circulation, can result from immunosuppressive therapy and favors the generation
of islet-reactive T cells, leading to islet destruction (Monti et al., 2008). It is thought that BMSCs may protect transplanted allogeneic
islets by negatively regulating persistent T-cell autoimmunity and controlling the activation and effector function of alloreactive
T cells. BMSCs may also suppress activation and proliferation of B cells, and prevent the differentiation and maturation of dendritic
cells, effectively preventing islet destruction. Further studies are necessary to determine the ability of BMSCs or other cell therapies to
prolong graft function or reverse graft failure.
Multiple clinical studies have examined the ability of bone marrow-derived cells to improve clinical outcome in both Type 1 and
Type 2 diabetes, and have established a safety profile for the use of these cells in diabetic patients. A Phase II, multicenter, random-
ized, double-blind, placebo-controlled study has been initiated to evaluate the safety and efficacy of adult human BMSCs for the
treatment of recently diagnosed Type 1 diabetes. The use of BMSCs as an adjunct therapy for islet transplantation warrants further
exploration and advancement toward clinical use.
Spinal Cord Injury
Spinal cord injury (SCI) results in disruption of the blood–brain barrier and initiates a cascade of inflammatory processes leading to
infiltration of immune cells and secondary cell death that extends beyond the site of initial injury (Silver and Miller, 2004). This
reactive process of secondary injury takes place in the days and weeks following SCI, and can result in exacerbation of neurological
dysfunction. An additional cause of spinal cord regeneration failure is the formation of the glial scar, which involves activation of
astrocytes in an attempt to restore the blood–brain barrier. These astrocytes produce inhibitory chondroitin sulfate proteoglycan,
a major barrier to regenerating axons. Other potential inhibitors in the glial scar are myelin-associated proteins, which also inhibit
neurite outgrowth and hinder repair.
BMSC therapy has been proposed to treat both acute and chronic SCI through multiple potential mechanisms of action (Wright
et al., 2011). The immunosuppressive properties of BMSCs may reduce the acute inflammatory response to SCI, reducing secondary
injury and cavitation. Direct transplantation of BMSCs after injury has been shown to increase preservation of spinal cord tissue and
decrease neuropathic pain. Direct transplantation of BMSCs into the cord may modify activation of astrocytes or encourage axonal
regeneration through downregulation of inhibitory components in the glial scar. BMSCs produce a number of growth factors,
including nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), which could promote axonal outgrowth
even in an inhibitory environment. Additionally, some reports suggest that BMSCs could act as bridges, guiding regenerating axons
across the injury cavity. BMSCs produce molecules including laminin, fibronectin, and collagen, which could decrease cavitation
and provide a permissive environment for growing axons (Ankeny et al., 2004).
As in most injury situations, some degree of inflammation is a necessary and beneficial component of the recovery process after
SCI (David and Kroner, 2011). Macrophages are known to phagocytose the myelin debris, clearing the way for functional regener-
ation, and to produce some protective cytokines and growth factors, which could enhance regeneration. Two subtypes of macro-
phages have been described with regards to their phenotype and activity: classically activated macrophages (M1) and
alternatively activated (M2). M1 macrophages are typically considered to be the product of activation with proinflammatory cyto-
kines IFNg and TNFa. Alternatively, activated macrophages are the product of activation with the cytokines interleukin-4 (IL-4) and
IL-13, and possess enhanced phagocytic capabilities and antiinflammatory activities, which are thought to contribute to their bene-
ficial effects after SCI. BMSCs and related cell types have been shown to drive macrophages toward the alternatively activated M2
phenotype and concurrently promote white matter sparing and reduce the effects of the inhibitory glial scar (Busch et al., 2011b;
Nakajima et al., 2012).
To date, the majority of cell therapy-focused clinical trials for SCI have utilized whole mononuclear cell preparations (MCPs)
from bone marrow, not cultured adherent BMSCs (Wright et al., 2011). MCPs are generally administered alongside granulocyte-
macrophage colony stimulating factor to mobilize the migration of these cells into the lesioned spinal cord and induce activation
resulting in the secretion of neurotrophic cytokines at the site of injury. Modest increases in neurological function have been re-
ported, but as relatively few patients have been treated thus far, it is difficult to determine if these results are due to an intrinsic
recovery process or directly attributable to the treatment itself.
Multiple Sclerosis
Multiple sclerosis (MS) is an immune-mediated disorder of the nervous system, which is characterized by inflammation and axonal
demyelination and degeneration (Odinak et al., 2011). The etiology of MS is uncertain, but it is likely that both genetic and envi-
ronmental factors contribute to disease onset and progression. Two aspects of the disease have the potential to be addressed by stem
cell therapy: prevention of further CNS damage via immunomodulation, and promotion of remyelination and repair. The basis for
the use of BMSCs in the treatment of MS comes largely from an animal model of MS, the experimental autoimmune encephalo-
myelitis (EAE) (Auletta et al., 2012). In this model, the combination of injected myelin immunogenic peptide and an adjuvant
generates widespread inflammation and demyelination in many regions of the brain and spinal cord. It has been demonstrated
that IV-infused BMSCs improve the clinical course and pathology scores in an EAE model (Zappia et al., 2005). This decreased
severity of the disease severity was paralleled by suppression of inflammation as indicated by T-cell anergy and decreasing demy-
elination and this response was associated with induction of tolerance toward the immunizing antigen. Intraventricularly injected
BMSCs have been observed to migrate to white matter lesions and induce upregulation of growth factors and proliferation of endog-
enous oligodendrocyte progenitor cells (Kassis et al., 2008). As a whole, these preclinical results suggest that not only do BMSCs
inhibit autoimmune attack, but they also provide significant neuroprotection despite their limited infiltration into the CNS.
Several studies have been undertaken to determine the safety and efficacy of BMSCs for the treatment of MS in the clinical setting
(Freedman et al., 2010). In a small study, 10 patients were given BMSCs via intrathecal administration and the only conclusion was
that the approach was clinically feasible (Connick et al., 2011). The preliminary results of a phase I/II study reported that a combi-
nation of intravenous and intrathecal administration of BMSCs given to 15 patients with MS resulted in no significant side effects
and revealed no unexpected pathology 1 year following injection (Slavin et al., 2008). This preliminary safety data suggest that
BMSCs can be considered relatively safe in the context of severe disease; however, additional studies are ongoing to determine
efficacy.
Stroke
Therapeutic options for patients suffering an ischemic stroke are extremely limited and agents which can exert neuroprotective
effects, induce neural plasticity, or encourage remodeling and neovascularization are of great interest. As in other injury paradigms,
the use of BMSCs was initially proposed as a cell replacement therapy; however, significant and reproducible transdifferentiation of
BMSCs into neural lineages in vivo have not been observed (Honmou et al., 2012). Following an ischemic stroke, some level of
spontaneous functional recovery is generally seen in both stroke patients and animal models, and it is possible that BMSCs can
harness and enhance this compensatory neural plasticity or remodeling in the days and weeks postinjury. Transplantation of BMSCs
in the hours to days following ischemia can reduce the size of the infarct and improve functional outcome measures in rodent
models of ischemic stroke. BMSCs are known to release growth factors and stimulate the release of beneficial factors from endog-
enous tissue. Intravenously delivered BMSCs also influence the ischemic tissue itself, reducing apoptosis of cells at the lesion
boundary and promoting proliferation of endogenous cells at the site of injury. BMSCs have been demonstrated to release
brain-derived neurotrophic factor (BDNF) and levels of this growth factor have been shown to increase at the site of ischemia in
rats receiving BMSC treatment (Nomura et al., 2005). BMSCs can also be engineered to express growth factors such as BDNF or
VEGF to be released over time at the injury site, providing a local and sustained neuroprotective, neurostimulatory, and/or angio-
genic therapy.
The proinflammatory environment after stroke leads to breakdown of the blood–brain barrier, which worsens the deficit
associated with the initial insult. Ischemic stroke stimulates adrenergic activation that promotes the release of immune cells
from the spleen, ultimately leading to a loss of splenic mass (Ajmo et al., 2009). Previous work has shown ischemic stroke
to be associated with the loss of splenocytes in conjunction with an increase in terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL)- þ apoptotic cells within the spleen, leading to decreased T-cell proliferation and cytokine produc-
tion culminating in a state of immunosuppression after stroke (Offner et al., 2006a,b). Separate experiments have shown
a peripheral increase in production of the proinflammatory cytokines TNFa, IFNg, IL-6, monocyte chemotactic protein-1
(MCP-1), and IL-2 after ischemic stroke (Offner et al., 2006a,b). After intravenous injection, BMSCs come into contact with
many organ systems including the spleen. Intravenous BMSC injection preserves the lost splenic mass (via inhibition of
CD8þ T-cell release) and potentially increases splenocyte proliferation resulting in production of antiinflammatory cytokines
such as IL-4 and IL-10 (Walker et al., 2010; Vendrame et al., 2006). The production of these cytokines could modulate the
proinflammatory response after injury in the lesion itself and in the penumbral regions of the stroke. These results support
the emerging concept that intravenous administration of bone marrow-derived cells can enhance stroke recovery through direct
effects on peripheral organs.
The safety, feasibility, and efficacy of autologous BMSCs administered intravenously have been examined in multiple Phase I
stroke clinical trials. MRIs have shown no tumor or abnormal cell growth in any patient to date and no severe adverse cell-
related effects have been reported. Bang and colleagues reported success in a 30-patient trial in patients suffering from severe middle
cerebral artery stroke in which 5 patients received 1 108 autologous BMSCs (Bang et al., 2005). While improvements were
observed in patients who received BMSCs, the treatment group was small and treatment procedures were not blinded. Additional
studies are necessary to conclusively determine if therapeutic intervention with BMSCs can improve clinical outcome in ischemic
stroke.
Potential Safety Issues Associated with BMSCs
Despite the success of BMSCs in preclinical models, several potential safety issues are associated with the use of BMSCs, particularly
in immunosuppressed patients. Given the strong capacity of BMSCs for immunomodulation, concerns have arisen that BMSCs
might interfere with immune responses against pathogens and therefore increase the risk of infection (Nauta and Fibbe, 2007).
Fortunately, no increases in infection have been observed in any of the clinical trials to date, in particular in those patients who
are already on immunosuppressive drugs. In fact, it was recently demonstrated that stimulated human BMSCs have potent antimi-
crobial effector function against bacteria, protozoal parasites, and viruses (Meisel et al., 2011).
In preclinical models, infused adherent stem cells can home to the stroma bed of preexisting tumors, with the implication that
through trophic support or immunomodulation these cells can promote tumor growth or block tumor clearance (Kuhn and Tuan,
2010). Conflicting data exist in the literature for this hypothesis without clarification of whether exogenously provided adherent
stem cells impact the endogenous endothelial and stromal populations involved in tumor initiation or growth; however, the
majority of reports do not reflect increased tumorigenic risk (Burt et al., 2008; Sensebe et al., 2012). Although human BMSCs
do not appear to be readily susceptible to chromosomal aberration in culture (Nauta and Fibbe, 2007), expanded BMSCs should
be tested for chromosomal stability and purity prior to clinical administration. A recent review article reports on completed clinical
studies with BMSCs covering more than 5000 patients treated in over 100 clinical studies bracketing 15 therapeutic areas including
both acute and chronic diseases (Ankrum and Karp, 2010). To date, with studies initiated over 16 years ago, no reports of tumor-
igenicity by donor product, or increased frequency of host tumorigenesis has been reported. While it is clear that only long-term
patient follow-up will provide a statistically valid evaluation of this association, near-term therapeutic decisions should be driven
by a careful analysis of patient risk/benefit considerations in specific disease settings.
Future Perspectives
The use of adult stem cells to treat inflammatory and autoimmune diseases offers novel and exciting prospects for the future. BMSCs
have immunomodulatory properties that make them uniquely suited to treat autoimmune disease and inflammation. Multiple
clinical studies have examined the ability of bone marrow-derived cells to improve clinical outcome and have established a safety
profile for the use of these cells in patients. Optimization of several parameters regarding BMSC therapy must be determined,
including the ideal time of administration, dosing regimen, route of administration, and identification of biomarkers that will iden-
tify the optimal time for intervention and will likely vary by indication. With the recent approval of BMSCs for the treatment of
GvHD, the field of adult stem cell therapy looks forward to clinical use in other areas. Based on the Phase II clinical trials underway
to establish the potency of BMSCs in MS, stroke, SCI, and diabetes, additional approvals are expected in the near future.
References
Aggarwal, S., & Pittenger, M. F. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 105, 1815–1822.
Ajmo, C. T., Jr., Collier, L. A., Leonardo, C. C., Hall, A. A., Green, S. M., Womble, T. A., Cuevas, J., Willing, A. E., & Pennypacker, K. R. (2009). Blockade of adrenoreceptors inhibits
the splenic response to stroke. Exp. Neurol., 218, 47–55.
Ankeny, D. P., Mctigue, D. M., & Jakeman, L. B. (2004). Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in
rats. Exp. Neurol., 190, 17–31.
Ankrum, J., & Karp, J. M. (2010). Mesenchymal stem cell therapy: two steps forward, one step back. Trends Mol. Med., 16, 203–209.
Asari, S., Itakura, S., Ferreri, K., Liu, C. P., Kuroda, Y., Kandeel, F., & Mullen, Y. (2009). Mesenchymal stem cells suppress B-cell terminal differentiation. Exp. Hematol., 37,
604–615.
Auletta, J. J., Bartholomew, A. M., Maziarz, R. T., Deans, R. J., Miller, R. H., Lazarus, H. M., & Cohen, J. A. (2012). The potential of mesenchymal stromal cells as a novel cellular
therapy for multiple sclerosis. Immunotherapy, 4, 529–547.
Bang, O. Y., Lee, J. S., Lee, P. H., & Lee, G. (2005). Autologous mesenchymal stem cell transplantation in stroke patients. Ann. Neurol., 57, 874–882.
Bartholomew, A., Sturgeon, C., Siatskas, M., Ferrer, K., Mcintosh, K., Patil, S., Hardy, W., Devine, S., Ucker, D., Deans, R., Moseley, A., & Hoffman, R. (2002). Mesenchymal stem
cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp. Hematol., 30, 42–48.
Burt, R. K., Loh, Y., Pearce, W., Beohar, N., Barr, W. G., Craig, R., Wen, Y., Rapp, J. A., & Kessler, J. (2008). Clinical applications of blood-derived and marrow-derived stem cells
for nonmalignant diseases. J. Am. Med. Assoc., 299, 925–936.
Busch, S. A., van Crutchen, S. T. J., Deans, R. J., & Ting, A. E. (2011a). Mesenchymal stromal cells as a therapeutic strategy to support islet transplantation in type 1 diabetes
mellitus. Cell Med. Part B Cell Transplant., 2, 43–53.
Busch, S. A., Hamilton, J. A., Horn, K. P., Cuascut, F. X., Cutrone, R., Lehman, N., Deans, R. J., Ting, A. E., Mays, R. W., & Silver, J. (2011b). Multipotent adult progenitor cells
prevent macrophage-mediated axonal dieback and promote regrowth after spinal cord injury. J. Neurosci., 31, 944–953.
Connick, P., Kolappan, M., Patani, R., Scott, M. A., Crawley, C., He, X. L., Richardson, K., Barber, K., Webber, D. J., Wheeler-Kingshott, C. A., Tozer, D. J., Samson, R. S.,
Thomas, D. L., Du, M. Q., Luan, S. L., Michell, A. W., Altmann, D. R., Thompson, A. J., Miller, D. H., Compston, A., & Chandran, S. (2011). The mesenchymal stem cells in
multiple sclerosis (MSCIMS) trial protocol and baseline cohort characteristics: an open-label pre-test: post-test study with blinded outcome assessments. Trials, 12, 62.
David, S., & Kroner, A. (2011). Repertoire of microglial and macrophage responses after spinal cord injury. Nat. Rev. Neurosci., 12, 388–399.
Dazzi, F., Szydlo, R. M., Apperley, J. F., & Goldman, J. M. (2002). Prognostic factors for acute graft-versus-host disease after donor lymphocyte infusions. Blood, 100, 2673–2674.
Di Nicola, M., Carlo-Stella, C., Magni, M., Milanesi, M., Longoni, P. D., Matteucci, P., Grisanti, S., & Gianni, A. M. (2002). Human bone marrow stromal cells suppress T-lymphocyte
proliferation induced by cellular or nonspecific mitogenic stimuli. Blood, 99, 3838–3843.
Ding, Y., Bushell, A., & Wood, K. J. (2010). Mesenchymal stem-cell immunosuppressive capabilities: therapeutic implications in islet transplantation. Transplantation, 89, 270–273.
Duffy, M. M., Ritter, T., Ceredig, R., & Griffin, M. D. (2011). Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell Res. Ther., 2, 34.
English, K., French, A., & Wood, K. J. (2010). Mesenchymal stromal cells: facilitators of successful transplantation? Cell Stem Cell, 7, 431–442.
English, K., Ryan, J. M., Tobin, L., Murphy, M. J., Barry, F. P., & Mahon, B. P. (2009). Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant
roles in human mesenchymal stem cell induction of CD4þCD25(High) forkhead box P3þ regulatory T cells. Clin. Exp. Immunol., 156, 149–160.
Freedman, M. S., Bar-Or, A., Atkins, H. L., Karussis, D., Frassoni, F., Lazarus, H., Scolding, N., Slavin, S., Le Blanc, K., & Uccelli, A. (2010). The therapeutic potential of
mesenchymal stem cell transplantation as a treatment for multiple sclerosis: consensus report of the international MSCT study group. Mult. Scler., 16, 503–510.
Ge, W., Jiang, J., Baroja, M. L., Arp, J., Zassoko, R., Liu, W., Bartholomew, A., Garcia, B., & Wang, H. (2009). Infusion of mesenchymal stem cells and rapamycin synergize to
attenuate alloimmune responses and promote cardiac allograft tolerance. Am. J. Transplant., 9, 1760–1772.
Honmou, O., Onodera, R., Sasaki, M., Waxman, S. G., & Kocsis, J. D. (2012). Mesenchymal stem cells: therapeutic outlook for stroke. Trends Mol. Med., 18, 292–297.
Kassis, I., Grigoriadis, N., Gowda-Kurkalli, B., Mizrachi-Kol, R., Ben-Hur, T., Slavin, S., Abramsky, O., & Karussis, D. (2008). Neuroprotection and immunomodulation with
mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis. Arch. Neurol., 65, 753–761.
Korsgren, O., Nilsson, B., Berne, C., Felldin, M., Foss, A., Kallen, R., Lundgren, T., Salmela, K., Tibell, A., & Tufveson, G. (2005). Current status of clinical islet transplantation.
Transplantation, 79, 1289–1293.
Krensky, A. M., Weiss, A., Crabtree, G., Davis, M. M., & Parham, P. (1990). T-lymphocyte-antigen interactions in transplant rejection. N. Engl. J. Med., 322, 510–517.
Kuhn, N. Z., & Tuan, R. S. (2010). Regulation of stemness and stem cell niche of mesenchymal stem cells: implications in tumorigenesis and metastasis. J. Cell Physiol., 222,
268–277.
Le Blanc, K. (2002). Mesenchymal stem cells. Basic science and future clinical use. Lakartidningen, 99, 1318–1321, 1324.
Le Blanc, K., Frassoni, F., Ball, L., Locatelli, F., Roelofs, H., Lewis, I., Lanino, E., Sundberg, B., Bernardo, M. E., Remberger, M., Dini, G., Egeler, R. M., Bacigalupo, A., Fibbe, W., &
Ringden, O. (2008). Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet, 371, 1579–1586.
Le Blanc, K., Rasmusson, I., Sundberg, B., Gotherstrom, C., Hassan, M., Uzunel, M., & Ringden, O. (2004). Treatment of severe acute graft-versus-host disease with third party
haploidentical mesenchymal stem cells. Lancet, 363, 1439–1441.
Le Blanc, K., & Ringden, O. (2007). Immunomodulation by mesenchymal stem cells and clinical experience. J. Intern. Med., 262, 509–525.
Liechty, K. W., Mackenzie, T. C., Shaaban, A. F., Radu, A., Moseley, A. M., Deans, R., Marshak, D. R., & Flake, A. W. (2000). Human mesenchymal stem cells engraft and
demonstrate site-specific differentiation after in utero transplantation in sheep. Nat. Med., 6, 1282–1286.
Mays, R. W., Van’t Hof, W., Ting, A. E., Perry, R., & Deans, R. (2007). Development of adult pluripotent stem cell therapies for ischemic injury and disease. Expert Opin. Biol. Ther.,
7, 173–184.
Maziarz, R. T., Devos, T., Bachier, C., Goldstein, S. C., Leis, J., Cooke, K. R., Perry, R., Deans, R. J., Van’t Hof, W. J., & Lazarus, H. M. (2012). Prophylaxis of acute GVHD using
multistem® stromal cell therapy: preliminary results after administration of single or multiple doses in a phase 1 trial. Biol. Blood Marrow Transplant., 18, S264–S265.
Meisel, R., Brockers, S., Heseler, K., Degistirici, O., Bulle, H., Woite, C., Stuhlsatz, S., Schwippert, W., Jager, M., Sorg, R., Henschler, R., Seissler, J., Dilloo, D., & Daubener, W.
(2011). Human but not murine multipotent mesenchymal stromal cells exhibit broad-spectrum antimicrobial effector function mediated by indoleamine 2,3-dioxygenase.
Leukemia, 25, 648–654.
Monti, P., Scirpoli, M., Maffi, P., Ghidoli, N., De Taddeo, F., Bertuzzi, F., Piemonti, L., Falcone, M., Secchi, A., & Bonifacio, E. (2008). Islet transplantation in patients with
autoimmune diabetes induces homeostatic cytokines that expand autoreactive memory T cells. J. Clin. Invest., 118, 1806–1814.
Nakajima, H., Uchida, K., Guerrero, A. R., Watanabe, S., Sugita, D., Takeura, N., Yoshida, A., Long, G., Wright, K. T., Johnson, W. E., & Baba, H. (2012). Transplantation of
mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury. J. Neurotrauma, 29, 1614–1625.
Nauta, A. J., & Fibbe, W. E. (2007). Immunomodulatory properties of mesenchymal stromal cells. Blood, 110, 3499–3506.
Nomura, T., Honmou, O., Harada, K., Houkin, K., Hamada, H., & Kocsis, J. D. (2005). I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem
cells protects against injury in a cerebral ischemia model in adult rat. Neuroscience, 136, 161–169.
Odinak, M. M., Bisaga, G. N., Novitskii, A. V., Tyrenko, V. V., Fominykh, M. S., Bilibina, A. A., Krugliakov, P. V., & Polyntsev, D. G. (2011). Transplantation of mesenchymal stem
cells in multiple sclerosis. Zh. Nevrol. Psikhiatr. Im. S. S. Korsakova, 111, 72–76.
Offner, H., Subramanian, S., Parker, S. M., Afentoulis, M. E., Vandenbark, A. A., & Hurn, P. D. (2006a). Experimental stroke induces massive, rapid activation of the peripheral
immune system. J. Cereb. Blood Flow Metab., 26, 654–665.
Offner, H., Subramanian, S., Parker, S. M., Wang, C., Afentoulis, M. E., Lewis, A., Vandenbark, A. A., & Hurn, P. D. (2006b). Splenic atrophy in experimental stroke is accompanied
by increased regulatory T cells and circulating macrophages. J. Immunol., 176, 6523–6531.
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., & Marshak, D. R. (1999). Multilineage potential
of adult human mesenchymal stem cells. Science, 284, 143–147.
Polchert, D., Sobinsky, J., Douglas, G., Kidd, M., Moadsiri, A., Reina, E., Genrich, K., Mehrotra, S., Setty, S., Smith, B., & Bartholomew, A. (2008). IFN-gamma activation of
mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur. J. Immunol., 38, 1745–1755.
Rasmusson, I., Ringden, O., Sundberg, B., & Le Blanc, K. (2003). Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes
or natural killer cells. Transplantation, 76, 1208–1213.
Ren, G., Zhang, L., Zhao, X., Xu, G., Zhang, Y., Roberts, A. I., Zhao, R. C., & Shi, Y. (2008). Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of
chemokines and nitric oxide. Cell Stem Cell, 2, 141–150.
Ringden, O., Uzunel, M., Sundberg, B., Lonnies, L., Nava, S., Gustafsson, J., Henningsohn, L., & Le Blanc, K. (2007). Tissue repair using allogeneic mesenchymal stem cells for
hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia, 21, 2271–2276.
Sensebe, L., Tarte, K., Galipeau, J., Krampera, M., Martin, I., Phinney, D. G., & Shi, Y. (2012). Limited acquisition of chromosomal aberrations in human adult mesenchymal stromal
cells. Cell Stem Cell, 10, 9–10. author reply 10–11.
Silver, J., & Miller, J. H. (2004). Regeneration beyond the glial scar. Nat. Rev. Neurosci., 5, 146–156.
Slavin, S., Kurkalli, B. G., & Karussis, D. (2008). The potential use of adult stem cells for the treatment of multiple sclerosis and other neurodegenerative disorders. Clin. Neurol.
Neurosurg., 110, 943–946.
Solari, M. G., Srinivasan, S., Boumaza, I., Unadkat, J., Harb, G., Garcia-Ocana, A., Feili-Hariri, M., et al. (2009). Marginal mass islet transplantation with autologous mesenchymal
stem cells promotes long-term islet allograft survival and sustained normoglycemia. J. Autoimmun., 32, 116–124.
Sordi, V. (2009). Mesenchymal stem cell homing capacity. Transplantation, 87, S42–S45.
Tolar, J., Le Blanc, K., Keating, A., & Blazar, B. R. (2010). Concise review: hitting the right spot with mesenchymal stromal cells. Stem Cells, 28, 1446–1455.
Tolar, J., Villeneuve, P., & Keating, A. (2011). Mesenchymal stromal cells for graft-versus-host disease. Hum. Gene Ther., 22, 257–262.
Vendrame, M., Gemma, C., Pennypacker, K. R., Bickford, P. C., Davis Sanberg, C., Sanberg, P. R., & Willing, A. E. (2006). Cord blood rescues stroke-induced changes in splenocyte
phenotype and function. Exp. Neurol., 199, 191–200.
Vija, L., Farge, D., Gautier, J. F., Vexiau, P., Dumitrache, C., Bourgarit, A., Verrecchia, F., & Larghero, J. (2009). Mesenchymal stem cells: stem cell therapy perspectives for type 1
diabetes. Diabete. Metab., 35, 85–93.
Walker, P. A., Shah, S. K., Jimenez, F., Gerber, M. H., Xue, H., Cutrone, R., Hamilton, J. A., Mays, R. W., Deans, R., Pati, S., Dash, P. K., & Cox, C. S., Jr. (2010). Intravenous
multipotent adult progenitor cell therapy for traumatic brain injury: preserving the blood brain barrier via an interaction with splenocytes. Exp. Neurol., 225, 341–352.
Wright, K. T., El Masri, W., Osman, A., Chowdhury, J., & Johnson, W. E. (2011). Concise review: bone marrow for the treatment of spinal cord injury: mechanisms and clinical
applications. Stem Cells, 29, 169–178.
Yagi, H., Soto-Gutierrez, A., Kitagawa, Y., Tilles, A. W., Tompkins, R. G., & Yarmush, M. L. (2010). Bone marrow mesenchymal stromal cells attenuate organ injury induced by LPS
and burn. Cell Transplant., 19, 823–830.
Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., Giunti, D., Ceravolo, A., Cazzanti, F., Frassoni, F., Mancardi, G., & Uccelli, A. (2005). Mesenchymal
stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood, 106, 1755–1761.
Assessment of Cellular Responses of Tissue Constructs in vitro in Regenerative
Engineering
Margaret AT Freeberg, Jacob G Kallenbach, and Hani A Awad, University of Rochester, Rochester, NY, United States
Biochemical and Biophysical Regulation of Cellular Responses 416

Characterizing Cellular Proliferation and Senescence 418
Quantifying Cellular Proliferation 418
Detection of Cellular Senescence 419
Characterizing Cellular Viability and Death 419
Live/Dead Staining 419
ATP/Metabolic Activity 420
Tetrazolium reduction 420
Resazurin reduction 420
Intracellular ATP quantification 420
Intracellular protease activity 420
Caspase activity 420
Characterizing Cellular Differentiation 421
Microscopy-Based Techniques 421
Confocal and multiphoton microscopy 422
Electron microscopy 422
FRET microscopy 422
Quantitative Gene Expression Analysis 423
DNA microarray 423
RNA sequencing 423
Quantitative Protein Analysis 425
Western blot 425
Enzyme-linked immunosorbent assays (ELISA) 425
Flow cytometry 425
Förster resonance energy transfer assays 425
Assessment of Mechanical Properties 425
Emerging Technologies 426
Further Reading 426
Glossary
Apoptosis A process of programmed cell death or suicide triggered by intrinsic (mitochondria-associated proteins) or extrinsic
(death receptor activation) pathways, characterized by an ordered sequence of events leading to chromatin degradation,
mitochondria disintegration, morphological changes and fragmentation, and ultimately inflammation-free phagocytosis of
the apoptotic bodies or fragments of the dying cell.
Cell cycle The ordered sequence of biological processes within a cell leading to the duplication of its genetic material (DNA)
and the production of two daughter cells; conventionally divided into a first gap (G1), a DNA synthesis (S) phase, a second gap
(G2), and a cell division or mitosis (M) phase.
Cell differentiation A multi-step process through which a stem cell loses its pluripotency and alters its phenotype to assume
a specialized function, characterized by overt changes such as cell shape and cell size, and covert changes such as altered gene
expression, metabolic activity, secretory phenotype, and responsiveness to external stimuli.
Cellular proliferation A process that results in a net increase of the number of cells through the balance of cell division and cell
death.
Necrosis A process of accidental or premature cell death by autolysis due to injurious events such as infection or trauma,
leading to the activation of an inflammatory response to eliminate the dead cell debris by phagocytosis.
Senescence A state of permanent and irreversible cell-cycle arrest associated with replicative aging in metabolically active and
viable cells, which is characterized by DNA damage, upregulation of cyclin-dependent kinase inhibitors, and an abnormal
secretory phenotype.

Regenerative Engineering j Assessment of Cellular Responses of Tissue Constructs in vitro 415
Abbreviations
3D Three-dimensional
AFM Atomic force microscopy
ATP Adenosine triphosphate
BrdU bromodeoxyuridine or 5-bromo-20 -deoxyuridine
Cas9 CRISPR-associated protein-9 nuclease
CRISPR Clustered regularly interspaced short palindromic repeats
ELISA Enzyme-linked immunosorbent assay
FRET Förster (fluorescence) resonance energy transfer
IHC Immunohistochemistry
ISH In situ hybridization
MSC Mesenchymal stem cells
MTS 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
MTT 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide
NAD Nicotinamide adenine dinucleotide
NADP Nicotinamide adenine dinucleotide phosphate
NGS Next generation sequencing
PCNA Proliferating cell nuclear antigen
PCR Polymerase chain reaction
RNA Ribonucleic acid
RNA-seq RNA sequencing
RNAi RNA interference
ROS Reactive oxygen species
RT-PCR Reverse transcription-polymerase chain reaction
SASP Senescence-associated secretory phenotype
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
WST-1 2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
X-gal 5-Bromo-4-chloro-3-indolyl-b-D-galactopyranoside
XTT 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
The replacement of terminally-diseased organs or massive tissue loss have been and continue to be critical needs in medicine. Tradi-
tionally, these surgical needs have been fulfilled using donated organs or tissue allografts. However, viable donor organs are limited
in their availability, carry numerous health risks, and require immunosuppressants for the life of the recipient. Processed allograft
tissues are readily available through tissue banks and can provide satisfactory short-term outcomes, but generally do not integrate
or mediate regeneration. Over the past 3 decades, tissue engineering and regenerative medicine, or regenerative engineering, have
emerged to design replacement tissues and organs that overcome the limitations of traditional sources. In the classical tissue engi-
neering paradigm, cells are seeded onto biomaterial scaffolds and stimulated biochemically and physically to grow a functioning
tissue or organ in vitro, in a simulated physiological environment such as a dish in an incubator or a more sophisticated bioreactor
system, to eventually be surgically implanted into a patient. The design of engineered tissues and organs must take into account the
different choices available for the three main building blocks of cells, signals, and scaffolds, which are beyond the scope of this
chapter. Regardless of these choices, this bottom-up approach inherently has a number of response variables that must be monitored,
optimized, and reproducibly-controlled; most importantly perhaps are related to cellular responses to their engineered environment.
When cells are dissociated from their physiological context (their native ECM) to be cultured in vitro, they can be induced to
assume one of four fates or phenotypes: proliferative, differentiated, senescent, or apoptotic. Likewise, when dissociated cells are
incorporated into a 3D biomaterial matrix in an engineered tissue construct, they can alter their phenotype depending on their inter-
actions with their engineered ECM substrate and the abundance of biological and biochemical signaling factors in their in vitro
culture environment. Monitoring the cellular responses to these biophysical and biochemical cues can be predictive of the function
of the engineered tissue. Assessment of cellular responses can be accomplished using invasive or destructive assays that require sacri-
ficial constructs in cross sectional studies, or using minimally- or non-invasive assays that can enable longitudinal, real-time moni-
toring of the functional maturation of the engineered tissue.
416 Regenerative Engineering j Assessment of Cellular Responses of Tissue Constructs in vitro
Biochemical and Biophysical Regulation of Cellular Responses
To create complex functional tissues, it is important to mimic the native tissue environment to restore function. Tissues and organs
are generally anisotropic, with spatially heterogeneous composition, morphology, nano- and micro-topographies, and biomechan-
ical properties that are optimized for function. The native and engineered tissue environments are composed of both biophysical
and biochemical cues that orchestrate cellular phenotype and drive altered responses. There is extensive research to understand how
biophysical and biochemical signals regulate cell fate and direct differentiation.
Built-in features of scaffold design such as substrate architecture and topography (surface patterning and fiber orientation)
and substrate stiffness can alter how cells perceive applied mechanical forces (tensile, compression, shear), interact with neigh-
boring cells, and respond to biophysical and biochemical cues to alter their phenotype (Fig. 1). Externally applied physical cues
can include mechanical, electrical, electromagnetic, acoustic and ultrasound, and photo stimulation. The effects of these
biophysical cues on stem cells have been extensively investigated in vitro. Surface chemistry and topography affect protein
adsorption from cell culture media, which consequently affect cell attachment mechanisms and impact cellular behaviors. In
addition, substrate or scaffold stiffness is an important biophysical cue that can direct cell differentiation. Cells adjust their cyto-
skeletal tension and stiffness to match their environmental substrate, therefore reduced cytoskeletal organization is typically
observed in cells grown on low stiffness substrates. This effect has mechanobiological consequences. For example, a substrate
stiffness of 0.1–1 KPa induces neurogenic differentiation, while a stiffness of 8–17 KPa induces myogenic differentiation, and
a 25–40 KPa stiffness induces osteogenic differentiation of stem cells. On the other hand, substrate stiffness < 0.1 KPa maintain
pluripotency of stem cells. Likewise, external mechanical forces of tension, compression or shear can either maintain cellular
Fig. 1 Biochemical and biophysical regulation of cellular responses. Biochemical cues include soluble molecules added to the culture media or can
include surface functionalization with proteins, peptides, or integrins. Physical cues include surface properties (topography, stiffness, alignment, etc.),
mechanical forces, electrical stimulation, application of ultrasound, and photostimulation. These cues can drive cellular responses, including differen-
tiation, proliferation, apoptosis, and senescence. Adapted from Ding, S. Kingshott, P., Thissen, H., Pera, M. and Wang, P. Y. (2017). “Modulation of
human mesenchymal and pluripotent stem cell behavior using biophysical and biochemical cues: A review.” Biotechnology and Bioengineering 114(2),
260–280.
pluripotency by enhancing proliferation and inhibiting differentiation or stimulating cell lineage differentiation. For example,
cyclic compression of MSC can promote cartilage formation or chondrogenesis, while shear stress from fluid flow can stimulate
MSC differentiation into endothelial cells to form vessel networks, whereas high intracellular tension can encourage MSC
toward osteoblasts. Even though specific mechanical forces are correlated with differentiated cell types, the mechanical environ-
ment needs to be maintained in order to maintain the differentiated cell phenotype. Electromagnetic stimulation is of particular
importance for cardiac and neural lineage cells that function to communicate via electrical pulses. Neurogenesis and cardiomyo-
genesis depend greatly on intensity, duration, and application method of the electromagnetic stimulus. Overall, electromagnetic
stimuli have been applied successfully to differentiate neurons and cardiomyocytes, and it remains an area of interesting
research to evaluate their effects on other cell types. Ultrasound is a mechanical wave (low intensity, high frequency) that
can modulate and pattern cellular behavior and tissue morphology. Besides its clinical imaging diagnostic value, ultrasound
has been shown to stimulate osteogenesis, increase proliferation, and increase differentiation of stem cells, and as such offers
exciting potential as a safe tissue engineering and regeneration approach. Photostimulation is likely wavelength- and cell
type-dependent as it has been shown to increase epidermal stem cell proliferation and migration, while decreasing proliferation
of bone marrow-derived MSC.
Biochemical cues are primarily soluble factors that are delivered as signals to guide cellular responses and include cytokines and
growth factors, genetic editors (RNAi, CRISPR/Cas9), proteins and proteases, and exosomes. In general, biochemical cues are easy to
deliver in culture media. However, it is often advantageous to tether biochemical cues to scaffolds to control their extended release
and their bioavailability for their target cells. This has been an area of intensive research in tissue engineering, where optimization
studies are typically performed in vitro. For example, osteogenic differentiation of MSC requires ascorbate, beta-glycerophosphate,
and bone morphogenetic proteins (BMPs) to stimulate the expression of the osteogenic genes Runx2, osteocalcin, and osteopontin,
whereas adipogenesis requires dexamethasone, indomethacin, and insulin. Transforming growth factor beta (TGF-b) is typically
added at varying doses to induce mesodermal lineages of bone, fat, cartilage, tendon, ligament, and cardiac differentiation. Further-
more, cardiomyocyte differentiation from pluripotent stem cells within embryoid bodies is promoted by culturing with non-
essential amino acids, L-glutamine, b-mercaptoethanol, and high concentrations (20%) of fetal bovine serum. Moreover, functional
insulin producing pancreatic b-cells were generated from stem cells through systematic addition and removal of activin, Wnt, fibro-
blast growth factor 10 (FGF10), retinoic acid, insulin-like growth factor 1 (IGF) and smoothened antagonists (SANT). Extracellular
matrix soluble and insoluble proteins are also important in controlling cellular behavior. For example, Matrigel, a gelatinous ECM
mixture from mouse sarcoma cells, supposedly maintains stem cell self-renewal in cell culture applications, but is an exogenous
murine mixture limited in its clinical applications. Fetal bovine serum (FBS) is ubiquitously used in cell culture as a biochemical
supplement because it contains copious biomolecules such as vitronectin, fibronectin, and albumin, as well as amino acids, sugars,
lipids and growth factors and cytokines. The adsorption of serum proteins on engineered materials critically influences cell adhesion
and cell migration. Engineered surface modifications, whether that be protein or polymer coatings on materials, have been shown to
affect MSC behavior. For example, protein coatings on polydimethylsiloxane (PDMS) surfaces primarily utilizing fibronectin and
Type I collagen have been shown to promote cell adhesion and growth in cell culture applications. Immobilized bioactive peptides
can directly influence cell phenotype through juxtacrine signaling.
Since biophysical and biochemical stimuli combine in native tissues, similar combinations need to be recapitulated for
growing artificially engineered tissues. Therefore, they systematically need optimization, and this bottom up approach often
employs empirical experimental strategies. This can be expensive, time-consuming, and highly variable due not only to the
need for sequential addition and subtraction of growth factors, which varies for different cell phenotypes, but also due to
the variability in biologically-derived culture media supplements such as sera. Furthermore, exogenous soluble factors and
animal derived products can elicit problems of contamination especially for clinically translatable cellular therapies. Thus,
a major emphasis of tissue engineering and regenerative medicine research has been, and continues to be focused on charac-
terizing the cellular responses to biophysical and biochemical stimuli in vitro prior to in vivo testing and clinical translation.
These cellular responses induce overt and covert alterations in cell phenotypes, which require specialized characterization
techniques.
Overt phenotypic changes such as changes in cell numbers and morphological changes including cytoskeletal organization and
contractility can be assessed using microscopy. Covert changes, as with cell differentiation for example, typically require biological
and biochemical assays to measure the associated transcriptional and translational phenomena. Some standard techniques measure
these changes by lysing the cells to measure altered gene expression, protein content and phosphorylation, or protein-DNA inter-
actions, to name a few examples. Other techniques can often rely non-destructive assays to characterize real time measurable
changes in cellular responses such as activatable ion channels (e.g., Ca2 þ flux) and receptor-ligand binding or changes induced
by the cells such as the cell-mediated unfolding of cryptic peptide sequences in structural proteins, as well as cell signaling (e.g.,
using Förster resonance energy transfer (FRET)-based approaches).
In practice, there are numerous techniques that can be employed to assess cellular proliferation and metabolic activity, multi-
potency or differentiation state, and whether the cells are being driven to pathologic phenotypes such as senescence or apoptosis.
Typically, a comprehensive assessment of cellular responses in engineered tissue constructs uses a combination of outcomes based
on gene expression, protein content and phosphorylation, enzymatic activity, imaging, and mechanical characterization. This
manuscript offers a survey of the state-of-the-art tools and assays for the assessment of cellular responses within engineered tissues
in vitro.
Characterizing Cellular Proliferation and Senescence
The cell cycle is an orchestrated sequence of events during which a cell duplicates its contents and divides into two daughter cells.
There are 4 phases of the cell cycle: G1, S, G2 and M phases (Fig. 2). The G1 phase, or Gap 1, is an interphase during which the cell
synthesizes mRNA and proteins necessary for transitioning into the subsequent step of DNA synthesis. The G1/S checkpoint in this
interphase orchestrates cell fate decisions, instructing the cells to become quiescent, differentiate, or proceed to DNA replication and
chromosome duplication in the S phase. Dysregulated G1/S transitioning can lead to transformation and cancer. Once DNA repli-
cation in the S phase is complete and DNA damage is detected and repaired, the cell cycle enters into a second interphase known as
G2 or Gap 2, in which the cells experience rapid growth and protein synthesis in preparation for mitosis in the M phase. Another
critical DNA damage control takes place within the G2/M checkpoint, which ensures that mitosis is not initiated before damage to
DNA during replication is repaired. All major checkpoint transitions in the cell cycle are orchestrated by cyclins, cyclin dependent
kinases (CDK) and a large family of cyclin and CDK regulators.
When cells exit the cell cycle they enter a phase known as G0 or the resting phase, in which they can acquire a quiescent pheno-
type or a non-cycling state. Tissue resident stem cells mostly exist in this state. Often cells in G0 can reversibly differentiate without
losing the ability to re-enter cell cycle such as the case with hepatocytes. However, if the cells irreversibly differentiate into a post-
mitotic phenotype such as osteocytes, myocytes, and mature neurons they cannot re-enter the cell cycle. Cells in G0 can sometime
assume another irreversible phenotype known as senescence, a degenerative and often aging-associated change in the cells accumu-
lating over many cycles of cell divisions, which often contributes to a pathology in the tissue. A third phenotype relates to a process
of programmed cell death known as apoptosis, which is characterized by overt morphological changes preceding death, including
membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation.
Apoptosis is a highly regulated process and has been linked to cell cycle since a number of tumor suppressor genes have been shown
to sensitize cells to programmed death.
Quantifying Cellular Proliferation

Detection of gene expression or antigens present exclusively in proliferating cells represent standard techniques for assessment of
cell proliferation. The nuclear protein Ki-67 is a bona fide proliferation marker, which is expressed during all phases of the cell cycle
but not during quiescence or in differentiated cells (Fig. 2). While its function remains largely unknown, recent discoveries sug-
gested a role for Ki-67 in organizing heterochromatin including compaction and long-range genomic interactions. Other active
cell cycle markers include the proliferating cell nuclear antigen (PCNA), which is a homotrimer scaffolding protein involved in
DNA replication and repair and chromatin remodeling. PCNA nuclear localization is particularly increased during the S phase
of the cell cycle. Both Ki-67 and PCNA can be detected in cell-seeded engineered tissue constructs using standard immunohisto-
chemistry techniques with a variety of antigen- and species-specific, commercially available antibodies. Other less commonly re-
ported nuclear antigen markers of cell proliferation, sometimes used as indices for cancer diagnosis, include type II DNA
topoisomerase and phosphohistone H3, both of which are essential for several proliferation events in DNA replication and chro-
matin modification and can be detected using immunohistochemistry.
Fig. 2 The cell cycle and cellular phenotypes. There are 4 phases of the cell cycle: G1 Phase, S phase, G2 phase, and M Phase. Ki-67 and PCNA are
nuclear antigens exclusively present in proliferating cells. Cells can exit the cell cycle to enter quiescence, or can differentiate or undergo senescence
or apoptosis. Differentiation can be assessed histologically by examining morphology or using IHC for phenotypic antigen markers. Senescence can
be detected by increased b-galactosidase. Apoptosis can be detected by probing for caspase activity.
Cell proliferation can also be measured by quantifying newly synthesized DNA by treating cells in cultures with the synthetic
thymidine analog bromodeoxyuridine (5-bromo-20 -deoxyuridine or BrdU). BrdU signal can be simply detected as it gets incorpo-
rated into newly synthesized DNA during the S phase using immunohistochemistry and visualized by a colorimetric, chemilumi-
nescent, or fluorescent reporter signal. More sophisticated analysis of the cell cycle phases using fluorescent-BrDU labeling can be
performed using flow cytometry.
Detection of Cellular Senescence

Cellular senescence is a phenotype associated with DNA damage, in which cells cease to divide but remain metabolically
active. Senescence often occurs when cells in culture have reached their maximum number of divisions, the Hayflick limit,
and is often associated with telomere shortening leading to DNA damage due to reduced telomerase activity. Environmental
stress factors in culture such as reactive oxygen species (ROS) can also induce senescence, sometimes downstream of
commonly-used growth factors, including TGF-b, or under supraphysiological oxygen conditions common in many tissue
engineering scenarios. In vivo, senescence is associated with aging-related pathologies, in which senescent cells produce
a pro-inflammatory secretome. This senescence associated secretory phenotype (SASP), which consists of inflammatory cyto-
kines, growth factors, and proteases is a bona fide markers panel for senescent activity, which remains understudied in tissue
engineering. SASP can be quantified at the gene expression level in the cells or at the secreted protein level using enzyme-linked
immunosorbent assays (ELISA), where innovations in multianalyte detection assays can provide comprehensive phenotypic
assessments.
Morphologically, senescent cells grow larger with flattened bodies and express b-galactosidase, an enzyme whose activity in cata-
lyzing the hydrolysis of b-galactosides can be detected using chromogenic substrates. The most popular substrate for detecting
b-galactosidase is the glycoside X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside). The X-gal assay relies on the activity
of b-galactosidase to induce release of a soluble indolyl group from X-gal, which can be subsequently oxidized to form a blue precip-
itate that can be visualized by eye in whole-mount constructs or using light microscopy at a higher magnification. Furthermore,
nuclear antigens belonging to a large family of CDK inhibitors, such as p21 and p16, have been associated with the induction
of senescence without detectable DNA damage, and therefore have also been used as bona fide markers of senescence, which
can be detected immunohistochemically. Moreover, senescent cells display components of the DNA damage response (DDR)
including phosphorylated gH2AX in their nuclei, which can be used as a means to assess the senescent state of the cells in vitro
using immunohistochemistry.
Characterizing Cellular Viability and Death
In assessing the function of a tissue engineered construct in vitro, it is critical to determine if cells are viable within their engineered
environment. There are numerous methods to evaluate cellular viability, which include assessment of cell and DNA damage, ATP
concentration, and metabolic activity.
Cells in engineered tissue can experience death by apoptosis or necrosis. Apoptosis is programed cell death, which plays an indis-
pensable role in morphogenesis and organogenesis during embryonic development, and is one of the key cellular responses to stress
within adult tissues or organs, often in the context of pathology. During apoptosis cells begin to retract and condense as they begin
to degrade their internal components. Apoptosis happens very rapidly, on the order of 20–60 minutes, making it very difficult to
measure. There a number of biochemical processes that occur between the onset of apoptosis and the end point of cell and nuclear
fragmentation. These processes include cytoskeletal reorganization, alterations (blebbing) in the membrane, proteolytic cleavage,
DNA degradation, and fragmentation into apoptotic bodies. These biochemical processes can be probed to detect apoptosis.
Necrosis is a distinct mode of cell death characterized as “accidental” or “uncontrolled” destruction of the cell and release of its
contents, which invokes an inflammatory response. Necrosis lacks the ordered sequence of events observed in apoptosis. Regardless
of the mechanism, cell death in engineered tissues in vitro can result from environmental stress factors including extreme hypoxia or
diffusion-limited nutrient bioavailability, and must be assessed as endpoint outcomes using sacrificial constructs or using real-time,
non-invasive assays.
There are numerous assays and techniques available to measure cell viability. Each assay has advantages and disadvantages.
Therefore, it is important to identify what best suits the experimental needs. Common viability assays include live/dead staining,
ATP/metabolic assays, and detection of protease activity. Additional assays, which will not be discussed herein, examine individual
cells to assess cell morphology, chromatin condensation, and detection of fragmented DNA. These are typically measured by light or
electron microscopy, flow cytometry, or using time-lapse microscopy.
Live/Dead Staining
This approach introduces a combination of two distinct fluorophores (green/red) to identify live and dead cells, respectively, within
an engineered construct. Typically, a membrane permeant, optically-silenced fluorophore, such as calcein-acetoxymethyl ester (AM)
can enter into the cytosol of a live cells, where AM is hydrolyzed by intracellular esterases to liberate the green fluorescent calcein
dye. The calcein fluorescence intensity is proportional to the number of viable cells because esterase activity is absent in dying cells.
The second fluorophore, typically a red-fluorescent ethidium homodimer, is not membrane permeant but has the ability to bind
DNA in dead cells where the cell and nuclear membranes are disrupted. Cell viability (green) or death (red) can then be visualized
using fluorescent or confocal microscopy, or quantitatively assessed using a spectrophotometer (plate reader) or a molecular
imaging instrument capable of measuring fluorescence in tissues.
ATP/Metabolic Activity
Methods that measure aspects of general cellular metabolism or enzymatic activity include: tetrazolium reduction, resazurin reduc-
tion, protease markers, and ATP detection. Metabolism based methods require incubation of a reagent with cells, which in the pres-
ence of metabolically active cells will react with a substrate to generate a colorimetric or fluorescent signal for detection. The signal is
proportional to the number of viable cells present, since dead cells lose their ability to convert the substrate to a measurable signal.
Tetrazolium reduction
Tetrazolium salt compounds are commonly used to detect viability. There are two basic categories of tetrazolium salts: (1) Cationic
salts that can permeate viable eukaryotic cells through electrostatic interactions with the anionic plasma membrane. Cationic tetra-
zolium salts include MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). (2) Anionic tetrazolium salts require an
electron-coupling reagent to permeate live cells. These include MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), and WST-1
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium). All of these assays depend upon the
action of dehydrogenase enzymes in metabolically active cells to reduce anabolic cofactors such as nicotinamide adenine dinucle-
otide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). In the presence of active dehydrogenase enzymes, the
colorimetric salt permeating into the cytosol of a metabolically active cell is reduced producing an intracellular purple precipitate
(formazan), which typically requires additional steps (such as solubilization) before it can be quantified by absorbance using a spec-
trophotometer or a plate reader. Tetrazolium reduction is toxic to cells and is an endpoint measure of viability, which can only be
performed in sacrificial constructs.
Resazurin reduction
Resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) is a cell permeable redox indicator. Similar to tetrazolium reduction, viable
cells with active metabolism can reduce resazurin to produce a pink and fluorescent precipitate (resorufin) which can be measured
colorimetrically or fluorescently using a spectrophotometer or a spectrofluorometer. As with tetrazolium salts, the reduction of resa-
zurin salts is induced by dehydrogenase enzymes in metabolically active and viable cells. Common resazurin reduction assays
include Alamar Blue.
Intracellular ATP quantification

Another cell viability assay utilizes of the critical role of intracellular ATP in respiration of living cells. Measurement of ATP concen-
tration employs bioluminescent detection using firefly luciferase. ATP detection methods require lysing the cells to release ATP,
which is subsequently reacted with luciferase to generate photons. When cells begin to die, they lose their membrane integrity
and ability to synthesize ATP, and as such dying cells contain little to no ATP. Additionally, endogenous ATPases rapidly deplete
any remaining ATP from the cytoplasm of a dying cell. Therefore, there is a tight linear correlation between cell number and the
concentration of ATP. The bioluminescence intensity can be measured by any plate reader or other instruments capable of detecting
luminescent signals.
Intracellular protease activity

Protease activity within living cells, including cytoplasmic aminopeptidase, can be exploited as markers for viability by intro-
ducing a cell permeant fluorogenic protease substrate such as glycyl-phenylalanyl-aminofuorocoumarin (GF-AFC). The
aminopeptidase-mediated cleavage of the GF amino acids liberates the fluorescent AFC, producing a signal proportional to
cell viability, which can be measured using a spectrofluorometer. Intracellular protease activity can also be exploited as tools
to assess cytotoxicity, such as lactate dehydrogenase, which is released by necrotic and apoptotic cells. In general, assessment
of intracellular protease activity is non-toxic to cells, and therefore, has the benefit of enabling longitudinal, non-destructive
assessments.
Caspase activity
A defining feature of apoptosis is the activation of caspase enzymes, which play critical roles in the programmed cleavage of protein
substrates and in the subsequent fragmentation of the cell. Caspase enzymes can be detected in cells in the early stages of apoptosis
using immunohistochemistry. Alternatively, cell-permeant, caspase-susceptible fluorogenic substrates can be used with intact cells
in engineered tissues or with tissue/cell lysates, and caspase activity as a measure of cell apoptosis can be quantified with
a spectrofluorometer.
Characterizing Cellular Differentiation

Microscopy-Based Techniques
The naked eye can view objects around 200 mm, but cannot resolve objects at the scale of typical animal cells (10–20 mm in diam-
eter) and intracellular organelles (500–5000 nm). Microscopy is a useful tool that allows for visualization of cells within viable or
fixed engineered tissues in vitro. The resolving power of a microscope is defined as the smallest detectable separation distance
between two points on a specimen, which depends on several factors including the wavelength of light (or electrons in an electron
microscopy), the optical and geometric features of the lenses, and sample preparation. Light microscopy can resolve features down
to 0.2 mm. The resolving power of scanning and transmission electron microscopy (SEM and TEM) is about 2 and 0.1 nm, respec-
tively (Fig. 3).
Microscopy techniques have advanced over the past decades to enable detection, measurement, and time-lapse monitoring of
numerous phenomena within living cells or engineered tissues at multiple scales. These techniques can be utilized to assess cellular
differentiation, by probing specific cellular phenotypes using immunohistochemistry techniques, or as a tool to monitor cellular
morphology and dynamics of cellular interactions within the scaffolds such as adhesion, spreading, and migration.
The use of light or fluorescence microscopy in assessing cellular responses in tissue engineered constructs is limited by the size of
the construct, its increasing opacity with maturation and compaction, and the refractive and diffractive features of the scaffold archi-
tecture. Dynamic observations on living cells within scaffolds are therefore restricted to superficial regions of interest, and often
utilize fluorescent reporters to tag cells or intracellular proteins. However, if the objective is to capture detailed mechanistic infor-
mation on cellular responses within the scaffolds, it is advantageous to perform cross-sectional, time-course studies on cryogenically
or chemically fixed engineered tissues using routine histology with the aid of a specific stains, antibodies for immunohistochem-
istry, or complementary nucleic acid probes for in situ hybridization. Brightfield histology is commonly performed by microscopic
examination of thinly cut sections of fixed tissue mounted on glass slides and subsequently stained with special chemical dyes to
give contrast to differentially visualize the various classes of extracellular matrix proteins and cells, as well as intracellular organelles.
There are numerous dyes with selective binding affinity to DNA, carbohydrates, lipids and proteins that can be used for primary,
counter, or differential staining. The choice of which stain to use can be made depending on the types of cells and extracellular
matrix expected and the questions asked.
Immunohistochemistry and in situ hybridization offer a level of molecular resolution not possible with histology. In immuno-
histochemistry, primary antibodies specific to intra- or extracellular antigens (proteins) are incubated with the tissue sections, and
then immunolabeled with appropriate secondary antibodies tethered to a chromogen or a fluorophore to visualize the antigen of
interest. When unbound antibodies are washed, the sections can be viewed with the aid of a microscope to give a binary determi-
nation of whether the antigen of interest is present in the tissue sample. Immunohistochemistry is a powerful diagnostic tool in
clinical pathology, and can be equally powerful in assessment of cellular responses and molecular evolution of engineered tissues.
In situ hybridization localizes the expression of genes of interest in populations of cells within engineered tissues. The principle of
Fig. 3 The resolving power of microscopy.

in situ hybridization takes advantage of the complementary nature of base pairing (A-T(U), C-G) in DNA and RNA. In this tech-
nique, a labeled (fluorescent, radioactive, or chromogenic) short sequence of nucleic acid bases (probe) designed to be complemen-
tary to a known sequence of DNA or RNA (gene of interest) is incubated with the tissue or section, and allowed to recombine with
the gene of interest. When unbound probe is washed away, the detection of a signal or lack thereof gives a binary determination of
whether the gene is expressed in the cells within the tissue samples, which can be done in whole mount preparations or with the aid
of microscopic examination of a tissue section.
While histology-based techniques including immunohistochemistry and in situ hybridization are valuable tools, whose use is
commonplace in biomedical sciences including tissue engineering, they are not without limitations. These imaging-based tech-
niques are for the most part non-quantitative. They are also sensitive to sampling, in the sense that only small tissue samples or
biopsies in thinly sliced sections are examined to assess cellular responses in thick 3D tissues. Furthermore, histology-based tech-
niques are highly empirical recipes, where nuanced procedural deviations in fixation, demineralization, or staining steps can lead to
difficulties or misinterpretations. Another limitation is that there are no universally accepted predictive marker or panel of markers
that would be conclusive regarding the functional maturity of engineered tissues. In that sense, histological-based assessments can
be biased to common paradigms because they target a priori selected antigens or genes of interest, and therefore can be an effective
tool for hypothesis testing, but a less effective discovery instrument. The cross-sectional nature of these destructive techniques gives
snapshots of discrete points in time, and therefore the functional assessment of the cellular responses in engineered tissues should
take into account the histological history (morphology, immunohistochemistry, and in situ hybridization), along with other rele-
vant outcomes. A word of caution is that histology imaging produces more information than meets the untrained eye, and therefore
the reading of histology slides should ideally be performed by trained pathologist.
Confocal and multiphoton microscopy

Visualizing tissue engineered constructs using the histology-based techniques mostly uses standard phase-contrast (bright-field)
microscopy or fluorescence microscopy. Both techniques are effective in imaging cells in monolayer cell cultures or thin tissue
sections on glass slides, but have very limited applications in 3D engineered constructs. While fluorescence microscopy can be
employed to detect the general location, number, morphology or viability of cells aided by genetic or chemical introduction of flu-
orophores, the quality of images weakens with depth, because the entire sample is evenly excited resulting in a blurred signal and
unfocused background. Confocal laser scanning microscopy (CLSM) is a specialized fluorescent microscopy technique that
increases the optical resolution by using a pinhole aperture to block out-of-focus light within the image. Then a composite image
is created by scanning across a field of view. This technique increases the resolution to about 200 nm (0.2 mm) and allows for 3D
imaging (z-stacks) to a depth of about 150–300 mm within the tissue construct, depending on the density and optical properties of
the tissue. However, CLSM can activate photo-sensitive transients within the cells resulting in cell and DNA damage or cell death,
and can therefore affect the cellular responses and functional outcomes. As such, CLSM should not be broadly considered a non-
invasive imaging technique and perhaps should be used in conjunction with culture media additives such as antioxidants, provided
that they do not influence the maturation of the tissue engineered constructs.
Multiphoton-excited fluorescence microscopy overcomes limitations of confocal microscopy to enable deeper light penetration
up to several hundred microns depth with reduced photodamage and exquisite resolution, which enables sharp 3D imaging of
intra- and extracellular structures and assessment of dynamic interactions of the cells with their engineered extracellular matrix.
Label-free multiphoton fluorescence microscopy can also take advantage of a nonlinear optical phenomenon known as second
harmonic generation (SHG), which allows discrimination of signals from specific structures (such as fibrous collagen) from the
bulk tissue-engineered constructs. SHG multiphoton microscopy can be particularly useful in imaging structural maturity and
assessing the compositional anisotropy of tissue engineered extracellular matrix.
Electron microscopy
Electron microscopy involves the use of accelerated electron beams, instead of light, to visualize the sample. This technique allows
for much greater magnification, resolving details at the nanoscale, which is useful to observe very small structures within the cell. It
is an endpoint imaging technique, which requires attention to specimen fixation and subsequent preparation protocols. In scanning
electron microscopy (SEM), the electron beam projected at a non-normal angle scans a region of interest, losing energy by a variety of
mechanisms, ultimately resolving surface features of the sample. In SEM, an ultrathin conductive coating (e.g., with gold), typically
applied by low vacuum sputter coating, is necessary for imaging non-conductive samples such as tissue engineered scaffolds. In
transmission electron microscopy (TEM) the electrons are transmitted through the specimen, and as such the specimens should be
sectioned to thin slices. TEM can achieve magnifications approaching 50 million times and resolutions at the sub-nanoscale
(< 1 Å or 0.1 nm). SEM has lower resolution compared to TEM, however, because the information retrieved in SEM are limited
to 3D surface features, it has the advantage of being performed on bulk engineered tissues without the need for sectioning. Further-
more, the advent of environmental scanning electron microscopy (ESEM) facilitates imaging unfixed hydrated biological samples,
producing images of good quality in low vacuum without the need for conductive coating.
FRET microscopy
Despite the unsurpassed high-resolution imaging of the electron microscope, one of its main limitations is the lack of compatible
labeling techniques necessary to interrogate the dynamic interactions between intracellular proteins in signal transduction pathways
and numerous vital cellular processes. FRET microscopy is an emerging microscopy technique that enables precise localization and
probing of the interactions between proteins in living cells at the molecular level, and could be of interest in mechanistic studies in
the area of tissue engineering. FRET microscopy is based on combining the principles of Förster (Fluorescence) Resonance Energy
Transfer phenomenon with high resolution fluorescence microscopy. This enables high resolution spatial and temporal assessment
of the weak and transient molecular interactions between proteins inside the living cell.
Quantitative Gene Expression Analysis

The central molecular biology dogma, which states that in the cell, DNA is transcribed into RNA, which is subsequently translated
into proteins, offers a powerful toolbox for the interrogation of cellular functions. RNA can be extracted from tissue engineered
constructs to assess changes in gene expression over time during the course of in vitro culture and in response to external stimuli.
Extraction of high quality RNA in sufficient quantities from bulk tissue constructs can be challenging. The extraction techniques are
nuanced and empirical, wherein subtle changes in various steps or reagents can lead to dramatic effects on purity and yield. This
purity and yield challenge arises from specific and non-specific physical and chemical interactions between the nucleic acids and
the biomaterials. In determining which extraction protocol to employ it is critical to think about the nature of these interactions
and always check for RNA quality and yield. The reverse transcription of RNA into complementary DNA and its subsequent ampli-
fication offer some of the most powerful techniques for the assessment of gene expression, which can be used to identify differen-
tiation state of cells or offer a mechanistic explanation of the cellular response to stimuli. Polymerase chain reaction (PCR) is used to
amplify a single copy or a few copies of a segment DNA generated from RNA by reverse transcription techniques. Reverse transcrip-
tion PCR or RT-PCR takes advantage of the complementary nature of deoxyribonucleotide-base pairing and is catalyzed by two
enzymes, reverse transcriptase and DNA polymerase. Reverse transcriptase, first discovered in RNA viruses, catalyzes the synthesis
of complementary DNA (cDNA) from RNA templates. DNA polymerase, an essential enzyme for DNA replication in mammalian
cells, catalyzes the copying of new DNA strands from existing ones through multiple rounds of replication. In RT-PCR, the ampli-
fication selectively amplifies a gene transcript using a specific primer sequence unique to the gene being probed. The essential steps
of PCR include thermal denaturation of the double-stranded DNA, annealing of the primer sequence to the complementary sites on
the denatured DNA strands, followed by extension of the annealed primers by DNA polymerase with the aid of mixed-in oligonu-
cleotides. This results in doubling the DNA product with every cycle of PCR. The amplified DNA product can then be visualized
using gel electrophoresis and DNA-binding dyes. Quantification of the PCR product on a gel can be done by imaging the gel
and quantifying the intensity of the gel band normalized to a housekeeping gene band (ubiquitously expressed gene across all treat-
ment groups), which allows for assessment of relative changes in gene expression across experimental groups (Fig. 4).
In comparison to classical RT-PCR methods, reverse transcription quantitative PCR (RT-qPCR), also known as quantitative real-
time PCR, uses probes made of sequence-specific oligonucleotides labeled with a fluorescent reporter to allow the real-time detec-
tion of probe hybridization with its complementary sequence on the DNA strand with every cycle of the amplification. This allows
for real-time quantification of the relative expression of the gene of interest, normalized to a housekeeping gene based on the fluo-
rescence intensity of the hybridized probe.
PCR-based techniques for assessment of relative gene expression are effective tools for hypothesis testing because they measure
a priori selected genes of interest. The same principles used in PCR can be used for discovery instruments such as DNA microarrays
and RNA sequencing.
DNA microarray
A DNA microarray is made of large numbers of microscopic spots, to which probes (DNA sequences corresponding to a specific gene
per spot) is attached. The probe at a spot can hybridize a target cDNA from an experimental sample if the gene it was designed to
detect is expressed, and the probe-target hybridization can be detected because the target cDNA is a priori labeled (e.g., with fluo-
rescence or chemiluminescence). The strength of the fluorescence or chemiluminescence signal at a spot on the microarray reflects
the amount of target sample hybridizing to the probe, which is representative of the relative expression of the gene identified by the
address of the spot on the microarray.
RNA sequencing
In tissue engineering, RNA sequencing (RNA-seq), which utilizes next-generation sequencing (NGS) technology for unbiased
reading of the whole transcriptome, can be used to assess the differential expression of genes (DEG) within purified RNA sample
from different engineered constructs or experimental conditions. While the utility of RNA-seq in tissue engineering might be
focused on the assessment of DEG, it is nevertheless a very powerful technique that can assess other important features of the tran-
scriptome. RNA-seq overcomes many of the practical limitations of microarrays. Most notably, RNA-seq does not require a priori
knowledge of the sequences of the gene of interest. Rather, once the sequences are determined using NGS, they can be aligned to
mapped genomes of different species to identify the genes significantly expressed and the levels of differential gene expression
between experimental conditions. This technique generates large sets of data whose analysis requires access to numerous knowledge
databases of gene functions, pathways, and networks and sophisticated statistical analysis techniques for unbiased interpretation of
the information. It is, therefore, a powerful discovery and hypothesis generating tool.
It should be noted that RNA extraction from bulk tissue and the aforementioned techniques cannot discriminate between the
different cell types contributing to differential gene expression in engineered composite tissues. Single cell sequencing, which is
technically feasible using NGS, can be enabled by additional sophisticated techniques such as laser capture microdissection, which
424
Regenerative Engineering j Assessment of Cellular Responses of Tissue Constructs in vitro
Fig. 4 Gene expression analysis methods. There are three methods of gene expression analysis (1) reverse transcription-polymerase chain reaction (RT-PCR), (2) DNA microarray, and (3) RNA sequencing
(RNA-seq). mRNA is isolated from a population of cells, which can be directly sequenced or reverse transcribed to generate cDNA. RNA-seq allows for detection of transcripts of thousands of genes from
a given sample. PCR methods result in quantification of expression of a single gene within a sample. The PCR amplification reaction involves three steps: heating to separate DNA strands, hybridization of
primer sequences, and DNA synthesis with DNA polymerase. Each cycle doubles the number of DNA transcriptions. PCR products can be quantified at the end of the amplification or in real-time (qPCR).
Lastly, multiple primers can be bound to a microarray that allows for detection of hundreds of genes. Fluorescence signal detection and normalization to a control sample allows for quantification of changes
in gene expression.
allows the isolation of single cell or a uniform population of cells in a tissue (e.g., from a histology slide) for gene expression anal-
ysis under direct microscopic visualization.
Quantitative Protein Analysis

Proteins perform many processes and functions within the cell including providing structural support and catalyzing enzymatic
reactions, and can be probed to assess cell phenotype. Tissue engineers can measure intracellular proteins, proteins expressed on
the surface of cells, and proteins secreted by cells into the extracellular matrix. Most of the methods utilize immunological detection
of proteins by introducing antibodies to probe for specific antigens on the target protein. These methods require lysing the tissue
and cells and are therefore used as endpoint destructive assays.
Western blot
A Western blot is a semiquantitative immunological method for detecting electrophoretically separated proteins. Isolated proteins
are first separated using a sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis (SDS-PAGE). After separation
using SDS-PAGE, the proteins are transferred to a nitrocellulose paper and incubated with a specific antibody that has been coupled
to a label (e.g., a radioactive isotope or a fluorescent dye), which can then be developed for detection. This method of protein detec-
tion provides qualitative and semi-quantitative data about the protein of interest relative to a ubiquitous housekeeping or loading
control protein not affected by the experimental conditions. The size of the protein band can be normalized by a loading control
band using image analysis software to provide semi-quantitative results.
Enzyme-linked immunosorbent assays (ELISA)

Enzyme-linked immunosorbent assays or ELISA is a common quantitative immunoassay used to measure the concentration of
intracellular or secreted peptides and proteins from tissue lysates and culture media using antibodies typically labeled with an
enzyme marker. The key step of ELISA is the immobilization of the antigens from the sample to a surface of a multiwell plate.
Protein detection is accomplished by measuring enzyme activity on a colorimetric substrate following an enzyme–antibody–
antigen reaction. The enzyme activity on the substrate causes a colorimetric change that is proportional to the concentration of
antigen (protein) and can be measured spectrophotometrically. In Direct ELISA, the antigen can be captured by direct adsorption
to the assay plate and labeled with a primary antibody. The second type of ELISA is a “sandwich” assay in which the antigen is
bound between two primary antibodiesdthe capture and the detection antibody. The third type is known as Competitive ELISA,
in which an unlabeled antibody is incubated with the sample presumably containing the antigen of interest, and then added to an
antigen-coated well. The next step after washing unbound antibodies is the addition of an enzyme-linked detection antibody,
specific to the primary antibody and detection is enabled by adding a colorimetric enzyme substrate.
Flow cytometry
Flow cytometry is a powerful laser-based tool used to sort cell populations based on surface or intracellular protein expression. The
most common use of flow cytometry in tissue engineering is the characterization of cell types (e.g., stem cells) prior to seeding on
biomaterial scaffolds. Protein detection is completed by fluorescently labeling the cellular proteins, commonly labeled using anti-
bodies coupled to fluorophores, and passing the cell suspension through a laser which excites the fluorophore to emit light at
varying wavelengths. The fluorescence counts can be measured to determine the number of cells expressing the protein(s) of
interest.
Förster resonance energy transfer assays

Förster or fluorescence resonance energy transfer or FRET-based assays are based on the principle of measuring the proximity of two
fluorophores and is commonly used to detect molecular interactions intramolecular distances. The principle mechanism of FRET is
energy transfer between the two fluorophores. A donor fluorophore is initially excited and can transfer energy to an acceptor fluo-
rophore. FRET can provide information about distances between domains of the protein to determine its conformational state and
protein interactions. Additionally, FRET-based assays can be used to measure proteolytic enzyme activity, interactions between
proteins such as integrins and extracellular proteins, and metabolic or signaling pathways in engineered tissues.
Assessment of Mechanical Properties

The mechanical properties of cells play a significant role in many biological processes. Cellular stiffness can be an indicator of actin
organization and differentiation status. Micro-indentation using an Atomic Force Microscope (AFM) is a common method used to
measure the stiffness of living cells. The general methodology is to indent a cell with an AFM tip of a specified geometry and measure
the applied force from the bending of the AFM cantilever. The small tip is made of silicon or silicon nitride and is made by nano-
fabrication techniques. The displacement of the cantilever can be measured with a laser. Mathematical modeling is used to fit the
force-displacement curve to determine cellular stiffness. In addition to measuring cellular stiffness, AFM can be used to scan the
surface of a tissue engineered construct to determine the nanotopography.
While the assessment of cellular mechanics can be informative of cellular phenotype, the assessment of construct biomechanics
can provide information about the functional properties of engineered tissues. In creating tissue constructs in vitro it is not only
important to mimic biological responses but also the mechanical phenotype of the native tissue. Typical tissue loading patterns
include: tension, compression, and shear. Simple tests assess mechanical properties in biologically-relevant modalities. Appropriate
biomechanical assessment of an in vitro constructs depends on the engineered tissue’s function. For example, tendon constructs can
be tested in uniaxial tension while engineered cartilage is tested in confined or unconfined compression or shear and engineered
heart valves can be tested in flexion. Engineered skin on the other hand can be stretched in biaxial loading configurations. These
types of biomechanical tests provide information about structural and material properties (force-deformation and stress–strain
constitutive laws, respectively), which can be critical to the function of the engineered tissue or organ.
Emerging Technologies
The ability to make real-time measurements of key responses of cells within an engineered tissue using non-invasive imaging tech-
niques in a high throughput system is a critical need for the heuristic science of tissue engineering. Genome editing using CRISPR/
Cas9 is an emerging technology that not only has therapeutic value, but can also enable the introduction of multiplex reporter
signals (e.g., fluorescence) as readouts of critical cellular functions or phenotypic gene expression and secretome markers, which
in theory can be detected using label-free, time-lapse fluorescence microscopy. Human microphysiological systems (tissue- and
organ-on-chip) can also provide a high throughput platform for screening the myriad combinations of stimuli that might need
to be optimized, investigate the interactions with other tissues and organs, and curtail the reliance on animal testing in the early
phases of development. The combination of the two technologies, genome editing and microphysiological systems, can be trans-
formative for the practice of tissue engineering.
Further Reading
Alberts, B. (2015). Molecular biology of the cell. New York: Garland Science, Taylor and Francis Group.
Berthiaume, F., Maguire, T. J., & Yarmush, M. L. (2011). Tissue engineering and regenerative medicine: History, progress, and challenges. Annual Review of Chemical and
Biomolecular Engineering, 2, 403–430.
Ding, S., Kingshott, P., Thissen, H., Pera, M., & Wang, P. Y. (2017). Modulation of human mesenchymal and pluripotent stem cell behavior using biophysical and biochemical cues:
A review. Biotechnology and Bioengineering, 114(2), 260–280.
Guilak, F., Butler, D. L., Goldstein, S. A., & Baaijens, F. P. T. (2014). Biomechanics and mechanobiology in functional tissue engineering. Journal of Biomechanics, 47(9), 1933–
1940. https://doi.org/10.1016/j.jbiomech.2014.04.019.
Ireland, R. G., & Simmons, C. A. (2015). Human pluripotent stem cell Mechanobiology: Manipulating the biophysical microenvironment for regenerative medicine and tissue
engineering applications. Stem Cells, 33(11), 3187–3196.
Sittampalam, G. S., Coussens, N. P., Brimacombe, K., et al. (Eds.). (2004). Assay Guidance Manual. Bethesda, MD: Eli Lilly & Company and the National Center for Advancing
Translational Sciences. https://www.ncbi.nlm.nih.gov/books/NBK53196/.
Vielreicher, M., Schürmann, S., Detsch, R., Schmidt, M. A., Buttgereit, A., Boccaccini, A., & Friedrich, O. (2013). Taking a deep look: modern microscopy technologies to optimize
the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine. Journal of the Royal Society Interface, 10, 20130263. https://doi.org/
10.1098/rsif.2013.0263.
Assessment of Tissue Constructs In Vivo in Regenerative Engineering
Anuradha Subramanian and Swaminathan Sethuraman, SASTRA Deemed University, Thanjavur, India
Introduction 427
Animal Models 427
Cardiovascular Tissue Construct 428
Nerve Tissue Constructs 428
Bone Tissue Construct 429
Osteochondral Interfacial Tissue Construct 429
Challenges in Assessment of In Vivo 430
References 430
Introduction
Transplantation procedures are clinically required due to the increasing prevalence of end-stage organ failure and loss of tissues due
to disease, disorder, and trauma. Shortage of donor organs and risk of immunosuppressing agents administered are the major limi-
tations of organ transplantation. Regenerative engineering is an interdisciplinary approach that combines trophic factors, cells and
extracellular matrix analog for the functional restoration of complex tissues and organ systems (Gu et al., 2014; Furth et al., 2007).
The basic requirement in tissue engineering medical products development is to characterize the safety and regenerating efficacy of
tissue construct using both in vitro and in vivo models. Though in vitro techniques are useful they are limited due to the nonphy-
siological culture condition, depletion of media with the accumulation of by-products, sudden exchange of media, diffusion limited
oxygen supply and inability of monolayer culture to recapitulate complex in vivo system provoke the importance of in vivo assess-
ment of tissue constructs (Slack et al., 2015). The preclinical assessment of toxicity and efficacy of tissue substitutes is required for
the translation of tissue constructs into clinics. Although various animal models are available and are used to envisage human
response, each animal model has its own limitations due to the difference in anatomy, physiology, and pathophysiology from
species to species (Ahern et al., 2009).
Animal models such as rodents, pigs, rabbits, dogs, sheep, and primates are widely used in various tissue-engineering applica-
tions (Fan et al., 2014; Fricain et al., 2013; Hu et al., 2013). Successful regeneration of any tissue mandates the fabrication of ideal
tissue substitute that is equivalent to structure, function, and mechanical strength of native tissue (Fan et al., 2009; Yu et al., 2008;
Millera et al., 2012). However, none of the animal models are suitable to assess all the desirable characteristics of ideal tissue
constructs while many animal models exist to evaluate the specific properties (Ahern et al., 2009; Angius et al., 2013). Hence,
the choice of animal models for the assessment of tissue constructs in regenerative engineering plays a vital role as the results
from preclinical models are often extrapolated to human responses.
Animal Models
Animals such as guinea pigs, goats, dogs, pigs, rodents, rabbits, and primates such as apes and monkeys are most widely used to test
the toxicity and efficacy in tissue engineering. An appropriate animal model should be determined based on the tissue constructs
and its applications (Cui et al., 2007). The choice of the animal model needs to reflect the appropriate aspect of human physiology
and pathological response (Rashid et al., 2004). For example, rodents are used for many tissue engineering applications to evaluate
the efficacy in regenerating skin, sciatic nerve, vascular conduit due to the ease of care, availability and the physiology of rats is more
similar to humans (Allen et al., 2014; Krupnick et al., 2002). However, the results obtained from rodent model require further
testing and evaluation in larger animal models as these results cannot be easily correlated to humans due to the difference in biome-
chanics and anatomical features with humans (Liebschner, 2004; Milano et al., 2006).
Each animal model has its own merits and limitations. Rabbits have been chosen for some studies due to its availability, cost-
effectiveness, and ease of maintenance. The anatomy, physiology, and immunological characteristics of pigs are equivalent to
humans (Summerfield et al., 2015; Guo and Yang, 2015). However, rapid growth rate, expensive animal maintenance including
care and feeding, complex surgical and anesthetic procedures with a mismatch in engineered constructs due to rapid growth of
animals are the potential drawbacks of pig models (Rashid et al., 2004). Dogs are much preferable animal models than pigs as
it has modest growth rate (Lim and Temenoff, 2009). Dog breeds such as beagles, foxhounds have been used as animal models
for various tissue applications. Short-term studies have been carried out in small animal models such as rodents, guinea pigs,
and rabbits up to 12 weeks whereas long-term studies have been performed in longer life-expectancy animals such as dogs, rabbits,
sheep, goats, pigs, etc. (Li et al., 2006; Chang et al., 2006). Selection of appropriate animal models to evaluate the specific properties

428 Regenerative Engineering j Assessment of Tissue Constructs In Vivo in Regenerative Engineering
of tissue construct is critical for the thorough investigation of the biomaterial substitutes. Hence, substitutes for cardiovascular,
nerve, bone, and osteochondral tissues require the search of appropriate animal models as follows.
Cardiovascular Tissue Construct
Cardiovascular disease is one of the leading causes of death worldwide. Biomaterials intended for cardiac or vascular applications
have to be extensively evaluated using in vivo preclinical animal models based on International Organization for Standardization
(ISO). The general requirements for the evaluation have been found in ISO 5840-1:2015 (Cardiovascular implants-cardiac valve
prosthesis) and ISO 7198:2016 (Cardiovascular implants and extracorporeal systems-vascular prosthesis-tubular vascular graft
and vascular patches). Prior to clinical evaluation, ISO requires that the tissue constructs should be tested in envisioned anatomical
sites using preclinical animal models.
Larger animal models such as dog, pig, sheep, and primates are preferable model for testing the therapeutic efficacy since it shows
slower heart rate that closely resemblance the cardiac tissue physiology to humans (Chong and Murry, 2014). Preclinical examina-
tion of cardiovascular implants such as heart valve, vascular grafts, and cardiac assist device should be based on the expected compli-
cations of device-on-host and host-on-device. For instance, implantation of heart valve prostheses may lead to paravalvular leak,
thrombosis, emboli formation, calcification, hemorrhage, infection, hemolytic anemia, etc., whereas the grafting of vascular tissue
substitutes results in anastomotic hyperplasia, disintegration, false aneurysm, infection, thrombosis, embolism, etc. (Zilla et al.,
2007). Complications of vascular stents are thrombosis, incomplete expansion, strut-based inflammation, proliferative restenosis,
and infections (Scott, 2006). Hence, blood compatibility and blood–material interaction should be evaluated for testing the safety
aspects of the cardiovascular implants.
Canine model is advantageous for assessing the cardiovascular regeneration due to the resemblance of cardiovascular physiology
to humans (Byrom et al., 2010). In addition, metabolic and hematological differences, white blood cells differential count and
platelet counts are also identical to humans (Levine et al., 2015). This model is mainly used to evaluate the thrombogenesis of stent
due to its hypercoagulation property compared humans (Bauer and Moritz, 2013). Fufukoshi et al., have fabricated robust type C
biotubes and implanted into the femoral arteries of beagles by end–end anastomosis. The implanted modified biotubes showed
higher external pressure resistance, excellent burst strength and 100% patency rate at 7 days of postimplantation compared to orig-
inal thinner wall biotubes (Furukoshi et al., 2016). Similarly, sheep is the choice of animal model for evaluating the cardiac valves
due to the close resemblance to the mechanical, hemodynamic flow property and calcification propensity of human (Driessen-Mol
et al., 2014; Schmidt et al., 2007). Specifically juvenile sheep < 6 months remain the best model of choice for the evaluation of
calcification of vascular grafts (Herijgers et al., 1999). However, sheep may not be a sensitive model for the assessment of valve
thrombosis due to the higher platelet count and its adhesiveness with respect to humans (Ratner, 2013). Pigs are more ideal models
for evaluating the pacemakers due to the similarities of electrophysiological parameters such as P and R wave amplitude, P-R inter-
vals, Q-T interval, QRS duration with humans (Ratner, 2013). Although larger animal models are ideal for preclinical evaluation of
vascular grafts, rat carotid and abdominal aorta replacement models have been used for the evaluation of in vivo performance of
vascular grafts due to ease of surgical exposure without temporary removal of visceral organs (Allen et al., 2014; Katsimpoulas et al.,
2015). Chan et al., had recently evaluated the efficacy of polycaprolactone electrospun vascular grafts in mouse carotid grafting
model using C57BL/6 transgenic mice. Use of transgenic mice for vascular grafting procedure showed higher neointimal hyperplasia
at proximal anastomosis and found to decrease towards the mid portion comparable to that of physiological response of humans to
vascular replacement (Chan et al., 2017). Since cardiovascular implants have direct contact with blood and soft tissues, evaluating
the consequence of blood–material and soft tissue–material interactions are necessary to extrapolate the human physiological and
pathological response of cardiovascular grafts.
Nerve Tissue Constructs
Regeneration of critical-sized defect (5–30 cm) in peripheral nerve repair demands the exogenous nerve conduits to reestablish the
outgrown neurites to the distal end of the axons (Kaplan et al., 2015). On the contrary, spinal cord injury remains challenging, as
these axons do not regenerate intrinsically (Geoffroy et al., 2017). Current strategies to repair nerve gaps up to 10 cm is nerve auto-
grafts whereas vascularized autografts are required for repairing the gaps of > 10 cm (Trehan et al., 2016; Palispis and Gupta, 2017).
Though autografts are gold standards, these grafts are not ideal due to the shortcomings such as donor site morbidity, size mismatch
and poor availability (Rinker and Vyas, 2014). Hence, the search for new biomaterials and novel strategies to repair the larger size
defects demands in vivo testing using animal models. Animal models for critical-sized defects have been chosen based on the rele-
vance to neurobiological regeneration profile of the humans since the critical size of the defect may vary from species to species
(Angius et al., 2013). Critical nerve defect of rat and rabbit, is 1.5 and 3 cm respectively, while pigs and humans have up to
4 cm (Kaplan et al., 2015). Mohammadi et al., bridged 10 mm sciatic nerve defect using cyclosporine-A releasing chitosan conduit
in rat sciatic model. Apart from histomorphometric and immunohistochemical analysis to confirm the regenerated axons and
Schwann cell-specific biomarker (S-100), functional recovery of regenerated nerves play vital role in the efficacy evaluation
(Mohammadi et al., 2014). Walking track analysis is a comprehensive test used to evaluate the sensory input, cortical integration,
and motor response by determining functional sciatic nerve index and muscle mass. Peptides have been self-assembled in aligned
Regenerative Engineering j Assessment of Tissue Constructs In Vivo in Regenerative Engineering 429
poly(lactide-co-glycolide) nanofibers and the efficacy of the scaffolds to regenerate the sciatic nerve was evaluated in a rat model for
16 weeks (Nune et al., 2017). Similarly electrophysiological assessment has been performed at third and sixth month of the implan-
tation of conductive polypyrrole/poly(D,L-lactic acid) nerve conduits in rat sciatic model by recording nerve conduction velocity on
lower leg triceps (Xu et al., 2014). Unlike humans, rodents have shorter nerve gap (< 10 mm), complete recovery after injury and
rapid peripheral axonal regeneration that limits the use of rat sciatic model used for the evaluation of biomaterial constructs. For the
evaluation of tissue construct in repairing spinal cord injury (SCI), rodents especially rats are widely used since it develops fluid-
filled cystic cavity immediately after injury, which mimics the human pathology. Out of various injury models, contusion SCI
model are preferable in order to mimic the pathophysiology of the humans (Sharif-Alhoseini et al., 2017). However, this biome-
chanical force based injury fail to create the lesion at specific axons. Complete transection of nerve is another model that mimic the
severe category of injury where no axons are spared between the two nerve stumps (Lee and Lee, 2013). Hence, this model is more
advantageous to evaluate the axonal regeneration potential of biomaterial scaffolds. Rabbits are considered as a poor animal model
for nerve tissue engineering applications due to hopping and variation in hind limb function than humans (Angius et al., 2013).
Despite the use of larger animal models such as dogs, sheep, pigs, and monkeys to evaluate the synthetic constructs with larger gaps,
expensive animal care and ethics restrict its potential use for functional evaluation. Among the various larger animal models, axot-
omy of nonhuman primates is equivalent to humans, which drives better comparison with probable human response (Hu et al.,
2007).
Bone Tissue Construct
Animal models explored for testing of repair and regeneration of bone substitutes are only boney vertebrates from fish to mammals.
Each model has its own merits and limitations in evaluating the specific biological activity of the tissue construct and tissue
responses based on the factors such as defect size, anatomy, and physiology, biology of animals (An et al., 2000). To elucidate
the surface osteoconductivity of the tissue substitute, small defects of maximum 2–5 mm and minimum of 1 mm (mice, rats,
rabbits) is considered whereas for porous scaffolds, the larger defect of 1–5 cm of maximum and minimum of 5–10 mm is
used, as the mass transport function is the major variable of porous substitutes (Reichert et al., 2009). Rodent models are generally
not preferred for assessing the tissue constructs for bone repair due to the life-long process of skeleton growth, absence of osteoclast
tunneling and limited trabecular bone content in comparison with bone biology of human (Miller et al., 1995). Rabbits are most
widely used in musculoskeletal and mandibular research studies owing to its ease of handling, larger trabecular bone mass, short
skeletal maturity with Haversian remodeling as humans (Jowsey, 1966). Biology and composition of bone for sheep, goat, dog, and
pig are equivalent to humans (Bagi et al., 2011). Tissue-engineered bone substitute using hydroxyapatite/tricalcium phosphate scaf-
fold seeded with adipose-derived stem cells demonstrated the reconstruction of bone in alveolar cleft model of mongrel dog
(Pourebrahim et al., 2013). Systemic variables such as age, sex, endocrine factors such as estrogen, parathyroid hormone PTH
and local variables such as type and size of defects interfere with the outcome of bone repair (Florencio-Silva et al., 2015). Hence,
critical-sized bone defects remain the better translational model for the evaluation of bone tissue constructs. Anatomy, morphology,
and remodeling with mineral density, mineral composition of bone in pigs show the close resemblance to human bones except the
denser trabecular network (Pearce et al., 2007). Further regeneration rate of bone in pigs (1.2–1.5 mm/day) is equivalent to humans
(1.0–1.5 mm/day) (Raschke et al., 1999). Among the large animals, multiple defects of 10 mm wide and 15 mm long created in
canine femoral multi-defect model aids in the intra-subject assessment of grafts (Cui et al., 2007). Similarly multiple critical-
sized cranial defects (20 mm diameter) in Dutch Texel sheep have been filled with different poly(trimethylene carbonate)-based
composite materials where one of defect left untreated (Zeng et al., 2017). Sheep is an excellent model to evaluate different
bone forming ability of tissue constructs by offering multiple surgical sites for critical-sized cranial defects.
Osteochondral Interfacial Tissue Construct

Degeneration of articular cartilage is a common disease in elder patients resulting in dysfunction of joints, long-term disability asso-
ciated with chronic pain. These defects progress to affect the underlying bone that necessitates the total joint replacement. However,
available therapeutic options are not fully curative to regenerate hyaline cartilage, and instead they develop fibrocartilage (Prado
et al., 2016). Ease of handling, caging, and maintenance costs of animals are the major reasons for using murine models. However,
rats and mice are not ideal models to predict the response of humans due to the smaller and thinner cartilage tissue which heals
better healing potential when compared to humans (Bagi et al., 2011). Cartilage thickness of murine, rabbit, canine, caprine, ovine,
porcine, equine, and human is 0.1; 0.3; 0.6–1.3; 0.7–1.5; 0.4–0.5; 1.5–2.0; 0.96–3.13, and 2.2–2.5 mm respectively (Chu et al.,
2010). Based on the thickness of cartilage, the critical size defect of rabbit (3 mm), dog (4 mm), sheep (6–7 mm), pig (6 mm), horse
(9 mm), and human (10 mm) also varies (Frisbie et al., 2006). Despite the advantage of early skeletal maturity in rabbits, sponta-
neous healing potential with difference in biomechanics is major limitations in correlating the results from rabbit to humans
(Hurtig et al., 2011; An and Freidman, 1998). Although canine models share many cartilage pathologies with humans such as osteo-
chondritis, osteoarthritis, canine knee joint may not model the human knee joint due to the anatomical difference (Proffen et al.,
2012). Characteristics such as joint size, biomechanics, weight, lack of intrinsic healing, trabecular bone thickness of porcine similar
to humans make the pigs as an ideal animal model (Chang et al., 2006). However, expensive animal maintenance and poor avail-
ability of skeletally mature animals are the major barriers of porcine model (Vasara et al., 2006). Performing preclinical evaluation
430 Regenerative Engineering j Assessment of Tissue Constructs In Vivo in Regenerative Engineering
of osteochondral tissue construct in equine model receives much attention due to the anatomical equivalence to human knee with
similar bone mineral density, larger defects with ease of assessment of repair tissues, poor intrinsic healing and fully extended stifle
during walk (Colbath et al., 2016; Bertuglia et al., 2016; Harrison et al., 2014).
Challenges in Assessment of In Vivo

Animal studies are performed to test tissue constructs to evaluate the response to interventions so that adequate data is available to
plan clinical trials. The safety and efficacy data obtained for substitutes in a homogenous animal population does not always prove
to be effective in humans also. Hence, the choice of appropriate animal model is essential to translate the research outcome from
animal to humans. Unresolved challenges remain in the translation of preclinical animal model to clinics. They are: (i) difference in
anatomical, physiological, and pathological response between animals and humans; (ii) lack of randomized or blind studies in
animal model; (iii) difficulty in systemic and local toxicity prediction; (iv) surgical limitation; (v) size mismatch; (vi) ethical
concerns; and (vii) differential healing response. Further, factors such as device exposure duration, nature of injury, injured tissue;
age and species may also influence the outcome. Most of the animal models are ideal for evaluation of complete safety and efficacy
of the tissue constructs. For example, animal models from murine to nonhuman primates shows extensive endothelialization in
lumen surface as healing response, which is absent in humans. The success rate for translation of tissue substitutes to clinics depends
on the choice of the animal models that in turn depends on the end-use application. Hence, the choice of appropriate animal
models is important to bridge the preclinical gap for translating the materials from lab to clinics.
References
Ahern, B. J., Parvizi, J., Boston, R., & Schaer, T. P. (2009). Preclinical animal models in single site cartilage defect testing: A systematic review. Osteoarthritis and Cartilage, 17,
705–713.
Allen, R. A., et al. (2014). Nerve regeneration and elastin formation within poly(glycerol sebacate)-based synthetic arterial grafts one-year post-implantation in a rat model.
An, Y. H., & Freidman, R. J. (1998). Animal models in orthopaedic research. Boca Raton, FL: CRC press.
An, Y. H., Woolf, S. K., & Friedman, R. J. (2000). Pre-clinical in vivo evaluation of orthopaedic bioabsorbable devices. Biomaterials, 21, 2635–2652.
Angius, D., et al. (2013). A systematic review of animal models used to study nerve regeneration in tissue-engineered scaffolds. Biomaterials, 33, 8034–8039.
Bagi, C. M., Berryman, E., & Moalli, M. R. (2011). Comparative bone anatomy of commonly used laboratory animals: Implications for drug discovery. Comparative Medicine, 61,
76–85.
Bauer, N., & Moritz, A. (2013). Coagulation response in dogs with and without systemic inflammatory response syndromedPreliminary results. Research in Veterinary Science, 94,
122–131.
Bertuglia, A., et al. (2016). Osteoclasts are recruited to the subchondral bone in naturally occurring post-traumatic equine carpal osteoarthritis and may contribute to cartilage
degradation. Osteoarthritis and Cartilage, 24, 555–566.
Byrom, M. J., Bannon, P. G., White, G. H., & Ng, M. K. C. (2010). Animal models for the assessment of novel vascular conduits. Journal of Vascular Surgery, 52, 176–195.
Chan, A. H. P., et al. (2017). Evaluation of synthetic vascular grafts in a mouse carotid grafting model. PLoS One, 12, 1–15.
Chang, C. H., et al. (2006). Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: A porcine model
assessed at 18, 24, and 36 weeks. Biomaterials, 27, 1876–1888.
Chong, J. J. H., & Murry, C. E. (2014). Cardiac regeneration using pluripotent stem cells-progression to large animal models. Stem Cell Research, 13, 654–665.
Chu, C. R., Szczodry, M., & Bruno, S. (2010). Animal models for cartilage regeneration and repair. Tissue Engineering. Part B, Reviews, 16, 105–115.
Colbath, A. C., et al. (2016). Equine models for the investigation of mesenchymal stem cell therapies in orthopaedic disease. Operative Techniques in Sports Medicine, 25, 41–49.
Cui, L., et al. (2007). Repair of cranial bone defects with adipose derived stem cells and coral scaffold in a canine model. Biomaterials, 28, 5477–5486.
Driessen-Mol, A., et al. (2014). Transcatheter implantation of homologous ‘off-the-shelf’ tissue-engineered heart valves with self-repair capacity: Long-term functionality and rapid
in vivo remodeling in sheep. Journal of the American College of Cardiology, 63, 1320–1329.
Fan, H., Liu, H., Toh, S. L., & Goh, J. C. H. (2009). Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model. Biomaterials, 30,
4967–4977.
Fan, H., Zeng, X., Wang, X., Zhu, R., & Pei, G. (2014). Efficacy of prevascularization for segmental bone defect repair using b-tricalcium phosphate scaffold in rhesus monkey.
Florencio-Silva, R., Sasso, G. R. D. S., Sasso-Cerri, E., Simões, M. J., & Cerri, P. S. (2015). Biology of bone tissue: Structure, function, and factors that influence bone cells. BioMed
Research International, 2015, 1–17.
Fricain, J. C., et al. (2013). A nano-hydroxyapatitedPullulan/dextran polysaccharide composite macroporous material for bone tissue engineering. Biomaterials, 34, 2947–2959.
Frisbie, D. D., Cross, M. W., & McIlwraith, C. W. (2006). A comparative study of articular cartilage thickness in the stifle of animal species used in human pre-clinical studies
compared to articular cartilage thickness in the human knee. Veterinary and Comparative Orthopaedics and Traumatology, 19, 142–146.
Furth, M. E., Atala, A., & Van Dyke, M. E. (2007). Smart biomaterials design for tissue engineering and regenerative medicine. Biomaterials, 28, 5068–5073.
Furukoshi, M., Moriwaki, T., & Nakayama, Y. (2016). Development of an in vivo tissue-engineered vascular graft with designed wall thickness (biotube type C) based on a novel
caged mold. Journal of Artificial Organs, 19, 54–61.
Geoffroy, C. G., Meves, J. M., & Zheng, B. (2017). The age factor in axonal repair after spinal cord injury: A focus on neuron-intrinsic mechanisms. Neuroscience Letters, 652,
41–49.
Gu, X., Ding, F., & Williams, D. F. (2014). Neural tissue engineering options for peripheral nerve regeneration. Biomaterials, 35, 6143–6156.
Guo, W., & Yang, S. (2015). Advantages of a miniature pig model in research on human hereditary hearing loss. J. Otol., 10, 105–107.
Harrison, S. M., Chris Whitton, R., Kawcak, C. E., Stover, S. M., & Pandy, M. G. (2014). Evaluation of a subject-specific finite-element model of the equine metacarpophalangeal joint
under physiological load. Journal of Biomechanics, 47, 65–73.
Herijgers, P., et al. (1999). Calcification characteristics of porcine stentless valves in juvenile sheep. European Journal of Cardiothoracic Surgery, 15, 134–142.
Hu, J., Zhu, Q. T., Liu, X. L., Xu, Y.b., & Zhu, J. K. (2007). Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with
autologous mesenchymal stem cells. Experimental Neurology, 204, 658–666.
Hu, N., et al. (2013). Long-term outcome of the repair of 50 mm long median nerve defects in rhesus monkeys with marrow mesenchymal stem cells-containing, chitosan-based
tissue engineered nerve grafts. Biomaterials, 34, 100–111.
Regenerative Engineering j Assessment of Tissue Constructs In Vivo in Regenerative Engineering 431
Hurtig, M. B., et al. (2011). Preclinical studies for cartilage repair: Recommendations from the international cartilage repair society. Cartilage, 2, 137–152.
Jowsey, J. (1966). Studies of Haversian systems in man and some animals. Journal of Anatomy, 100, 857–864.
Kaplan, H. M., Mishra, P., & Kohn, J. (2015). The overwhelming use of rat models in nerve regeneration research may compromise designs of nerve guidance conduits for humans.
Journal of Materials Science. Materials in Medicine, 26, 1–5.
Katsimpoulas, M., et al. (2015). Investigation of the biomechanical integrity of decellularized rat abdominal aorta. Transplantation Proceedings, 47, 1228–1233.
Krupnick, A. S., et al. (2002). A novel small animal model of left ventricular tissue engineering. The Journal of Heart and Lung Transplantation, 21, 233–243.
Lee, D., & Lee, J. K. (2013). Animal models of axon regeneration after spinal cord injury. Neuroscience Bulletin, 29, 436–444.
Levine, D. N., et al. (2015). A novel canine model of immune thrombocytopenia: Has ITP gone to the dogs? British Journal of Haematology, 167, 110–120.
Li, X., Feng, Q., Liu, X., Dong, W., & Cui, F. (2006). Collagen-based implants reinforced by chitin fibres in a goat shank bone defect model. Biomaterials, 27, 1917–1923.
Liebschner, M. A. K. (2004). Biomechanical considerations of animal models used in tissue engineering of bone. Biomaterials, 25, 1697–1714.
Lim, J. J., & Temenoff, J. S. (2009). Tendon and ligament tissue engineering: Restoring tendon/ligament and its interfaces. Fundamentals of Tissue Engineering and Regenerative
Medicine. https://doi.org/10.1007/978-3-540-77755-7_20.
Milano, G., et al. (2006). Comparison between different femoral fixation devices for ACL reconstruction with doubled hamstring tendon graft: A biomechanical analysis. Arthroscopy:
The Journal of Arthroscopic & Related Surgery, 22, 660–668.
Miller, S. C., Bowman, B. M., & Jee, W. S. (1995). Available animal models of osteopeniadSmall and large. Bone, 17, 117–123.
Millera, J. S., et al. (2012). Rapid casting of patterned vascular networks for perfusable engineered 3D tissues. Nature Materials, 11, 768–774.
Mohammadi, R., Heydarian, H., & Amini, K. (2014). Effect of local administration of cyclosporine A on peripheral nerve regeneration in a rat sciatic nerve transection model. Chinese
Journal of Traumatology (English Edition), 17, 12–18.
Nune, M., Subramanian, A., Krishnan, U. M., Kaimal, S. S., & Sethuraman, S. (2017). Self–assembling peptide nanostructures on aligned poly(lactide–co–glycolide) nanofibers for
the functional regeneration of sciatic nerve. Nanomedicine, 12, 219–235.
Palispis, W. A., & Gupta, R. (2017). Surgical repair in humans after traumatic nerve injury provides limited functional neural regeneration in adults. Experimental Neurology, 290,
106–114.
Pearce, A. I., Richards, R. G., Milz, S., Schneider, E., & Pearce, S. G. (2007). Animal models for implant biomaterial research in bone: A review. European Cells and Materials, 13,
1–10.
Pourebrahim, N., et al. (2013). A comparison of tissue-engineered bone from adipose-derived stem cell with autogenous bone repair in maxillary alveolar cleft model in dogs.
International Journal of Oral and Maxillofacial Surgery, 42, 562–568.
Prado, M. P., Kennedy, J. G., Raduan, F., & Nery, C. (2016). Diagnosis and treatment of osteochondral lesions of the ankle: Current concepts. Revista Brasileira de Ortopedia, 51,
489–500.
Proffen, B. L., McElfresh, M., Fleming, B. C., & Murray, M. M. (2012). A comparative anatomical study of the human knee and six animal species. The Knee, 19.
Raschke, M. J., et al. (1999). Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis. Bone, 24, 81–88.
Rashid, S. T., Salacinski, H. J., Hamilton, G., & Seifalian, A. M. (2004). The use of animal models in developing the discipline of cardiovascular tissue engineering: A review.
Ratner, B. D. (2013). Biomaterials science: An introduction to materials in medicine. In Academic Press.
Reichert, J. C., et al. (2009). The challenge of establishing preclinical models for segmental bone defect research. Biomaterials, 30, 2149–2163.
Rinker, B., & Vyas, K. S. (2014). Clinical applications of autografts, conduits, and allografts in repair of nerve defects in the hand: Current guidelines. Clinics in Plastic Surgery, 41,
533–550.
Schmidt, D., Stock, U. A., & Hoerstrup, S. P. (2007). Tissue engineering of heart valves using decellularized xenogeneic or polymeric starter matrices. Philosophical Transactions of
the Royal Society of London. Series B, Biological Sciences, 362, 1505–1512.
Scott, N. A. (2006). Restenosis following implantation of bare metal coronary stents: Pathophysiology and pathways involved in the vascular response to injury. Advanced Drug
Delivery Reviews, 58, 358–376.
Sharif-Alhoseini, M., et al. (2017). Animal models of spinal cord injury: A systematic review. Spinal Cord.
Slack, G. C., et al. (2015). Necessity of latency period in craniofacial distraction: Investigations with in vitro microdistractor and clinical outcomes. Journal of Plastic, Reconstructive
& Aesthetic Surgery, 68, 1206–1214.
Summerfield, A., Meurens, F., & Ricklin, M. E. (2015). The immunology of the porcine skin and its value as a model for human skin. Molecular Immunology, 66, 14–21.
Trehan, S. K., Model, Z., & Lee, S. K. (2016). Nerve repair and nerve grafting. Hand Clinics, 32, 119–125.
Vasara, A. I., et al. (2006). Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation. Osteoarthritis and Cartilage, 14,
1066–1074.
Xu, H., et al. (2014). Conductive PPY/PDLLA conduit for peripheral nerve regeneration. Biomaterials, 35, 225–235.
Yu, D., Li, Q., Mu, X., Chang, T., & Xiong, Z. (2008). Bone regeneration of critical calvarial defect in goat model by PLGA/TCP/rhBMP-2 scaffolds prepared by low-temperature rapid-
prototyping technology. International Journal of Oral and Maxillofacial Surgery, 37, 929–934.
Zeng, N., van Leeuwen, A. C., Grijpma, D. W., Bos, R. R. M., & Kuijer, R. (2017). Poly(trimethylene carbonate)-based composite materials for reconstruction of critical-sized cranial
bone defects in sheep. Journal of Cranio-Maxillo-facial Surgery, 45, 338–346.
Zilla, P., Bezuidenhout, D., & Human, P. (2007). Prosthetic vascular grafts: Wrong models, wrong questions and no healing. Biomaterials, 28, 5009–5027.
Bioengineered Kidney and Bladder
DS Koslov and A Atala, Wake Forest Baptist Medical Center, Winston–Salem, NC, USA
Introduction 432
Cells Used in Regenerative Medicine 433
Adult Stem Cells 433
Embryonic Stem Cells 434
Somatic Cell Nuclear Transfer 434
Altered Nuclear Transfer 435
Single Cell Embryo Biopsy 435
Cells from Arrested Embryos 435
Amniotic Fluid and Placental Stem Cells 435
Induced Pluripotent Stem Cells 436
Biomaterials 436
Acellular Matrices 436
Synthetic Biomaterials 437
Biologic Biomaterials 438
Vascularization and Innervation 438
Bioreactors 438
Renal-Specific Studies 438
Cell-Based Renal Therapy 439
Renal Precursor Therapy 439
Factor-Based Renal Therapy 439
Materials-Based Renal Therapy 440
Organ Decellularization and Repopulation 440
Conclusion 440
References 440
Glossary
Blastocyst Early developmental stage, typically 70–100 cells. It contains the inner cell mass, which will develop to the
trilaminar germ layers, and trophoblast, which will become the placenta.
Embryo Zygote derivative that develops into offspring; in humans, this is defined from fertilization to the end of the first
8 weeks gestation.
Embryoid Body In vivo aggregation of pluripotent stem cells, which can undergo differentiation to cells of all three germ layers.
A source of difficulty in clinical stem cell use.
Extracellular Matrix (ECM) Complex array of proteins, polysaccharides, growth factors, and proteoglycans found in animal
tissue that provide support, regulate intercellular communication, divide tissues, and guide development as an embryo.
Commonly referred to as connective tissue, it is essential in tissue growth and function.
Myelomeningocele Congenital defect with open spinal canal with protrusion of meninges and spinal nerves through the
defect. Patients may have associated defects and neural damage to structures such as the bladder.
Stem Cells Population of immature cells that remain undifferentiated and can become several types of mature cells. Several
types exist based on the location and range of mature cells they can become.
Introduction
Organ transplantation has long been a therapy for patients suffering from diseased and terminally damaged organs, even dating to
antiquity. Initial contributions to transplantation came from the joined efforts of Charles Lindbergh and Alexis Carrel, a Nobel Prize
Laureate in Medicine, in ex vivo organ preservation. Transplantation came to the forefront in 1954 when Joseph Murray performed
the first whole organ transplant between identical twins. Murray continued his work in the 1960s when he successfully transplanted
a kidney between nonrelated patients. However, lack of good immune suppression, inability to monitor and control rejection, and
donor shortages spurred physicians and scientists to look for alternative sources of transplantable tissues.

Regenerative Engineering j Bioengineered Kidney and Bladder 433
Soon, synthetic materials were introduced to substitute for whole organ transplantation. Tetrafluoroethylene (Teflon) and sili-
cone, for example, introduced a wide array of devices that could be applied for human use. While these devices provided structural
replacement for organs, providing function was the next challenge. As new techniques for cell harvesting, culture, and expansion
were developed, studies of the extracellular matrix (ECM) and its interaction with cells led the way to a better understanding of
cell and tissue growth and differentiation. The advent of human bone marrow transplant in the 1970s was the first in development
of several ways to combine devices and materials with cell biology concepts, ultimately creating a new field called tissue engineering.
As scientists from different fields came together with the common goal of tissue replacement, the field of tissue engineering became
more formally established. More recently, the fields of cell transplantation and tissue engineering have been combined with stem
cell biology, creating what is now known as ‘regenerative medicine.’
While organ transplantation remains a mainstay of treatment for patients with severely compromised organ function, the
number of patients in need of treatment far exceeds the organ supply, and this shortfall is expected to worsen as the global pop-
ulation ages. However, recent advances in regenerative medicine suggest that it can provide new alternatives to donor organs. In
the past two decades, scientists have attempted to grow native and stem cells, engineer tissues, and design treatment modalities
using regenerative medicine techniques for almost every tissue of the human body. The genitourinary system has been at the fore-
front in the advancement of bioengineering. Both basic science and clinical experience have shown advances in both bladder and
kidney bioengineering, two organs that are targeted by congenital, neurological, traumatic, and degenerative processes. This article
will discuss the basics of tissue engineering including cell isolation and biomaterial selection, and then specific experiences in both
urologic and renal tissue regeneration.
Cells Used in Regenerative Medicine
Bioengineering relies on appropriate selection of cells to populate the new tissues. Ideally, the cells would be easily derived in an
ethically acceptable manner, expand easily in culture, and be safe to use in the clinical setting. By definition, stem cells regenerate
indefinitely in an undifferentiated state, and can mature into one of many types of cells (Hipp and Atala, 2008). They come in three
main categories: adult, embryonic, or amniotic fluid and placental. More recently, a new population of induced pluripotent stem
cells (iPSCs) has been introduced. Embryonic stem cells (ESCs) are obtained from the inner cell mass of a blastocyst and are plurip-
otent. Fetal and neonatal amniotic fluid and placenta taken during amniocentesis or chorionic villus sampling contain multipotent
cells that may be useful in cell therapy applications. Adult stem cells, on the other hand, are usually isolated from organ or bone
marrow biopsies. When cells are used for tissue reconstitution, donor tissue is dissociated into individual cells that are either
implanted directly into the host or expanded in culture and then attached to a support matrix or reimplanted after expansion
(Hipp and Atala, 2008). Many techniques for generating stem cells have been studied over the past few decades, and the main
ones are discussed in detail below. Table 1 summarizes the unique forms of generating pluripotent stem cells.
Adult Stem Cells
Adult stem cells are unipotent undifferentiated cells located within specific organs and tissues including muscle (Crisan et al., 2008),
gastrointestinal tract (Weiner, 2008), brain (Taupin, 2006), bone marrow (Till and McCulloch, 1964), and skin (Jensen et al.,
2008), and replace cells lost in their respective locations only. Apart from those located in bone marrow, these stem cells are small
in number and difficult to maintain in culture (Mimeault and Batra, 2008). An advantage of using native adult stem cells is that they
can be obtained from the specific organ to be regenerated, and then expanded and used in the same patient without rejection. Major
Table 1 Methods of generating pluripotent stem cells
Method Advantage Limitation
Somatic cell nuclear transfer Customized stem cells Requires oocytes

Has been shown to work in nonhuman primates Has not been shown to work in humans
Single cell embryo biopsy Patient specific to embryo Allogeneic cell types
Does not destroy or create embryos Are not known if single cells are totipotent
Has been done in humans Requires coculturing with a previously established
human ESC line
Arrested embryos Cells obtained from discarded embryos Allogeneic cell types
Has been done in humans Quality of cell lines might be questionable
Altered nuclear transfer Customized stem cells Ethical issues surround embryos with no potential
Modified genome
Has not been done with human cells
Reprogramming Customized stem cells Retroviral transduction
No embryos or oocytes needed Oncogenes
Has been done with human cells
434 Regenerative Engineering j Bioengineered Kidney and Bladder
advances in cell culture techniques have been made in the past decade and make autologous adult stem cells attractive targets for
clinical applications.
In the bladder, autologous cells can be isolated for engineered tissue repopulation. Progenitor cells in the bladder have been
identified in larger quantities near the neck of the bladder and trigone (Nguyen et al., 2007), and procedures have been developed
to expand urothelial cells in vitro. These cells can subsequently be seeded on to a matrix and reintroduced to the host with varying
degrees of success. Even cells derived from a diseased organ can be expanded and reimplanted in a scaffold and will reacquire
normal form and function (Lai et al., 2002).
Within the adult stem cell group exists a unique subpopulation, the mesenchymal stem cells (MSCs)/multipotent adult progen-
itor cells, which have the ability to differentiate into several cell types. Originating in the bone marrow, MSCs are multipotent,
demonstrating the ability to differentiate developmentally if injected into a blastocyst (da Silva Meirelles et al., 2008), as well as
to integrate into pulmonary structures (Nolen-Walston, 2008), spleen (in’t Anker et al., 2003), muscle (Crisan et al., 2008; Luttun
et al., 2006), liver (Mimeault and Batra, 2008; Ikeda et al., 2008), and gastrointestinal tract (Jiang et al., 2002). These cells are easier
to maintain in culture than other adult progenitor cells (Caplan, 2007). Notably, MSCs have demonstrated renoprotection in
several models of acute kidney injury. MSCs were found to be increased in numbers in peripheral circulation after acute kidney
ischemia (Kale et al., 2003) and were seen to relocate to the kidney (Togel et al., 2005a), likely in response to increased levels of
stromal cell-derived factor-1 (Togel et al., 2005b). The MSCs were traced to the kidney (Lange et al., 2005), and administration of
MSCs in postischemic renal injury hastened renal function (Lange et al., 2005; Kunter, 2007; Morigi et al., 2004; Lin et al., 2003).
They have also shown to have positive effects on chronic renal failure models (Choi et al., 2009). It has been established that MSCs
that move to the kidney after injury do not integrate into the organ, rather they deliver microvesicles in a paracrine manner, which
provides renoprotective molecules such as insulinlike growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF).
Furthermore, MSC may reduce inflammatory reactions after acute kidney damage by inhibiting several T-lymphocyte activities,
keep dendritic cells in an immature state, reduce interleukin (IL)-2 production, and augment CD4þCD25þ T-regulatory cells (Aggar-
wal et al., 2005). Therapeutic use of MSC in kidney-related injury may yield an effective manner of cell-based therapy. Beyond adult
and MSCs, pluripotent cells also pose a promising source for tissue regeneration.
Embryonic Stem Cells
Pluripotent cells were found in the inner cell mass of the human embryo in 1981, and given the name human embryonic stem cells
(hESCs). These cells are able to differentiate into all cells of the human body, excluding placental cells, and self-propagate in an
undifferentiated state. These cells have great therapeutic potential, but their use is limited by both biological and ethical factors
(Landry and Zucker, 2004). Several studies have demonstrated that tissues of all germ layers arise from ESC (Zhang et al., 2001;
Reubinoff et al., 2000; Levenberg et al., 2002; Kehat et al., 2001; Assady et al., 2001; Chung et al., 2006), and protocols exist to
expand these cells ex vivo (Thompson et al., 1998). Therapeutic value is questionable, however, as these cells form embryoid bodies
in vitro and teratomas in vivo and may induce an inflammatory immune response. Additionally, use of discarded embryos to isolate
hESC is an area of ethical controversy.
Somatic Cell Nuclear Transfer
Somatic cell nuclear transfer (SCNT) is the process of transplanting nuclei from adult cells into oocytes or blastocysts and allowing
them to grow and differentiate, producing pluripotent cells. Figure 1 illustrates SCNT. This process has both reproductive and
Figure 1 Somatic Cell Nuclear Transfer (SCNT) to produce replacement tissues using a patient’s cells.
therapeutic implications. Both have the same initial process, removing an adult cell nucleus, placing into an oocyte, and stimulating
it to grow with electricity or chemicals. This will produce an embryo genetically identical to the donated cell nucleus: if planted in
a uterus, a clone will develop. If the embryo is used for tissue development, it is considered therapeutic. The resultant cells are
immunoidentical to the donor and capable of identical growth to a naturally formed embryo (Hochedlinger et al., 2002; Brambink
et al., 2006). The interest of SCNT is to employ the capacity to develop nonimmunogenic cells that can develop into any tissue to
replace the damaged organ.
In SCNT, a viable embryo is not always developed. A new study demonstrates that leaving the oocyte’s genetic material and
simply adding the somatic cell genome will produce a triploid blastocyst that can lead to a viable embryo that can develop all three
germ layers (Noggle et al., 2012). Several studies to date have demonstrated the capacity to remove cell nuclei from animal model
cells, place in blastocysts and expand ex vivo, replace in the organism, and produce anatomic and functional renal units (Lanza et al.,
2002; Atala et al., 1995; Yoo et al., 1996). Reproductive SCNT is banned in most countries due to ethical dilemmas, while thera-
peutic SCNT is an important form of research.
Altered Nuclear Transfer
Altered nuclear transfer takes a genetically modified somatic cell nucleus and transplants it into an oocyte. The mutation allows
development to a certain point, and then halts progression. This technique allows development of pluripotent stem cells using
a blastocyst without the controversy of hESC, has been shown to work in mice, but needs further studies to understand how the
mutation affects development (Meissner et al., 2006; Benahmed et al., 2001).
Single Cell Embryo Biopsy
hESC research is an area of ethical controversy in that it results in the destruction of embryos; a method of isolating these cells
without destroying the embryo would be advantageous. In 2006, Chung and colleagues were the first to report the generation of
mouse ESC lines in this manner (Becker and Chung et al., 2006). Their method was based on obtaining a single cell embryo biopsy
for preimplantation genetic diagnosis. Cells were taken from eight-cell blastomeres rather than from blastocysts. The cells differen-
tiated into derivatives of all three embryonic germ layers in vitro, as well as into teratomas in vivo, and were considered pluripotent. In
addition, the mouse embryos that resulted from the biopsied blastomeres developed to term without a reduction in their develop-
mental potential.
Cells from Arrested Embryos
hESC lines can also be derived from arrested embryos from in vitro fertilization (IVF) clinics (Zhang et al., 2006a). Only a few of the
zygotes produced in the process of IVF will develop to the morula and blastocyst stages, and the ones that stop dividing are consid-
ered dead embryos and are usually discarded (Hardy, 1993; Geber and Sampaio, 1999; Landry et al., 2004). As not all of these cells
in the discarded embryos are abnormal (Martinez et al., 2002), this may prove to be a source of hESCs. More studies are needed to
characterize the full proliferation and differentiation potential of ESCs derived from arrested embryos.
Amniotic Fluid and Placental Stem Cells
A fourth stem cell population is the amniotic fluid and placental-derived stem cell (AFPS). Aspirates taken from amniotic sac yield
a heterogeneous population of cells (von Koskull et al., 1981; Medina-Gomez, 1982), mostly fetal in origin. Yet a unique form of
stem cell can be isolated from the amniotic fluid and placenta called AFPS. These cells are less differentiated than adult stem cells but
more so than ESCs, can form all three germ layers, and notably do not form teratomas in vivo. They also can differentiate to cells of
all three germ layers including myogenic, neurallike, adipogenic, hepatic, and osteogenic (De Coppi et al., 2007b), as well as mature
cartilage (Kolambkar et al., 2007), kidney (Perin et al., 2007), lung (Warburton, 2008). They can be obtained via chorionic villus
sampling or amniocentesis and have the potential to be banked for self or allogenic use. A major advantage of isolating cells from
gestational tissue is that it allows for autologous reimplantation, effectively bypassing the possibility of immune rejection.
Several techniques have been developed to isolate and maintain AFPS in vitro, allowing a more thorough understanding of this
cell population. Remarkably, AFPS is capable of propagating rapidly while keeping normal karyotypes even in late passages (Atala,
2009). Telomere length, and cell cycle check points (Brown et al., 2002) are normal, and expression of stem cell markers SSEA4 and
OCT4 are seen (De Coppi et al., 2007b), which indicate an ESC-like cell, but not exactly identical. These cells do form all germ layers
in vitro, but not teratomas in vivo, making them a more appealing therapeutic option for regeneration (Thompson et al., 1998).
Compared to ESCs, iPSCs, and MSCs, AFPS grow more rapidly and there are established protocols to induce the less immature cells
to differentiate into too many cell types (De Coppi et al., 2007b). ESCs are more plastic than MSC/AFPS, but AFPS express OCT4,
which is correlated with ESC plasticity (De Coppi et al., 2007b). There are implications for use in bladder muscle (De Coppi et al.,
2007a), nerve (De Coppi et al., 2007b), kidney (Perin et al., 2010), lung (Carraro et al., 2008), heart (Bollini et al., 2011) and heart
valve (Weber et al., 2011), diaphragm (Fuchs et al., 2004), bone (De Coppi et al., 2007b), and cartilage (Kolambkar et al., 2007).
Of note, amniotic fluid stem cells (AFSC) have shown to reduce renal damage postischemia, mediated by released antiapoptotic
effects, activated Akt, IL-6, and VEGF release, and this effect can be enhanced by preconditioning AFPS with glial cell line-derived
neurotrophic factor (GDNF) (Rota et al., 2012).
Induced Pluripotent Stem Cells
iPSCs, a recent development, pose an alternative source of pluripotent cells for use in tissue regeneration. Nobel Prize laureate Shi-
nya Yamanaka created iPSC through somatic cell reprogramming (Takahashi and Yamanaka, 2006). These cells maintain the
morphology and growth properties of ESC and express ESC marker genes while avoiding the ethical and immunological problems
associated with the use of ESC. A variety of cells can be used to produce iPSCs, including keratinocytes, mesenchymal cells in fat,
oral mucosa, dental pulp, peripheral blood, and cord blood (Zhou and Ding, 2010). iPSCs, in turn, have successfully been seeded
on to scaffolds and differentiated to other cell types (Xie et al., 2011). iPSCs have typically been produced by viral vectors integrating
key transcription factors such as Oct3/4, Sox2, Klf4, and c-Myc into somatic cells. This does, however, potentially increase the risk of
tumor formation. Alternative methods of iPSC generation, such as plasmid transfection and direct protein delivery, are under study
and efficacy is uncertain (Zhou and Ding, 2010). The establishment of safe methods of iPSC generation for clinical applications is
an ongoing process and iPSCs may be applicable for medical treatment in the near future.
While selection of appropriate cells for bioengineering is crucial, and an area of development, it is only one branch of designing
an organ. Appropriate material structure for the cells to grow on is equally significant.
Biomaterials
Cell growth and migration is heavily influenced by their microenvironment, including the ECM, soluble factors, and physical
stimuli. The ECM is a dynamic environment that provides structure and guidance cues to cells, a barrier between different compart-
ments or cell types, and intercellular communication regulation. It also aids in embryologic development, tissue repair, and wound
healing. The main components of ECM are structural proteins, collagens, elastins, and proteoglycans (Aitken and Bagli, 2009). In
the urinary bladder, the composition and architecture of native ECM influences development, and provides function and protection
against urine and potential pathogens. Replacement of biomaterials thus must perform several roles. They ideally would guide cell
growth, maintain visceral structure and correct cellular alignment, facilitate vascularization and innervation, provide resilience to
the stresses of the normal bioenvironment, and be degradable with nontoxic degradation products. In the bladder, the material
must coordinate both urothelial and detrusor smooth muscle growth to facilitate stretching and relaxing for normal urination.
In the kidney, it must facilitate blood filtration and urine draining.
Several types of biomaterials have been explored: biologics including alginate and collagen, decellularized tissue matrices such as
bladder or gastrointestinal submucosa, and synthetic materials such as polylactic acid, polyglycolic acid, and poly(lactic-co-glycolic
acid). The first type discussed is native acellular matrices, which have been isolated and used in both in vitro and in vivo studies.
Figure 2 demonstrates several types of biomaterials.
Acellular Matrices
Acellular matrices have shown promise in various settings. Matrices are isolated most often from bladder acellular matrix or small
intestine submucosa (SIS), and decellularized by chemical and mechanical techniques (Sutherland et al., 1996). Of note, these
matrices do maintain their biological properties, and facilitate cell ingrowth and structure through type 1 collagen, glycosamino-
glycans, and other bioactive proteins and factors including VEGF, fibronectin, transforming growth factor-b1, and fibroblast growth
factor (FGF) (Chun et al., 2007; Yang et al., 2010; Kanai et al., 1999). Acellular matrices have been successful in guiding cell migra-
tion and growth in bladder regeneration models, both with and without preseeding with urothelial and smooth muscle cells (Ober-
penning et al., 1999; Kropp et al., 1996; Zhang et al., 2006b; Reddy et al., 2000). Rat trials have shown vascularization with acellular
matrices (Sievert et al., 2007), but larger pig and dog models need cellularization in order to develop successful blood supply
(Zhang et al., 2006b; Brown et al., 2002). Without cell seeding, cellular matrices have shown normal urothelial growth and
abnormal smooth muscle growth (Atala, 1996, 1998). Similar findings were shown in a clinical trial conducted on patients with
exstrophy–epispadias complex and bladders with poor capacity and compliance using SIS as the matrix for bladder augmentation
(Cainoe et al., 2012). Follow-up studies demonstrated integration of the matrix into the bladder, normal urothelial growth,
decreased smooth muscle:collagen ratio, and no significant increase in capacity and compliance. Other biomaterials of interest
are synthetics.
Figure 2 Scanning electron microscopic image of biomaterials used in tissue engineering. Upper left, collagen sponge; upper right, acellular matrix
from pig bladder submucosa; lower, polyglycolic acid matrix.
Synthetic Biomaterials
Synthetic biomaterial polymers (polyglycolic acid, polylactic acid, poly-lacto-co-glycolic acid, and recently silk fibroin) are also an
area of development (Pariente et al., 2001). These materials are quickly formed, thermoplastic, and thus malleable to many shapes,
and have biodegradation by-products that can be rapidly cleared from the body. Synthetics are formed into sponges or meshes to
achieve the appropriate high surface area to volume and porosity that are required for adequate tissue growth. Usually, permanent
synthetic materials used for bladder reconstruction succumb to mechanical failure and urinary stone formation, and use of degrad-
able materials leads to fibroblast deposition, scarring, graft contracture, and a reduced reservoir volume over time. A significant clin-
ical trial comparing collagen and a collagen–Polyglycolic acid (PGA) hybrid in patients with myelomeningocele-related
neuropathic bladder needing cystoplasty demonstrated that use of the composite led to improved compliance and capacity
compared to collagen alone. Optimal outcomes were seen in patients treated with a scaffold seeded with autologous urothelial
and smooth muscle cells, anastomosed to existing bladder, and covered with omentum (Atala et al., 2006). Results of this trial
are shown in Figure 3. Beyond acellular matrices and synthetic materials, biologics such as collagen and alginate have been focused
on as well.
Figure 3 Construction of engineered bladder. Left, cell-seeded bladder scaffold material; center, the seeded scaffold is anastomosed to native
bladder; right, implant covered with fibrin glue and omentum.
Biologic Biomaterials
Biologic materials such as collagen and alginate are advantageous in that they are less immunogenic. Collagen, a structural protein
found throughout the body, is minimally inflammatory and has already been approved by the United States Food and Drug Admin-
istration (USFDA) for wound care (Cen et al., 2008). Collagen can be manipulated with chemicals and physical methods to increase
intermolecular cross-linking, which makes it resilient (Li, 1995). Additionally, collagen has been shown to guide cell activity and
phenotype via specific interactions (Silver et al., 1992). Collagen and alginate have been used in bioprinting with inject techniques,
and are used as ‘ink’ to print organs into designed three-dimensional structures that allow appropriate cell and tissue growth
(Boland et al., 2006; Campbell, 2007).
Vascularization and Innervation
Designing a bladder that truly replaces the lost tissues requires adequate blood supply and innervation. The bladder derives its
blood source primarily from branches of the internal iliac artery, and this needs to be reestablished in the bioengineered tissue,
a formidable task, and there is a limited distance to which cells can be located from a blood supply (Folkman et al., 1992). It
has been demonstrated that new vessels develop in two manners: vasculogenesis, which is a de novo development of a vessel
and capillaries from mesoderm (Drake et al., 1998), while angiogenesis is a two-stage process where capillaries are synthesized
from existing vessels. During angiogenesis, under the guidance of factors VEGF and FGF (Battegay, 1995), endothelial cells separate,
the basement membrane of the vessel degrades by enzymes, endothelial cells migrate and proliferate to form the edge of a new
capillary, a lumen is formed, a new basement membrane is synthesized, and ultimately, pericytes and vascular smooth muscle
form (Folkman et al., 1992; Ferrara et al., 2007; Yamanaka et al., 2002). To engage this process, various methods of integrating
VEGF and FGF into the biomaterial and cell constructs have shown increased vascularization and reduced graft shrinkage (Gerber
et al., 1998; Askari et al., 2004).
Innervation of the bladder is as important and perhaps more complicated than its vasculature. A complex arrangement of
afferent and efferent nerves with sympathetic and parasympathetic as well as central and peripheral components allows the bladder
to work in synergy with the urethra to fill and void at low pressures. To regain lost function, this component must also be present.
Neurotrophic growth factors include nerve growth factor (NGF), GDNF, and even VEGF. Excessive levels of NGF can cause hyper-
innervation and hypersensitivity, while low levels will not promote adequate branching and axonal growth (Schnegelsberg et al.,
2010; Piquilloud et al., 2007). A combination of NGF with VEGF or GDNF integrated into engineered tissues will likely prove to be
the optimal way to lead to regenerative bladder innervation (Madduri, 2009; Kikuno et al., 2009; Nitta et al., 2010), and prove that
appropriate neuronal growth will rely on biomaterial physical alignment (Corey et al., 2007).
Bioreactors
Cell phenotype and tissue formation depends on physical stimuli to influence cellular alignment, cell proliferation, remodeling and
production of ECM components. Bioreactors recreate physiologic conditions ex vivo, which provide these stimuli to engineered
tissues in the form of forces including compression, shear stresses, pulsatile flow, and electrical stimulation. This process has
been proved to be important in the engineered blood vessels (Seliktar et al., 2000), cartilage (Elder and Athanasiou, 2009), and
ligaments (Moreau et al., 2008). In bladder bioengineering, simulating normal physiological conditions of bladder cycling (filling
and emptying) may provide the additional mechanical properties required to improve functional outcome after implantation
(Hubschmid et al., 2005; Wallis et al., 2008; Haberstroh et al., 2002; Farhart and Yeger, 2008). It has been shown that this stretching
effect on bladders positively influenced in vitro cell and tissue growth, cellular alignment, and changes in gene expression (e.g., colla-
gens, uroplakin II). Further investigations are needed to decide on the importance of ex vivo bioreactors in bladder engineering.
Another approach is the in vivo bioreactor, where bioengineered tissues are temporarily placed into an organism to promote
growth and development, and then transplanted into the true recipient. This in vivo preconditioning may further support tissue
development, enhance vascularization of the engineered tissue, and prevent fibrosis and loss of contractility. In an experiment
from Baumert et al., SIS was seeded with urothelial and smooth muscle cells and placed into a porcine omentum. Three weeks after
placement in this in vivo bioreactor, the scaffolds showed a multilayered urothelium and organized outer layers composed of SMCs
and fibroblasts, and rich tissue vascularization. The constructs could also be transferred to the site of bladder replacement without
compromising the blood supply (Baumert et al., 2007). While promising, this required a two-stage procedure and the potential
benefits over current strategies need to be further investigated before they can be applied in the clinic.
Renal-Specific Studies
While significant advances have been made in bladder bioengineering, there is also research primarily applicable to the kidney. The
kidney is a complex organ with multiple cell types and an integrative functional anatomy that renders it one of the most difficult
organ to reconstruct. It is the target of many congenital and acquired diseases including diabetes, hypertension, polycystic kidney
disease, as well as ischemia and toxic injury, which leave patients with acute or permanent renal damage. The United States Renal
Data System reported 594 374 patients with end stage renal disease (ESRD) in 2010, with 116 946 new patients in that same year
(USRDS, 2012). While renal transplantation remains the cornerstone in treating ESRD, many will spend years on dialysis. While
effective, dialysis is a temporary solution, and regenerative medicine approaches have been heavily studied to address renal bioen-
gineering. Solutions need to address the myriad of functions the kidney performs, including filtering blood, excreting waste, regu-
lating systemic pH and water balance, and hormone production. The approaches discussed are cell based, renal precursor based,
factor based, and materials based.
Cell-Based Renal Therapy
As discussed above, MSCs have demonstrated the ability to aid in renal recovery after ischemic and toxic injury. Administration of
these cells after acute injury has been renoprotective (Togel et al., 2005), but has no effect on fibrotic changes (Stokman et al., 2008).
Renal Precursor Therapy
Many advances in renal regeneration are based on embryology. The kidney derives from intermediate mesoderm of the urogenital
ridge in the fetus in a three-stage process. Located along the posterior abdominal wall, the first structures to appear are the
pronephros. Developing caudal to cranial from the urogenital ridge starting at day 22 of gestation, these structures are composed
of pronephric tubules and duct. Caudal to the pronephros, the mesonephros develop and are embryologically functional, but ulti-
mately degenerate with some remnant cells migrating to the developing adrenogonadal primordia, which become adrenal glands
and gonads. The final embryologic structures to develop are metanephros, which develop from metanephric mesenchyme and
epithelial ureteric bud interactions, leading to a renal vesicle, and progressively nephrons, podocytes, and capillaries of the glomer-
ulus are developed (Maezawa et al., 2012). Thus the kidney is derived from many sites, leading to its intricate form and function.
Regenerative approaches keep this complexity in focus to replace these organs.
Several groups have worked on transplanting renal precursors, both allogeneic and xenogeneic, and most prominently have
found that the immature structures were less susceptible to host rejection, and recruited vasculature from host structures. These
structures were found to produce urine and fulfill other endocrine roles of the kidney.
Early studies of renal precursors repeatedly demonstrated that transplanting more premature metanephros were less likely to be
rejected, were capable of producing urine, and could do so in extrarenal locations. A series of experiments demonstrated that E13–
E16 metanephroi could be transplanted into neonatal mice recipients. They were able to show vascularization, maturation, and
tubular extension to host medulla in donated tissues. They were also filtering. These results were not reproduced in adult recipient
mice (Woolf et al., 1990). Subsequent studies demonstrated function in metanephroi placed outside the renal parenchyma,
including subcapsular (Abrahamson et al., 1994) and omental placement (Rogers et al., 1998), which was able to prolong survival
in anephric rats by an average of 58 h compared to controls (controls were those not receiving transplant and those with transplant
without ureteroureterostomy to host ureter) (Rogers and Hammerman, 2004). Transplanted metanephroi were shown to produce
urine as well. Of note, human (7–8 week) and pig (3.5–4 week) precursors can grow, undergo complete nephrogenesis, and
produce urine in immunodeficient mice (Dekel et al., 2003). The same group also indicated that vascularization of these structures
was of host origin, and that the less immunogenicity observed may be accounted for by this fact. Recently, animal models with
blood loss-induced anemia after being transplanted with xenogeneic metanephroi have shown host MSC recruitment and subse-
quent erythropoietin (EPO) production. Anemic mice received extrarenal metanephros and anemic cat received extrarenal pig meta-
nephros; both recipients demonstrated increased EPO levels of host MSC origin (Matsumoto, 2012). This study demonstrates that
metanephros can serve as nonimmunogenic scaffold for host tissue migration and differentiation to EPO-producing cells, but that
these cells are multipotent mesenchymal cells, and not pluripotent, as demonstrated by ESC and induced pluripotent cell failure to
integrate and produce EPO.
Transplantation of embryonic structures is promising, due to the observed decreased immunogenicity, and possible reconstitu-
tion of all renal functions, including filtration, urine production, and EPO production. Additional studies in 2012 indicate that
metanephros transplant can even increase blood pressure in an induced hypotension model in anephric rats, used to simulate
dialysis-associated hypotension (Yokote et al., 2012). Embryonic renal structures may play a key role in recovering renal loss in
patients in the future.
Factor-Based Renal Therapy
A separate approach to renal regeneration is administration of specific growth factors that have shown positive effects on renal
repair. In animal studies, epidermal growth factor and hepatocyte growth factor have improved survival in acute renal injury
(Hammerman and Miller, 1994). In human trials, IGF-1 has shown to improve renal function in patients with ESRD (Vijayan
et al., 1999). Potential problems with exogenous administration of growth factors are their possible myriad of effects in other
tissues, difficulty in maintenance of therapeutic levels, and dose-dependent responses. Very specific delivery systems need to be
established for growth factor-based therapy.
Materials-Based Renal Therapy
Previous efforts in tissue engineering of the kidney have been directed toward the development of extracorporeal renal support
systems made of biologic and synthetic components. One approach has been development of an extracorporeal device with
a membrane and a single renal cell type to form a monolayer of cells to perform the endocrine and metabolic renal functions,
and it could be used with conventional hemodialysis (Aebischer et al., 1987; Ip and Aebischer, 1989; Humes et al., 1999; Tirana-
thanagul, 2005). Many researchers have used porcine and canine renal epithelial cell lines in this model. Problems arose with
sodium transport, multilayer growth, and necrosis (Fujita et al., 2002). Membrane material and coating strongly influenced mono-
layer adhesion and function (Zhang et al., 2009; Chun et al., 2007; Sato et al., 2005). A similar study looked at human renal epithe-
lial cells grown on a fiber cartridge for uremic dogs. It was shown to excrete ammonia and convert 1,25-dihydroxyvitamin D3, but
not adequately transport electrolytes (Humes et al., 2002). Devices described as above have been used in phase I and II clinical trials
with some evidence of assistance in recovery in patients with acute kidney injury (Humes et al., 2004; Tumlin et al., 2008). Primary
problems using this method lie in exposure of renal cells to nonphysiologic environments. Unique cell culture techniques, however,
are being studied to target this dilemma (Minuth et al., 2005; Schumacher et al., 2002; Minuth and Strehl, 2007).
Research into implantable renal replacement devices have been conducted, but this poses a formidable challenge due to the
foreign body response generated. Experiments in rats with this approach have been done, but are far from clinical trials at this point
(Minuth and Strehl, 2007).
Organ Decellularization and Repopulation
While use of entirely manufactured renal replacement devices has been attempted as described, others sought to decellularize ex vivo
organs and repopulate the scaffold with new cells to generate transplantable viscera. The kidney has complex anatomy that derives
from differing germ cell layers, and this approach would ideally preserve vascular and parenchymal anatomy to see if the residual
extracellular matrices would provide niches for cell growth and retain the signaling capacity to guide stem cell differentiation.
Optimal decellularization techniques would rapidly remove all native cells while maintaining organ structure and matrix integrity,
and several groups have recently made advances in using this approach. Bonandrini et al. (2014) showed with scanning electron
microscopy and microcomputerized tomography that structures were conserved after decellulizing rat kidneys with sodium dodecyl
sulfate; afterward they were able to seed the matrix with murine embryonic stem cells and the cells showed signs of dedifferentia-
tion. Orlando et al. showed that decellularized porcine kidneys could be reimplanted unseeded in live pigs and maintain renal struc-
ture after 2 weeks in vivo (Orlando et al., 2012). Ross et al. were able to demonstrate that residual renal matrices after
decellularization from rats could guide xenobiologic precursor cells (mouse ESCs) to differentiate into mature, tissue-
appropriate cells (Ross et al., 2009). Song et al. were able to remove cells from three species (rat, pig, human), reseed them with
human umbilical venous endothelial cells, and rat neonatal kidney cells, and create units that filter and produce urine in recipient
rats after growth in a perfusion bioreactor (Song et al., 2013). These exciting advancements in both manufactured and repopulated
kidneys show promise as a way to provide renal replacement for the current demand for transplant that far exceeds the supply of
donors.
Conclusion
The field of regenerative medicine has made advances in kidney and bladder bioengineering. A wide array of studies, both in the
laboratory and clinic, have developed techniques to replace bladder tissue when augmentation is needed. For the kidney, studies in
the role of MSCs have shown promise in repairing acute and chronic injury, as well as in the fields of factor-based, and material-
based renal therapy. The future of bioengineering and regenerative medicine is promising, with urology and nephrology leading
much of the new findings and developments.
References
Abrahamson, D. R., et al. (1994). Glomerular development in intraocular and intrarenal grafts of fetal kidneys. Lab. Invest., 64, 629–639.
Aebischer, P., Ip, T. K., Panol, G., & Galletti, P. M. (1987). The bioartificial kidneydprogress towards an ultrafiltration device with renal epithelial-cells processing. Life Supp. Syst.,
5, 159–168.
Aggarwal, S., et al. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 105, 1815–1822.
Aitken, K. J., & Bagli, D. J. (2009). The bladder extracellular matrix. Part 1: architecture, development and disease. Nat. Rev. Uro., 6, 596–611.
in’t Anker, P. S., et al. (2003). Mesenchymal stem cells in human second-trimester bone marrow, liver, lung and spleen exhibit similar immunophenotype but a heteogeouns
multilineage differentiation potential. Haematologica, 88, 845–852.
Askari, A., et al. (2004). Cellular, but not direct, adenoviral delivery of vascular endothelial growth factor results in improved left ventricular function and neovascularization in dilated
ischemic cardiomyopathy. J. Am. Coll. Cardiol., 43, 1908–1914.
Assady, S., et al. (2001). Insulin production by human embryonic stem cells. Diabetes, 50, 1691–1697.
Atala, A. (2009). Amniotic fluid-derived pluripotential cells (Chapter 16). In Essentials of Stem Cell Biology (pp. 145–150). Elsevier Inc.
Atala, A. (1998). Autologous cell transplantation for urologic reconstruction. J. Urol., 159(1), 2–3.
Atala, A. (1996). This month in investigative urology: commentary on the replacement of urologic associated mucosa. J. Urol., 156(2 Pt 1), 338–339.
Atala, A., et al. (1995). Renal cell growth in vivo after attachment to biodegradable polymer scaffolds. J. Urol., 153, 4.
Atala, A., et al. (2006). Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 9518, 1241–1246.
Battegay, E. J. (1995). Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J. Mol. Med., 73, 333–346.
Baumert, H., Simon, P., Hekmati, M., et al. (2007). Development of a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for bladder tissue engineering. Eur.
Urol., 52, 884–890.
Becker, S., & Chung, Y. (2006). Embryonic stem cells from single blastomeres. Methods Enzymol., 418, 108–116.
Benahmed, F., et al. (2001). Multiple regulatory regions control the complex expression patter of the mouse Cdx2 homeobox gene. Neurourol. Urodyn., 20, 157–165.
Boland, T., et al. (2006). Application of inkjet printing to tissue engineering. Biotechnol. J., 1, 910–917.
Bollini, S., et al. (2011). In vitro and in vivo cardiomyogenic differentiation of amniotic fluid stem cells. Stem Cell Rev., 7, 364–380.
Bonandrini, B., et al. (2014). Recellularization of well-preserved acellular kidney scaffold using embryonic stem cells. Tissue Eng. Part A, 20(9-10), 1486–1498.
Brambink, T., et al. (2006). ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. Proc. Natl. Acad. Sci. U.S.A., 103,
933–938.
Brown, A., et al. (2002). 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model. Biomaterials, 23, 2179–2190.
Cainoe, P., et al. (2012). Bladder augmentation using acellular collagen biomatrix: a pilot experience in exstrophic patients. Pedatr. Surg. Int., 28, 421–428.
Campbell, P. G. (2007). Tissue engineering with the aid of inkjet printers. Expert Opin. Biol. Ther., 7, 1123–1127.
Caplan, A. I. (2007). Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J. Cell Physiol., 213, 341–347.
Carraro, G., et al. (2008). Human amniotic fluid stem cells can integrate and differentiate into epithelial lung lineages. Stem Cells, 26, 2902–2911.
Cen, L., et al. (2008). Collagen tissue engineering: development of novel biomaterials and applications. Pediatr. Res., 63, 492–496.
Choi, S., et al. (2009). The role of mesenchymal stem cells in the functional improvement of chronic renal failure. Stem Cells Dev., 18, 521–529.
Chun, S. Y., et al. (2007). Identification and characterization of bioactive factors in bladder submucosa matrix. Biomaterials, 28, 4251–4256.
Chung, Y., et al. (2006). Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature, 439, 216–219.
Corey, J. M., et al. (2007). Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth. J. Biomed. Mater. Res. A, 83a, 636–645.
Crisan, M., et al. (2008). A reservoir of brown adipocyte progenitors in human skeletal muscle. Stem Cells, 26, 2425–2433.
De Coppi, P., et al. (2007a). Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent
compensatory hypertrophy of surviving smooth muscle cells. J. Urol., 117, 369–376.
De Coppi, P., et al. (2007b). Isolation of amniotic stem cell lines with potential for therapy [see comment] Nat. Biotechnol., 38, 405–413.
Dekel, B., et al. (2003). Human and porcine precursors as a new source for transplantation. Nat. Med., 9, 53–60.
Drake, C. J., et al. (1998). Morphogenesis of the first blood vessels. Ann. N. Y. Acad. Sci., 857, 155–179.
Elder, B. D., & Athanasiou, K. A. (2009). Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration. Tissue Eng. Part B Rev., 15, 43–53.
Farhat, W. A., & Yeger, H. (2008). Does mechanical stimulation have any role in urinary bladder tissue engineering? World J. Urol., 26, 301–305.
Ferrara, N., et al. (2007). The biology of vascular endothelial growth factor. Endocr. Rev., 18, 4–25.
Folkman, J., et al. (1992). Angiogenesis and inflammation. In J. I. Gallin, I. M. Goldstein, & R. Snyderman (Eds.), Inflammation: Basic Principles and Clinical Correlates (pp. 821–
839). New York: Raven Press.
Folkman, J., et al. (1973). Self-regulation of growth in three dimensions. J. Exp. Med., 138, 745.
Fuchs, J. R., et al. (2004). Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes. J. Pediatr. Surg., 39, 834–838.
Fujita, Y., Kakuta, T., Asano, M., Itoh, J., Sakabe, K., Tokimasa, T., & Saito, A. (2002). Evaluation of Naþ active transport and morphological changes for bioartificial renal tubule cell
device using Madin-Darby canine kidney cells. Tissue Eng., 8, 13–24.
Gerber, H. P., et al. (1998). Vascular endothelial growth factor induces expression of the antiapoptotic proteins BCL-2 and A1 in vascular endothelial cells. J. Biol. Chem., 273,
13313–13316.
Haberstroh, K. M., Kaefer, M., Depaola, N., et al. (2002). A novel in vitro system for the simultaneous exposure of bladder smooth muscle cells to mechanical strain and sustained
hydrostatic pressure. J. Biomech. Eng., 124, 208–213.
Hammerman, M. R., & Miller, S. B. (1994). Therapeutic use of growth-factors in renal-failure. J. Am. Soc. Nephrol., 5, 1–11.
Hardy, K. (1993). Development of human blastocysts in vitro. In B. Bavister (Ed.), Preimplantation Embryo Development (pp. 184–199). New York: Springer.
Hipp, J., & Atala, A. (2008). Sources of stem cells for regenerative medicine. Stem Cell Rev., 4, 3–11.
Hochedlinger, K., et al. (2002). Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature, 415, 1035–1038.
Hubschmid, U., Leong-Morgenthaler, P. M., Basset-Dardare, A., et al. (2005). In vitro growth of human urinary tract smooth muscle cells on laminin and collagen type 1-coated
membranes under static and dynamic conditions. Tissue Eng., 11, 161–171.
Humes, H. D., Buffington, D. A., MacKay, S. M., Funke, A. J., & Weitzel, W. F. (1999). Replacement of renal function in uremic animals with a tissue-engineered kidney. Nat.
Biotech., 17, 451–455.
Humes, H. D., Fissell, W. H., Weitzel, W. F., Buffington, D. A., Westover, A. J., MacKay, S. M., & Gutierrez, J. M. (2002). Metabolic replacement of kidney function in uremic animals
with a bioartificial kidney containing human cells. Am. J. Kidney Dis., 39, 1078–1087.
Humes, H. D., Weitzel, W. F., Bartlett, R. H., Swaniker, F. C., Paganini, E. P., Luderer, J. R., & Sobota, J. (2004). Initial clinical results of the bioartificial kidney containing human
cells in ICU patients with acute renal failure. Kidney Int., 66, 1578–1588.
Ikeda, E., et al. (2008). Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease. Differentiation, 76, 495–505.
Ip, T. K., & Aebischer, P. (1989). Renal epithelial-cell-controlled solute transport across permeable membranes as the foundation for a bioartificial kidney. Artif. Organs., 13, 58–65.
Jensen, U. B., et al. (2008). A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus. J. Cell Sci., 121, 609–617.
Jiang, Y., et al. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418, 41–49 [see comment]; [erratum appears in Nature 2007 447(7146),
879–880].
Kale, S., et al. (2003). Bone marrow stem cells contribute to repair of ischemically injured renal tubule. J. Clin. Invest., 112, 42–49.
Kanai, N., Fujita, Y., Kakuta, T., & Saito, A. (1999). The effects of various extracellular matrices on renal cell attachment to polymer surfaces during the development of bioartificial
renal tubules. Artif. Organs., 23, 114–118.
Kehat, I., et al. (2001). Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes [see comment] J. Clin. Invest., 108,
407–414.
Kikuno, N., et al. (2009). Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced
neurogenic rat bladder. BJU Int., 103, 1424–1428.
Kolambkar, Y. M., et al. (2007). Chondrogenic differentiation of amniotic fluid-derived stem cells. J. Mol. Histol., 38, 405–413.
Kropp, B. P., et al. (1996). Regenerative urinary bladder augmentation using small intestinal submucosa: urodynamic and histopathologic assessment in long-term canine bladder
augmentation. J. Urol., 155, 2098–2104.
Kunter, U. (2007). Mesenchymal stem cells prevent progressive experimental renal failure but maldifferentiate into glomerular adipocytes. J. Am. Soc. Nephrol., 18, 1754–1764.
von Koskull, H., et al. (October 1981). Glial and neuronal cells in amniotic fluid of anencephalic pregnancies. Prenat. Diagn., 1(4), 259–267.
Lai, J. Y., Yoon, C. Y., Yoo, J. J., Wulf, T., & Atala, A. (2002). Phenotypic and functional characterization of in vivo tissue engineered smooth muscle from normal and pathological
bladders. J. Urol., 168(4 Pt 2), 1853–1857.
Landry, D. W., & Zucker, H. A. (2004). Embryonic death and the creation of human embryonic stem cells. J. Clin. Invest., 114, 1184–1186.
Lange, C., et al. (2005). Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int., 68, 1613–1617.
Lanza, R. P., et al. (2002). Generation of histocompatible tissues using nuclear transplantation. Nat. Biotechnol., 20, 589–596.
Levenberg, S., et al. (2002). Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A., 99, 4391–4396.
Li, S. T. (1995). Biologic biomaterials: tissue derived biomaterials (collagen). In J. D. Bronzino (Ed.), The Biomedical Engineering Handbook (pp. 627–647). Boca Raton, FL: CRS
Press.
Lin, F., et al. (2003). Hematopoeitic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice. J. Am. Soc. Nephrol., 14, 1188–1199.
Luttun, A., et al. (2006). Differentiation of multipotent adult progenitor cells into functional endothelial and smooth muscle cells. Curr. Protoc. Immunol. Chapter 22: Unit 22F.9.
Madduri, S. (2009). Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro. Neurosci. Res., 65(1), 88–97.
Maezawa, Y., Kreidberg, J., & Quaggin, S. (2012). Embryology of the kidney. In Brenner and Rector’s the Kidney (ninth ed., pp. 2–30).
Martinez, F., Rienzi, L., Iacobelli, M., et al. (2002). Caspase activity in preimplantation human embryos is not associated with apoptosis. Hum. Reprod., 17, 1584–1590.
Matsumoto, K. (2012). Xenotransplanted embryonic kidney provides a niche for endogenous mesenchymal stem cell differentiation into erythropoietin-producing tissue. Stem Cells,
30(6), 1228–1235.
Medina-Gomez, P. (1982). Cell morphology in long-term cultures of normal and abnormal amniotic fluids. Hum. Genet., 60(4), 310–313.
Meissner, A., et al. (2006). Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature, 439, 212–215.
Mimeault, M., & Batra, S. K. (2008). Recent progress on tissue-resident adult stem cell biology and their therapeutic implications. Stem Cell Rev., 4, 27–49.
Minuth, W. W., Schumacher, K., & Strehl, R. (2005). Renal epithelia in long term gradient culture for biomaterial testing and tissue engineering. Biomed. Mater. Eng., 15, 51–63.
Minuth, W. W., & Strehl, R. (2007). Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and
pharmaceutical research. Biomed. Mater., 2, R1–R11.
Moreau, J. E., Bramono, D. S., Horan, R. L., et al. (2008). Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments. Tissue Eng. Part A,
14, 1161–1172.
Morigi, M., et al. (2004). Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J. Am. Soc. Nephrol., 15, 1794–1804.
Nguyen, M. M., et al. (2007). Urothelial progenitor cells: regional differences in the rat bladder. Cell Prolif., 40(2), 157–165.
Nitta, M., et al. (2010). Reconstitution of experimental neurogenic bladder dysfunction using skeletal muscle-derived multipotent stem cells. Transplantation, 89, 1043–1049.
Noggle, S., et al. (2012). Human oocytes reprogram somatic cells to a pluripotent state. Science, 478, 70–75.
Nolen-Walston, R. D. (2008). Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration. Am. J. Physiol. Lung Cell Mol. Physiol., 294, L1158–
L1165.
Oberpenning, F., et al. (1999). De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nat. Biotechnol., 17, 149–155.
Orlando, G., et al. (2012). Production and implantation of renal extracellular matrix scaffolds from porcine kidneys as a platform for renal bioengineering investigations. Ann. Surg.,
256(2), 363–370.
Pariente, J. L., Kim, B. S., & Atala, A. (2001). In vitro biocompatibility assessment of naturally derived and synthetic biomaterials using normal human urothelial cells. J. Biomed.
Mater. Res., 55, 33–39.
Perin, L., et al. (2010). Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis. PLoS ONE, 5, e9357.
Perin, L., et al. (2007). Renal differentiation of amniotic fluid stem cells. Cell Prolif., 40, 936–948.
Piquilloud, G., et al. (2007). Variations in glial cell line-derived neurotrophic factor release from biodegradable nerve conduits modify the rate of functional motor recovery after rat
primary nerve repairs. Eur. J. Neurosci., 26, 1109–1117.
Reddy, P. P., et al. (2000). Regeneration of functional bladder substitutes using large segment acellular matrix allografts in a porcine model. J. Urol., 164, 936–941.
Reubinoff, B. E., et al. (2000). Neural progenitors from human embryonic stem cells. Nat. Biotechnol., 18, 399–404 [see comment]; [erratum appears in Nat. Biotechnol. 200
18(5), 559].
Rogers, S. A., & Hammerman, M. R. (2004). Prolongation of life in anephric rats following de novo renal organogenesis. Organogenesis, 1, 22–25.
Rogers, S. A., et al. (1998). Transplantation of developing metanephroi into adult rats. Kidney Int., 54, 27–37.
Ross, E. A., et al. (2009). Embryonic stem cells proliferate and differentiate when seeded into kidney scaffolds. J. Am. Soc. Nephrol., 20, 2338–2347.
Rota, C., et al. (2012). Human amniotic fluid stem cell preconditioning improves their regenerative potential. Stem Cells Dev., 11, 1911–1923.
Sato, Y., Terashima, M., Kagiwada, N., Aung, T., Inagaki, M., Kakuta, T., & Saito, A. (2005). Evaluation of proliferation and functional differentiation of LLC-PK1 cells on porous
polymer membranes for the development of a bioartificial renal tubule device. Tissue Eng., 11, 1506–1515.
Schnegelsberg, B., et al. (2010). Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity, and changes in urinary bladder function. Am. J.
Physiol. Regul. Integr. Comp. Physiol., 298, r534–r547.
Schumacher, K., Strehl, R., de Vries, U., & Minuth, W. W. (2002). Advanced technique for long term culture of epithelia in a continuous luminal–basal medium gradient.
Seliktar, D., Black, R. A., Vito, R. P., et al. (2000). Dynamic mechanical conditioning of collagen-gel blood vessel constructs induces remodeling in vitro. Ann. Biomed. Eng., 28,
351–362.
Sievert, K. D., et al. (2007). Tissue engineering for the lower urinary tract: a review of a state of the art approach. Eur. Urol., 52, 1580–1589.
Silver, F. H., et al. (1992). Cell growth on collagen: a review of tissue engineering using scaffolds containing extracellular matrix. J. Long Term Eff. Med. Implant., 2, 67–80.
da Silva Meirelles, L., Caplan, A. I., & Nardi, N. B. (2008). In search of the in vivo identity of mesenchymal stem cells. Stem Cells, 26, 2287–2299.
Song, J. J., et al. (2013). Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat. Med., 19(5), 646–651.
Stokman, G., Leemans, J. C., Stroo, I., Hoedemaeker, I., Claessen, N., Teske, G. J. D., Weening, J. J., & Florquin, S. (2008). Enhanced mobilization of bone marrow cells does not
ameliorate renal fibrosis. Nephrol. Dial. Transplant., 23, 483–491.
Sutherland, R. S., et al. (1996). Regeneration of bladder urothelium, smooth muscle, blood vessels and nerves into an acellular tissue matrix. J. Urol., 56, 571–577.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.
Taupin, P. (2006). Therapeutic potential of adult neural stem cells. Recent Patents CNS Drug Discov., 1, 229–303.
Thompson, J. A., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147 [see comment]; [erratum appears in Science 1998
282(5395), 1827].
Till, J. E., McCulloch, E. A., & Siminovitch, L. (1964). A stochastic model of stem cell proliferation, based on the growth of spleen colony forming cells. Proc. Natl. Acad. Sci. USA,
51, 29–36.
Tiranathanagul, K., Eiam-Ong, S., & Humes, H. D. (2005). The future of renal support: high-flux dialysis to bioartificial kidneys. Crit. Care Clin., 21, 379–394.
Togel, F., et al. (2005). Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am. J. Physiol. Ren.
Physiol., 289, F31–F42.
Togel, F., et al. (2005a). Renal SDF-1 signals mobilization and homing of CXCR4-positive cells to the kidney after ischemic injury. Kidney Int., 67, 1772–1784.
Togel, F., Hu, Z. M., Weiss, K., Isaac, J., Lange, C., & Westenfelder, C. (2005b). Administered mesenchymal stem cells protect against ischemic acute renal failure through
differentiation-independent mechanisms. Am. J. Physiol. Ren. Physiol., 289, F31–F42.
Tumlin, J., Wali, R., Williams, W., Murray, P., Tolwani, A. J., Vinnikova, A. K., Szerlip, H. M., Ye, J. M., Paganini, E. P., Dworkin, L., Finkel, K. W., Kraus, M. A., & Humes, H. D.
(2008). Efficacy and safety of renal tubule cell therapy for acute renal failure. J. Am. Soc. Nephrol., 19, 1034–1040.
United States Renal Data System, USRDS. (2012). Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States. Bethesda, MD: National
Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases.
Vijayan, A., Franklin, S. C., Behrend, T., Hammerman, M. R., & Miller, S. B. (1999). Insulin-like growth factor I improves renal function in patients with end-stage chronic renal
failure. Am. J. Physiol. Regul. Integr. Comp. Physiol., 276, R929–R934.
Wallis, M. C., Yeger, H., Cartwright, L., et al. (2008). Feasibility study of a novel urinary bladder bioreactor. Tissue Eng. Part A, 14, 339–348.
Warburton, D. (2008). Stem/progenitor cells in lung development, injury repair, and regeneration. Proc. Am. Thorac. Soc., 5, 703–706.
Weber, B., et al. (2011). Prenatally harvested cells for cardiovascular tissue engineering: fabrication of autologous implants prior to birth. Placenta, 32(Suppl. 4), S316–S319.
Weiner, L. P. (2008). Definitions and criteria for stem cells. Methods Mol. Biol., 438, 3–8.
Woolf, A. S., et al. (1990). Creation of a functioning chimeric mammalian kidney. Kidney Int., 38, 991–997.
Xie, C., Hu, J., Ma, H., et al. (2011). Three-dimensional growth of IPS cell-derived smooth muscle cells on nanofibrous scaffolds. Biomaterials, 32, 4369–4375.
Yamanaka, M., et al. (2002). Loss of anti-apoptotic genes in aging rat crura. J. Urol., 168, 2296–3000.
Yang, B., et al. (2010). Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng. Part C Methods, 16,
1201–1211.
Yokote, S., et al. (2012). The effect of metanephros transplantation on blood pressure in anephric rats with induced acute hypotension. Nephrol. Dial. Transplant., 27(9),
3449–3455.
Yoo, J. J., et al. (1996). Creation of functional kidney structures with excretion of kidney-like fluid in vivo. Pediatrics, 98(Suppl.), 605.
Zhang, H., Tasmin, F., Ying, J. Y., & Zink, D. (2009). The impact of extracellular matrix coatings on the performance of human renal cells applied in bioartificial kidneys. Biomaterials,
30, 2899–2911.
Zhang, S. C., et al. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells [see comment] Nat. Biotechnol., 19, 1129–1133.
Zhang, X., et al. (2006a). Derivation of human embryonic stem cells from developing and arrested embryos. Stem Cells, 24, 2669–2676.
Zhang, Y., et al. (2006b). Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystecomy model. BJU Int., 98,
1100–1105.
Zhou, H., & Ding, S. (2010). Evolution of induced pluripotent stem cell technology. Curr. Opin. Hematol., 17, 276–280.
Bioengineering Scaffolds for Regenerative Engineering
Zichen Qian, Daniel Radke, Wenkai Jia, Mitch Tahtinen, Guifang Wang, and Feng Zhao, Michigan Technological University,
Houghton, MI, United States
Introduction 444
Scaffolding Materials 445
Synthetic Materials 446
Hybrid Materials 447
Scaffold Fabrication Technologies 448
Decellularization 448
Mechanical impact of decellularization protocol 449
Biological impact of decellularization protocol 449
Polymeric Scaffold Fabrication 450
Highly porous scaffold fabrication 450
Electrospinning 451
Bioprinting of 3-D Scaffolds 452
Stereolithography 452
Solid free-form fabrication 453
Bioprinted scaffolds 453
Applications of Scaffolds in Tissue Engineering 454
Introduction 454
Spinal Cord/Neuron Repair 454
Wound Healing 454
Bone Regeneration 455
Vascular Scaffolds 455
Summary and Future Directions 456
References 456
Further Reading 461
Introduction
Tissue engineering has been a rising field since the mid-1980s when the term was used loosely to cover everything from pros-
thetics to tissue manipulation. Today, the field of tissue engineering has evolved into applying precise engineering principles
to clinical problems (Winterswijk and Nout, 2007). Cells, scaffolds, and signals are often called the tissue engineering triad,
which are combined through engineering technologies to achieve the replacement or regeneration of damaged tissues (O’Brien,
2011). The purpose of scaffolding is to mimic the structure and function of extracellular matrix (ECM) in native tissues to fully, or
partially replace, damaged or diseased tissues. Ideally, scaffolds should be able to execute physiological functions including
mechanical support, cell attachment and survival, and nutrient/waste transportation once implanted. They should also disappear
upon the completion of tissue regeneration (Minuth et al., 1997; Chan and Leong, 2008; Stock and Vacanti, 2001; Hutmacher,
2001).
Fabrication of scaffolds should meet several criteria regardless of target tissue types. The scaffolds need be biocompatible, biode-
gradable, mechanically compatible, and bioactive. Metals, ceramics, and polymers are the three fundamental biomaterials, in which
metals and ceramics are often utilized for the manufacturing of stiff tissues such as bone scaffolds and dental scaffolds (Graney et al.,
2016; Chróscicka et al., 2016; Denry and Holloway, 2014; Kim et al., 2016a; Zhao et al., 2017; Capek et al., 2016; Yu et al., 2017;
Wang et al., 2016). For scaffold engineering, polymeric materials have gained popularity since it is easier to produce synthetic poly-
mers with specific properties and shape them into desired architectures. Drawbacks of synthetic polymers include the lack of bioac-
tivity and tissue integration capability, compared with natural proteins, which could result in the rejection of the scaffold. In
addition, local pH can be distorted by some acid polymer degradation by-products and result in cell necrosis (O’Brien, 2011).
On the other hand, fabrication of natural polymers into scaffolds with desired mechanical properties is challenging. To overcome
the disadvantages of synthetic and natural polymers, a combination of the two materials is widely accepted as a strategy to balance
scaffold properties (Grayson et al., 2004).
Numerous fabrication methods have been developed to tailor “tissue specificity” of scaffolds to fulfill diverse needs. The most
straightforward method of fabricating such scaffolds would be decellularizing similar tissues from another source to remove most of
the antigens while preserving the mechanical and biochemical properties (Liao et al., 2008; Rieder et al., 2005). Studies are trying to
manipulate antigen-removed xenogeneic tissues for human use to widen the tissue donor availability; however, the sources of

Regenerative Engineering j Bioengineering Scaffolds for Regenerative Engineering 445
tissues are still quite limited. Fortunately, tissue decellularization is not the only option. Fabrication technologies including salt
leaching, gas foaming, phase separation, and electrospinning can generate three-dimensional (3-D) variable architectures that
mimic the nature of different tissue microenvironments. Nevertheless, it remains difficult to recreate complex microarchitectures
such as the interface between different tissues (Hollister, 2005). With the development of 3-D printing technologies, computer-
aided bioprinting of specialty tissues with precise geometry has become possible (Nowicki et al., 2016). By careful selection of
printing techniques and materials, it is possible to fabricate functional scaffolds with tailored architecture and mechanical proper-
ties to replace a variety of tissues.
This article will provide a broad overview of scaffolding materials and fabrication techniques. Beyond providing a review of
general principles, specific examples of regenerative applications that utilize scaffolds fabricated through different techniques are
also examined and discussed.
Scaffolding Materials
Material selection is critical for scaffold design due to the bioscaffold material having to be nontoxic and supportive to cell adhesion,
proliferation, and migration (Chan and Leong, 2008). The scaffold should also provide proper mechanical support to included cells
and match the mechanical properties of native tissues and organs. Based on the material sources and compositions, scaffold mate-
rials can be classified into natural materials, synthetic materials, and hybrid materials. Hybrid materials are designed to integrate
different classes of materials to balance the scaffold properties to meet various requirements (Grayson et al., 2004).
Natural Materials
The natural ECM is a collection of molecules secreted by cells that contains complex components including collagen, laminin, hya-
luronic acid (HA), and fibronectin. ECM provides an ideal environment for cells to proliferate and carry out their functions. ECM
can mechanically support cells to form an integrated and functional tissue and provide biomolecules to maintain cell motility and
viability. Ideally, tissue-engineered scaffolds should play a similar function to natural ECM, which supplies mechanical and molec-
ular support for cell and tissue regeneration. Materials isolated from physiological systems have great potential for scaffold fabri-
cation due to their structural and biochemical similarity to native tissues. These natural materials are biodegradable and can provide
biological signaling for cell attachment, proliferation, and differentiation. Proteins derived from ECM are much more popular in the
tissue engineering field compared with decellularized ECM. A single ECM component can be extracted from different species, which
overcomes the donor limitation. Scaffolds from single component are more reproducible and hold better potential to be manufac-
tured in large scale. Collagen is the most popular protein-based scaffolding material in tissue engineering. Other naturally derived
proteins such as fibrin and HA have also been used for engineering different tissues.
Collagen is one of the main protein components in ECM, providing support to connective tissues and maintaining tissue integ-
rity. There are 28 types of collagen that have been identified, in which collagen types I, II, III, and V compose the main part of the
bone, cartilage, tendon, and skin. Collagen as a scaffold component possesses advantages such as high mechanical strength,
biocompatibility, degradability, and broader accessibility. The collagen fibrils in scaffolds carry integrin binding sites, such as integ-
rins a2b1 and a1b2 (Heino, 2000), which are beneficial for cell adhesion and migration (Heino, 2000). The porous structure created
in the collagen scaffold allows cells to penetrate and further promote tissue regeneration (Glowacki and Mizuno, 2008). These char-
acteristics make collagen a popular scaffold material in tissue engineering (Cen et al., 2008). For instance, bone and cartilage tissues
are supportive tissues that require scaffolds to bear high mechanical loads, which inspired researchers to apply collagen when engi-
neering bone and cartilage tissues. Combining chondrocytes with a collagen scaffold could promote organized cartilage defect heal-
ing within 12 months (De Franceschi et al., 2005). Applying a collagen I sponge scaffold as a transient cartilage template showed
improved scaffold stiffness and chondrocyte vitality. The chondrocytes deposited collagen I, collagen X, and alkaline phosphatase,
indicating the collagen scaffold could be potentially utilized in endochondral bone regeneration (Oliveira et al., 2010). Due to its
abundant sources, bioproperties, and mechanical strength, collagen scaffolds have also been widely used in engineering other
tissues, such as cardiac (Boland et al., 2004; Boccafoschi et al., 2005; Park et al., 2009), skin (Ma et al., 2003; Gautam et al.,
2014), and nerve (Bozkurt et al., 2007) tissues.
Fibrin is a nonglobular fibrous protein that contributes to blood clotting. It is also a fibrous network polymer formed by poly-
merization of fibrinogen in the presence of thrombin, resulting in the porous structure of a fibrin clot that mimics ECM (Barsotti
et al., 2011). Fibrinogen can be isolated from blood plasma of the patient, which reduces the risk of immune rejection and disease
transmission. Fibrin contains cadherin, integrin, and platelet binding sites (Mosesson, 2005), through which the cell adhesion,
migration, and proliferation are promoted (Ehrbar et al., 2005). Fibrin has been widely applied as a carrier for cells and growth
factors in tissue engineering applications. With the introduction of thrombin, fibrinogen solution undergoes a cascade of polymer-
ization to form a hydrogel, meanwhile encapsulating cells and absorbing growth factors. Applying fibrin gel as a cell carrier could
promote seeding efficiency and increase ECM accumulation in the extracellular space (Ye et al., 2000). In a study that applied fibrin
as a myofibroblast carrier for cardiovascular regeneration, the addition of fibrin decreased the loss of collagen and promoted ECM
formation, indicating fibrin could promote tissue regeneration by promoting the accumulation of ECM components (Mol et al.,
2005). A similar study compared the performance of fibrin with collagen as an arterial media equivalent (Grassl et al., 2002). Results
showed that fibrin could promote smooth muscle cells to secrete more than three times the amount of collagen than the collagen gel
446 Regenerative Engineering j Bioengineering Scaffolds for Regenerative Engineering
group, resulting in a mechanically stronger arterial media. This study indicates the feasibility of applying fibrin in cardiovascular
engineering. Fibrin scaffolds have also been tested for repairing spinal cord injury by carrying the neurotrophic factors, such as
neurotrophin-3, which can be incorporated into a fibrin scaffold for sustained release and resulted in significantly enhanced sprout-
ing of neural fibers near the transplantation site. All these studies show that fibrin scaffolds function as excellent cell or growth factor
carriers for tissue engineering applications (Johnson et al., 2009). The challenge for applying a fibrin scaffold is that fibrin degrades
easily. Adding protease inhibitors to the fibrin scaffold could help alleviate this problem, but the added concentration of protease
inhibitors needs to be minimized to avoid interrupting cellular activities (Mol et al., 2005; Shaikh et al., 2008).
Beyond natural proteins, polysaccharides are also extensively studied as scaffolding materials. Polysaccharides are long-chain
polymeric carbohydrates composed of monosaccharide units, which are bonded together by glycosidic linkages. Polysaccharides
can be obtained from abundant and relatively cost-effective sources, including animal, vegetal, and microbial. Polysaccharides
are biocompatible and generally have good hemocompatibility. They can be processed into special scaffolds to meet the diverse
requirements of different tissue types (Mano et al., 2007). Cellulose, starch, and chitin are representative examples of widely
used polysaccharides. However, cellulose requires further modification for use in tissue engineering applications due to its water
sensitivity and low cell adhesion on the scaffold surface (Kalia et al., 2011). Also, starch lacks dimensional stability and mechanical
strength and is difficult to process, making it not applicable as a pure scaffold (Lu et al., 2009). These limitations confine the appli-
cations of cellulose and starch. Chitin can be found in far-ranging sources such as the cuticle of insects, the cell wall of fungi, and the
shell of crustacean (Ozdil and Aydin, 2014). Although chitin is insoluble in common solvents, its deacetylated form, chitosan, can
be easily dissolved in dilute acids (pH < 6). Moreover, chitosan has been proved to be biologically renewable, biodegradable,
biocompatible, and nonantigenic (Di Martino et al., 2005). Due to its structural similarity with various glycosaminoglycans
(GAGs) found in articular cartilage, chitosan has been used to modulate chondrocyte morphology and differentiation for cartilage
tissue engineering (Suh and Matthew, 2000). Results from in vivo studies showed that chitosan hydrogel could support cartilage
matrix accumulation and promote cartilage regeneration (Hoemann et al., 2005; Hao et al., 2010). Moreover, chitosan can promote
osteoblast proliferation and mineralized ECM deposition by upregulating associated osteogenesis gene expression (Mathews et al.,
2011). Therefore, chitosan has been fabricated into sponge or highly porous composite scaffolds for bone regeneration (Seol et al.,
2004; Zhang et al., 2008; Venkatesan and Kim, 2010; Li et al., 2005a).
Another naturally derived material that has been extensively used is HA, which is a linear GAG with high molecular weight and
biodegradability (Fakhari and Berkland, 2013). HA is a component of most connective, epithelial, and neural tissues. It associates
with cell surface receptors. For example, association with integrins modulates cellular behaviors such as cell attachment, motility,
and proliferation (Evanko et al., 1999). HA can be massively produced by animal tissue extraction, bacterial synthesis, and enzy-
matic synthesis from nucleotide sugars (Boeriu et al., 2013). The nonlinear viscoelastic property endues HA the function of lubri-
cation and shock absorption (Zhang and Christopher, 2015). It can also function as space-filling materials and increase the
viscoelastic property of bulk materials, which makes it an attractive material in cartilage (Yoo et al., 2005) and bone engineering
(Mano et al., 2007). HA-based scaffolds can stimulate matrix deposition when seeded with preadipocytes, indicating that HA scaf-
folds are able to be applied when generating volume-retaining tissue (Stillaert et al., 2008). Another experiment applied constructed
HA scaffolds seeded with preadipocytes as an implant for soft tissue regeneration (Stillaert et al., 2008). The scaffold successfully
integrated with native tissue, showing the capability of HA scaffolds in adipose tissue regeneration. The high solubility of HA at
room temperature and under acidic pH values is the main obstacle for keeping its structural integrity. Adding cross-linkers could
help solve this problem (Allison and Grande-Allen, 2006; Zheng Shu et al., 2004).
Besides the several materials introduced earlier, there are many other types of natural material that are widely applied as mate-
rials for tissue engineering scaffolds, such as GAGs, alginate, or silk fibroin. Due to the naturally derived nature of these scaffolds,
they have reduced immunological reaction and can provide a suitable microenvironment for cell vitality, migration, proliferation,
and differentiation. However, the degradation rate of natural scaffolds is difficult to control. Their mechanical strength is usually not
strong enough to support certain tissue functions. Optimization of processing and design strategies is required for the application of
natural materials, which will be discussed later.
Synthetic Materials
Although natural materials provide excellent properties including biocompatibility, biodegradability, and bioactivity, issues such as
immunogenicity, reproducibility of fabrication, impaired mechanical properties, and control of degradation rate need to be
resolved for practical applications (Lutolf and Hubbell, 2005). Compared with biological materials, synthetic materials are
much more controllable and easy to process. Synthetic materials have been widely investigated as tissue engineering scaffolding
materials due to their flexibility in physical, chemical, and mechanical properties modulation. They can be modified to have
different properties that fulfill the criteria of a variety of implantation sites with decent reproducibility (Bracaglia and Fisher,
2015). Conventional categories of synthetic materials include ceramic, metal, and polymeric materials. Those materials have
been extensively studied for tissue engineering applications.
Synthetic polymers provide highly tunable mechanical strength and controllable degradation rate, which make them appealing
candidates for both soft and stiff tissue engineering scaffolds. Linear aliphatic polyesters, such as poly(lactic acid) (PLA), poly(gly-
colic acid) (PGA), and their copolymers, poly(lactic acid-co-glycolic acid) (PLGA), are the most frequently used polymers as tissue
engineering scaffolds due to their biodegradability and biocompatibility (Armentano et al., 2010). The degradation products are
nontoxic components and can be removed by natural metabolism. Besides, they have been approved by US Food and Drug
Administration (FDA) for clinical use. Another well-accepted aliphatic polyester used in tissue engineering is the poly(3 -caprolac-
tone) (PCL) (Woodruff and Hutmacher, 2010). PCL has a relatively low degradation rate compared with PLA and PLGA, which
provides prolonged support for tissues that require a longer regeneration process. In particular, PCL has been proved to carry a degra-
dation rate similar to the rate of new bone formation (Lam et al., 2009), which makes it attractive for bone tissue engineering.
Mesenchymal stem cells (MSCs) cultured on PCL supplied with osteogenic factors are observed to form mineralization and collagen
I deposition on the scaffold (Yoshimoto et al., 2003). PCL also shows the ability to induce chondrogenesis of MSCs seeded on its
scaffold to differentiate toward chondrocytes and enhance secretion of cartilage-specific ECM (Li et al., 2005b), which designates its
capability in engineering cartilages. In addition, there are some other synthetic polymers that have been widely applied as tissue
engineering scaffolds, such as poly(propylene fumarate) (PPF) and poly(glycerol sebacate) (PGS). PPF is a good candidate for
bone engineering scaffolds. It is biodegradable, biocompatible, and osteoconductive and can be reinforced by cross-linking (Shi
et al., 2005; Zhu et al., 2010). PGS is biocompatible, biodegradable, and cost-effective. Its flexibility makes it popular for engi-
neering soft tissues (Motlagh et al., 2006; Masoumi et al., 2013; Ravichandran et al., 2012), such as skin and cardiovascular tissues.
Broadly used ceramic materials consist of calcium phosphate (CaP) ceramics, CaP cements, and bioactive glasses. These mate-
rials have their unique advantages, but are not appearing to be very attractive for engineering most tissue types due to their slow
degradation rate. However, they are preferred in constructing bone and dental scaffolds due to their superior load bearing, wear
properties, and integration promoting effect on human tissues (Rahaman et al., 2011). Within the variety of ceramics, CaP
compounds (LeGeros, 2002) and bioactive glass (Rahaman et al., 2011; Thomas et al., 2005) are exceptional for fabrication of
bone engineering scaffolds due to their osteoconductive properties. Hydroxyapatite (HAp), beta-tricalcium phosphate, biphasic
CaP, carbonated apatite, and amorphous CaP are the most common CaP ceramics that have been explored in bone tissue engi-
neering (Habraken et al., 2007).
Currently, metal scaffolds are mainly applied in treating orthopedic diseases. Typical metal scaffolds contain stainless steel,
cobalt-based alloys, titanium-based alloys, and magnesium-based alloys (Yang et al., 2001). Metal scaffolds possess strong mechan-
ical strength and thus can provide robust tissue mechanical support as bone replacement. Moreover, metal scaffolds have unique
properties that allow for distinctive scaffold designs. For example, titanium-based alloys are able to be fabricated into shape
memory scaffolds, which means that they can regain their original shape after deformation. This shape memory property allows
large strain while bearing loading and returning to original position when relaxed. To take advantage of this property, Ni–Ti alloy
has been widely used as orthodontic plates and staples for bone fractures (Wang et al., 2016). The controllable degradation property
is another benefit of certain metal alloys. Compared with biodegradable polymers, biodegradable metals can provide higher
mechanical strength, and they are applicable in supporting tissue architectures (Farraro et al., 2014). However, the degradation
by-products of biodegradable metals could potentially cause inflammation. Controlling the degradation rate and by-product
removal should be taken into consideration when designing metal-incorporated scaffolds (Lietaert et al., 2013). Some metal scaf-
folds face mechanical mismatch problems that can damage surrounding tissues and causes foreign body reactions (Jung et al.,
2015). To solve these problems, metal scaffolds are fabricated into porous structures to decrease material stiffness, while increasing
cell infiltration and tissue regeneration (Yang et al., 2001).
Synthetic materials provide better plasticity and stronger mechanical strength than naturally derived materials. Their degradation
rate and mechanical strength are more controllable through different synthesis approaches. The reproducibility also makes them
promising as tissue engineering scaffolds. However, the synthetic materials often lack biocompatibility and bioactivity, which
should be resolved during the scaffold design and fabrication (Seo and Park, 2010).
Hybrid Materials
As described earlier, both natural and synthetic materials have their pros and cons that need to be resolved for a good scaffold
design. It is usually difficult for one type of material to fulfill the complex requirements for tissue regeneration. Novel design strat-
egies for different combinations of biomaterials with diverse properties to complement the gaps of each other are necessary. The
addition of natural materials into synthetic scaffolds would provide molecular signals to the scaffold and increase the biocompat-
ibility, while the addition of synthetic materials into natural scaffolds could significantly increase the mechanical strength (Chan
and Leong, 2008). The hybrid of materials can be achieved by blending, coating, or chemical grafting of needed moieties on poly-
mers. For example, a hybrid of PLGA and collagen for cartilage tissue engineering took advantage of PLGA as mechanical supporting
skeleton while collagen provided cell binding sites to allow integration (Dai et al., 2010). As the result anticipated, chondrocytes
showed even distribution and deposited abundant ECM on the scaffold. In another study, ECM-derived components were
combined with PCL that led to favorable biological and mechanical properties for cardiovascular tissue engineering
(Heydarkhan-Hagvall et al., 2008). Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was coated on decellularized porcine aortic
heart valves, giving rise to significantly enhanced mechanical strength and well-maintained biological properties (Hong et al.,
2009). The coating of fibrin on PLA increased the elasticity of the scaffold, enhanced cell proliferation, cell adhesion, and even
cell distribution (Gamboa-Martínez et al., 2011). Collagen-coated co-poly(L-lactide/epsilon-caprolactone) (PLCL) tube was applied
to treat urethral defects (Kanatani et al., 2007). After being implanted in an animal model, the tube was functionalized through
epithelialization and pericyte wrappings, which benefited from the incorporation of collagen. Polypyrrole (PPy) has applications
as neural conduits based on its electric conductivity. The incorporation of HA into PPy improved the biocompatibility of the scaf-
fold while preserving its original conductivity for neural scaffold designs (Cen et al., 2004). Research studied the mechanical and
biological properties of collagen–PCL blends that were applied for engineered skin. With a small amount of PCL, the strength of
acellular collagen increased up to 10% compared with a purely collagen scaffold, while the biocompatibility of the scaffolds was not
affected (Powell and Boyce, 2009).
The promising next generation of tissue engineering scaffolds should have the integration of desired strength, degradability, and
bioactivity. The materials should not only contain growth factors facilitating cell attachment, proliferation, and differentiation but
also provide suitable mechanical strength for supporting tissue function. The hybridized materials offer flexibility and controlla-
bility for fulfilling these requirements.
Scaffold Fabrication Technologies
Different strategies have been explored for creating tissue engineering scaffolds. Decellularization of tissues to generate scaffolds is
the most straightforward method to create tissue-specific scaffolds. To successfully obtain a functional scaffold from decellulariza-
tion, delicate cell removal processes are usually required to preserve the mechanical properties and bioactivities postdecellulariza-
tion. Nonetheless, the sources of donor tissues are far behind demand (Liao et al., 2008; Gilbert et al., 2006). Various natural and
synthetic polymers have been processed with different strategies to recreate the porosity and bioactivity of the target tissues and to
widen the selection other than decellularized tissue scaffolds. As aforementioned, polymeric materials provide highly tunable
mechanical strength and controllable degradation rate by their chemical composition. Different fabrication techniques, including
particulate leaching, gas foaming, phase separation, and electrospinning, have been developed to create pores and microarchitec-
tures that mimic spatial structures of specific tissues (Zhang et al., 2005; Annabi et al., 2009; Zhang et al., 2012; Davidenko et al.,
2012; Chew et al., 2008). The fabricated scaffolds must be porous to support nutrient and oxygen transportation and encourage cell
penetration, adhesion, and proliferation to promote regeneration. The structure and size of the pores can be roughly controlled by
these methods, but recreation of detailed microstructures is not possible. With the development of 3-D printing technologies, more
precisely controlled fabrication processes are introduced to manipulate the scaffold microarchitectures through the computer-
directed nozzle dispersion (Nowicki et al., 2016).
Decellularization
Tissue engineering scaffolds can be fabricated from harvested tissues and organs through the removal of the native cellular compo-
nents. The remaining ECM provides an ideal environment to accommodate tissue cells. ECM is specialized for the local context of its
environment but is composed of the same general components, even across species (Bernard et al., 1983a, 1983b; Constantinou
and Jimenez, 1991; Exposito et al., 1992). Therefore, the goal of decellularization is to effectively remove all cellular and nuclear
materials, while minimizing the impact on the ECM, to produce a scaffold that will maximize compatibility with native tissues
and lessen host immune response. The most common decellularization procedures today typically incorporate a series or combi-
nation of treatment methods, but ultimate efficacy depends upon the context of the tissue. Nevertheless, the standard procedure
involves treatment with a nonionic detergent to disrupt lipid–lipid and lipid–protein interactions, rupturing the membranes of cells
and removing their attachments to the ECM while minimally affecting the protein–protein interactions of the ECM itself. This is
followed by a brief enzymatic treatment to disrupt the peptide bonds of genetic materials that would otherwise provoke a host
response, usually in combination with mechanical agitation to wash out the resulting cellular debris (Gilbert et al., 2006). This
process is illustrated in Fig. 1. It was originally thought that a decellularization protocol with fast lysing and removal of cells would
be preferential for reducing the exposure time of the tissue to the detergent and thus minimizing the damage of the detergent on the
Fig. 1 Decellularization of tissues to create scaffolds. The tissue from the donor is treated with detergent to break the lipid bilayer of the cell
nucleus. DNA is removed by an extensive wash with DNase treatment if necessary. Detergent is neutralized post decellularization to ensure the
biocompatibility of the decellularized scaffolds. Illustration is created based on modification of Servier Medical Art under a creative commons 3.0
unported licenses.
remaining ECM. In comparing a nonionic detergent-based decellularization protocol (Triton X-100) with faster anionic detergent-
based protocol (sodium dodecyl sulfate, SDS, and sodium deoxycholate, SDC) of porcine aortic heart valves and pericardia, it was
found that the lessened exposure time of the SDC-based protocol did not result in better preservation of structural ECM compo-
nents (Roosens et al., 2016). Both protocols led to similar alterations to the ECM, but cell nuclear staining revealed DNA fragments
still present in all SDC treated tissues that were not caught with staining of cellular mass. Gel electrophoresis confirmed the presence
of these DNA fragments after the SDC protocol and the absence of any such DNA fragments after the Triton X-100 protocol. Despite
the preservation of ECM tissue structure under these protocols, porcine heart valves decellularized with the SDC protocol showed
a significant reduction in soluble collagen that was conserved in the Triton X-100 protocol. It was also found that the pericardia were
less susceptible to the deterioration of soluble collagen following decellularization protocols (Roosens et al., 2016). Triton X-100
remains one of the most popular detergents for decellularization protocols, having proved itself both thorough with cell removal
and gentle on ECM. Given that one of the primary advantages of a decellularized ECM scaffold is its mechanical similarity to native
tissue, considerable interest was drawn to analyzing how decellularization alters the ECM that is left behind.
Mechanical impact of decellularization protocol

The mechanical properties of the scaffold after decellularization depend upon the chemicals and methods used during the decel-
lularization procedure (Liao et al., 2008). The disruption or loss of collagen has been implicated as the major contributor to the
alteration of mechanical strength following decellularization, while the disruption of elastin alters mechanical resilience (Liao
et al., 2008; Grauss et al., 2005). Ideally, a minimal impact on the ECM structure and mechanics is desired, but this is not always
possible. Even the most delicate treatments will alter the mechanical properties slightly due to the absence of the cellular compo-
nents, though such slight alterations are usually without statistical significance. Generally, it is preferable to sacrifice a small
mismatch in mechanics than to risk a host response provoked by lingering genetic materials. The mechanical impact of the decel-
lularization protocol primarily depends on how the protocol alters the collagen and elastin of the ECM, which is in turn dependent
upon the context of the tissue to be decellularized. For example, porcine aortic heart valves decellularized with a Triton X-100 based
protocol (1% concentration) increased in net extensibility by around 100%, based on circumferential and radial maximum stretch
ratios at 60 N/m equibiaxial tension (Liao et al., 2008). This corresponded with a reduction in tissue area and thickness by about
one standard deviation of the sample (n ¼ 18) according to morphological analysis using scanning electron microscopy (SEM).
Small angle light scattering analysis confirmed the preservation of the collagen fiber structure, but the Triton X-100 treatment
did not preserve the regional alignments found in the untreated aortic valves and resulted in a more homogeneous fiber structure.
SEM analysis calculated a normalized orientation index and found decellularization caused a decrease in areas that were relatively
highly aligned in the native aortic valves, but this decrease was not statistically significant. Decellularization resulted in a major loss
of tissue stiffness and a shift from a nearly linear moment–curvature response to a nonlinear relationship, causing a 46%–80%
reduction in stiffness. Histological analysis confirmed the absence of any cellular material after decellularization; however, the
well-organized collagen crimp structure of the fibrosa was disrupted and the spongiosa was depleted (Liao et al., 2008). Bovine
pericardial tissue decellularized with a Triton X-100 treatment (1% concentration) did not exhibit any significant change in thick-
ness or microstructure and did not alter stress-relaxation behavior when treated with Triton X-100 alone (Mendoza-Novelo et al.,
2011). The complete decellularization procedure resulted in an increase in maximal stress retention of approximately 20% when
compared to untreated tissue and reduced the native tissue modulus without statistical significance. No statistical difference was
observed in the ultimate tensile stress between the native and decellularized pericardial tissue. Pellegata et al. observed that porcine
aortic and carotid arterial segments ranging from 2 to 11 mm in lumen diameter decellularized by an SDC-based protocol (4%
concentration) displayed no significant difference in Young’s modulus, compliance, ultimate circumferential stress, burst pressure,
or suture retention strength when compared to native tissues. There was however a significant decrease in ultimate strain and an
increase in residual stress after relaxation (Pellegata et al., 2013). HE staining revealed no alterations to elastic laminae structure
or organization, but the absence of cellular components between laminae layers was thought to have altered the mechanical inter-
action between them.
Biological impact of decellularization protocol

In addition to altering the structurally derived mechanical properties of the scaffold, the method of decellularization alters both the
chemically and physically derived biological interactions with the decellularized ECM scaffold (Rieder et al., 2005). The first heart
valves engineered through decellularization of porcine heart valves were disastrous failures. Incomplete decellularization induced
a severe inflammatory response that led to the development of a fibrous sheath on the interior and exterior graft surfaces, blocking
cell repopulation and endothelialization and ultimately leading to graft failure (Simon et al., 2003). The development of this dense
fibrous sheath began as early as 2 days after implantation. Histological analysis of grafts explanted post failure indicated a severe
foreign body reaction started at the exterior of the graft with dense neutrophil granulocyte and macrophage reactions present within
the fibrous sheath, which infiltrated into the leaflet tissue. Lymphocytes were present after 1 year. Inexplicable calcium deposits were
discovered within the wall of the conduit but not the leaflet, and analysis of preimplant samples revealed the presence of similar
calcium deposits and dense patches of remnant cells. Evaluation of immunogenicity and thrombogenicity of porcine pulmonary
leaflets, decellularized by four different decellularization protocols, revealed significant differences that correlated with alterations
to the ECM architecture (Zhou et al., 2010). b-thromboglobulin (b-TG) and thrombin–antithrombin complex (TAT) were used to
determine thrombogenicity and revealed no significant differences in and between native and decellularized tissues according to
TAT. However, b-TG revealed a significant difference between untreated tissue and those treated with SDC or SDS, which displayed
similar b-TG levels, and a very significant difference between untreated tissue and those treated with trypsin/EDTA or trypsin/EDTA/
Triton X-100, which displayed similar b-TG levels. Two-photon laser scanning microscopy revealed the SDC protocol left collagen
and elastin structures almost completely unaffected, the SDS protocol caused elastin fibers to become compact and microcurled and
collagen fibers to become compact and lose structure detail, the trypsin/EDTA protocol revealed much fewer elastin and collagen
fibers with the collagen fibers appearing more loose and wavy, and the trypsin/EDTA/Triton X-100 protocol kept elastin fibers intact
while the collagen fibers lost their wavelike structure and became misaligned (Zhou et al., 2010). Many studies only test decellu-
larized scaffolds for immediate biocompatibility and successful end-stage regeneration. As such, mechanisms linking altered ECM
structure to the development and progression of immunologic response have not been thoroughly investigated, though previous
studies have examined ECM structures fabricated from a variety of protocols and noted distinct acute and chronic host response
and remodeling (Badylak and Gilbert, 2008). Residual chemicals from decellularization protocols have also recently been impli-
cated in significantly altering immunogenicity, though specific mechanisms have yet to be identified (Poornejad et al., 2016). It
is difficult to identify which protocols will have what effect on which tissues. SDC and Triton X-100 remain two of the primary
detergents used, SDC requires less treatment time to remove cellular components, but research has shown this does not necessarily
translate to better preservation of ECM. On the other hand, Triton X-100 is viewed as the safe alternative, requiring longer exposure
times but lessening the impact to the ECM, though again studies have shown this is not always the case and depends on the tissue
being decellularized. Literature searches to discover what methods have generated what results with the tissue of interest are key.
These existing protocols can then be modified to attain specifically desired results, such as experimentally adjusting concentration
or exposure time of a particular treatment for a particular tissue to balance the cell removal, ECM preservation, immunogenicity,
and mechanical properties.
Polymeric Scaffold Fabrication

Highly porous scaffold fabrication
As aforementioned, pore size and porosity are critical for scaffolds to allow mass transportation and cell penetration. Thermally
induced phase separation has been developed as a simple and cost-effective fabrication method to generate porous structures in
3-D scaffolds (Fig. 2A). Generally, the polymer solution is frozen at low temperature to achieve liquid–solid phase separation.
The polymer-lean phase is then removed by evaporation or extraction, and the polymer-rich phase is left to form a polymer skeleton
with porous structures (Zeng et al., 2015; Whang et al., 1995). The cooling temperature and cooling rate are used to control the pore
size from several micrometers to around 100 mm (Haugh et al., 2009; Peters et al., 2014). In addition, uniaxial aligned geometry of
the pores can also be created by introducing a uniaxial temperature gradient (Zhang et al., 2012; Davidenko et al., 2012). Unfor-
tunately, through this method, homogenous interconnectivity, porosity, and pore size are difficult to be reproduced. The pore-to-
pore variation is impossible to regulate (Matsuba et al., 2003).
As an alternative, the porogen leaching method is able to produce more uniform porous structures in scaffolds, which disperse
a porogen (usually sodium chloride salt granules) into the water-insoluble polymer through solvent casting followed by porogen
leaching in water, leaving a matrix with interconnective porous structure (Fig. 2B) (Tran et al., 2011; Goldstein et al., 2001).
Biocompatible solvents are required during the polymer casting process; otherwise, the residue of solvent retained in the scaffolds
could cause cytotoxicity. As pore size and structure of the scaffold are dependent on the crystal structure of the porogen, a variety of
porogens other than salts have been investigated. Spherical porogens are more favorable since they create well-controlled intercon-
nectivity compared with cubic shapes, due to the contact geometry (Zhang et al., 2005). For example, gelatin microspheres with
uniform particle size have been fabricated and adapted as a porogen (chloroform as solvent) for fabricating PCL scaffolds. The
generated pore size can be modified by tuning the size of the gelatin microspheres, while porosity can be controlled by the amount
of gelatin microsphere added during fabrication. The resulting scaffolds have less creep deformation and better interconnectivity
than sodium chloride control (Draghi et al., 2005). Another study used poly(ethylene glycol) (PEG) as a porogen to control the
pore properties of a polymer scaffold, such as PCL. The size of the pore was determined by employment of different molecular
weight PEG, and the porosity was manipulated through changing the ratio of PEG added into the blended materials (Columbus
et al., 2014).
Gas foaming has been utilized as another method to create porous scaffolds due to its organic, solvent-free, and inexpensive
nature (Fig. 2C). Like salt leaching, a foaming agent is added into the polymer solution to create bubbles, which is then dried
to obtain the interconnected porous scaffold. Effervescent salt-based gas foaming is more cost-efficient, but surfactants are usually
added to stabilize the foam. Dense gas CO2 foaming is more controllable and surfactant-free, but cosolvents are needed to enhance
the solubility of CO2. Through dense gas CO2 foaming, the pore size can be controlled by adjusting the temperature and pressure
(Annabi et al., 2010).
A sole method introduced previously has its own limitations, and multiple methods are combined to improve the properties of
porous scaffolds during the fabrication process. For example, to increase the interconnectivity and also include large pores in the
PLLA scaffold fabricated by phase separation, salt particulates (200–250 mm) were added into the polymer solution prior to phase
separation. After salt leaching, the microstructure of PLLA was significantly improved, resulting in better nutrient transportation and
cell proliferation (Tu et al., 2003). Importantly, the fabrication methods discussed in this section are not highly equipment-
dependent and can be processed at low costs. Moreover, the porous structure of the scaffolds is relatively controllable and repro-
ducible that is essential for industrial mass production of scaffolds.
Fig. 2 Fabrication methods of highly porous polymeric scaffolds. (A) Temperature gradient is introduced to the freezing process of the polymer
solution, inducing phase separation. The polymer-lean phase is then extracted with vacuum, resulting in pores after drying. (B) Particulate leaching of
polymer/porogen mixture. Water soluble porogen is removed by water leaching, leaving void spaces between insoluble polymer structures. (C) Gas
foaming agent is added into polymer solution to create a foamy polymer solution. The foam is then dried to form spongy structures. SEM images
were modified from (A) Li, X.-T., Zhang, Y. and Chen, G. Q. (2008) Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials.
Biomaterials 29(27), 3720–3728. https://doi.org/10.1016/j.biomaterials.2008.06.004, with permission from Elsevier. (B) Chatterjee, K., Hung, S.,
Kumar, G. and Simon, C. G. (2012). Time-dependent effects of pre-aging 3D polymer scaffolds in cell culture medium on cell proliferation. Journal of
Functional Biomaterials 3(2), 372–381. https://doi.org/10.3390/jfb3020372, with permission from MDIP. (C) Martín-de León, J., Bernardo, V. and
Rodríguez-Pérez, M. A. (2016). Low density nanocellular polymers based on PMMA produced by gas dissolution foaming: fabrication and cellular
structure characterization. Polymers 8(7), 265. doi:10.3390/polym8070265, with permission from MDIP.
Electrospinning
Highly porous scaffolds can be created via methods discussed in the earlier section, but those techniques are not sufficient to repro-
duce the nanofibrous microstructure of ECM in native tissues. Electrospinning has been adapted to fabricate fibrous tissue-
engineered scaffolds due to the favorable features of fibers such as large surface area, interconnectivity, adequate porosity, and
good mechanical stability. Synthetic, natural, and hybrid polymeric materials have been utilized to make fibrous scaffolds. A general
setup for electrospinning includes a syringe pump with flow control, a high-voltage power supplier, and a grounded collector. Poly-
mer fibers are drawn from a Taylor cone, formed at the droplet of the flowing polymer, under an electric field, and deposited on the
collector to form a fiber layer. The fiber layers can then be folded or glued together to form scaffolds. Various parameters such as
voltage, flow rate, and collector distance/structure of electrospinning can be tuned to obtain fibers with diameters from nanoscale to
microscale (Liu et al., 2017; Chen et al., 2015; Santos et al., 2013; Paskiabi et al., 2015; Kim and Kim, 2014; Sekiya et al., 2013; Qi
et al., 2016).
With the standard setting of electrospinning, random and aligned electrospun fibers can be produced (Fig. 3A). Alignment is an
attractive feature of scaffolds that facilitate regeneration of highly oriented tissues such as nerve and muscle (Kim et al., 2016b; Aviss
et al., 2010). Several strategies including electrostatic and mechanical methods have been used to govern the electrospun fiber align-
ment (spiral and linear, Fig. 3B and C), by which the collector is either placed in aligned electric field or spinning at a certain rate to
regulate alignment of electrospun fibers. Linear alignment of the fibers can be collected by the rotating reel setup, and the fiber size is
controllable by the speed of revolution (Chew et al., 2008). Radial alignment can be produced by a novel tethered pyroelectrody-
namic spinning system. This method is independent from high-voltage source and nozzle-free, which produces spiral fibrous scaf-
folds resembling the cochlear morphology (Coppola et al., 2014; Mecozzi et al., 2016). Aligned electrospun fibers (axial/radial) are
proved to regulate cell morphology, cell migration, and cellular differentiation both in vitroand in vivo, which can promote tissue
regeneration by directing the cell growth with desired orientation (Chew et al., 2008; Kim et al., 2008; Kutikov et al., 2015). For
instance, the aligned scaffold could guide the axonal growth where two damaged nerve ends need to be bridged. The neurite exten-
sion benefited from the alignment guidance of the scaffolds (Yao et al., 2009).
Fig. 3 (A) General electrospinning setup with syringe pump flow control, high-voltage power source, grounded fiber collector, and humidity control
box. (B–D) Different collector designs with their corresponding collected fiber organization.
3-D fibrous scaffolds mimicking native ECM can be achieved from electrospinning techniques by using a tubular spinning
collector or simply wrapping the electrospun scaffold in tubular shape. For example, PLGA–collagen–elastin tubular scaffolds
were synthesized for mimicking native arteries with enhanced physical strength and cell ingrowth (Stitzel et al., 2006). By stacking
electrospun fibrous scaffolds with different materials and different orientation of the fibers, complex structures can be constructed
through multiple different layers in one conduit. As an example, 3-D spiral scaffolds were achieved by simply wrapping an aligned
electrospun fibrous scaffold in the direction perpendicular to the fiber alignment (Kutikov et al., 2015). Such morphology mimics
the orientation of the cortical bone, which is favorable for fabrication of cortical bone replacement. The space between the fibers
inside the created spiral scaffold allows cell penetration and calcium deposition to realize reformation of the bone tissue (Kutikov
et al., 2015).
Bioprinting of 3-D Scaffolds

Existing industrial and commercial printing technologies have been adapted to a biological medium as a method to reliably fabri-
cate 3-D biological scaffolds. This process has proved to be much more efficient than the tedious growth and manipulation of cell
sheets but remains subject to the constraints and dependencies of existing printing technologies. These adapted printing methods
can produce either acellular or cellularized scaffolds, though certain methods are unable to accommodate the inclusion of cells by
nature of their fabrication process. For example, printers that utilize a sintering laser to melt or cure material may end up inducing
cell death through hyperthermia. Rapid prototyping methods follow the same principle as cell-sheet stacking, creating a scaffold
from the bottom up by fabricating one layer at a time, and do so with much greater precision and speed (Fig. 4A). Bioprinting
methods deposit a biological “ink” following the principle of additive manufacturing to generate a 3-D structure. This “bioink,”
as it has come to be known, is a mixture of biological and cellular components suspended within a delivery medium such as gelatin,
collagen, HA, alginate, or PEG, though any biocompatible material possessing viscosity suitable for the printing method may be
used (Nowicki et al., 2016). These 3-D bioprinting methods primarily differ in their composition of bioink and method of depo-
sition, though a simple extrusion method is beginning to emerge as standard practice. Among these broad categories, a few tech-
niques stand out for their potential in fabricating biological scaffolds.
Stereolithography
Stereolithography (SLA) uses a deflected or projected laser to cure and harden exposed areas of a photoreactive polymer at the
surface of a reservoir of material. 3-D constructs can be fabricated by curing successive layers of polymer, as illustrated by
Fig. 4 Demonstration of general 3-D printing workstation with controlled nozzle dispenser (A). (B) Stereolithography is based on photo-initiated
polymerization by shining a laser at desired location to polymerize locally to form features inside polymer precursor solution. (C) Solid free-form
fabrication (SFF) deposit polymers into desired structures. (D) Combined strategy of SFF with another nozzle depositing bioink protected cells to form
cell integrated scaffolds. The printed cellular scaffold is usually immersed in medium to sustain the viability of the cells.
Fig. 4B. This technology has previously been used industrially to create rapid prototypes and functional models, but theoretically,
any photopolymer of suitable viscosity can be used as fabrication material. Biodegradable polymers such as diethyl fumarate and
PPF, HAp, and even photocurable ceramic suspensions have been used to fabricate tissue engineering scaffolds for bone regener-
ation (Christenson et al., 2007; Cooke et al., 2003; Langton et al., 1997; Leukers et al., 2005). The limitations of SLA include
the geometric fidelity of the fabricated construct, which may be compromised by rehydration (Matsuda and Magoshi, 2002),
and its restriction to homogenous materials (Bártolo, 2011). Powder-fusion printing (PFP) is similar to SLA in resolution and prin-
ciple, though it utilizes a granular material to be bound together through laser sintering (Yang et al., 2002). This broadens the avail-
able fabrication material to include metals and plastics, but does not overcome the homogenous material hurdle and severely
hinders the ability to include biological materials during fabrication.
Solid free-form fabrication

Though SLA and PFP are suitable for the rapid fabrication of tissue engineering scaffolds, they are unsuitable for the fabrication of
biologically inclusive materials, requiring that cells and biological components be seeded onto the scaffolds post production. Solid
free-form fabrication (SFF) utilizes a precise Cartesian coordinate positioning system similar to those used to guide the sintering
lasers of SLA and PFP but directs the position of a nozzle to deposit material as depicted in Fig. 4C (Sears et al., 2016). In the
previous industrial applications, this material has been a polymer feedstock forced through a heated nozzle or drawn from a heated
reservoir, but this method can also print biologically relevant polymers to produce precision lattice structures (Zein et al., 2002). SFF
has been used to print hydrogels that utilized a semi-interpenetrating network of PEG and alginate with silicate nanoplatelets to give
the gel zero-shear viscosity above 10 kPa s, granting shape retention after printing and shear-thinning properties that allowed for
extrusion (Compton and Lewis, 2014; Hong et al., 2015). Due to the dependency upon material viscosity for the rate of flow
and subsequent deposition, the machine must be calibrated specifically to individual materials to allow for proper printing results
(Billiet et al., 2014; Fedorovich et al., 2008; Khalil et al., 2005).
Bioprinted scaffolds
The most common and affordable method, originating from conversions of commercially available polymer 3-D printers based off
of industrial designs, is microextrusion printing (Murphy and Atala, 2014). It is essentially a simplified, scaled down version of SFF,
often utilizing only a single deposition nozzle. This provides an advantage over inkjet and laser printing methods in that it can
deposit very high cell densities. Laser-assisted printing is capable of much higher resolution; however, it requires rapid gelation
kinetics to achieve high shape fidelity, which results in low overall flowrate and relatively long print times (Guillotin and Guillemot,
2011). A subset of extrusion printing known as fused deposition modeling (FDM) allows for the precise fabrication of graded
microstructures with controlled porosity and layer geometry, owing to its scalability and computer control software (Nowicki et al.,
2016). FDM can be expanded upon for even greater control through coaxial extrusion, which simply allows for multiple bioinks or
other material reservoirs to be simultaneously deposited through the same nozzle (Jia et al., 2016). This allows for rapid and
controlled layering of different materials for the manipulation of micro and bulk structural properties or even integrated blending
or encapsulation of materials with distinct properties. As shown in Fig. 4D, this allows cells to be embedded into the scaffold during
fabrication. Coaxial extrusion has allowed for the direct 3-D bioprinting of perfusable vascular constructs and endothelialized
myocardium (Zhang et al., 2016a). SFF may also be combined with existing scaffold fabrication procedures to aid in the production
of specialized devices. Ainola et al. utilized SFF to produce a microfibrous scaffold encapsulated by an electrospun nanofibrous
mesh to create a 3-D scaffold for cartilage repair that would confine MSCs and chondrocytes to the damaged site and impede
the ingrowth of fibroblasts to prevent fibrous scarring (Ainola et al., 2015). Zhang et al. (2016b) developed an in-house SFF 3-
D printing method to fabricate a magnetothermal bioceramic scaffold for simultaneous hyperthermal anticancer therapy and
bone regeneration. The scaffold could be raised to 42 C through the application of a magnetic field therapy that successfully
reduced the viability of local osteosarcoma cells without compromising the regenerative promotion of the scaffold during
in vitro testing. Together, those studies demonstrated the potential of 3-D printing in biomanufacturing tissue-engineered scaffolds
with high controllability.
Applications of Scaffolds in Tissue Engineering

Introduction
Applications investigated in this section primarily look to replace the customary practice of autografts. In neural, skin, bone, and
cardiovascular tissue autografts are used frequently to repair large injuries. The major downfall of autografts is the requirement
for a second surgical site where a donor tissue will be harvested. This donor site is prone to morbidity and infection. Moreover,
autografts are not always accessible for all patients such as those who have undergone multiple surgeries or the elderly who do
not have viable tissue to use. Thus, engineered tissues are critically needed to replace autografts.
Spinal Cord/Neuron Repair

In peripheral neurons, Schwann cells proliferate and align to form bands of Büngner that guide sprouting axonal growths, though
the chance of a functional regeneration decreases with the severity of the injury and distance of the resulting gap. Furthermore,
injuries to peripheral nerves typically involve scarring of connective tissues, resulting in aberrant sprouts believed to be responsible
for spontaneous neuropathic pain symptoms (Deumens et al., 2010). Nerve gaps greater than 2 cm require surgical intervention.
The current gold standard of treatment is an autologous nerve graft, but results are limited since the tension generated by suturing
nerve segments larger than a few millimeters severely impedes axon regeneration. Limited supply of donor nerves and the require-
ment of a second surgery site that is prone to infection and morbidity are additional limiting factors (Deumens et al., 2010; Jenkins
et al., 2015; Gu et al., 2014). Bioengineered scaffolds offer an alternative intervention able to integrate the multiple mechanisms
involved in successful and functional neuron repair.
An ideal neural engineered scaffold would incorporate neuroprotective and neuroregenerative drugs (Kabu et al., 2015); biomi-
metic mechanical properties (Yao et al., 2016); topographical and physical guidance cues (Mobasseri et al., 2015); factors for the
recruitment, differentiation, and growth of stem cells to avoid scar formation (Ajioka et al., 2014); angiogenic factors for sustainable
growth and effective integration (Álvarez et al., 2014); and functional priming via electric stimulation (Akhavan et al., 2016).
Current neural scaffolds focus primarily on providing physical guidance to developing axons through the use of channels or
conduits fabricated from polymers or biologically derived materials, often placing emphasis on the surface patterning to promote
alignment (Pawar et al., 2015; Archibald et al., 1991; Zhu et al., 2015; Vaysse et al., 2015). More recently, interest has been drawn to
the integration of neural scaffolds with neural stem cells (NSCs) (Sharifi et al., 2016). Studies have shown that scaffolds constructed
from ECM derived from central nervous system (CNS) tissues demonstrated the ability to differentiate NSCs into neurons while
ECM derived from non-CNS tissues did not (Crapo et al., 2013; Duan et al., 2016). Proper differentiation of stem cells, either seeded
on the scaffold or recruited endogenously, will speed regeneration and reduce the risk of glial scarring.
Wound Healing
Wound healing involves a set of interrelated physiological events including inflammation, proliferation, and remodeling (Stadel-
mann et al., 1998). However, pathological conditions such as diabetes mellitus can disrupt the wound healing process and result in
chronic wounds (Blakytny and Jude, 2006). Tissue-engineered skin grafting scaffolds have been included to treat chronic cutaneous
wounds. The optimal scaffold should be nonimmunogenic and biodegradable while incorporating, or being able to recruit, cells
and growth factors that can synthesize granulation tissue, allow for reepithelialization, and absorb harmful free radicals that
promote inflammation. Furthermore, the scaffold must be able to facilitate the contraction of the wound and reduce scar tissue
formation (Zhong et al., 2010).
Several skin substitutes have already been approved by FDA to treat diabetic foot ulcers and venous leg ulcers (Hu et al., 2006;
Marston et al., 2003; Landsman et al., 2011). These skin substitutes are constructed with matrix scaffolds seeded with fibroblasts
and/or keratinocytes to resemble the contents of the skin. For example, Demagraft is a cryopreserved fibroblast-derived skin substi-
tute fabricated by seeding fibroblasts on bioabsorbable PGA mesh to constantly provide growth factors and matrix proteins that
promote diabetic wound healing (Naughton et al., 1997). Unfortunately, the average time to fabricate these skin substitutes by har-
vesting the patients’ own cells is long, and the transportation of living cell products is difficult (Zelen et al., 2016). These drawbacks
inspired the development of acellular skin grafts from synthetic and natural polymers such as PLGA, collagen, alginate, and chito-
san, to avoid the complications of live-cell products (Zong et al., 2003; Singer and Clark, 1999; Mayol et al., 2014; O’Meara et al.,
2015). Those scaffolds are helpful in assisting wound closure. Many growth factors play a role in this process, notably vascular endo-
thelial growth factor and basic fibroblast growth factor, which are known to induce cell proliferation and angiogenesis. Embedding
these growth factions into scaffolds offers the opportunity to further accelerate the wound healing process in vivo by allowing faster
penetration of inflammatory cells and promoting mesenchymal cell recruitment (Breen et al., 2008; Losi et al., 2013; Sun et al.,
2014). The incorporation of stem cells directly into scaffolds is another promising method that offers abundant growth factors while
reducing scarring. An MSC sheet with built-in vascular networks has been created and was shown to accelerate wound healing,
modulate inflammation, and reduce scarring through the therapeutic functions of MSCs and vascularization (Chen et al., 2017).
Together, these studies have demonstrated the clinical potential of tissue-engineered scaffolds in promoting wound healing. The
in vivo diabetic models have confirmed that those therapeutic scaffolds are able to heal both diabetic and nondiabetic chronic
wounds (Dash et al., 2013; Chereddy et al., 2015).
Bone Regeneration
The ideal bone tissue engineering scaffold should not only meet the required mechanical properties but also have osteoconductive
(stimulate bone cells), osteoinductive (stimulate undifferentiated cells), and osteogenic (stimulate both mechanisms) potentials
(Albrektsson and Johansson, 2001; Giannoudis et al., 2011; Khan et al., 2008; Yi et al., 2016; Srouji et al., 2006; El-Ghannam,
2005). Since bone tissue is majorly constructed by collagen I and HAp, a variety of bone tissue-engineered scaffolds have been
designed based on the variation of these two materials (Polo-Corrales et al., 2014; Aravamudhan et al., 2013; Schneider et al.,
2010; Oh et al., 2011; Niu et al., 2012). While collagen serves as a continuous organic template, CaP, as inorganic constituents,
not only strengthen the scaffold but also provide osteoconductivity to the scaffold. Collagen promotes osteoblast adhesion and
calcium deposition, which is critical for bone tissue formation and regeneration (Thitiset et al., 2013). Through mineralization
and cross-linking, collagen as a scaffold can successfully approach the mechanical strength of cancellous bone that is critical for
repairing load-bearing bones (Dhand et al., 2016). HAp or other CaP ceramics are chemically and mechanically compatible
with the bones (Kalita et al., 2007; Lc, 2009; Panseri et al., 2012). To promote osteoinductivity, highly porous CaP ceramics
have been fabricated to encourage cell integration. Meanwhile, the CaP materials can release Ca2 þ ions to create a suitable micro-
environment to promote osteogenic differentiation and mineral deposition (Rahaman et al., 2011). Researchers have investigated
the effects of incorporating different cell types and growth factors into the scaffolds to stimulate osteogenesis. For example, incor-
poration of osteoblasts differentiated from MSCs can enhance the mechanical properties of engineered bone-like tissue (Naito et al.,
2011). Beside osteogenic properties, a good bone scaffold should promote angiogenesis to form and maintain a healthy bone
during regeneration (He et al., 2013).
In summary, to mimic the bone tissue microenvironment and promote regeneration, mechanically qualified scaffolds with
incorporated growth factors are needed to promote both osteogenesis and angiogenesis. Most scaffolds fulfilling the criteria are still
at proof-of-concept stage, and in vivo testing is on demand in order to apply them clinically.
Vascular Scaffolds
Cardiovascular disease remains the leading cause of death worldwide (Pashneh-Tala et al., 2015). Modern treatments such as
stents and angioplasty only provide temporary relief to occluded blood vessels (Alfonso et al., 2003; Moussavian et al., 2001).
Autografts may suffer from a mismatch of size, vessel structure, or mechanics that limit their suitability as a replacement and carry
the complications of donor-site morbidity associated with a second surgery. In some cases, such as in systemic vascular diseases
like atherosclerosis, autografts may already be just as unsuitable as the vessel they are intended to replace. Synthetic vascular grafts
have shown to be at least as effective as autografts in clinical studies; however, they must be paired with anticoagulants, such as
a surface conjugation of heparin, to avoid thrombus and occlusion (Devine et al., 2004; Begovac et al., 2003; Pulli et al., 2010;
Gutowska et al., 1995). This makes synthetic materials unsuitable for replacing small-diameter blood vessels, such as those
used to bypass coronary blockages, where even a small degree of thrombus or hyperplasia can completely occlude the lumen (Seifu
et al., 2013).
For this reason, research has shifted toward facilitating integration of the vascular graft with the native tissues of the host. The
components of the ECM are generally conserved across species, and so, blood vessels harvested from animal sources can be decel-
lularized to remove the immune-response-triggering cells and genetic material, leaving a scaffold that very closely mimics the native
host tissue and provides an ideal environment for vascular reconstruction (Gilbert et al., 2006). However, decellularization proce-
dures are still being refined, and the effects on bulk tissues have not been rigorously investigated, but it has been shown that
different tissues respond differently to the various steps of any given decellularization protocol (Badylak and Gilbert, 2008).
Each decellularization procedure must be custom-tailored to the tissue at hand. A procedure too intense may alter the ECM structure
or organization resulting in a change to its natural function via alteration of the physical or chemical interaction with cells, or it may
leave behind traces of cytotoxic detergents that impede cellular regrowth and host integration. If the procedure is too weak, it may
leave behind foreign cells or genetic materials capable of provoking a host response that could in turn potentially compromise the
entire graft.
Decellularization procedures can also be used to extract ECM from cultured cells, to establish a more reproducible tissue source,
and have demonstrated the ability to integrate with the native host tissue and even remodel and grow with native blood vessels in
animal studies (Syedain et al., 2016).
However, decellularized ECM is only a scaffold and still requires an appropriate blood-contacting surface that can avoid forma-
tion of thrombus but still promote native cell ingrowth. ECM components like elastin have shown to be effective blood-contacting
surfaces for maintaining hemocompatibility and regulating cellular regrowth but are structurally weak on their own (Yokoyama
et al., 2017). These blood-contacting surfaces may be reinforced with synthetic materials for mechanical strength (McCarthy
et al., 2015) or fabricated with controlled architecture to impart more durable mechanics (Xing et al., 2017). The ultimate goal,
however, is to replace the diseased blood vessel with as close an approximation to its natural state as possible. For this, biodegrad-
able and naturally derived materials show the best potential (Wu et al., 2012).
Summary and Future Directions
The development of tissue-engineered scaffolds has been dramatically accelerated in the last decade. Novel technologies now allow
the potential to regenerate most tissues. In this article, new materials were reviewed for creating special scaffolds with unique appli-
cations. More prospective composites are being investigated to combine both natural and synthetic materials to achieve a balance
between mechanical properties and bioactivities. Emerging technologies such as electrospinning, particulate leaching/phase sepa-
ration, tissue decellularization, and 3-D printing have been explained in detail.
It is generally accepted that the scaffolds need to be vascularized, or otherwise be able to promote vascularization, to further
foster tissue regeneration. While more functional scaffolds are being created, a lot of studies have shifted toward developing vascu-
larization strategies for engineered scaffolds. Scaffold vascularization could be realized by coculturing endothelial cells and mural
cells to form vasculatures inside the scaffold prior to implantation. Other vascularization strategies including creating microchan-
nels, embedding angiogenic growth factors, and assembling endothelialized tissue constructs are being extensively studied to
improve the survival and functions of implanted constructs (Thein-Han and Xu, 2013; Chen et al., 2014; Baldwin et al., 2014;
Hasan et al., 2014; Kolesky et al., 2014; Laschke and Menger, 2016). 3-D printing of vascular channels inside scaffolds has been
proved to support cell metabolic functions within the constructs (Miller et al., 2012).
With the development of 3-D bioprinting technology, creating large scaffolds for whole-organ replacement may become
possible. However, the challenges of printing an organ-size scaffold that resembles the complex architecture, mimics the mechanical
properties, and restores the function of target organ remain unsolved. To overcome the challenges, highly computer-aided printing
system with microsize printing nozzles have been used to recreate the anatomical architecture and mechanical strength of the
femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains (Hinton et al., 2015). Another challenge
of bioprinting large scaffolds is to sustain the already-printed structures while the whole construct is printing. A sacrificial polymer
framework can be printed during the process of scaffold printing. It can then be removed after the printed scaffold is reinforced
(Kang et al., 2016). Last but not the least, large organ-size scaffolds have the problem of reseeding cells into the core of the scaffold.
Bioreactor system with perfusion functions can help aid cell penetration, but incorporating cells during the printing process might
be a better solution, which strongly relies on a superior design of bioink to sustain and protect the cells while printing (Adam et al.,
2016; Ott et al., 2008; Hinton et al., 2015). The next generation of tissue-engineered scaffolds of organ size will be based on the 3-D
bioprinting technology that enables the clinical possibility of using biomimetic synthetic organs to fulfill the transplantation
demands.
References
Adam, E. J., Alexandra, L. R., & Ramille, N. S. (2016). Advancing the field of 3D biomaterial printing. Biomedical Materials, 11(1), 014102.
Ainola, M., et al. (2015). A bioactive hybrid three-dimensional tissue-engineering construct for cartilage repair. Journal of Biomaterials Applications, 30(6), 873–885.
Ajioka, I., et al. (2014). Enhancement of neuroblast migration into the injured cerebral cortex using laminin-containing porous sponge. Tissue Engineering. Part A, 21(1–2),
193–201.
Akhavan, O., et al. (2016). Rolled graphene oxide foams as three-dimensional scaffolds for growth of neural fibers using electrical stimulation of stem cells. Carbon, 97, 71–77.
Albrektsson, T., & Johansson, C. (2001). Osteoinduction, osteoconduction and osseointegration. European Spine Journal, 10(Suppl. 2), S96–S101.
Alfonso, F., et al. (2003). A randomized comparison ofrepeat stenting with balloon angioplasty in patients with in-stent restenosis. Journal of the American College of Cardiology,
42(5), 796–805.
Allison, D. D., & Grande-Allen, K. J. (2006). Review. Hyaluronan: a powerful tissue engineering tool. Tissue Engineering, 12(8), 2131–2140.
Álvarez, Z., et al. (2014). Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold. Biomaterials, 35(17), 4769–4781.
Annabi, N., et al. (2009). Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro. Biomaterials, 30(27), 4550–4557.
Annabi, N., et al. (2010). Controlling the porosity and microarchitecture of hydrogels for tissue engineering. Tissue Engineering. Part B, Reviews, 16(4), 371–383.
Aravamudhan, A., et al. (2013). Cellulose and collagen derived micro-nano structured scaffolds for bone tissue engineering. Journal of Biomedical Nanotechnology, 9(4), 719–731.
Archibald, S. J., et al. (1991). A collagen-based nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman
primates. Journal of Comparative Neurology, 306(4), 685–696.
Armentano, I., et al. (2010). Biodegradable polymer matrix nanocomposites for tissue engineering: a review. Polymer Degradation and Stability, 95(11), 2126–2146.
Aviss, K. J., Gough, J. E., & Downes, S. (2010). Aligned electrospun polymer fibres for skeletal muscle regeneration. European Cells & Materials, 19, 193–204.
Badylak, S. F., & Gilbert, T. W. (2008). Immune response to biologic scaffold materials. Seminars in Immunology, 20(2), 109–116.
Baldwin, J., et al. (2014). In vitro pre-vascularisation of tissue-engineered constructs a co-culture perspective. Vascular Cell, 6, 13.
Barsotti, M. C., et al. (2011). Fibrin as a scaffold for cardiac tissue engineering. Biotechnology and Applied Biochemistry, 58(5), 301–310.
Bártolo, P. J. (2011). Stereolithographic processes. In Stereolithography (pp. 1–36). Berlin: Springer.
Begovac, P., et al. (2003). Improvements in GORE-TEX® vascular graft performance by Carmeda® bioactive surface heparin immobilization. European Journal of Vascular and
Endovascular Surgery, 25(5), 432–437.
Bernard, M. P., et al. (1983a). Nucleotide sequences of complementary deoxyribonucleic acids for the pro alpha 1 chain of human type I procollagen. Statistical evaluation of
structures that are conserved during evolution. Biochemistry, 22(22), 5213–5223.
Bernard, M. P., et al. (1983b). Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2 (I) identifies structurally
conserved features of the protein and the gene. Biochemistry, 22(5), 1139–1145.
Billiet, T., et al. (2014). The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials, 35(1), 49–62.
Blakytny, R., & Jude, E. (2006). The molecular biology of chronic wounds and delayed healing in diabetes. Diabetic Medicine, 23(6), 594–608.
Boccafoschi, F., et al. (2005). Biological performances of collagen-based scaffolds for vascular tissue engineering. Biomaterials, 26(35), 7410–7417.
Boeriu, C. G., et al. (2013). Production methods for Hyaluronan. International Journal of Carbohydrate Chemistry, 2013, 14.
Boland, E. D., et al. (2004). Electrospinning collagen and elastin: preliminary vascular tissue engineering. Frontiers in Bioscience, 9(1422), e32.
Bozkurt, A., et al. (2007). In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix. Tissue Engineering, 13(12),
2971–2979.
Bracaglia, L. G., & Fisher, J. P. (2015). Extracellular matrix-based biohybrid materials for engineering compliant, matrix-dense tissues. Advanced Healthcare Materials, 4(16),
2475–2487.
Breen, A. M., et al. (2008). The use of therapeutic gene eNOS delivered via a fibrin scaffold enhances wound healing in a compromised wound model. Biomaterials, 29(21),
3143–3151.

Capek, J., et al. (2016). Highly porous, low elastic modulus 316L stainless steel scaffold prepared by selective laser melting. Materials Science and Engineering: C, 69, 631–639.
Cen, L., et al. (2004). Assessment of in vitro bioactivity of hyaluronic acid and sulfated hyaluronic acid functionalized electroactive polymer. Biomacromolecules, 5(6), 2238–2246.
Cen, L., et al. (2008). Collagen tissue engineering: development of novel biomaterials and applications. Pediatric Research, 63(5), 492–496.
Chan, B. P., & Leong, K. W. (2008). Scaffolding in tissue engineering: general approaches and tissue-specific considerations. European Spine Journal, 17(Suppl. 4), 467–479.
Chen, W., et al. (2014). Prevascularization of biofunctional calcium phosphate cement for dental and craniofacial repairs. Dental Materials, 30(5), 535–544.
Chen, Y., et al. (2015). Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells
through Wnt/b-catenin signaling. BMC Cell Biology, 16, 22.
Chen, L., et al. (2017). Pre-vascularization enhances therapeutic effects of human mesenchymal stem cell sheets in full thickness skin wound repair. Theranostics, 7(1), 117–131.
Chereddy, K. K., et al. (2015). Combined effects of PLGA and vascular endothelial growth factor promote the healing of non-diabetic and diabetic wounds. Nanomedicine:
Nanotechnology, Biology and Medicine, 11(8), 1975–1984.
Chew, S. Y., et al. (2008). The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation. Biomaterials, 29(6), 653–661.
Christenson, E. M., et al. (2007). Biodegradable fumarate-based polyHIPEs as tissue engineering scaffolds. Biomacromolecules, 8(12), 3806–3814.
Chróscicka, A., et al. (2016). Synthetic calcite as a scaffold for osteoinductive bone substitutes. Annals of Biomedical Engineering, 44, 2145–2157.
Columbus, S., Krishnan, L. K., & Kalliyana Krishnan, V. (2014). Relating pore size variation of poly (3-caprolactone) scaffolds to molecular weight of porogen and evaluation of
scaffold properties after degradation. Journal of Biomedical Materials Research. Part B: Applied Biomaterials, 102(4), 789–796.
Compton, B. G., & Lewis, J. A. (2014). 3D-printing of lightweight cellular composites. Advanced Materials, 26(34), 5930–5935.
Constantinou, C. D., & Jimenez, S. A. (1991). Structure of cDNAs encoding the triple-helical domain of murine a2 (VI) collagen chain and comparison to human and Chick
homologues. Use of polymerase chain reaction and partially degenerate oligonucleotides for generation of novel cDNA clones. Matrix, 11(1), 1–9.
Cooke, M. N., et al. (2003). Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth. Journal of Biomedical Materials Research. Part B:
Applied Biomaterials, 64(2), 65–69.
Coppola, S., et al. (2014). Tethered pyro-Electrohydrodynamic spinning for patterning well-ordered structures at micro- and nanoscale. Chemistry of Materials, 26(11), 3357–3360.
Crapo, P. M., et al. (2013). Effects of biologic scaffolds on human stem cells and implications for CNS tissue engineering. Tissue Engineering. Part A, 20(1–2), 313–323.
Dai, W., et al. (2010). The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering. Biomaterials, 31(8), 2141–2152.
Dash, S. N., et al. (2013). Towards reaching the target: clinical application of mesenchymal stem cells for diabetic foot ulcers. Rejuvenation Research, 17(1), 40–53.
Davidenko, N., et al. (2012). Biomimetic collagen scaffolds with anisotropic pore architecture. Acta Biomaterialia, 8(2), 667–676.
De Franceschi, L., et al. (2005). Transplantation of chondrocytes seeded on collagen-based scaffold in cartilage defects in rabbits. Journal of Biomedical Materials Research. Part A,
75(3), 612–622.
Denry, I., & Holloway, J. A. (2014). Low temperature sintering of fluorapatite glass-ceramics. Dental Materials: Official Publication of the Academy of Dental Materials, 30(2),
112–121.
Deumens, R., et al. (2010). Repairing injured peripheral nerves: bridging the gap. Progress in Neurobiology, 92(3), 245–276.
Devine, C., McCollum, C., & Trial, T. N. W. F.-P. (2004). Heparin-bonded Dacron or polytetrafluorethylene for femoropopliteal bypass: five-year results of a prospective randomized
multicenter clinical trial. Journal of Vascular Surgery, 40(5), 924–931.
Dhand, C., et al. (2016). Bio-inspired in situ crosslinking and mineralization of electrospun collagen scaffolds for bone tissue engineering. Biomaterials, 104, 323–338.
Di Martino, A., Sittinger, M., & Risbud, M. V. (2005). Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials, 26(30), 5983–5990.
Draghi, L., et al. (2005). Microspheres leaching for scaffold porosity control. Journal of Materials Science: Materials in Medicine, 16(12), 1093–1097.
Duan, H., et al. (2016). Functional hyaluronate collagen scaffolds induce NSCs differentiation into functional neurons in repairing the traumatic brain injury. Acta Biomaterialia, 45,
182–195.
Ehrbar, M., et al. (2005). Endothelial cell proliferation and progenitor maturation by fibrin-bound VEGF variants with differential susceptibilities to local cellular activity. Journal of
Controlled Release, 101(1), 93–109.
El-Ghannam, A. (2005). Bone reconstruction: from bioceramics to tissue engineering. Expert Review of Medical Devices, 2(1), 87–101.
Evanko, S. P., Angello, J. C., & Wight, T. N. (1999). Formation of hyaluronan-and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth
muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology, 19(4), 1004–1013.
Exposito, J., et al. (1992). Sea urchin collagen evolutionarily homologous to vertebrate pro-alpha 2 (I) collagen. Journal of Biological Chemistry, 267(22), 15559–15562.
Fakhari, A., & Berkland, C. (2013). Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomaterialia,
9(7), 7081–7092.
Farraro, K. F., et al. (2014). Revolutionizing orthopaedic biomaterials: the potential of biodegradable and bioresorbable magnesium-based materials for functional tissue engineering.
Journal of Biomechanics, 47(9), 1979–1986.
Fedorovich, N. E., et al. (2008). Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. Tissue Engineering. Part A, 14(1), 127–133.
Gamboa-Martínez, T. C., Ribelles, J. G., & Ferrer, G. G. (2011). Fibrin coating on poly (L-lactide) scaffolds for tissue engineering. Journal of Bioactive and Compatible Polymers,
26(5), 464–477.
Gautam, S., et al. (2014). Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering. Materials Science
and Engineering: C, 34, 402–409.
Giannoudis, P. V., Jones, E., & Einhorn, T. A. (2011). Fracture healing and bone repair. Injury, 42(6), 549–550.
Gilbert, T. W., Sellaro, T. L., & Badylak, S. F. (2006). Decellularization of tissues and organs. Biomaterials, 27(19), 3675–3683.
Glowacki, J., & Mizuno, S. (2008). Collagen scaffolds for tissue engineering. Biopolymers, 89(5), 338–344.
Goldstein, A. S., et al. (2001). Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials, 22(11), 1279–1288.
Graney, P. L., et al. (2016). In vitro response of macrophages to ceramic scaffolds used for bone regeneration. Journal of the Royal Society Interface, 13(120).
Grassl, E., Oegema, T., & Tranquillo, R. (2002). Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent. Journal of Biomedical Materials
Research, 60(4), 607–612.
Grauss, R. W., et al. (2005). Histological evaluation of decellularised porcine aortic valves: matrix changes due to different decellularisation methods. European Journal of Cardio-
Thoracic Surgery, 27(4), 566–571.
Grayson, W., Ma, T., & Bunnell, B. (2004). Human mesenchymal stem cells tissue development in 3D PET matrices. Biotechnology Progress, 20(3), 905–912.
Gu, X., Ding, F., & Williams, D. F. (2014). Neural tissue engineering options for peripheral nerve regeneration. Biomaterials, 35(24), 6143–6156.
Guillotin, B., & Guillemot, F. (2011). Cell patterning technologies for organotypic tissue fabrication. Trends in Biotechnology, 29(4), 183–190.
Gutowska, A., et al. (1995). Heparin release from thermosensitive polymer coatings: in vivo studies. Journal of Biomedical Materials Research. Part A, 29(7), 811–821.
Habraken, W. J., Wolke, J. G., & Jansen, J. A. (2007). Ceramic composites as matrices and scaffolds for drug delivery in tissue engineering. Advanced Drug Delivery Reviews,
59(4–5), 234–248.
Hao, T., et al. (2010). The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels. Osteoarthritis and Cartilage,
18(2), 257–265.
Hasan, A., et al. (2014). Microfluidic techniques for development of 3D vascularized tissue. Biomaterials, 35(26), 7308–7325.
Haugh, M. G., Murphy, C. M., & O’Brien, F. J. (2009). Novel freeze-drying methods to produce a range of collagen–glycosaminoglycan scaffolds with tailored mean pore sizes.
Tissue Engineering. Part C, Methods, 16(5), 887–894.
He, X., et al. (2013). Integration of a novel injectable nano calcium sulfate/alginate scaffold and BMP2 gene-modified mesenchymal stem cells for bone regeneration. Tissue
Engineering. Part A, 19(3–4), 508–518.
Heino, J. (2000). The collagen receptor integrins have distinct ligand recognition and signaling functions. Matrix Biology, 19(4), 319–323.
Heydarkhan-Hagvall, S., et al. (2008). Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials, 29(19), 2907–2914.
Hinton, T. J., et al. (2015). Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Science Advances, 1(9),
e1500758.
Hoemann, C., et al. (2005). Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. Osteoarthritis and Cartilage, 13(4), 318–329.
Hollister, S. J. (2005). Porous scaffold design for tissue engineering. Nature Materials, 4(7), 518–524.
Hong, H., et al. (2009). Fabrication of a novel hybrid scaffold for tissue engineered heart valve. Journal of Huazhong University of Science and Technology. Medical Sciences, 29,
599–603.
Hong, S., et al. (2015). 3D printing of highly stretchable and tough hydrogels into complex, cellularized structures. Advanced Materials, 27(27), 4035–4040.
Hu, S., et al. (2006). Evaluation of Apligraf® persistence and basement membrane restoration in donor site wounds: a pilot study. Wound Repair and Regeneration, 14(4), 427–433.
Hutmacher, D. W. (2001). Scaffold design and fabrication technologies for engineering tissuesdstate of the art and future perspectives. Journal of Biomaterials Science. Polymer
Edition, 12(1), 107–124.
Jenkins, P. M., et al. (2015). A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells. Nanoscale Research Letters,
10(1), 264.
Jia, W., et al. (2016). Direct 3D bioprinting of perfusable vascular constructs using a blend bioink. Biomaterials, 106, 58–68.
Johnson, P. J., Parker, S. R., & Sakiyama-Elbert, S. E. (2009). Controlled release of neurotrophin-3 from fibrin-based tissue engineering scaffolds enhances neural fiber sprouting
following subacute spinal cord injury. Biotechnology and Bioengineering, 104(6), 1207–1214.
Jung, H.-D., et al. (2015). Fabrication of mechanically tunable and bioactive metal scaffolds for biomedical applications. JoVE (Journal of Visualized Experiments), 106, e53279.
Kabu, S., et al. (2015). Drug delivery, cell-based therapies, and tissue engineering approaches for spinal cord injury. Journal of Controlled Release, 219, 141–154.
Kalia, S., et al. (2011). Cellulose-based bio- and nanocomposites: a review. International Journal of Polymer Science, 2011.
Kalita, S. J., Bhardwaj, A., & Bhatt, H. A. (2007). Nanocrystalline calcium phosphate ceramics in biomedical engineering. Materials Science and Engineering: C, 27(3), 441–449.
Kanatani, I., et al. (2007). Fabrication of an optimal urethral graft using collagen-sponge tubes reinforced with copoly (L-lactide/ε-caprolactone) fabric. Tissue Engineering, 13(12),
2933–2940.
Kang, H.-W., et al. (2016). A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nature Biotechnology, 34(3), 312–319.
Khalil, S., Nam, J., & Sun, W. (2005). Multi-nozzle deposition for construction of 3D biopolymer tissue scaffolds. Rapid Prototyping Journal, 11(1), 9–17.
Khan, Y., et al. (2008). Tissue engineering of bone: material and matrix considerations. JBJS, 90(Suppl. 1), 36–42.
Kim, M. S., & Kim, G. (2014). Three-dimensional electrospun polycaprolactone (PCL)/alginate hybrid composite scaffolds. Carbohydrate Polymers, 114, 213–221.
Kim, Y. T., et al. (2008). The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials, 29(21), 3117–3127.
Kim, J.-A., et al. (2016a). Effect of the biodegradation rate controlled by pore structures in magnesium phosphate ceramic scaffolds on bone tissue regeneration in vivo. Acta
Kim, J. I., et al. (2016b). A controlled design of aligned and random nanofibers for 3D bi-functionalized nerve conduits fabricated via a novel electrospinning set-up. Scientific
Reports, 6, 23761.
Kolesky, D. B., et al. (2014). 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Advanced Materials, 26(19), 3124–3130.
Kutikov, A. B., et al. (2015). Templated repair of long bone defects in rats with bioactive spiral-wrapped electrospun amphiphilic polymer/hydroxyapatite scaffolds. ACS Applied
Materials & Interfaces, 7(8), 4890–4901.
Lam, C. X., et al. (2009). Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo. Journal of Biomedical Materials Research Part A, 90(3), 906–919.
Landsman, A. S., et al. (2011). A retrospective clinical study of 188 consecutive patients to examine the effectiveness of a biologically active cryopreserved human skin allograft
(TheraSkin ®) on the treatment of diabetic foot ulcers and venous leg ulcers. Foot & Ankle Specialist, 4(1), 29–41.
Langton, C., et al. (1997). Development of a cancellous bone structural model by stereolithography for ultrasound characterisation of the calcaneus. Medical Engineering & Physics,
19(7), 599–604.
Laschke, M. W., & Menger, M. D. (2016). Prevascularization in tissue engineering: current concepts and future directions. Biotechnology Advances, 34(2), 112–121.
Lc, C. (2009). Next generation calcium phosphate-based biomaterials. Dental Materials Journal, 28(1), 1–10.
LeGeros, R. Z. (2002). Properties of osteoconductive biomaterials: calcium phosphates. Clinical Orthopaedics and Related Research, 395, 81–98.
Leukers, B., et al. (2005). Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing. Journal of Materials Science: Materials in Medicine, 16(12), 1121–1124.
Li, Z., et al. (2005a). Chitosan–alginate hybrid scaffolds for bone tissue engineering. Biomaterials, 26(18), 3919–3928.
Li, W.-J., et al. (2005b). A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials, 26(6), 599–609.
Liao, J., Joyce, E. M., & Sacks, M. S. (2008). Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet. Biomaterials, 29(8),
1065–1074.
Lietaert, K., et al. (2013). Open cellular magnesium alloys for biodegradable orthopaedic implants. Journal of Magnesium and Alloys, 1(4), 303–311.
Liu, X., et al. (2017). Electrospinnability of poly lactic-co-glycolic acid (PLGA): the role of solvent type and solvent composition. Pharmaceutical Research, 1–12.
Losi, P., et al. (2013). Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice. Acta Biomaterialia, 9(8), 7814–7821.
Lu, D., Xiao, C., & Xu, S. (2009). Starch-based completely biodegradable polymer materials. Express Polymer Letters, 3(6), 366–375.
Lutolf, M. P., & Hubbell, J. A. (2005). Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnology, 23(1),
47–55.
Ma, L., et al. (2003). Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials, 24(26), 4833–4841.
Mano, J. F., et al. (2007). Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. Journal of the Royal
Society Interface, 4(17), 999–1030.
Marston, W. A., et al. (2003). The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers. Results of a prospective randomized trial. Diabetes Care,
26(6), 1701–1705.
Masoumi, N., et al. (2013). Valvular interstitial cell seeded poly (glycerol sebacate) scaffolds: toward a biomimetic in vitro model for heart valve tissue engineering. Acta Biomaterialia,
9(4), 5974–5988.
Mathews, S., et al. (2011). Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the
associated genes. Cell Proliferation, 44(6), 537–549.
Matsuba, G., et al. (2003). Kinetics of phase separation and crystallization in poly(ethylene-ran-hexene) and poly(ethylene-ran-octene). Polymer, 44(24), 7459–7465.
Matsuda, T., & Magoshi, T. (2002). Preparation of vinylated polysaccharides and photofabrication of tubular scaffolds as potential use in tissue engineering. Biomacromolecules,
3(5), 942–950.
Mayol, L., et al. (2014). Design and characterization of a chitosan physical gel promoting wound healing in mice. Journal of Materials Science: Materials in Medicine, 25(6),
1483–1493.
McCarthy, C. W., et al. (2015). Fabrication and short-term in vivo performance of a natural elastic lamina–polymeric hybrid vascular graft. ACS Applied Materials & Interfaces, 7(30),
16202–16212.
Mecozzi, L., et al. (2016). Spiral formation at the microscale by [small mu]-pyro-electrospinning. Soft Matter, 12(25), 5542–5550.
Mendoza-Novelo, B., et al. (2011). Decellularization of pericardial tissue and its impact on tensile viscoelasticity and glycosaminoglycan content. Acta Biomaterialia, 7(3),
1241–1248.
Miller, J. S., et al. (2012). Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nature Materials, 11(9), 768–774.
Minuth, W. W., Sittinger, M., & Kloth, S. (1997). Tissue engineering: generation of differentiated artificial tissues for biomedical applications. Cell and Tissue Research, 291(1),
1–11.
Mobasseri, A., et al. (2015). Polymer scaffolds with preferential parallel grooves enhance nerve regeneration. Tissue Engineering. Part A, 21(5–6), 1152–1162.
Mol, A., et al. (2005). Fibrin as a cell carrier in cardiovascular tissue engineering applications. Biomaterials, 26(16), 3113–3121.
Mosesson, M. (2005). Fibrinogen and fibrin structure and functions. Journal of Thrombosis and Haemostasis, 3(8), 1894–1904.
Motlagh, D., et al. (2006). Hemocompatibility evaluation of poly (glycerol-sebacate) in vitro for vascular tissue engineering. Biomaterials, 27(24), 4315–4324.
Moussavian, M., Casterella, P. J., & Teirstein, P. S. (2001). Restenosis after angioplasty. Current Treatment Options in Cardiovascular Medicine, 3(2), 103–113.
Murphy, S. V., & Atala, A. (2014). 3D bioprinting of tissues and organs. Nature Biotechnology, 32(8), 773–785.
Naito, H., et al. (2011). The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue. Tissue Engineering. Part A,
17(17–18), 2321–2329.
Naughton, G., Mansbridge, J., & Gentzkow, G. (1997). A metabolically active human dermal replacement for the treatment of diabetic foot ulcers. Artificial Organs, 21(11),
1203–1210.
Niu, L.-N., et al. (2012). Intrafibrillar silicification of collagen scaffolds for sustained release of stem cell homing chemokine in hard tissue regeneration. FASEB Journal, 26(11),
4517–4529.
Nowicki, M. A., et al. (2016). 3D printing of novel osteochondral scaffolds with graded microstructure. Nanotechnology, 27(41), 414001.
O’Brien, F. J. (2011). Biomaterials & scaffolds for tissue engineering. Materials Today, 14(3), 88–95.
Oh, S.-A., et al. (2011). Collagen three-dimensional hydrogel matrix carrying basic fibroblast growth factor for the cultivation of mesenchymal stem cells and osteogenic differ-
entiation. Tissue Engineering. Part A, 18(9–10), 1087–1100.
Oliveira, S. M., et al. (2010). An improved collagen scaffold for skeletal regeneration. Journal of Biomedical Materials Research Part A, 94(2), 371–379.
O’Meara, S., Martyn-St James, M., & Adderley, U. J. (2015). Alginate dressings for venous leg ulcers. Cochrane Database of Systematic Reviews, 8.
Ott, H. C., et al. (2008). Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nature Medicine, 14(2), 213–221.
Ozdil, D., & Aydin, H. M. (2014). Polymers for medical and tissue engineering applications. Journal of Chemical Technology and Biotechnology, 89(12), 1793–1810.
Panseri, S., et al. (2012). Magnetic hydroxyapatite bone substitutes to enhance tissue regeneration: evaluation in vitro using osteoblast-like cells and in vivo in a bone defect. PLoS
One, 7(6), e38710.
Park, I. S., et al. (2009). A collagen/smooth muscle cell-incorporated elastic scaffold for tissue-engineered vascular grafts. Journal of Biomaterials Science, Polymer Edition, 20(11),
1645–1660.
Pashneh-Tala, S., MacNeil, S., & Claeyssens, F. (2015). The tissue-engineered vascular graftdpast, present, and future. Tissue Engineering. Part B, Reviews, 22(1), 68–100.
Paskiabi, F. A., et al. (2015). Optimizing parameters on alignment of PCL/PGA nanofibrous scaffold: an artificial neural networks approach. International Journal of Biological
Macromolecules, 81, 1089–1097.
Pawar, K., et al. (2015). Biomaterial bridges enable regeneration and re-entry of corticospinal tract axons into the caudal spinal cord after SCI: association with recovery of forelimb
function. Biomaterials, 65, 1–12.
Pellegata, A. F., et al. (2013). Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering. BioMed Research
International, 2013.
Peters, B.-H., Molnár, F., & Ketolainen, J. (2014). Structural attributes of model protein formulations prepared by rapid freeze-drying cycles in a microscale heating stage. European
Journal of Pharmaceutics and Biopharmaceutics, 87(2), 347–356.
Polo-Corrales, L., Latorre-Esteves, M., & Ramirez-Vick, J. E. (2014). Scaffold design for bone regeneration. Journal of Nanoscience and Nanotechnology, 14(1), 15–56.
Poornejad, N., et al. (2016). The impact of decellularization agents on renal tissue extracellular matrix. Journal of Biomaterials Applications, 31(4), 521–533.
Powell, H. M., & Boyce, S. T. (2009). Engineered human skin fabricated using electrospun collagen–PCL blends: morphogenesis and mechanical properties. Tissue Engineering.
Part A, 15(8), 2177–2187.
Pulli, R., et al. (2010). Midterm results from a multicenter registry on the treatment of infrainguinal critical limb ischemia using a heparin-bonded ePTFE graft. Journal of Vascular
Surgery, 51(5), 1167–1177. e1.
Qi, H., et al. (2016). Bioactivity assessment of PLLA/PCL/HAP electrospun nanofibrous scaffolds for bone tissue engineering. Life Sciences, 148, 139–144.
Rahaman, M. N., et al. (2011). Bioactive glass in tissue engineering. Acta Biomaterialia, 7(6), 2355–2373.
Ravichandran, R., et al. (2012). Minimally invasive injectable short nanofibers of poly (glycerol sebacate) for cardiac tissue engineering. Nanotechnology, 23(38), 385102.
Rieder, E., et al. (2005). Tissue engineering of heart valves decellularized porcine and human valve scaffolds differ importantly in residual potential to attract monocytic cells.
Circulation, 111(21), 2792–2797.
Roosens, A., et al. (2016). Impact of detergent-based Decellularization methods on porcine tissues for heart valve engineering. Annals of Biomedical Engineering, 1–13.
Santos, L. G., et al. (2013). Electrospun membranes of poly(lactic acid) (PLA) used as scaffold in drug delivery of extract of Sedum dendroideum. Journal of Nanoscience and
Nanotechnology, 13(7), 4694–4702.
Schneider, R. K., et al. (2010). The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional
collagen scaffolds. Biomaterials, 31(3), 467–480.
Sears, N. A., et al. (2016). A review of three-dimensional printing in tissue engineering. Tissue Engineering. Part B, Reviews, 22(4), 298–310.
Seifu, D. G., et al. (2013). Small-diameter vascular tissue engineering. Nature Reviews Cardiology, 10(7), 410–421.
Sekiya, N., et al. (2013). Efficacy of a poly glycolic acid (PGA)/collagen composite nanofibre scaffold on cell migration and neovascularisation in vivo skin defect model. Journal of
Plastic Surgery and Hand Surgery, 47(6), 498–502.
Seo, Y.-K., & Park, J.-K. (2010). Tissue engineered scaffold utilizing the reinforced technique. Biotechnology and Bioprocess Engineering, 15(4), 527–533.
Seol, Y.-J., et al. (2004). Chitosan sponges as tissue engineering scaffolds for bone formation. Biotechnology Letters, 26(13), 1037–1041.
Shaikh, F. M., et al. (2008). Fibrin: a natural biodegradable scaffold in vascular tissue engineering. Cells, Tissues, Organs, 188(4), 333–346.
Sharifi, F., et al. (2016). Polycaprolactone microfibrous scaffolds to navigate neural stem cells. Biomacromolecules, 17(10), 3287–3297.
Shi, X., et al. (2005). Rheological behaviour and mechanical characterization of injectable poly (propylene fumarate)/single-walled carbon nanotube composites for bone tissue
engineering. Nanotechnology, 16(7), S531.
Simon, P., et al. (2003). Early failure of the tissue engineered porcine heart valve SYNERGRAFT® in pediatric patients. European Journal of Cardio-Thoracic Surgery, 23(6),
1002–1006.
Singer, A. J., & Clark, R. A. F. (1999). Cutaneous wound healing. New England Journal of Medicine, 341(10), 738–746.
Srouji, S., Kizhner, T., & Livne, E. (2006). 3D scaffolds for bone marrow stem cell support in bone repair. In , 1. Regenerative Medicine (pp. 519–528), 4.
Stadelmann, W. K., Digenis, A. G., & Tobin, G. R. (1998). Physiology and healing dynamics of chronic cutaneous wounds. American Journal of Surgery, 176(2A Suppl), 26S–38S.
Stillaert, F., et al. (2008). Human clinical experience with adipose precursor cells seeded on hyaluronic acid-based spongy scaffolds. Biomaterials, 29(29), 3953–3959.
Stitzel, J., et al. (2006). Controlled fabrication of a biological vascular substitute. Biomaterials, 27(7), 1088–1094.
Stock, U. A., & Vacanti, J. P. (2001). Tissue engineering: current state and prospects. Annual Review of Medicine, 52(1), 443–451.
Suh, J.-K. F., & Matthew, H. W. (2000). Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials, 21(24), 2589–2598.
Sun, X., et al. (2014). bFGF-grafted electrospun fibrous scaffolds via poly(dopamine) for skin wound healing. Journal of Materials Chemistry B, 2(23), 3636.
Syedain, Z., et al. (2016). Tissue engineering of acellular vascular grafts capable of somatic growth in young lambs. Nature Communications, 7, 12951.
Thein-Han, W., & Xu, H. H. K. (2013). Prevascularization of a gas-foaming macroporous calcium phosphate cement scaffold via coculture of human umbilical vein endothelial cells
and osteoblasts. Tissue Engineering. Part A, 19(15–16), 1675–1685.
Thitiset, T., et al. (2013). Development of collagen/demineralized bone powder scaffolds and periosteum-derived cells for bone tissue engineering application. International Journal of
Molecular Sciences, 14(1), 2056–2071.
Thomas, M. V., Puleo, D. A., & Al-Sabbagh, M. (2005). Bioactive glass three decades on. Journal of Long-Term Effects of Medical Implants, 15(6).
Tran, R. T., et al. (2011). A new generation of sodium chloride porogen for tissue engineering. Biotechnology and Applied Biochemistry, 58(5), 335–344.
Tu, C., et al. (2003). The fabrication and characterization of poly(lactic acid) scaffolds for tissue engineering by improved solid–liquid phase separation. Polymers for Advanced
Technologies, 14(8), 565–573.
van Winterswijk, P. J., & Nout, E. (2007). Tissue engineering and wound healing: an overview of the past, present, and future. Wounds, 19(10), 277–284.
Vaysse, L., et al. (2015). Micropatterned bioimplant with guided neuronal cells to promote tissue reconstruction and improve functional recovery after primary motor cortex insult.
Venkatesan, J., & Kim, S.-K. (2010). Chitosan composites for bone tissue engineeringdan overview. Marine Drugs, 8(8), 2252–2266.
Wang, X., et al. (2016). Topological design and additive manufacturing of porous metals for bone scaffolds and orthopaedic implants: a review. Biomaterials, 83, 127–141.
Whang, K., et al. (1995). A novel method to fabricate bioabsorbable scaffolds. Polymer, 36(4), 837–842.
Woodruff, M. A., & Hutmacher, D. W. (2010). The return of a forgotten polymerdpolycaprolactone in the 21st century. Progress in Polymer Science, 35(10), 1217–1256.
Wu, W., Allen, R. A., & Wang, Y. (2012). Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery. Nature Medicine, 18(7), 1148–1153.
Xing, Q., Qian, Z., Tahtinen, M., Yap, A. H., Yates, K., & Zhao, F. (2017). Aligned nanofibrous cell-derived extracellular matrix for anisotropic vascular graft construction. Advanced
Healthcare Materials, 6(10), 1601333.
Yang, S., et al. (2001). The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Engineering, 7(6), 679–689.
Yang, S., et al. (2002). The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue Engineering, 8(1), 1–11.
Yao, L., et al. (2009). Orienting neurite growth in electrospun fibrous neural conduits. Journal of Biomedical Materials Research. Part B: Applied Biomaterials, 90B(2), 483–491.
Yao, S., et al. (2016). Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth. Nanoscale, 8(19), 10252–
10265.
Ye, Q., et al. (2000). Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering. European Journal of Cardio-Thoracic Surgery, 17(5), 587–591.
Yi, H., et al. (2016). Recent advances in nano scaffolds for bone repair. Bone Research, 4, 16050.
Yokoyama, U., et al. (2017). Arterial graft with elastic layer structure grown from cells. Scientific Reports, 7(1), 140.
Yoo, H. S., et al. (2005). Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering. Biomaterials, 26(14), 1925–1933.
Yoshimoto, H., et al. (2003). A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials, 24(12), 2077–2082.
Yu, W., et al. (2017). In vitro and in vivo evaluation of MgF2 coated AZ31 magnesium alloy porous scaffolds for bone regeneration. Colloids and Surfaces B: Biointerfaces, 149,
330–340.
Zein, I., et al. (2002). Fused deposition modeling of novel scaffold architectures for tissue engineering applications. Biomaterials, 23(4), 1169–1185.
Zelen, C. M., et al. (2016). Treatment of chronic diabetic lower extremity ulcers with advanced therapies: a prospective, randomised, controlled, multi-centre comparative study
examining clinical efficacy and cost. International Wound Journal, 13(2), 272–282.
Zeng, S., et al. (2015). Characterization of silk fibroin/chitosan 3D porous scaffold and in vitro cytology. PLoS One, 10(6), e0128658.
Zhang, Z., & Christopher, G. F. (2015). The nonlinear viscoelasticity of hyaluronic acid and its role in joint lubrication. Soft Matter, 11(13), 2596–2603.
Zhang, J., et al. (2005). A comparative study of porous scaffolds with cubic and spherical macropores. Polymer, 46(13), 4979–4985.
Zhang, Y., et al. (2008). Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering. Biomaterials, 29(32), 4314–4322.
Zhang, Q., et al. (2012). Preparation of uniaxial multichannel silk fibroin scaffolds for guiding primary neurons. Acta Biomaterialia, 8(7), 2628–2638.
Zhang, Y. S., et al. (2016a). Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomaterials, 110, 45–59.
Zhang, Y., et al. (2016b). 3D-printed bioceramic scaffolds with a Fe3O4/graphene oxide nanocomposite interface for hyperthermia therapy of bone tumor cells. Journal of Materials
Chemistry B, 4(17), 2874–2886.
Zhao, B., et al. (2017). Corrosion resistance characteristics of a Ti-6Al-4V alloy scaffold that is fabricated by electron beam melting and selective laser melting for implantation
in vivo. Materials Science and Engineering: C, 70(Part 1), 832–841.
Zheng Shu, X., et al. (2004). In situ crosslinkable hyaluronan hydrogels for tissue engineering. Biomaterials, 25(7–8), 1339–1348.
Zhong, S. P., Zhang, Y. Z., & Lim, C. T. (2010). Tissue scaffolds for skin wound healing and dermal reconstruction. Wiley Interdisciplinary Reviews: Nanomedicine and Nano-
biotechnology, 2(5), 510–525.
Zhou, J., et al. (2010). Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity. Biomaterials, 31(9), 2549–2554.
Zhu, X., et al. (2010). Effects of composite formulation on mechanical properties of biodegradable poly (propylene fumarate)/bone fiber scaffolds. International Journal of Polymer
Science, 2010.
Zhu, W., et al. (2015). Highly aligned nanocomposite scaffolds by electrospinning and electrospraying for neural tissue regeneration. Nanomedicine: Nanotechnology, Biology and
Medicine, 11(3), 693–704.
Zong, X., et al. (2003). Structure and morphology changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber membrane. Biomacromolecules, 4(2),
416–423.
Further Reading
Chatterjee, K., et al. (2012). Time-dependent effects of pre-aging 3D polymer scaffolds in cell culture medium on cell proliferation. Journal of Functional Biomaterials, 3(2),
372–381.
Li, X.-T., Zhang, Y., & Chen, G.-Q. (2008). Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials. Biomaterials, 29(27), 3720–3728.
Martín-de León, J., Bernardo, V., & Rodríguez-Pérez, M.Á. (2016). Low density Nanocellular polymers based on PMMA produced by gas dissolution foaming: fabrication and cellular
structure characterization. Polymer, 8(7), 265.
Biomaterials for Tissue Engineering and Regenerative Medicine
Ohan S Manoukian and Naseem Sardashti, University of Connecticut Health, Farmington, CT, United States; and University of
Connecticut, Storrs, CT, United States
Teagen Stedman, University of Connecticut Health, Farmington, CT, United States
Katie Gailiunas, Anurag Ojha, Aura Penalosa, Christopher Mancuso, and Michelle Hobert, University of Connecticut, Storrs, CT,
United States
Sangamesh G Kumbar, University of Connecticut Health, Farmington, CT, United States; and University of Connecticut, Storrs, CT,
United States
Introduction 462
Natural Biomaterials 463
Collagen 463
Alginate 464
Chitosan 465
Silk 466
Cellulose 469
Bacterial Cellulose 469
Fibrin 470
Gelatin 471
Polyglycolic Acid 473
Polylactic Acid 474
Poly Lactic-co-Glycolic Acid 475
Polycaprolactone 476
Polyetheretherketone 478
Polyethylene Glycol 478
Polymethyl Methacrylate 479
Conclusion 480
Acknowledgments 480
References 481
Further Reading 482
Introduction
Biomaterials are natural or synthetic materials intended to interface with biological systems without causing harm to the body.
Biomaterials are utilized in many applications that intend to evaluate, treat, or replace any tissue or organ in the body. Depending
on the intended application, materials are selected from metals, polymers, ceramics, and composites that are biocompatible. The
design must have the ability to mimic properties of tissues and perform the intended biological function.
Biomedical engineering is a multidisciplinary field that combines aspects such as biomaterials, bioinformatics, biomechanics,
instrumentation, biology, computer science, and medicine to solve medical problems. Biomaterials are essential to biomedical engi-
neering because optimal integration between the body and biomedical device is essential for a successful design. If the immune
system rejects the apparatus, it will fail. Regardless of the structural or functional design of the biomedical device, if the material
releases toxins or is unstable in the body, it will not be a successful solution. Recent advancements in materials science, such as
nanotechnology, have allowed subfields of biomedical engineering including tissue engineering and drug delivery to improve
healthcare.
There are many applications and scenarios where these biomaterials can be utilized to assist the recovery process for patients.
These applications cover a broad range and a single biomaterial is usually not limited to one application. Furthermore, creating
composites may enhance the desired properties and minimize the weaknesses of the combined materials. Given the large spectrum
of regenerative medicine applications, biomaterials range from full-size polyetheretherketone (PEEK) cranial implants down to the
nanostructures such as nanofibers for fabricating scaffolds. Bone screws, vertebral body replacements, micro-scale scaffolds, nano-
scale scaffolds, and drug delivery systems are among the most popular applications for biomaterials. New materials and applica-
tions are constantly being investigated and researched in this dynamic and rapidly evolving field. This article is meant to serve
as an introduction to Biomaterials for Regenerative Medicine, highlighting the properties and applications of various natural and
synthetic biomaterials commonly used in tissue engineering and regenerative medicine.

Regenerative Engineering j Biomaterials for Tissue Engineering and Regenerative Medicine 463
Natural Biomaterials
Natural biomaterials have existed for centuries, and are presently used in a variety of biomedical applications. These natural bioma-
terials have versatile capabilities due to their optimal biocompatibility, biodegradability, and remodeling abilities. This allows for
their application in the repair or replacement of damaged tissues and organs within the body. Additionally, natural biomaterials
have the ability to support cell migration, proliferation, differentiation, and adhesion. These characteristics are vital for tissue engi-
neering as the naturally derived biomaterials will promote the attachment and migration of the cells from the surrounding envi-
ronment, and in turn encourage tissue regeneration (Ha et al., 2013; Ige et al., 2012).
Although natural biomaterials have numerous advantages, they still pose potential problems. A limitation of natural
biomaterials is the immunogenic response that can result following implantation. Another disadvantage, especially for natural
polymers, is the tendency to decompose at temperatures below their melting point. This limits their use in implants as the
natural biomaterials lack the versatility to fabricate a range of shapes and sizes. Due to the unpredictability of an in vivo
source such as an animal, lot-to-lot variability is an additional concern with these biomaterials (Ige et al., 2012; Bartis and
Pongrácz, 2011).
Naturally derived biomaterials can be organized into several groups such as protein-based biomaterials, polysaccharide-based
biomaterials, and decellularized tissue-derived biomaterials. Protein-based biomaterials include collagen, gelatin, and silk. In addi-
tion, polysaccharide-based biomaterials are comprised of cellulose and chitosan. Finally, examples of decellularized tissue-derived
biomaterials include decellularized blood vessels and liver (Ha et al., 2013). Several natural biomaterials will be further discussed in
the following sections.
Collagen
Collagen is the most abundant, natural polymer found in the body, and it accounts for a third of the body’s proteins. This is caused
by the abundance of three amino acids: glycine, proline, and hydroxyproline. These amino acids provide a basis for collagen’s triple
helical structure to create the macroscopic fibers seen in the extracellular matrix (ECM) of tissue, bone, etc. (see Fig. 1). Collagen’s
high resistance to tensile forces maintains and stabilizes structures within tissues (Jenkins et al., 2003). As the most governing
protein in the extracellular matrix, collagen accounts for nearly 75% of the dry weight of skin (Mizuno et al., 2003). Collagen’s
mechanical properties, biocompatibility, and compatibility with other polymers makes it an ideal material for tissue regeneration
(see Fig. 2).
As previously stated, collagen is highly biocompatible. Collagen’s ability to conduct electricity makes the protein ideal for many
tissue engineering purposes. The more conductive a polymer is, the better cell attachment and cell proliferation on the scaffold
(Jenkins et al., 2003). Collagen’s conductivity, approximately 0.3 Siemens/m, can be enhanced by combining the material with
other polymers, and thus improve its tissue engineering functionality (MacDonald et al., 2008).
The degradation of collagen can be attributed to collagenase, which is an enzyme naturally found in the human body. Collage-
nase is responsible for the degradation of collagen molecules, which generally occurs in a short period of time. The degradation time
is contingent upon the molecular weight of the collagen molecules, which vary throughout the body.
The length scales of collagen type I

Nano Micro Macro
α-Helices Fibrils Tissues

Structure
Tropocollagen Fibers Organs
Resistance to
Self Cell Resistance to Tissue
Function compressive strain
assembly binding tensile strain structure
(composites)
Fabrication Electrospinning Moldules Molding
Mechanical
Modification Crosslinking Composites
stimulation
Fig. 1 The hierarchical structure of collagen type I leads to specific biological functions and characteristics across length scales. Reprinted with
permission from Walters, B. D. and Stegemann, J. P. (2014) Strategies for directing the structure and function of three-dimensional collagen bioma-
terials across length scales. Acta Biomaterialia 10 (4): 1488–1501.
464 Regenerative Engineering j Biomaterials for Tissue Engineering and Regenerative Medicine
Fig. 2 Scanning electron microscopy (SEM) images of electrospun collagen-based fibers. (A, B) Mesh like and aligned collagen fibers, respectively.
(C) A 50–50 collagen–chitosan fiber mesh. Reprinted with permission from Walters, B. D. and Stegemann, J. P. (2014) Strategies for directing the
structure and function of three-dimensional collagen biomaterials across length scales. Acta Biomaterialia 10 (4): 1488–1501.
Collagen has been very useful in numerous applications and research studies. In a study conducted by Cho et al., the use of pheo-
chromocytoma (PC12) cells showed that collagen helps nerve growth with its ability to conduct electricity through carbon nano-
tubes (Cho and Borgens, 2010). The study shows more improvement in cell viability for scaffolds electrically stimulated than the
control scaffolds that were not electrically stimulated. The neurite length in electrically stimulated scaffolds was longer and had
a higher density of developed neural cells when compared with scaffolds of the control. Studies such as these indicate that collagen
can be used as a valuable platform for tissue regeneration.
Alginate
Alginate is a natural occurring polymer found in brown seaweed. It is a commonly used biomaterial known for its ability to be made
into a gel and retain a structure similar to the ECM. It is commonly used for wound healing, drug delivery, and tissue engineering
(Manoukian et al., 2016a). Alginate has great biocompatibility and produces little to no immunogenic response. The studies that
did see an immunogenic response were likely caused by impurities present in the polymer. Since alginate is retrieved from a natural
source, it acquires many impurities from the environment. Studies that used highly purified alginate saw no immunogenic response,
and thus only purified alginate should be introduced to the body. The chemical structure of alginate can be seen in Fig. 3 (Lee and
Mooney, 2012).
This naturally occurring polymer is not degradable in mammals due to the lack of the enzyme alginase, which degrades alginate.
However, one solution to this problem is to make alginate gels that are ionically cross-linked since then they will dissolve. An alter-
native approach would be to partially oxidize the alginate chains. Gels can also be made out of G-blocks or gene fragments that are
retrieved from alginate. The G-blocks are oxidized and cross-linked with adipic acid dihydrazide (AAD) to form gels. This allows for
degradable soft gels that dissolve much slower than most gels. The more AAD in the gel, the slower the degradation (Lee and
Mooney, 2012).
Due to its generally slow degradation, alginate is most suited for applications that will require longer periods of time. For
example, alginate has been used as a biomaterial in myocardial repair. In the study, an alginate hydrogel was used to deliver
stem cells to an infarcted rat heart. This method of delivery saw much better results regarding scar size and microvascular density
than methods that directly injected the cells.
Fig. 3 Alginate structure and the egg-box model of hydrogel formation. Reproduced from Lee, K.Y., Mooney, D.J. (2012). Alginate: Properties and
biomedical applications. Progress in Polymer Science 371, 106–126.
When designing any cell culture scaffold for tissue regeneration, it is important to mimic the meshwork of the native ECM. The
ECM is an elaborate cellular environment that is responsible for a variety of cell functions such as cell-to-cell communication, and
for providing structural support. Synthesizing a scaffold similar to the native ECM will ensure optimal interaction with the native
cells as well as the necessary structural support (Geckil et al., 2010). Alginate’s structure is similar to the ECM of cardiac tissue, which
makes it a desirable biomaterial for cardiac tissue engineering. Future research intends to design alginate scaffolds that mimic the
native ECM and inspire native cardiac tissue functions. A schematic design of engineering cardiac tissue can be seen in Fig. 4
(Ruvinov and Cohen, 2016).
Alginate has also been used with hydroxyapatite to produce a scaffold intended for bone regeneration. The alginate was used in
combination with the hydroxyapatite to create porous microspheres. A porous scaffold allows for greater integration of cells and
a more stable bone structure as materials begin to degrade (Rossi et al., 2012). In terms of drug delivery, alginate is commonly
used due to its gelling ability. Hydrogels are readily dissolvable, allowing them to release drugs in a predictable manner. Alginate
was used as a neuro-bridge to treat spinal cord injuries by enriching the polymer with two growth factors known to enhance spinal
cord repair. The alginate scaffold released these growth factors and was seen to increase the number of surviving neurons in spinal
cord injuries (Grulova et al., 2015).
Chitosan
Chitosan is the principal derivate from the polysaccharide polymer chitin, which can be found in the skeleton and internal struc-
ture of invertebrates, such as in the exoskeleton of shellfish. It is the second most popular natural polysaccharide after cellulose,
but their structure differs by replacing hydroxyl for an acetamido group in the C-2 position (see Fig. 5). Chitosan is obtained by
partial deacetylation of chitin, that is, the removal of proteins and the dissolution of calcium carbonate (Dutta et al., 2004). The
degree of deacetylation of chitin can vary in order to modify its solubility and solution properties. To obtain chitosan, the typical
degree of acetylation is less than 0.35, which contributes to its nontoxic characteristics (Kumar, 2000). In addition to its nontoxic
properties, chitosan is a naturally abundant and renewable biomaterial with important properties for regenerative medicine such
as biocompatibility and biodegradation. It has good biological performance and it degrades into products that are easily
absorbed. Physically, chitosan has poor mechanical properties, which is why it is often used as part of a composite (Dutta
et al., 2004).
Chitosan is a biodegradable polymer with a semicrystalline structure. Porous chitosan scaffolds have been used for implanta-
tion, and the results showed that a connective tissue matrix formed within the implant’s pore spaces. Additionally, there was
Fig. 4 Three-dimensional engineered nanowired cardiac tissue. (A) Isolated cardiomyocytes that were cultured in either alginate or alginate-
nanowired composites. (B) The cardiomyocytes in the alginate scaffolds (top) only form small clusters with nonsynchronous behavior while the
alginate-nanowired scaffold exhibit synchronization throughout the scaffold. Reproduced from Ruvinov, E., Cohen, S. (2016). Alginate biomaterial for
the treatment of myocardial infarction: Progress, translational strategies, and clinical outlook: From ocean algae to patient bedside. Advanced Drug
Delivery Reviews 96, 54–76.
CH2OH CH2OH
H O H O H
H O H NaOH
OH H OH H
H deacetylation
H NHCOCH3 H NHCOCH3
n
CH2OH CH2OH
H O H O H
H O H
OH H OH H
H
H NH2 H NH2
n
Fig. 5 Chemical structure of chitosan. Reprinted with permission from Dutta, P. K., Duta, J. D., Tripathi, V. S. (2004). Chitin and chitosan: Chem-
istry, properties and applications. Journal of Scientific & Industrial Research 63, 20–31.
very low incidence of chitosan-specific reaction, including cellular and serological reactions (VandeVord et al., 2002). Several studies
showed that chitosan’s degradation was related to the degree of acetylation and the polymer’s molecular mass. In vertebrates, lyso-
zyme and bacterial enzymes typically degrade this biomaterial. In vivo studies have also reported ways in which chitosan degrades
with different administration methods. In oral administration, degradation occurs mainly in the gastrointestinal track, while in
intravenous administration, it is likely to degrade in the liver and kidney (Kean and Thanou, 2010).
In several studies, chitosan has shown promising results as a scaffolding material for tissue engineering. It is easily processed into
porous scaffolds, films, and beads. Chitosan’s applications include bone tissue, central nervous system, and articular cartilage.
Depending on the application, composites are made to improve and complement the biomaterial properties. For example, in
bone tissue engineering, a microporous chitosan/calcium phosphate composite scaffold showed enhanced osteoblast attachment
that increased scaffold strength while maintaining biocompatibility. Moreover, a chitosan-glycosaminoglycan (GAG) composite
has been successfully used to aid articular cartilage repair (Dutta et al., 2004).
In terms of wound healing, the deacetylation of chitin into chitosan showed significant improvement in its interaction with
mammalian tissues. The cells involved include osteoblasts, fibroblasts, macrophages, and keratinocytes, which aid the tissue regen-
eration and repair process (Madihally and Matthew, 1999). Moreover, chitin and its derivatives have also shown to increase extra-
cellular lysozyme activity, inhibit fibroplasia, and promote tissue growth for better healing (Kumar, 2000). In some circumstances,
wound healing incorporates the use of artificial skin. Chitosan has been utilized in two ways to address this injury. First, chitosan is
used for skin replacement since it has characteristics similar to those of GAGs, which is an essential component in skin. Second, it is
used in solution to aid healing and fibroplasia, which is the formation of fibrous tissue in wounds (Kumar, 2000). Overall, there are
several studies in which chitosan showed excellent potential as tunable porous scaffolds for tissue engineering purposes. An
example of this is shown in Fig. 6.
Silk
Silk is a natural fiber of fibroin protein produced by arthropods such as silkworms, spiders, flies, mites, or scorpions. Silkworm silk is
used in biomedical applications due to its mechanical properties, biocompatibility, and ease of production. Silk is purified easily,
using enzymes or alkali to remove sericin, leaving fibroin behind (see Fig. 7). The molecular weight and dispersity index will vary
depending on the source and methods of processing (Zhang et al., 2009). Silk is promising for tissue engineering because of its
elasticity, strength, and biocompatibility (Kundu et al., 2013). The silk most commonly used in biomaterials is known as mulberry
silk (Kundu et al., 2013). It is produced by the silkworm Bombyx mori. Mulberry silk has been used as surgical sutures for many years,
and recently the biomedical applications have greatly increased (Yang et al., 2007).
Natural fibers can be twisted into rope, braided, or woven. They can also be fabricated into various forms such as hydrogels,
lyophilized powders, porous scaffolds, native silk mats, and silk microparticles (see Fig. 8). They can be dissolved in certain salt
solutions and then regenerated into other forms such as films, electrospun fibers, porous scaffolds, or hydrogels (Kundu et al.,
2013). Silk fibroin is water soluble, which allows for water-based processing in mild conditions without harsh chemicals (Kundu
et al., 2013). Topographic patterns can be added to silk film surfaces using lithography. These patterns encourage alignment and
adhesion of fibroblasts and epithelial cells (Lawrence et al., 2009). Natural silk is tough and ductile, with a high strength-to-density
ratio. Due to its toughness, strength, and strain-hardening tendency, silk is a promising material for load bearing scaffolds in tissue
engineering (Kundu et al., 2013).
Silk is found naturally in the form of fibers consisting of a core protein, silk fibroin, coated with the protein sericin. The glue-like
sericin causes unwelcome immunological responses in the human body, so natural silk is often purified to remove the sericin.
Fig. 6 SEM images of chitosan scaffold cross sections, which were fabricated by the freeze-gelation method. This process was done under three
different conditions: (A) at a freezing temperature of 80 C, a 0.2 concentration of acetic acid in the chitosan solution, and a 95% concentration of
ethanol in the rinse buffer, (B) has nearly identical conditions except it had a 0% concentration of ethanol in the rinse buffer, (C) had similar condi-
tions to (A) as well aside from the difference of a 0.8 concentration of acetic acid in the chitosan solution, and (D) differs from the original conditions
shown in (A) with a 20 C freezing temperature. Reproduced from Hsieh, C. Y., et al. (2007). Analysis of freeze-gelation and cross-linking processes
for preparing porous chitosan scaffolds. Carbohydrate Polymers 67 (1): 124–132.
Fig. 7 Chemical structure of silk fibroin.
Purified silk fibroin demonstrates mild foreign body responses (Yang et al., 2007). Antigenicity and immunogenicity of silk has
been tested in rats, pigs, and humans, resulting in only low levels of inflammation and a minimal immune response. Silk sutures
are removed after a period of time, but a long-term immune response has yet to be investigated (Kundu et al., 2013).
Silk has a tailorable degradation, which ranges from months to years. The degradation rate depends on the processing during the
formation of the material (Zhang et al., 2009). Purified silk fibroin fibers begin degrading 6 months after implantation, losing most
tensile strength at the end of 1 year. At end of 2 years, they no longer exist at the site (Yang et al., 2007). Processed silk degrades faster
than unpurified silk fibers. The degradation rate depends on the secondary structure of the silk fiber or scaffold, which results during
processing. Moreover, macrophage-led biodegradation indicates bioresorbability of silk. During in vitro experiments, osteoblasts
and osteoclasts have also been shown to degrade silk (Kundu et al., 2013).
Silk has been applied in a variety of disciplines such as vascular grafts, bone, cartilage, ligament, skin, and many others. In
vascular grafts, electrospun nanofibers have shown the potential to serve as blood-vessel transplants since they have the ability
for cell attachment and the necessary mechanical properties for blood flow (Zhang et al., 2009). In bone tissue engineering, electro-
spun silk matrices support marrow stromal cell attachment and extracellular matrix formation. When growth factors are added to an
electrospun scaffold, bone formation is improved (Zhang et al., 2009). Porous silk fibroin scaffolds (see Fig. 9) have the toughness
Fig. 8 Biomaterials fabricated from silk fibroin: (A) hydrogels; (B) lyophilized powder; (C) 3D porous scaffolds; (D) native silk mat; (E) silk micro-
particles. Reproduced from Kundu, B., Raijkhowa, R., Kundu, S. C., Wang, X. (2013). Silk fibroin biomaterials for tissue regenerations. Advanced Drug
Delivery Reviews 654, 457–470.
Fig. 9 Applications of silk. Reprinted by permission from Macmillan Publishers Ltd.: Nature Protocols, advance online publication, 22 September
2011. Alam, J., Rahman, W., Mazid, R. A. and Khan, M. R. (2015). Gamma-irradiated gelatin-based films modified by HEMA for medical application.
International Journal of Polymer Analysis and Characterization 20(5), 426-434. https://doi.org/10.1038/nprot.2011.379.
and biocompatibility ideal for bone regeneration. Silk hydrogels also work well, and incubating the scaffolds with cells before
implantation increases vascularization (Kundu et al., 2013). When electrospun silk matrices are treated with microwave-induced
argon plasma, they show promise as a cartilage repair material. Cartilage tissue, including the menisci of the knees, undergo
very slow and frequently inadequate and insufficient natural repair and regeneration due to their avascular nature, often necessi-
tating surgical and suture repair (Beamer et al., 2015; Masoudi et al., 2015; Beamer et al., 2017). They have been shown to improve
human neonatal knee articular cartilage chondrocyte adhesion and proliferation (Zhang et al., 2009). Silk-chitosan composite
sponges are also known to support the growth of chondrocytes (Kundu et al., 2013).
Silk scaffolds can also be used in ligament tissue engineering. Woven silk fibers form a lightweight, strong, elastic material
with mechanical properties similar to those of a human’s anterior cruciate ligament (ACL). Modifying braided silk scaffolds
with short polypeptides increases collagen production when seeded with mesenchymal stem cells (MSCs). Incorporating
microporous silk sponges into a knitted silk mesh mimics a ligament’s extracellular matrix. At 16 weeks postimplantation,
the silk partially degraded, allowing for ingrowth of regenerated ligament tissue (Fan et al., 2008). Twisted silk scaffolds
or silk-coated poly-lactic-co-glycolic acid (PLGA) nanofibers are both options for ligament regeneration (Kundu et al.,
2013). Additionally, knit silk-collagen sponges can be good scaffolds for tendon regeneration when incorporated with
stromal cell-derived factor 1 (SDF-1) alpha. It attracts fibroblasts and reduces the accumulation of inflammatory agents
(Shen et al., 2010).
An additional application for silk is in skin, which is a complex, layered organ. Composite scaffolds best mimic the extracellular
matrix of skin by combining silk with collagen I or chitin (Kundu et al., 2013). Silk matrices can be electrospun and functionalized
to release molecules such as proteins, DNA, or antibiotics. Functionalized electrospun matrices are used as scaffolds for cell cultures.
Scaffolds that release drugs improve biocompatibility and are capable of reducing inflammation and encouraging the migration,
proliferation, and differentiation of cells. Silver nanoparticles can be deposited on silk electrospun matrices to be used as wound
dressings with antimicrobial properties. When collagen I is added to the silk matrix, it causes keratinocytes to adhere and spread
(Zhang et al., 2009).
Cellulose
Cellulose is a naturally occurring biomaterial that is commonly used in the biomedical field. It is extremely abundant, easily renew-
able, and biodegradable. Due to inter- and intramolecular hydrogen bonding between the hydroxyl groups of the neighboring cellu-
lose chains, cellulose is insoluble in water, despite being hydrophilic, and is difficult to dissolve with common organic solvents (Eo
et al., 2016). This lack of solubility makes cellulose an ideal biomaterial for tissue engineering purposes since it is sustainable under
physiological conditions. Other applications include designing grafts, drug release, aiding in ion exchange, and wound healing. A
chemical structure of cellulose can be seen in Fig. 10 (Kang et al., 2015; Torres et al., 2012).
Similar to other natural polymers made up of alginates and chitosan, cellulose polymers have sufficient biocompatibility. In
addition, cellulose also has adequate mechanical properties, which are necessary to withstand physiological conditions (Torres
et al., 2012). Cellulose has also shown to have antiinflammatory and anticancer effects (Eo et al., 2016). Naturally, cellulose
does not degrade in humans due to the lack of the enzyme cellulase, which breaks down cellulose. Degradation is one of the bigger
problems cellulose faces as a biomaterial. Fortunately, biodegradation can be improved in vitro through periodate oxidation (Torres
et al., 2012).
Bacterial Cellulose
Bacterial cellulose (BC), also known as microbial cellulose, is a biodegradable, natural cellulose that is synthesized by bacteria. The
diameter of BC fibers is 20–100 nm. Bacterial cellulose has high water retention due to being very hydrophilic and having a high
surface area to mass ratio. It also has great mechanical strength, exhibits high crystallinity, and is relatively inexpensive to produce
(Fu et al., 2013; Mano et al., 2007). The molecular structure is shown in Fig. 11.
BC can be synthesized by several types of bacteria, including Cluconacetobacter xylinus. When modified, it can form a material
similar to cartilage that is easily molded (Kowalska-Ludwicka et al., 2013). BC can also be produced by the fermentation of Aceto-
bacter xylinum. A limitation is that BC nanofibrils pack tightly to create a dense mesh, which limits opportunities for cell ingrowth.
This can be remedied by incorporating porogens during fermentation (Zaborowska et al., 2010). It can also be produced by Glu-
conacetobacter xylinus and is highly permeable (Fu et al., 2012).
OH
HO 3 OH 1 6′
2 4′
5′ O
O
O
O 2′
4 5 HO 3′ OH 1′
6
OH
Fig. 10 Schematic representation of cellulose structure.
Fig. 11 Chemical structure of bacterial cellulose (Ulery et al., 2011).
Bacterial cellulose was synthesized to make a biocompatible version of naturally occurring cellulose. BC is generally nontoxic,
and low cytotoxicity is observed (Fu et al., 2013). When modified, it has chemical and physical properties suitable for reconstructive
surgery (Kowalska-Ludwicka et al., 2013). Endothelial cells, chondrocytes, and smooth muscle cells have been shown to adhere to
BC scaffolds (Zaborowska et al., 2010). In previous studies, bacterial cellulose did not elicit an immunogenic response aside from
temporary inflammation, and thus is biocompatible (Torres et al., 2012). BC is also biodegradable by enzymes found in nature but
not in the body. Buffers can be added to encourage bioabsorption (Fu et al., 2013).
A common application for bacterial cellulose is found in skin. BC is permeable and absorbent enough to drain fluid from the
wound. It is also easily removed after a certain period of recovery, but is still able to conform to the body for extended periods of
time (see Fig. 12). Modifying BC with nitrogen plasma improved cell affinity and increased porosity. Additionally, incorporating
human epidermal growth factor and collagen increased the proliferation of human fibroblasts. A composite of BC and collagen I
makes a superior wound dressing with a high biocompatibility and tensile strength. Silver nanoparticles can also be incorporated
into the composite for antimicrobial effects (Fu et al., 2013). Moreover, the transparency of BC is good for wound bandages as it
absorbs heat and reduces pain, which is especially helpful for burn patients (Kowalska-Ludwicka et al., 2013).
Fibrin
Fibrin is a natural biopolymer formed during the last leg of the coagulation cascade after a division of fibrinogen by the action of
thrombin. The coagulation cascade is initiated by an injury or after blood comes in contact with foreign material. Thrombin,
produced in the last step of the cascade, divides fibrin peptides A & B to form fibrin monomers (see Fig. 13). The monomers
form a fibrin network stabilized through crosslinking by factor XIIIa (see Fig. 14) (Ariëns et al., 2002). Along with platelets and
red blood cells, fibrin clots blood to stop bleeding. Fibrin produces a matrix in which cells such as leukocytes, fibroblasts, and endo-
thelial cells attach, move, grow, and organize to generate different functions (see Fig. 15) (Shats et al., 1997).
Fig. 12 (A) pure bacterial cellulose; (B) wet BC-ClAlPc; (C) dry BC-ClAlPc; (D) SEM image of BC-ClAlPc (Clark and Deswarte, 2011).
Fig. 13 Fibrogen structure. Reprinted with permission from Brown, A. C., and Barker, T. H. (2014). Fibrin-based biomaterials: modulation of macro-
scopic properties through rational design at the molecular level. Acta Biomaterialia 10 (4): 1502–1514.
Fig. 14 Fibrin polymerization. Reprinted with permission from Brown, A. C., and Barker, T. H. (2014). Fibrin-based biomaterials: modulation of
macroscopic properties through rational design at the molecular level. Acta Biomaterialia 10 (4): 1502–1514.
As a naturally produced polymer in the human body, fibrin is highly biocompatible. Fibrin fights off materials that are not
biocompatible with the body. Fibrin gel, formed by fibrino-peptides to be insoluble, can be degraded with plasmin-mediated fibri-
nolysis (Sidelmann et al., 2000). Unlike collagen-based hydrogel, which has fast degradation rate, fibrin has a controllable degra-
dation rate, which is beneficial in tissue engineering. Different types of tissue regeneration require respective degradation rates of the
regenerative vehicle, and thus fibrin’s tunable degradation is important (Kjaergard and Weis-Fogh, 1994). Fibrin developed inject-
able scaffolds are used for the treatment of damaged cardiac and cartilage tissues (Chien et al., 2012). Additionally, fibrin gel-based
cell carriers protect cells from environmental forces experienced during the cell delivery process while improving cell viability and
tissue growth (Christman et al., 2004).
Gelatin
Gelatin consists of a mixture of water and peptide fragments. These peptide fragments are typically derived from animal tissue. This
process involves the reduction of crosslinks in collagen as well as the removal of any impurities that may be present. Since gelatin is
a derivative of collagen, it can be manipulated in ways that affect the materials crosslink density and chemical reactivity. These vari-
ables can be modified for the intended function and application. In addition to its low production cost, gelatin has excellent
biocompatibility and is easy to manufacture. Some of the common applications associated with gelatin are drug delivery, hydrogels,
and scaffolds.
Gelatin has been utilized in the pharmaceutical industry as a medium for drug delivery, most commonly in the form of oral
capsules. Although effective, regenerative medicine is now focused on methods that allow for more localized delivery. Microspheres
Fig. 15 Fibrin PLLA/PLGA composite gels facilitate graft neovascularization and perfusion. (A) Low-power images of fluorescein isothiocyanate
(FITC)-Dextran (green) and bright-field images to emphasize the graft area. (B) High-magnification image of the graft area which show the thorough
penetration of FITC-Dextran and neovessels 10 days post-implantation. (C) Quantification of the FITC-Dextran and neovessel penetration into the graft
area and graft area size (D) showed that the cellularized or noncellularized fibrin and PLLA/PLGA composites provided the most support for graft
neovascularization and perfusion (n ¼ 3–6). Reprinted with permission from Brown, A. C., and Barker, T. H. (2014). Fibrin-based biomaterials:
modulation of macroscopic properties through rational design at the molecular level. Acta Biomaterialia 10 (4): 1502–1514.
are an example of a matrix that has shown a lot of potential as a targeted delivery system. There are advantages of using microspheres
in applications such as drug delivery, bone tissue engineering, and regeneration. Absorption and desorption of substances as well as
the kinetic release of the loaded drug components become variables that can be further modified to better suit the intended function
(Hossain et al., 2015). Gelatin microspheres containing calcitonin gene-related peptide (CGRP) or substance P promoted bone
growth in a rabbit osteoporotic bone defect model. The rate of bone growth was positively correlated with the dose administered
(Chen et al., 2016).
Aside from microspheres, nanoparticles are also an area of interest. Gelatin nanoparticles were infused with polyethylene glycol
and then incubated with breast cancer or BT-20 cells to determine uptake. The results of this study showed that this method may be
effective as a long-circulating delivery system in vivo. Furthermore, the nanoparticles have the potential to encapsulate hydrophilic
macromolecules and are internalized by tumor cells. Although drug delivery is the most common application for this biomaterial, it
is not the only one. A two-component polymer system was created using gelatin films incorporated with 2-hydroxyethyl methac-
rylate (HEMA) monomer. This material was tested mechanically to determine whether it exhibited properties similar to skin tissue.
Once this was confirmed, an additional test was done on burn victims, which resulted in an accelerated rate of healing (Alam et al.,
2015).
Synthetic biomaterials are not of natural origin and are synthesized for an intended purpose. These biomaterials have several advan-
tages such as high reproducibility, availability, and tunability. Through slight changes during fabrication, it is easy to tune or modify
numerous characteristics such as the mechanical properties, degradation rate, and composition of the synthetic biomaterial. A limi-
tation of synthetic biomaterials is that they structurally differ from native tissues and organs. This can affect their biocompatibility
and ability to promote tissue remodeling. Synthetic materials also often lack sites for cell adhesion, which may limit their
regenerative capabilities in vivo. Since they are not of natural origin, synthetic biomaterials are more susceptible to eliciting an
immune response and toxicity after implantation. Although this is a valid concern, there are still several synthetic biomaterials
that are highly biocompatible and integrate well with the body (Ha et al., 2013; Ige et al., 2012; Bartis and Pongrácz, 2011).
Synthetic biomaterials are comprised of metals, ceramics, nonbiodegradable polymers, and biodegradable polymers. They are
commercialized and used in clinical settings for treatments such as metal hip implants, plastic intraocular lenses, and many other
products utilized in the medical field (Ha et al., 2013). The following sections will detail several synthetic biomaterials and how they
are applied in biomedical disciplines.
Polyglycolic Acid
Polyglycolic acid (PGA) was one of the initial, degradable polymers researched for biomedical application. Since the 1970s, PGA
has been used as the degradable suture DEXON due its material characteristics including a melting point (Tm) greater than 200 C,
a glass transition temperature (Tg) between 35 C and 40 C, and a very high tensile strength. The majority of recent studies utilize
PGA as a filler material integrated into other degradable polymers. PGA is commonly incorporated into scaffolds for various tissue
engineering applications such as bone, tendon, cartilage, tooth, and spinal regeneration. Despite these applications, PGA has limi-
tations as its rapid degradation compromises its mechanical strength, and could potentially cause an undesirable inflammatory
response due to the resulting increase of glycolic acid (Ulery et al., 2011). The chemical structure of PGA can be seen in Fig. 16.
PGA has proved to be highly biocompatible in most of its applications. Even though there are some reports of potential immu-
nogenic responses when utilizing PGA, most applications have not caused any inflammatory reaction. Additionally, PGA is known
for its hydrolytic instability. Hydrolytically unstable polymers are materials that have chemical bonds in their backbone that are
susceptible to hydrolysis without an external influence. In the case of PGA, its hydrolytic instability can be attributed to the ester
linkage in its backbone. PGA’s random hydrolysis led to the combination with other polymers such as polylactic acid (PLA) to
control its degradation rate. In addition to degradation by hydrolysis, PGA also undergoes enzymatic degradation in vivo (Ulery
et al., 2011; Clark and Deswarte, 2011).
In practice, PGA has been used in an effort to enhance facial nerve regeneration. This was done by placing bone marrow stem
cells into a PGA tube, and observing for neural regenerative effects. PGA is suitable for neural regeneration because it is absorbable
and has FDA approval for nerve grafting (Anderson et al., 2015). Nerve grafting is a very complex process, and is still in the early
stages of research. In the studies conducted thus far, PGA has been shown promising results for producing nerve graft structures
(Costa et al., 2013).
PGA has also been utilized in wound healing and adhesives. Polyglycolic acid sheets were used in conjunction with fibrin glue
spray as an open wound healing material for soft tissues as well as bone surfaces during oral surgery. The PGA adheres to the wound
successfully and helps prevent postoperative bleeding as well as inspire epithelialization. These sheets were first used only for soft
tissues, and have since been used on hard tissues as well. These PGA sheets can be seen in Fig. 17 (Sakaguchi et al., 2015). As an
adhesive, PGA was combined with fibrin sealant to create a very successful tissue adhesive. The combination of the PGA and fibrin
created a much stronger sealant than any other biomaterial combination (Shinya et al., 2009).
n
Fig. 16 PGA structure.
Fig. 17 (A) Sheets of PGA were cut into small pieces (5–10 mm wide) and (B) approximately 3–10 pieces were used to cover each wound.
Polylactic Acid
Lactic acid is a naturally occurring organic acid that can be produced by chemical synthesis or fermentation. Typically, this process
consists of hydrolysis of lacronitrile by strong acids (Figs. 18 and 19). These processes allow for polylactic acid (PLA) to be derived
from renewable resources. Currently, PLA is one of the most produced polymers on the market. Primary production of PLA happens
through fermentation and is environmentally friendly (Rasal et al., 2010). PLA is a thermoplastic, meaning it can easily cast into
many shapes and has potential for 3D printing (Stratton et al., 2017). It is also highly biocompatible, and can be easily absorbed
by the body.
When lactic acid polymers are manufactured, they can be created in a way that allows for different degradation rates. Hydrolysis
kinetics is a multivariable concept that determines how fast implants will degrade. Density, bond strength, H2O concentration, and
hydrophilicity all play roles in this degradation process. By changing the degradation rate, the polymer can be designed to release
embedded medicine (James et al., 2016). The fastest rates of release are associated with implants that will undergo bulk degrada-
tion, while the slower ones only degrade at the surface. Surface degradation is beneficial in applications where sustained release of
given compounds are needed (James et al., 2016).
The ability to modify the surface of PLA allows for it to be a versatile biomaterial. Chemical modification, plasma treatments,
entrapment, and surface coatings can all be used to change the way PLA will function in the body. These methods can be used to
modify variables such as hydrophilicity and biocompatibility. However, many have yet to be perfected and can cause some draw-
backs due to undesired changes. In biomedical application, PLA’s resorbability makes it an ideal polymer for short-term fixation
such as in fracture repair. This type of fixation shows promise for adolescents that are still growing. These devices are ideal for
craniofacial surgical operations, an area that is very active as a child grows (Holmes et al., 2004). Conventional biostable devices
would not be ideal in this situation, as they would not function properly in the dynamic environment (Fig. 20) (Peltoniemi et al.,
2002).
O O
OH OH
OH OH
H CH3 CH3 H
L-Lactic acid D-Lactic acid

Fig. 18 Lactic acid enantiomers. Reprinted with permission from Holmes, R. E., Cohen, S. R., Cornwall, G. B., Thomas, K. A., Kleinhenz, K. K.,
Beckett, M. Z. (2004). MacroPore resorbable devices in craniofacial surgery. Clinics in Plastic Surgery 313, 393–406.
HO O O
C CH O
OH n
H3C CH3
Lactic acid Poly(lactic acid) or polylactide
−H2O Ring opening

O polymerization
CH3
O
H3C
O
Lactide
Fig. 19 Hydrolysis of lactic acid to create poly(lactic acid). Reprinted with permission from Holmes, R. E., Cohen, S. R., Cornwall, G. B., Thomas,
K. A., Kleinhenz, K. K., Beckett, M. Z. (2004). MacroPore resorbable devices in craniofacial surgery. Clinics in Plastic Surgery 313, 393–406.
Bone and cartilage

regeneration
Bone fixation devices 3D scaffolds
Applications
Ligament Vehicle for long

reconstruction term drug delivery
Injectable micropheres
for drug delivery
Fig. 20 Poly(lactic acid) applications for regenerative medicine.
Poly Lactic-co-Glycolic Acid

Poly lactic-co-glycolic acid (PLGA) is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA) (Fig. 21). It is one of the
preferred materials for biomedical applications because it can be easily optimized for a variety of uses. PLGA can be modified in
terms of its shape, size, degradation rate, and mechanical properties to meet the needs of a given application (Makadia and Siegel,
2011). The copolymer is a biocompatible and biodegradable material with minimal systemic toxicity. As PLGA degrades, it
produces metabolic monomers, lactic acid, and glycolic acid, which are metabolized and eliminated from the body (Danhier
et al., 2012). Currently, PLGA is widely used in sutures, drug delivery, and tissue engineering scaffolds (Lee et al., 2016).
Although PLGA is considered a biocompatible, synthetic polymer, its degradation products may cause inflammatory and foreign
body reactions upon implantation. These reactions are initiated by a pH decrease in surrounding tissues, which results from the
accumulation of lactic and glycolic acid during degradation (Wei Ji et al., 2012). Composition, crystallinity, average molecular
weight, size, and shape are all factors that affect degradation of PLGA. Polymer composition is the most important factor when dis-
cussing degradation because the PLA/PGA ratio determines the amount of glycolic acid, which in turn affects its degree of hydro-
philicity. The rate of degradation is directly proportional to the glycolic acid concentration (Makadia and Siegel, 2011). Degradation
times of 50:50, 75:25, and 85:12 PLGA are 1–2 months, 4–5 months, and 5–6 months, respectively (Ulery et al., 2011).
Size and shape have similar effects on the rate of degradation in that larger surface areas cause increased degradation of the
matrix. In addition, PLGA’s bulk degradation is reported to be quicker than its surface degradation, which causes drug delivery
to be faster in devices with high surface area to volume ratios. Finally, molecular weight is directly related to the polymer chain
O O
H+ O
O
O
OH OH
CH3 O
x y HO
OH
PLGA Lactic acid Glycolic acid
Metabolic
pathways
Fig. 21 PLGA structure and hydrolysis.

Fig. 22 Silk/PLGA-based scaffold for ligament/tendon tissue engineering. (A) Transmission Electron Microscopy (TEM) and (B) SEM images of
electrospun bFGF-containing PLGA fibers which show the distribution of proteins as indicated by the black arrows. (C) SEM image of FGF coating
with electrospun fibers (eF) on microfibrous knitted silk scaffolds (mF). (D) Bone marrow stromal cell (BMSC)-seeded scaffold prior to rolling into
cylindrical ligament/tendon constructs and (E) after 7 days of culture. Reproduced from Sahoo, S., Toh, S. L., Goh, J. C. (2010). A bFGF-releasing
silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials 31(11), 2990–2998.
length, which means that the higher the molecular weight, the lower the degradation rates since the polymer chain is proportionally
larger (Makadia and Siegel, 2011).
PLGA provides a great variety of options when it comes to drug delivery applications. Its degradation is faster than many poly-
mers, but more importantly, its degradation rate can be controlled. PLGA can be packed in any shape and size such as nanospheres,
macrospheres, and millimeter-sized implants (Makadia and Siegel, 2011). It is used in the controlled administration of drugs,
peptides, and proteins. PLGA has been employed to deliver chemotherapeutics, proteins, vaccines, antibiotics, analgesics, antiin-
flammatory drugs, and siRNA (Ulery et al., 2011). PLGA can also be applied in tissue regeneration to engineer scaffolds that
promote cell culture, proliferation, and differentiation until the regenerated tissue has stabilized (Danhier et al., 2012; Pan and
Ding, 2012). In recent years, studies have shown that mesenchymal stem cells have the ability to differentiate into several tissues
including bone, tendon, ligament, and fat. In one study, Sahoo et al. demonstrated the use of a silk/PLGA-based scaffold for liga-
ment/tendon tissue engineering. The scaffold promoted cell proliferation and enhanced the production of collagen that resulted in
strong structures that proved to have characteristics to aid ligament/tendon repair (see Fig. 22) (Sahoo et al., 2010). Finally, PLGA
scaffolds also provide optimal conditions for bone regeneration. These structures are characterized by interconnected pores and
large surface areas that allow tissue ingrowth. Preferred pore sizes range from 100 to 350 mm with more than 90% porosity
(Yoshimoto et al., 2003). Several techniques are used in the fabrication of PLGA scaffolds, including gas foaming, microsphere sin-
tering, poregen leaching, electrospinning, and polymer printing (Ulery et al., 2011). For example, PLGA foams were successfully
used to culture osteoblasts derived from rat mesenchymal cells; showing proper mineralization and three-dimensional bone forma-
tion (Yoshimoto et al., 2003).
Polycaprolactone
Polycaprolactone (PCL) is a synthetic, biodegradable polymer. PCL nanofibers can be aligned to mimic an extracellular matrix struc-
ture (Manoukian et al., 2017). The average diameter of the nanofibers is 500–900 nm (Engel et al., 2008). PCL was first synthesized
in the 1930s, but was not commonly used due to its slow degradation rate and inability to bear heavy loads. It regained popularity
after the emergence of tissue engineering in the 1990s (Woodruff and Hutmacher, 2010). The molecular structure is shown in
Fig. 23. When combined with collagen type I, it is capable of withstanding a high-pressurized environment for an extended period
n n
Fig. 23 Chemical structure of PCL (Costa et al., 2013).
of time. The addition of collagen increases the burst pressure and allows PCL to become stronger as a result (Sell et al., 2009).
Natural biomaterials can be added to PCL to enhance polycaprolactone’s mechanical properties and stability while avoiding the
need to crosslink and introduce harmful crosslinking agents (Cooper et al., 2011).
PCL was determined to be biocompatible after both short-term and long-term studies did not elicit any adverse reactions from
the host tissue. Fibrous scaffolds of electrospun PCL blended with gelatin demonstrate exceptional biocompatibility with bone
marrow stromal cells. PCL composites also support the growth of adipose-derived stem cells and human coronary artery endothelial
cells (Sell et al., 2009). Chitosan–PCL composite scaffolds have also been shown to be compatible with Schwann cells in nerve
studies (Cooper et al., 2011).
It takes more than a year for polycaprolactone to degrade noticeably, and the total degradation time is up to 4 years. PCL is bio-
degraded by bacteria and fungi. In the human body, degradation is a two-step process. During the first year, ester groups are hydro-
lytically cleaved. Next, intracellular degradation occurs (Woodruff and Hutmacher, 2010). Degradation is ultimately dependent on
the molecular weight and crystallinity of the polymer or composite (Lam et al., 2008b). The slow degradation of this synthetic poly-
mer is beneficial for neural regeneration as nerves regenerate slowly (Cooper et al., 2011).
PCL is a versatile polymer that has been implemented into various biomedical research studies. For example, PCL or PCL/
hydroxyapatite scaffolds have been created using a precision extrusion deposition process. They support the growth and migration
of primary fetal bovine osteoblasts and have the correct mechanical properties, pore size, and interconnectivity for bone tissue engi-
neering (Shor et al., 2007). Nonwoven meshes of PCL incorporated with beta-tricalcium phosphate nanoparticles form a composite
that mimics the structure of bone tissue at the bone–cartilage interface (Erisken et al., 2008). PCL scaffold porosity can be created
and tuned using selective laser sintering to make scaffolds similar to trabecular bone (Eshraghi and Das, 2010). Electropsun scaf-
folds of PCL/collagen type I composites have also been successfully implanted as an artery in a rabbit model. The composite demon-
strated mechanical properties such as tensile strength and elasticity comparable to native arteries. Moreover, the PCL/collagen type 1
composite did not cause platelet adhesion which prevented undesirable clotting (Sell et al., 2009). Nanofiber scaffolds of PCL,
collagen type I, and collagen type II support growth and attachment of human coronary artery endothelial cells (Sell et al.,
2009) (Fig. 24). In terms of neural regeneration, aligned chitosan–PCL composite fibrous scaffolds resulted in Schwann cells
Fig. 24 Electrospun PCL/collagen composite scaffolds: (A) The gross appearance of scaffold; SEM images of (B) the entire PCL/collagen composite
(18x), (C) the scaffold surface (6000x), and (D) the scaffold’s cross-sectional appearance (4000x). Reproduced from Sell, S. A., McClure, M. J., Garg,
K., Wolfe, P. S., Bowlin, G. L. (2009). Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.
Advanced Drug Delivery Reviews 61 (12), 1007–1019.
aligning in a bipolar fashion along the direction of the aligned fibers, and thus making these scaffolds appropriate for the recon-
struction of nervous tissue (Cooper et al., 2011).
Polyetheretherketone
Polyetheretherketone (PEEK) is a thermoplastic polymer that was commercialized in the early 1980s and later proposed as a mate-
rial to be used for medical applications (Panayotov et al., 2016). Typically PEEK composite materials have been used as a replace-
ment for metallic and ceramic implants, which have a higher modulus of elasticity (Kurtz and Devine, 2007). PEEK was first
introduced to the medical field as a fracture fixation, and it remains to be used for this purpose. Despite several years and advance-
ments, PEEK is still one of the most popular biomaterials on the market due to its versatile capabilities (Manoukian et al., 2016b).
The chemical stability of PEEK has been of interest to medical device companies since its introduction in 1998. At room temper-
ature, the only solvent capable of dissolving PEEK is 98% sulfuric acid (Ferguson et al., 2006). Its modulus of elasticity is nearly
identical to cortical bone, which allows it to be an optimal candidate for an interbody device in vertebral fusion applications.
Furthermore, PEEK is highly resistant to gamma and electron beam radiation, which allows for easy sterilization. The free radicals,
which are produced from these sterilization methods, have a life span of about 20 min. Due to this short lifetime, PEEK will not be
considered as a source of secondary electron emission. PEEK is readily visible under MRI because of its natural radiolucency
(Johansson et al., 2016).
PEEK has been incorporated into numerous practices such as dental reconstruction, spinal surgery, and many others as shown in
Fig. 25. A major drawback often observed with titanium and titanium alloy implants is stress shielding. Due to the significant differ-
ence in elastic modulus between titanium and bone the strains that they face when stresses are applied are different. This can cause
bone loss and ultimately implant failure. Recently PEEK has been proposed as an alternative because of its favorable elastic modulus
(Fig. 26). However, PEEK does not have a high resistance to mechanical strength which means it would need to undergo modifi-
cations to better suit the desired functionality. PEEK has also been proposed as the implant material for vertebral fusion surgery
since its stiffness is similar to bone and will allow the graft to heal uniformly. In addition, PEEK is radiolucent which will allow
the doctor to ensure that the implant is properly positioned for optimal healing.
Polyethylene Glycol
Polyethylene glycol (PEG) is a linear or branched polyether with a general structure that is terminated with hydroxyl groups (see
Fig. 27) (Roberts et al., 2002). Some relevant characteristics for its use in the biomedical field include solubility in water and organic
solvents, controlled solubility, nontoxic, and hospitable to biological materials (Harris, 1992). PEG is widely used for biomedical
applications because it is a nontoxic polymer that yields nonimmunogenicity and nonantigenicity. It does not harm active cells or
proteins, and thus it does not cause immunogenic responses when introduced in the body (Alcantar et al., 2000). Tunable
Cranial reconstruction
Hip implants 3D scaffolds
Applications
Bone reconstruction Tooth

replacement
Vertebral body
replacements
Fig. 25 Medical applications of polyetheretherketone.

Temporary PEEK abutments
(A) (B) (C)
PEEK dental implants
(D) (E) (F)
Fig. 26 Examples of PEEK dental implants and temporary abutments (A) PEEK abutment (DENTIN Implants Technologies LTD); (B) PEEK abutment
(Nobel Biocare); (C) PEEK abutment (SGC Dental); (D) PEEK Perso A implant (SisoMM); (E) Win! PEEK implant (Champions Implants); (F) Biopik
(Biopikimplants). Reprinted with permission from Panayotov, I. V., Orti, V., Cuisinier, F., Yachouh, J. (2016). Polyetheretherketone (PEEK) for medical
applications. Journal of Materials Science: Materials in Medicine 277, 1–11.
HO−(CH2−CH2−O)n−H)
Fig. 27 Chemical structure of poly(ethylene glycol) (PEG).
biodegradability is one of the most important characteristics in the biomedical applications of PEG. However, PEG is nonreactive
and it requires functionalization with cross-linked groups in order to create insoluble networks. Addition of acrylate, thiol, amine,
maleimide, or vinyl sulfone reactive groups has been used to functionalize PEG (Zustiak and Leach, 2010). Cross-linked polymerics
networks are used to create hydrogels for biomedical applications such as tissue engineering diagnostics, wound dressing, drug
delivery, and barrier materials to regulate biological adhesions (Ahmed, 2015).
PEG is commonly used in the form of hydrogels. Hydrogels are cross-linked polymer networks characterized by their ability to
absorb and retain water inside their structure (Ahmed, 2015). A hydrogel’s water content makes it ideal for tissue engineering appli-
cations since their physical properties are similar to soft tissues. In one study, a polyethylene glycol (PEG) hydrogel, composed of
PEG vinyl sulfone (PEG-VS) cross-linked with PEG-diester-dithiol, was used to create three-dimensional hydrogel matrices with
tunable mechanical properties and degradation. Influential parameters included molecular weight, polymer density, and the
distance between thiol and ester group in the cross-linker. Additionally, the hydrogels were modified with the fibronectin-
derived cell-adhesive peptide, arginylglycylaspartic acid (RGD), to aid cell viability in 3-D culture, and to show cell responses for
soft tissue regeneration (Zustiak and Leach, 2010) (see Fig. 28). Mahoney and Anseth et al. developed hydrogels by photopolyme-
rizing methacrylate groups covalently linked to PEG macromers. Scaffolds were created in order to support neural cell growth and
function. In their approach, they demonstrated that neural precursor cells tolerate photoencapsulation and culture in PEG hydrogels
with only 10% cell death in 24 h (Mahoney and Anseth, 2006).
Surface coating of materials is a technique used when developing artificial implants. The goal is to produce biocompatible inter-
faces between body fluids and materials such as polymers, ceramics, and metals (Alcantar et al., 2000). PEG is widely used for this
purpose due to its biocompatibility, water solubility. Alcantar, Aydil, and Israelachvili et al. developed a method that allows grafting
of PEG onto activated silica films, which can be deposited in various materials such as metals and plastics. The produced films were
smoother, more hydrophilic, and absorbed less proteins (Alcantar et al., 2000).
Polymethyl Methacrylate
Polymethyl methacrylate (PMMA) is a lightweight, synthetic polymer that is an economical alternative to polycarbonate when
extremely high strength is not necessary. An advantage is that PMMA does not contain potential harmful subunits like
bisphenol-A found in polycarbonate. Moreover, the synthetic polymer is easier to handle, process, and less expensive than polycar-
bonate. In practice, PMMA is often used for craniofacial tissue defects such as skin and dentures (Pielichowski and Njuguna, 2005).
Fig. 28 Cell viability in degradable 3D PEG hydrogels that contained 100 mM RGD. A live/dead assay was preformed for 24 hours following cell
culture, and the findings showed no significant differences in cell viability. Reproduced from Zustiak, S. P., Leach, J. B. (2010). Hydrolytically
degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. Biomacromolecules 115, 1348–1357.
PMMA has great mechanical properties and low toxicity. While being popular for hip-joint transplantations because of its
inert properties, PMMA displays slow degradation (Ganesh et al., 1997). Therefore, creating a polymer blend of polycaprolactone
with PMMA produces a polymer material that is better suited for biomaterial applications. In vitro studies conducted by So-Ra
Son et al. 2013 used MTT assays to examine cytotoxicity and proliferation of MG-63 osteoblast cells on blended scaffolds of PCL/
PMMA (Son et al., 2013). The study found the blended polymer material suitable for osteoblast cell proliferation. Further
evidence of confocal images and expression of proliferation cell nuclear antigen confirmed proliferation and expression of cells
in the 7:3 PCL:PMMA blended polymer environment. PMMA is a nonbiodegradable polymer utilized in applications that require
permanent, mechanically stable structures such as bone tissue regeneration (Jessy and Ibrahim, 2014). At high temperatures, the
long chain backbone of PMMA separates and reacts with itself to change properties. PMMA can undergo thermal degradation and
thermal oxidative degradation in the presence of oxygen, as well as photodegradation, oxidative degradation, and UV degrada-
tion. Thermal degradation causes changes in properties of the polymer such as reduced ductility, chalking, color changes, and
cracking. PMMA depolymerization at 300–400 C produces volatile monomers of methyl methacrylate (MMA) (Holland and
Hay, 2001).
PMMA is used in biomaterial applications such as bone cement, lenses, bone substitutes, and drug delivery systems. It is used to
remove wrinkles and scars on skin tissue permanently. In dental implants, polymer material PMMA is substituted for missing dental
roots. Similar to the physical and mechanical qualities of human dentine, PMMA has a low modulus of elasticity, thermal and elec-
trical passiveness, and ideal porosity (Leigh, 1975).
Conclusion
Biomedical engineering is a board field that mainly represents aspects of biomaterials, bioinformatics, biomechanics, bio-
instrumentation to aid in medicine and health. In this article, we discuss the use of biomaterials that facilitate the interaction
between the body and materials present on the surface level of biomedical devices. This is important in the successful use of
biomedical devices because if the immune system rejects the biomaterials present on the device, the device will fail and cause
a severe immune response in the human body. With advancements in nanotechnology and materials science, there have been
further studies of biomaterials for tissue engineering and drug delivery methods. In this article we discuss both natural and synthetic
polymers used in biomaterials and their characteristics. The polymers examined in this article are collagen, chitosan, silk, bacterial
cellulose, fibrin, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polyetheretherketone (PEEK), polyeth-
ylene glycol (PEG), and polymethyl methacrylate (PMMA). Understanding their properties has allowed biomedical engineers to
make vast improvements in the field of biomaterials, and the lives of patients.
Acknowledgments
Authors acknowledge funding support from the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health
(award number R01EB020640), Connecticut Regenerative Medicine Research Fund (15-RMB-UCHC-08) and the National Science Foundation
Graduate Research Fellowship award in support of this work.
References
Ahmed, E. M. (2015). Hydrogel: Preparation, characterization, and applications: A review. Journal of Advanced Research, 6, 105–121.
Alam, J., Rahman, W., Mazid, R. A., & Khan, M. R. (2015). Gamma-irradiated gelatin-based films modified by HEMA for medical application. International Journal of Polymer
Analysis and Characterization, 20(5), 426–434.
Alcantar, N. A., Aydil, E. S., & Israelachvili, J. N. (2000). Polyethylene glycol-coated biocompatible surfaces. Journal of Biomedical Materials Research, 51, 343–351.
Anderson, M., et al. (2015). Peripheral nerve regeneration strategies: Electrically stimulating polymer based nerve growth conduits. Critical Reviews™ in Biomedical Engineering,
43, 2–3.
Ariëns, R. A., Lai, T.-S., Weisel, J. W., Greenberg, C. S., & Grant, P. J. (2002). Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood, 100(3),
743–754.
Bartis, D., & Pongrácz, J. (2011). In Three dimensional tissue cultures and tissue engineeringTeach. Mater. Med. Biotechnol. Master’s Program, Univ. Pećs Univ., Debrecen, (p. p.
vol. 15).
Beamer, B. S., et al. (2015). Analysis of a new all-inside versus inside-out technique for repairing radial meniscal tears. Arthroscopy: The Journal of Arthroscopic & Related Surgery,
31(2), 293–298.
Beamer, B. S., et al. (2017). Changes in contact area in meniscus horizontal cleavage tears subjected to repair and resection. Arthroscopy: The Journal of Arthroscopic & Related
Surgery, 33(3), 617–624.
Chen, J., et al. (2016). Gelatin microspheres containing calcitonin gene-related peptide or substance P repair bone defects in osteoporotic rabbits. Biotechnology Letters, 1–8.
Chien, C. S., Ho, H. O., Liang, Y. C., Ko, P. H., Sheu, M. T., & Chen, C. H. (2012). Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for
tissue engineering of cartilage. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 100(4), 948–955.
Cho, Y., & Borgens, R. B. (2010). The effect of an electrically conductive carbon nanotube/collagen composite on neurite outgrowth of PC12 cells. Journal of Biomedical Materials
Research Part A, 95(2), 510–517.
Christman, K. L., Vardanian, A. J., Fang, Q., Sievers, R. E., Fok, H. H., & Lee, R. J. (2004). Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and
induces neovasculature formation in ischemic myocardium. Journal of the American College of Cardiology, 44(3), 654–660.
Clark, J. H., & Deswarte, F. (2011). Introduction to chemicals from biomass. Wiley.
Cooper, A., Bhattarai, N., & Zhang, M. (2011). Fabrication and cellular compatibility of aligned chitosan-PCL fibers for nerve tissue regeneration. Carbohydrate Polymers, 85(1),
149–156.
Costa, H. J. Z. R., et al. (2013). Mesenchymal bone marrow stem cells within polyglycolic acid tube observed in vivo after six weeks enhance facial nerve regeneration. Brain
Research, 1510, 10–21.
Danhier, F., Ansorena, E., Silva, J. M., Coco, R., Le Breton, A., & Préat, V. (2012). PLGA-based nanoparticles: An overview of biomedical applications. Journal of Controlled Release,
161(2), 505–522.
Dutta, P. K., Duta, J. D., & Tripathi, V. S. (2004). Chitin and chitosan: Chemistry, properties and applications. Journal of Scientific & Industrial Research, 63, 20–31.
Engel, E., Michiardi, A., Navarro, M., Lacroix, D., & Planell, J. A. (2008). Nanotechnology in regenerative medicine: The material side. Trends in Biomaterials, 26(1), 39–47.
Eo, M. Y., Fan, H., Cho, Y. J., Kim, S. M., & Lee, S. K. (2016). Cellulose membrane as a biomaterial: From hydrolysis to depolymerization with electron beam. Biomaterials Research,
20(1), 16.
Erisken, C., Kalyon, D. M., & Wang, H. (2008). Functionally graded electrospun Polycaprolactone and Beta-Tricalcium phosphate nanocomposites for tissue engineering applications.
Biomaterials, 29(30), 4065–4073.
Eshraghi, S., & Das, S. (2010). Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two-dimensional, and three-dimensional orthogonally
oriented porous architectures produced by selective laser sintering. Acta Biomaterialia, 6(7), 2467–2476.
Fan, H., Liu, H., Wong, E. J. W., Toh, S. L., & Goh, J. C. H. (2008). In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold.
Biomaterials, 29(23), 3324–3337.
Ferguson, S. J., Visser, J. M., & Polikeit, A. (2006). The long-term mechanical integrity of non-reinforced PEEK-OPTIMA polymer for demanding spinal applications: Experimental and
finite-element analysis. European Spine Journal, 15(2), 149–156.
Fu, L., et al. (2012). Skin tissue repair materials from bacterial cellulose by a multilayer fermentation method. Journal of Materials Chemistry, 22(48), 12349–12357.
Fu, L., Zhang, J., & Yang, G. (2013). Present status and applications of bacterial cellulose-based materials for skin tissue repair. Carbohydrate Polymers, 92(2), 1432–1442.
Ganesh, K., Latha, R., Kishore, K., George, B., & Ninan, K. (1997). Stabilization of thermal degradation of poly (methyl methacrylate) by polysulfide polymers. Journal of Applied
Polymer Science, 66(11), 2149–2156.
Geckil, H., Xu, F., Zhang, X., Moon, S., & Demirci, U. (2010). Engineering hydrogels as extracellular matrix mimics. Nanomedicine, 5(3), 469–484.
Grulova, I., et al. (2015). Delivery of alginate scaffold releasing two trophic factors for spinal cord injury repair. Scientific Reports, 5, 13702.
Ha, T. L. B., Quan, T. M., Vu, D., Si, D., & Andrades, J. (2013). Naturally derived biomaterials: Preparation and application. Regenerative Medicine and Tissue Engineering,
247–274.
Harris, J. M. (Ed.). (1992) (1st edn.), XXI. Poly(ethylene glycol) chemistry (p. 385). USA: Springer.
Holland, B., & Hay, J. (2001). The kinetics and mechanisms of the thermal degradation of poly (methyl methacrylate) studied by thermal analysis-Fourier transform infrared
spectroscopy. Polymer, 42(11), 4825–4835.
Holmes, R. E., Cohen, S. R., Cornwall, G. B., Thomas, K. A., Kleinhenz, K. K., & Beckett, M. Z. (2004). MacroPore resorbable devices in craniofacial surgery. Clinics in Plastic
Surgery, 31(3), 393–406.
Hossain, K. M. Z., Patel, U., & Ahmed, I. (2015). Development of microspheres for biomedical applications: A review. Progress in Biomaterials, 4(1), 1–19.
Ige, O. O., Umoru, L. E., & Aribo, S. (2012). Natural products: A minefield of biomaterials. ISRN Materials Science, 2012.
James, R., Manoukian, O. S., & Kumbar, S. G. (2016). Poly (lactic acid) for delivery of bioactive macromolecules. Advanced Drug Delivery Reviews, 107, 277–288.
Jenkins, C. L., Bretscher, L. E., Guzei, I. A., & Raines, R. T. (2003). Effect of 3-hydroxyproline residues on collagen stability. Journal of the American Chemical Society, 125(21),
6422–6427.
Jessy, R. S., & Ibrahim, M. H. (2014). Biodegradability and biocompatibility of polymers with emphasis on bone scaffolding: A brief review. International Journal of Scientific and
Research Publications, 4, 610.
Johansson, P., Jimbo, R., Naito, Y., Kjellin, P., Currie, F., & Wennerberg, A. (2016). Polyether ether ketone implants achieve increased bone fusion when coated with nano-sized
hydroxyapatite: A histomorphometric study in rabbit bone. International Journal of Nanomedicine, 11, 1435.
Kang, H., Liu, R., & Huang, Y. (2015). Graft modification of cellulose: Methods, properties and applications. Polymer, 70, A1–A16.
Kean, T., & Thanou, M. (2010). Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 62(1), 3–11.
Kjaergard, H., & Weis-Fogh, U. S. (1994). Important factors influencing the strength of autologous fibrin glue; the fibrin concentration and reaction timedComparison of strength
with commercial fibrin glue. European Surgical Research, 26(5), 273–276.
Kowalska-Ludwicka, K., et al. (2013). Modified bacterial cellulose tubes for regeneration of damaged peripheral nerves. Archives of Medical Science, 9(3), 527–534.
Kumar, M. N. V. R. (2000). A review of chitin and chitosan applications. Reactive & Functional Polymers, 46, 1–27.
Kundu, B., Raijkhowa, R., Kundu, S. C., & Wang, X. (2013). Silk fibroin biomaterials for tissue regenerations. Advanced Drug Delivery Reviews, 65(4), 457–470.
Kurtz, S. M., & Devine, J. N. (2007). PEEK biomaterials in trauma, orthopedic, and spinal implants. Biomaterials, 28(32), 4845–4869.
Lam, C. X. F., Savalani, M. M., Teoh, S.-H., & Hutmacher, D. W. (2008b). Dynamics of in vitro polymer degradation of Polycaprolactone-based scaffolds: Accelerated versus
simulated physiological conditions. Biomedical Materials, 3(3).
Lawrence, B. D., Marchant, J. K., Pindrus, M. A., Omeneto, F. G., & Kaplan, D. L. (2009). Silk film biomaterials for cornea tissue engineering. Biomaterials, 30(7), 1299–1308.
Lee, K. Y., & Mooney, D. J. (2012). Alginate: Properties and biomedical applications. Progress in Polymer Science, 37(1), 106–126.
Lee, P., et al. (2016). Osteochondral scaffold combined with aligned nanofibrous scaffolds for cartilage regeneration. RSC Advances, 6(76), 72246–72255.
Leigh, J. A. (1975). Use of PMMA in expansion dental implants. Journal of Biomedical Materials Research Part A, 9(4), 233–242.
MacDonald, R. A., Voge, C. M., Kariolis, M., & Stegemann, J. P. (2008). Carbon nanotubes increase the electrical conductivity of fibroblast-seeded collagen hydrogels. Acta
Madihally, S. V., & Matthew, H. W. (1999). Porous chitosan scaffolds for tissue engineering. Biomaterials, 20(12), 1133–1142.
Mahoney, M. J., & Anseth, K. (2006). Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. Biomaterials, 27, 2265–2274.
Makadia, H. K., & Siegel, S. J. (2011). Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymer, 3(3), 1377–1397.
Mano, J. F., et al. (2007). Natural origin biodegradable systems in tissue engineering and regenerative medicine: Present status and some moving trends. Journal of the Royal
Society Interface, 4(17), 999–1030.
Manoukian, O. S., et al. (2016a). Bioactive nanofiber dressings for wound healing. In Wound healing biomaterials–Volume 2: Functional biomaterials (p. 451).
Manoukian, O. S., et al. (2016b). Tissue-engineered medical products. In Biomaterials and nanotechnology for tissue engineering (p. 289).
Manoukian, O. S., et al. (2017). Electrospun nanofiber scaffolds and their hydrogel composites for the engineering and regeneration of soft tissues. Biomedical nanotechnology:
Methods and protocols, 261–278.
Masoudi, A., et al. (2015). Biomechanical evaluation of an all-inside suture-based device for repairing longitudinal meniscal tears. Arthroscopy: The Journal of Arthroscopic &
Related Surgery, 31(3), 428–434.
Mizuno, K., Hayashi, T., & Bächinger, H. P. (2003). Hydroxylation-induced stabilization of the collagen triple helix further characterization of peptides with 4 (R)-hydroxyproline in the
Xaa position. Journal of Biological Chemistry, 278(34), 32373–32379.
Pan, Z., & Ding, J. (2012). Poly(lactide-co-glycolide) porous scaffolds for tissue engineering and regenerative medicine. Interface Focus, 2, 366–377.
Panayotov, I. V., Orti, V., Cuisinier, F., & Yachouh, J. (2016). Polyetheretherketone (PEEK) for medical applications. Journal of Materials Science: Materials in Medicine, 27(7), 1–11.
Peltoniemi, H., et al. (2002). The use of bioabsorbable osteofixation devices in craniomaxillofacial surgery. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and
Endodontology, 94(1), 5–14.
Pielichowski, K., & Njuguna, J. (2005). Thermal degradation of polymeric materials. iSmithers Rapra Publishing.
Rasal, R. M., Janorkar, A. V., & Hirt, D. E. (2010). Poly (lactic acid) modifications. Progress in Polymer Science, 35(3), 338–356.
Roberts, M. J., Bentley, M. D., & Harris, J. M. (2002). Chemistry for peptide and protein PEGylation. Advanced Drug Delivery Reviews, 54(4), 459–476.
Rossi, A. L., et al. (2012). Ultrastructure of regenerated bone mineral surrounding hydroxyapatite–alginate composite and sintered hydroxyapatite. Bone, 50(1), 301–310.
Ruvinov, E., & Cohen, S. (2016). Alginate biomaterial for the treatment of myocardial infarction: Progress, translational strategies, and clinical outlook: From ocean algae to patient
bedside. Advanced Drug Delivery Reviews, 96, 54–76.
Sahoo, S., Toh, S. L., & Goh, J. C. (2010). A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells.
Biomaterials, 31(11), 2990–2998.
Sakaguchi, Y., et al. (2015). Polyglycolic acid sheets with fibrin glue can prevent esophageal stricture after endoscopic submucosal dissection. Endoscopy, 47(04), 336–340.
Sell, S. A., McClure, M. J., Garg, K., Wolfe, P. S., & Bowlin, G. L. (2009). Electrospinning of collagen/biopolymers for regenerative medicine and cardiovascular tissue engineering.
Advanced Drug Delivery Reviews, 61(12), 1007–1019.
Shats, E., Nair, C., & Dhall, D. (1997). Interaction of endothelial cells and fibroblasts with modified fibrin networks: Role in atherosclerosis. Atherosclerosis, 129(1), 9–15.
Shen, W., et al. (2010). The effect of incorporation of exogenous stromal cell-derived factor-1 alpha within a knitted silk-collagen sponge scaffold on tendon regeneration.
Biomaterials, 31(28), 7239–7249.
Shinya, N., et al. (2009). Improvement of the tissue-adhesive and sealing effect of fibrin sealant using polyglycolic acid felt. Journal of Investigative Surgery, 22(5), 383–389.
Shor, L., Güçeri, S., Wen, X., Gandhi, M., & Sun, W. (2007). Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions
in vitro. Biomaterials, 28(35), 5291–5297.
Sidelmann, J. J., Gram, J., Jespersen, J., & Kluft, C. (2000). In Seminars in thrombosis and hemostasis, vol. 26 (06). Fibrin clot formation and lysis: Basic mechanisms (pp. 605–
618). New York, NY, USA: Thieme Medical Publishers, Inc.
Son, S.-R., Linh, N.-T. B., Yang, H.-M., & Lee, B.-T. (2013). In vitro and in vivo evaluation of electrospun PCL/PMMA fibrous scaffolds for bone regeneration. Science and
Technology of Advanced Materials, 14(1), 015009.
Stratton, S., et al. (2017). Polymeric 3D printed structures for soft-tissue engineering. Journal of Applied Polymer Science. https://doi.org/10.1002/app.45569.
Torres, F. G., Commeaux, S., & Troncoso, O. P. (2012). Biocompatibility of bacterial cellulose based biomaterials. Journal of Functional Biomaterials, 3(4), 864–878.
Ulery, B. D., Nair, L. S., & Laurencin, C. T. (2011). Biomedical applications of biodegradable polymers. Journal of Polymer Science Part B: Polymer Physics, 49(12), 832–864.
VandeVord, P. J., Matthew, H. W. T., DeSilva, S. P., Mayton, L., Wu, B., & Wooley, P. H. (2002). Evaluation of the biocompatibility of a chitosan scaffold in mice. Journal of
Biomedical Materials Research, 59(3), 585–590.
Wei Ji, F. Y., Seyednejad, H., Chen, Z., Hennink, W. E., Anderson, J. M., Jeroen, J. A. J., & van den Beucken, J. J. P. (2012). Biocompatibility and degradation characteristics of
PLGA-based electrospun nanofibrous scaffolds with nanoapatite incorporation. Biomaterials, 3, 6604–6614.
Woodruff, M. A., & Hutmacher, D. W. (2010). The return of a forgotten polymerdPolycaprolactone in the 21st century. Progress in Polymer Science, 35(10), 1217–1256.
Yang, Y., et al. (2007). Development and evaluation of silk fibroin-based nerve grafts used for peripheral nerve regeneration. Biomaterials, 28(36), 5526–5535.
Yoshimoto, H., Shin, Y., Teraia, H., & Vacanti, J. P. (2003). A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials, 24,
2077–2082.
Zaborowska, M., Bodin, A., Bäckdahl, H., Popp, J., Goldstein, A., & GAtenholm, P. (2010). Microporous bacterial cellulose as a potential scaffold for bone regeneration. Acta
Zhang, X., Reagan, M. R., & Kaplan, D. L. (2009). Electrospun silk biomaterial scaffolds for regenerative medicine. Advanced Drug Delivery Reviews, 61, 988–1006.
Zustiak, S. P., & Leach, J. B. (2010). Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties. Biomacromolecules,
11(5), 1348–1357.
Further Reading
Lam, C. X. F., Hutmacher, D. W., Schantz, J.-T., Woodruff, M. A., & Teoh, S. H. (2008a). Evaluation of Polycaprolactone scaffold degradation for 6 months in vitro and in vivo.
Journal of Biomedical Materials Research, 90A(3), 906–919.
Biomimetic Approaches for Regenerative Engineering
Nirmalya Tripathy, University of Washington, Seattle, WA, United States
Rafiq Ahmad, Jeong Eun Song, and Gilson Khang, Chonbuk National University, Jeonju-si, Jeollabuk-do, Republic of Korea
Introduction 483
Biomimetic Materials 484
Fibrin 484
Collagen 484
Glycosaminoglycans 485
Self-assembling polypeptides 485
Synthetic Materials 486
Mechanisms of hydrogel formation 486
Hybrid Materials 487
Fabrication of Biomimetic Scaffold 487
Prefabricated preparation of scaffolds 488
Porous scaffolds by conventional approaches 488
Fibrous scaffolds by electrospinning 488
Rapid prototyping-based microfabrication 489
Modular hierarchical assemblies 489
Scaffold Functionalization 490
Biomolecules Delivery 491
Importance of Bioreactors 492
Biomimetic Scaffolds for Regenerative Engineering 492
Bone tissue 492
Cardiovascular tissue 493
Conclusions and Future Prospects 494
Acknowledgments 494
Further Reading 495
Introduction
In general terms, “Biomimetics” aims toward understanding biological fundamentals and implementing for developing bio-
medically relevant technologies and functional biomaterials mimicking physicochemical, mechanical, and biological characteristics
of natural material, for example, for biomedical devices or as scaffolds for tissue regeneration. In regenerative medical therapy, tissue
engineering (TE) approaches direct the cells to differentiate at the desired time and place while maintaining the right phenotype.
Thus a biomimetic approach to the generation of engineered tissues enables the assembly of functional tissues using biologically
derived design requirements. These include synthesis to optimization of certain compositions or properties similar to those of the
extracellular matrix (ECM), novel processing technologies to achieve structural features mimicking the ECM on various levels, strat-
egies to emulate cell–ECM interactions, and biologic delivery strategies. This article outlines current biomimetic materials
approaches in TE, with improved cellular/tissue functions and regenerative outcomes, showing the biomimetic materials signifi-
cance for TE and regeneration. Biomimetic scaffolding materials, natural or synthetic, biodegradable or permanent, are tuned
into 3D architectures suitable for cell seeding and cultivation. Biomaterial choices are mostly guided by the requirement for enough
mechanical strength and restore cell signaling for the tissues. They also act as informational templates for cells by implementing
patterning, ligand binding, and sustained release of cytokines. Each tissue consists of its own challenges for designing scaffold.
For example, bone tissues consist of dense mineralized matrix that can withstand significant compressive loads, requires scaffolds
that are capable for providing mechanically stable framework with interconnected large pores for cell infiltration. Another instance
is cardiac muscle, dense syncytium of electromechanically coupled cells that transmit force and deformations in multiple directions,
requires soft and elastic scaffolds. As well, custom-designed scaffolds were also finding utility in developing tissue models for drug
screening and further disease studies without usage of in vivo models such as 3D printed cardiac tissue, neural constructs for predic-
tive drug screening.

484 Regenerative Engineering j Biomimetic Approaches for Regenerative Engineering
Biomimetic Materials
Natural Materials
Fibrin
Fibrinogen (Fg) is a monomeric and has the 340 kDa molecular weight and aggregates to produce fibrin polymer subsequently
thrombin cleavage. Thrombin acts on fibrinogen to cleave the fibrin peptides on fibrinogen that put off physicochemical polymer-
ization or self-assembly of the molecule. The resulting network is chemically cross-linked by the blood transglutaminase factor XIII
a, and its complex fibril structure and cross-linked character depend upon the details of its formation. Fibrin plays a significant
responsibility in healing and tissue repair in adults, but less so throughout embryonic development. As a result, moderately few
morphogenetic signals concerned in development interrelate particularly with fibrin, although they are most vigorous when immo-
bilized in a 3D matrix. Fibrin has been used as a scaffold protein for the immobilization of adhesion and growth factors. For
instance, heparin-binding growth factors have been integrated to fibrin scaffolds, and bind with heparin, then it is immobilized
via a heparin-binding peptide enzymatically integrated into the fibrin matrix. Fibrin, not like collagen or fibronectin, is not asso-
ciated with mature tissue structure, but somewhat as a temporary repair stage ECM component. This matrix fibrous has been
used in the form of fibrin glue as a tool for surgeons. And the fibrin glue formulations have important amounts of fibronectin which
may unintentionally improve cell migration within the fibrin matrix. Fibrin has been used clinically as a scaffold matrix for the
release of keratinocytes, providing mesenchymal stem cells transfected with growth factors, for spinal cord repair and repair of
peripheral nerves.
Even though fibrin is not an ECM in the common sense, because cells in the local environment do not make it, the material is
nonetheless a crucial member of the body’s repertoire of matrices and serves the function of a conditional matrix, being remodeled
and replaced with ECM molecules. In difference to fibrillar collagen matrices, where cell migration taking both through mechanisms
that are reliant and independent of proteolytic degradation, cell migration in fibrin is almost completely dependent upon cell-
connected proteolytic activity like plasmin and MMPs. This difference in cellular behavior from fibrillar collagen perhaps results
from the slighter mesh size of the fibrin matrices and the stronger fibril–fibril connections, owing to the nature of network forma-
tion and covalent stabilization. A number of proteins are naturally integrated into fibrin matrix throughout coagulation, such as
fibronectin and alpha-2-plasmin inhibitor, and these factors covalently cross-linked into the matrix by factor XIIIa. Other biomol-
ecules, such as FGF-2, bind noncovalently to fibrin and are capable of providing growth factor-specific bioactivity, such as poten-
tiating of endothelial cell (EC) proliferation, even when bound. On the other hand, fibrin matrices remain naturally badly active for
the majority cell types, leading to their functionalization with ECM peptides, recombinant ECM protein domains, or growth factors.
Similar to collagen matrices as described earlier, engineered growth factors or ECM fragments can be included into fibrin nonco-
valently by linking with a fibrin-binding domain to the recombinant protein. For instance, the native knob: pocket connections
throughout fibrin assembly can be demoralized to delay the release of recombinant proteins. However, to have even better incor-
poration and controlled release, biomolecules can be covalently integrated. In a powerful approach, the factor XIIIa enzymatic
substrate sequence of alpha-2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu, can be linked with bioactive peptides or
recombinant proteins, allowing these molecules to be covalently included during the fibrin’s natural polymerization process via
factor XIIIa-stimulated cross-linking. For instance, VEGF, fibronectin fragments, and parathyroid hormone 1–34 have been engi-
neered and covalently included to be released in a manner that depends on local cell-induced proteolysis. In the case of the
PTH1–34 fusion, the hormone is active only when released: modification of its N-terminus with the factor XIIIa substrate fusion
inhibits the activity of the hormone and this activity is regained after cell-induced proteolysis between the fusion and the
PTH1–34 domain. This engineered peptide in fibrin is currently in human clinical evaluation for local bone repair.
Collagen
Collagen is a natural component of ECM existing in many tissues, like skin, bone, tendon, ligament, and other connective tissues.
One of the fundamental hypotheses in collagen research, as connected to biomaterials, is that evolutionary bioengineering has
formed a material that has perfect properties for biological applications. An essential characteristic of this type of biomaterial is
its exceptional assembled structure, widespread happening in nature, and possible to complete humiliate in biological environ-
ments; thus, collagen has been extensively used as a practical biomaterial in TE. Collagen is made up 89% of natural matrix and
32% of the volumetric composition of bone. Collagen scaffolds formed according to an identical protocol have exposed exceptional
biological performance owing to their high porosity and permeability. Earlier studies noted that scaffolds need a high porosity and
surface area combined with a good permeability and pore interconnectivity for cell migration and nutrient perfusion through the
cell culturing procedure. The major drawback of collagen as a scaffold substance for bone TE is that it has comparatively poor
mechanical properties. Collagen type I, the mainly abundant protein in mammals, has a triple helical structure made of three poly-
peptide chains containing repeating Gly-X-Y triplets in which the X and Y positions are regularly engaged by proline and 4-
hydroxyproline, correspondingly, the latter of which is extremely significant for intermolecular hydrogen bonding. Collagen can
be readily purified from skin, tendon, and placenta. This ECM matrix protein can be reconstituted to form a fibrillar matrix by rising
the pH and temperature of a forerunner solution. Broadly used, collagen type I is regularly treated with proteases to get rid of small
nonhelical telopeptides that are located at the ends of the triple-helical domain and that responsible to most of the protein’s
cross-species immunogenic nature. However, for clinical application of this material, concerns of immunogenicity and disease
transmission stay behind. To evade these risks, methods for the recombinant expression of collagen have been urbanized in several
eukaryotic expression systems, and recombinant human collagen types I and III are commercially accessible.
Regenerative Engineering j Biomimetic Approaches for Regenerative Engineering 485
The in vivo application of collagen gels is imperfect by a short age in mechanical strength; many methods have been explained in
order to form collagen matrices that own adequate mechanical proprieties to at least partially oppose cell-induced contraction. For
example, chemical glycation can regulate, in convenient manner, the elastic character of collagen gels. Collagen hydrogels cross-
linked enzymatically or compressed to comparatively high density. Heat and chemical treatments have also been urbanized to
create cross-linked collagen sponges for bone and cartilage repair, even though these materials turn into so thick that they are
no longer actually hydrogels. Fascinatingly, the dipole moment of fibrillar collagen gives rise to the possibility to get everlasting
microscopic and even macroscopic alignment of fibrillar collagen matrices. For example, collagen fibril alignment under a strong
magnetic field has been established to inform special individuality for inducing directed cell migration, such as neurites which culti-
vate preferentially in the route of fibril alignment. Collagens bind with several cellular receptors that alter the cells’ performance;
collagen gel bioactivity can be improved with growth factors that have been engineered to attach the matrix in a noncovalent
manner. Collagen binding sequences resultants from collagenase, von Willebrand factor or fibronectin have been recombinantly
merged to growth factors in order to holdup their discharged from collagen scaffolds. For instance, collagen injection of vascular
endothelial growth factor is capable to recover cardiac function after an acute myocardial infarction.
Glycosaminoglycans
Glycosaminoglycans (GAGs), linear polymers containing two repeating disaccharide units, is a significant part of human physi-
ology. As GAGs are a form of biopolymers, few issues needs to be resolved sequentially for enhancing their wide applicability.
For instance, GAGs and other biopolymers can have dissimilar molecular weights, purity, structure, and even charge distribution
depending on the source. The ECM structural proteins have increased biomechanical and biochemical functions owing to the
long unbranched polysaccharides, the GAGs. These polysaccharides, generally, are components of proteoglycans of the ECM, aside
from in the case of hyaluronic acid, and it is not covalently linked to a protein core and is entwined inside the extracellular space.
These strongly anionic polymers absorb water, which provides compressive strength to the ECM, while the GAGs also directly affect
tissue organization via connections with cell-surface receptors.
Hyaluronic acid (HA) is a linear polysaccharide that contains alternating units of a repeating disaccharide, b-1,3-N-acetyl-D-
glucosamine and b-1,4-D-glucuronic acid. HA is a nonsulfated GAG, and it originates throughout the body, from the vitreous of
the eye to the ECM of cartilage tissues. HA can be modified in many ways to alter the properties of the resulting materials, including
modifications leading to hydrophobicity and biological activity. HA can be altered in several ways to alter the properties of the
resulting materials, including modifications leading to hydrophobicity and biological activity. Hyaluronic acid can be isolated
from animal tissue, such as the rooster comb, and can be biotechnologically formed using Streptococcus bacterium. Because the
material absorbs enormous amounts of water at equilibrium, it forms a hydrogel that is nonfibrillar, and owing to entanglement
connected with its high molecular weight, up to several million Daltons, the gel dissolves only very gradually. Numerous chemical
hyaluronic acid derivatives have been prepared and by controlling the functional group (e.g., pendant hydrophobic groups), the
type of covalent bond (e.g., stable or hydrolytically sensitive), and the cross-linking density, it is promising to create a wide range
of physically various materials. A number of changes of the carboxyl and hydroxyl groups of hyaluronic acid have been created, to
crosslink the material into an elastic gel that resists suspension or rendering the polymer controllably more hydrophobic and thus
less soluble. Hydrogels and hyaluronic acid have been used for various applications, including keratinocyte transfer for dermal
wound healing and chondrocyte transplantation for cartilage repair. Although hyaluronic acid interacts with at least three cell-
surface receptors (CD44, RHAMM, and ICAM-1), its biological activity can be considerably increased by the integration of other
functional biomolecules. For instance, hyaluronic acid gels have been functionalized with peptides or protein fragments resulting
from fibronectin to get better fibroblast proliferation and wound healing. Likewise, cellular infiltration into the hydrogel can be
enhanced by using MMP-sensitive cross-linkers, since hyaluronic acid gel degradation is principally due to hyaluronidase while
cell migration is ambitious principally by the activities of various MMPs.
Self-assembling polypeptides
Inspired by the understanding of protein self-assembly, significant progress has been prepared using supramolecular self-assembly
of biomolecules to form nanofibrillar matrices in situ. These approaches use noncovalent intermolecular interactions to fabricate
higher order structures by self-assembly of oligomeric peptide, nucleotide, and nonbiological amphiphilic building blocks. Many of
these systems necessitate nonphysiologic circumstances for self-assembly, numerous can gel at cell-tolerable conditions. Zhang and
coworkers developed a class of nanofibrillar gels with very high water content (> 99%) cross-linked by assembly of self-
complementary amphiphilic peptides in physiological medium. These ionic self-complementary peptides are characterized by
a periodic repetition of alternating ionic hydrophilic and hydrophobic amino acids. Upon exposure to aqueous solutions with
neutral pH, they form stable b-strand and b-sheet structures, partitioning the side chains to two sides, one polar and the other
nonpolar. They self-assemble to form nanofibers with the nonpolar residues inside and positively and negatively charged residues
forming complementary ionic interactions, like a checkerboard. A number of similar scaffolds have been reported and customized
to deliver growth factors and cells. For instance, gels of RAD self-assembling peptide have been used to encapsulate several growth
factors to accelerate dermal wound healing and myocardial repair.
Self-assembled hydrogel peptides are also being used as a tool for three-dimensional cell culture. For example, several types of
cells like chondrocytes and neural stem cells have been cultured within these scaffolds. However, these gels biomechanically system-
atize cells in a three-dimensional fashion; they show no specific cell interaction because they are not equipped with any specific
biofunctional ligands. To engineer receptor-mediated biospecificity, active and functional motifs from the ECM have been working
to significantly enhance their interactions with cells and tissues. Self-assembling oligomeric amphiphiles permit integration of
specific biomolecular signals without inhibiting the self-assembling properties and nanofiber formation. The scaffolds used for
the laminin-derived peptide Ile-Lys-Val-Ala-Val, encapsulated neural progenitor cells were observed to differentiate into neurons.
Following this trend, a range of self-assembling peptides with various functional motifs such as cell adhesion sites or protease sensi-
tive sequences have been produced. Another type of polypeptides that forms hydrogels is derived from the Val-Pro-Gly-X-Gly penta-
peptide repeat (where X is a guest amino acid other than proline) found in human tropoelastin. Called elastin-like-polypeptides
(ELPs), the chains are soluble in aqueous solution, but as the solution temperature is raised, ELPs become insoluble and aggregate
at a critical temperature, termed the inverse transition temperature. By changing the composition and chain length of guest residues,
the transition temperature can be accurately tuned between 0 C and 100 C for specific applications such as recombinant protein
purification or cell culture. For example, in vitro studies reported that ELPs have been established to support the synthesis and reten-
tion of cartilaginous matrix from encapsulated chondrocytes and adult stem cells. Likewise to self-assembling peptides, ELPs have
also been customized with ECM ligands derivative from fibronectin, in order to support better cell attachment.
Synthetic Materials
The immense opportunities for biomaterials design and functionality enabled by mimicking nature continue to stretch the limits of
imagination. As both biological understanding and engineering capabilities develop, more sophisticated biomedical materials can
be synthesized that have multifaceted chemical, biological, and physical characteristics designed to achieve specific therapeutic
goals. The biological materials defined earlier assist as a point of departure for biomimicry, the use of synthetic materials to summa-
rize salient materials features of natural ECM molecules, such as in situ crosslinking, demonstration of adhesion ligands, binding of
growth factors, and susceptibility to cell-derived proteases. Synthetic analogs have several advantages. The methods used for matrix
formation and functionalization are becoming progressively straightforward and easy to control. Several reactions can be performed
under gentle, repeatedly physiological, circumstances that allow integration of cells or biological molecules with slight loss of func-
tion or viability. The use of completely synthetic materials removes the purification problems that can happen with naturally deriv-
ative materials as well as decreases the potential for an immune response or pathogen transmission. Lastly, polymeric-constructed
hydrogels, which represent a huge class of synthetic materials used for this application, mimic the extremely hydrated viscoelastic
properties of the natural ECM, permit for transport by diffusion and interstitial flow, and can present soluble, affinity-bound, or
covalently bound biological features. Synthetic cell-responsive hydrogels for use in TE can be designed from a number of hydro-
philic synthetic polymers or polysaccharides, with poly(ethylene glycol) (PEG), poly(vinyl alcohol), poly(hydroxyethyl methacry-
late), alginate derivatives, and others, and have been reviewed comprehensively in the literature.
Mechanisms of hydrogel formation

The mechanical and biological influence of hydrogels can be controlled spatially, so specific signals can be exhibited in a pattern that
mimics the morphological gradients found in development and wound healing. The external tools like temperature and light
further expand hydrogel manipulation to the temporal scale, so variations can be made to the structure and demonstration of
biophysical signals on demand. By using light, biological signals can be committed to or released from a hydrogel, and structural
crosslinks can be made or broken to correspondingly harden or deteriorate a material in real time. Light that has entered skin tissue
and reached an underlying hydrogel has been used to expose cell-adhesive peptides, which was found to increase the number of
inflammatory cells and increase fibrosis in a PEG implant. The vinyl sulfone adapted multi-arm PEG macromers (PEG-VS) to permit
a Michael addition reaction among the acrylated PEG and a thiol group, which is utmost often offered as a free cysteine on a peptide
or protein. And the PEG-VS are first functionalized with mono-cysteine species, like cell adhesion ligands, growth factor, and their
ligands. Polymers can be cross-linked into hydrogel networks covalently by numerous mechanisms, which can be largely grouped
into chain-growth polymerizations, such as photopolymerization, and step growth polymerizations, such as Michael addition reac-
tions. An example of photopolymerization contains photo-introduced reactions between PEG diacrylate molecules or among thi-
olacrylates or thiolenes. The functionalized PEG-VS are then cross-linked into a biodegradable gel network consuming peptides that
contain a protease-sensitive substrate sequence lined by cysteine encompassing domains. Michael adding reactions are not restricted
to acrylates and also happen between maleimide and thiol groups, as has been revealed for the crosslinking of a p-maleimidophenyl
altered dextran.
Freshly, sequential copper-free click chemistry has been useful for the creation of hydrogels and their succeeding patterning
with biomolecules. Exactly, an azide can react with an alkyne to practice a triazole using a di-fluorinated cyclooctyne moiety
throughout hydrogel formation. Orthogonal thiol-ene photocoupling such as with peptides comprising the photoreactive allyl
ester Fmoc-Lys-OH can be used to pattern biomolecules. Anseth’s group has used this click chemistry pattern to prepare PEG
hydrogels with MMP-cleavable sequences and patterned combination of Arg-Gly-Asp (RGD) peptides to deliver localized cell
attachment. In addition to Michael adding reactions and click chemistry, step-wise polymerizations can also be attained through
enzyme-catalyzed crosslinking of peptide-functionalized materials. Sperinde and Griffith reported that transglutaminase to cross-
link multifunctional glutaminyl PEG with polypeptides comprising alternating lysine and phenylalanine residues. Otherwise,
a mixture of multi-arm PEG molecules, each conjugated with one of two different counter-reactive peptide substrates for factor
XIIIa, can be cross-linked in the occurrence of this transglutaminase. Comparable to the method used to covalently functionalize
fibrin matrices, biofunctional peptides labeled with one of this factor XIIIa. In addition to the covalent crosslinking mechanisms
defined earlier, hydrogels can also be designed by physical or ionic connections between molecules. This behavior is detected in
the self-assembly of peptide amphiphiles into fibrillar b-sheet structures, as defined earlier, and throughout the complexation of
polymers or polysaccharides through ions.
Hybrid Materials
Two or more materials combined and made composite material by merging often ones that have very dissimilar properties. The
two materials work together to provide the composite unique properties. Though, within the composite you can simply tell the
different materials separately as they do not dissolve or mix into each other. The major benefit of composite materials is that they
are light as well as strong. By choosing a suitable combination of matrix and strengthening material, a new material can be made
that accurately meets the requirements of a particular application. Composites also deliver design flexibility because many of them
can be molded into complex shapes. Nanocomposites, high performance materials, display uncommon property combinations
and unique design potentials. Nanocomposite is a multiphase solid material and it has one of the phases, has one, two, or three
dimensions of < 100 nm, or structures having nano-scale recurrence distances between the different phases that make up the mate-
rial. Nanocomposites are found in nature, for example in the structure of the abalone shell and bone. The use of nanoparticle-rich
materials long predates the understanding of the physical and chemical nature of these materials. Jose-Yacaman et al. investigated
the origin of the depth of color and the resistance to acids and bio-corrosion of Maya blue paint, attributing it to a nanoparticle
mechanism. From the mid-1950s nanoscale organo-clays have been used to control flow of polymer solutions (e.g., as paint vis-
cosifiers) or the constitution of gels (e.g., as a thickening substance in cosmetics, keeping the preparations in homogeneous form)
although the resulting product is more efficient, the raw materials are often expensive. Although most materials used in TE scaf-
folds this far are in their pure form (single component), one polymer often does not meet all the needs for various TE applications.
Natural bone matrix is a typical example of organic/inorganic composite material consisting of collagen and mineral (apatites).
This natural composite material has an excellent balance between strength and toughness, superior to either of its individual
components. As an example of biomimetic scaffolds, polymer/inorganic composite scaffolds will be reviewed below as advanta-
geous scaffolds for bone TE.
The term “composite” is usually referred for those materials in which the different phases are separated on a scale larger than the
atomic, and properties such as the elastic modulus are significantly changed in comparison with those of a homogeneous material.
The engineering of composites and nanocomposites draws on traditional characterization and processing technologies. Research
describing structures comprising nanoparticles appears to trust on methods that are being pushed to the limit of resolution. Nano-
composites contrast from conventional composite materials due to the extraordinarily high surface to volume ratio of the reinforc-
ing phase and its exceptionally high aspect ratio. Similar to the major inorganic component of natural bone, the inorganic
compound such as hydroxyapatite or a calcium phosphate (CaP) in a composite scaffold delivers good osteoconductivity while
the polymer offers the constant structure and design flexibility to attain the high porosity and high surface area, which are essential
for anchorage-dependent cells such as osteoblasts to survive and differentiate. By blending and phase separation techniques, poly-
mer/inorganic composite scaffolds have been established with enhanced mechanical properties and osteoconductivity. The HAP-
comprising scaffolds progress osteoblastic cell seeding consistency and show significantly improved expression of mature bone
marker genes such as osteocalcin (OC) and bone sialoprotein (BSP) above plain polymer scaffolds. Bone tissue formation
throughout the scaffold has been established. Some other groups fabricated poly(lactic-co-glycolic acid) (PLGA)/HAP or PLGA/
PCL/HAP composite scaffolds using a technique called salt leaching. Though the salt-leaching technique has restrictions in making
highly porous and well-connected pore structures, they also constantly established the enhanced osteoconductivity over the PLGA
scaffolds. Another report shows that HAP in the composite scaffolds significantly progresses the protein adsorption capacity,
inhibits apoptotic cell death, and offers a more favorable microenvironment for bone tissue regeneration.
Considering that protein-scaffold and cell-scaffold contacts occur at the scaffold pore surfaces, a biomimetic method has been
established to grow bone-like apatite nanoparticles on prefabricated porous polymer scaffolds in a simulated body fluid to effi-
ciently modify the internal pore wall surfaces with bone-like apatite without changing the bulk structures and properties of the scaf-
folds. The apatite produced via the biomimetic procedure in an SBF is partially carbonated HAP more similar to the natural bone
apatite than the stoichiometric HAP crystals. The moderately carbonated apatites must degrade quicker than the stoichiometric HAP
crystals and assist as a better scaffold constituent in terms of new bone tissue remodeling. The growth of apatite crystals is signif-
icantly affected by the polymer materials, porous structure, and ionic concentration of the SBF as well as the pH value. To addition-
ally mimic the nanofibrous organic collagen and the incompletely carbonated nanoapatite of the bone matrix, macroporous
nanofibrous scaffolds were examined for bone-like apatite deposition. A huge number of nanoapatite particles were designed,
and even a uniform and dense layer of nano-apatite was used to shelter the entire internal pore wall surfaces without clogging
the macro-pores after a sensibly long time of incubation in an SBF.
Fabrication of Biomimetic Scaffold

Previous TE scaffolds encompassing fibrous biodegradable polymer fabrics were formed using textile technologies. There are several
polymers like poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and other semicrystalline can be processed into fibers by textile
technologies. PGA nonwoven scaffold is widely used in TE research. In a living system, cells collect a various signals and instructions
through communication with the adjacent cells within tissues and, more significantly, cellular behaviors are directed by complex
biochemical and biophysical information offered by the ECM, as demonstrated by cell fate determination and contact direction
phenomenon. Originally cells located in a 3D cushioned network are protected against external mechanical stress. Furthermore, the
ECM delivers a proper niche for cell adhesion, migration, proliferation, and differentiation due to molecular connections between
specific cell membrane receptors and signaling cues from surrounding ECM resources. Consequently, for successful tissue regener-
ation at a target site, the architecture of scaffolds must emulate the natural design of the ECM and instruct cellular behavior while
sufficiently housing the cells. TE scaffolds have been arranged using variety of materials and numerous processing conditions;
employment of appropriate fabrication approaches is important for successful regeneration of target tissue.
Prefabricated preparation of scaffolds

Polymeric biomaterial plays a significant role in today’s health care technology. Polymer hydrogels were the leading experimentally
designed biomaterials for human use. Until recently, conventional methods to develop prefabricated scaffolds have targeted mostly
on introduction of open pores and interlinked channels inside biodegradable scaffolds to induce the viability of injected cells.
Several processing techniques have been established to fabricate biodegradable 3D foams for scaffolds. And there are two important
methods, salt leaching and gas foaming, that have been extensively used because these methods are straightforward, cost-effective,
and easy to scale up. Plentiful scaffolds prepared from these techniques have resulted in effective clinical outcomes and are ulti-
mately expected to enter the commercial market. As an alternative, electrospinning permits nanoscaled fibrous design of scaffolds
that mimic functional collagen structures. Electrospun nanofibers are characterized by multifaceted fibrous and interconnective
porous structures, which make it possible to fabricate extremely structured scaffolds for making cellular growth. Microfabrication
techniques are constructed on rapid prototyping (RP) methods including stereolithography, fused deposition modeling, shape
deposition manufacturing, selective laser sintering, and 3D bioplotting, and these are measured as effective directions to fabricate
custom-made scaffolds with a specified anatomical outline for hard tissue regeneration. Newly, modular assembly approaches have
been used to develop biomimicking hybrid tissues with a synthetic architecture loaded with inductive biological signals and spec-
ified cells.
Porous scaffolds by conventional approaches

Hierarchical computational techniques have been used to design of 3D anatomic scaffolds with porous architecture that maintains
the function and mass transport. SFF has allowable fabrication of scaffolds with well-ordered architecture from polymer, ceramic,
hydrogel, and even metal biomaterials. Porous scaffolds have considerably better mechanical properties than scaffolds processed
using other methods. These enhanced mechanical properties are particularly important for bone-TE, which has much greater stiff-
ness and strength than other tissues. However, even for soft tissue, traditional methods are followed to develop scaffolds that are not
sufficient mechanically. The need for enough scaffold mechanical properties, joined with a wide range of scaffold base-material
properties, necessitates the capacity to control scaffold mechanical properties through architecture topology design. Even though
preliminary steps have been made in linking CTD and SFF, future work should determine how closely designed scaffolds can
achieve desired mechanical properties as a function of material and SFF processing method.
Biocompatible and biodegradable polymeric foams have been harnessed as temporal structural supports to regenerate various
tissues such as bone, cartilage, nerve, ligament, skin, and liver. An open porous geometry with interconnected channels is a require-
ment for high density cell growth within the scaffold as well as the mass transport of nutrients, oxygen, and metabolic waste; a high
cell density and efficient mass transport contribute to cell viability, proliferation, and ultimate treatment into functional tissues.
Bioerodible aliphatic polyesters including PLA, PGA, and their copolymer, PLGA, have been extensively used to fabricate biodegrad-
able scaffolds because of their convenient hydrolysis into nontoxic components in vivo. A wide range of biodegradable scaffolds
with diverse morphologies have been fabricated by conventional methods such as porogen leaching, gas foaming, emulsion/freeze
drying, and expansion in supercritical fluid. In particular, a porogen leaching method was primarily engaged to create a highly
porous scaffold with large interconnectivity. In this method, particulate materials at a micrometric level including inorganic salts,
carbohydrates, and paraffin spheres are usually embedded into a polymer/solvent paste, followed by elimination through washing
after solvent evaporation.
The level of porosity and the overall pore size and also the surface properties of the microspheres were delicately tuned. The
mean pore size of the microspheres was adjusted from 7.9 to 29.4 mm by varying PLGA concentration in oil phase during double
emulsion process. Moreover, the hydrophilic amino groups were introduced on the microsphere surface to facilitate the cellular
adhesion and proliferation. When chondrocytes were cultured on the super porous microspheres, large amounts of deoxyribonu-
cleic acids, collagen, and GAGs were produced, implying a high rate of cell growth and expression of a cartilage-specific phenotype.
These porous spherical scaffolds could potentially be used to distribute therapeutic cells to damaged sites by injection.
Fibrous scaffolds by electrospinning

Electrospinning is a well-known technique for the fabrication of nanoscale fibers. It continues to be studied widely due to its range
of advantages such as high surface-to-volume ratio, tunable porosity, and ease of surface functionalization. Certainly, the resulting
fibers are enormously useful for applications in TE, drug delivery, and wound dressings. This is an area with vast potential for
controlled release research. As electrospun fibers mimic the ECM of tissues in terms of scale and morphology, there is the potential
for them to be used as scaffolds. Electrospinning is a flexible tool for developing ultrafine fibers with a wide range of diameters from
a few micrometers to several nanometers. The externally applied Coulomb forces are similar to the mechanical forces applied in
traditional spinning. The processing flexibility of this technique ensures fiber production from a broad range of precursor materials
that consist of synthetic polymers, semiconductors, natural polymers, ceramics, and their combinations. The final product produced
by electrospinning is often in the form of a 2D nonwoven mesh characterized by arbitrarily oriented nano/micrometer-sized fibers.
Electrospun fibrous meshes have attractive characteristics such as a wide surface-to-volume ratio and a highly interconnective
porous architecture, and can be assembled into aligned fibers, making these meshes ideal for application in nanoscaled devices.
Compared to the conservative methods producing porous materials, through past quite a few years, the electrospinning tech-
nique has received more attention since as a simple and powerful means to create nanometer-sized elements. With regard to
biomedical applications such as TE scaffolds, drug delivery carriers, and wound care devices, these fibrous materials allow delicate
modulation of cellular characteristic and fine control of drug release. Because of their structural and morphological resemblance to
the native ECM, electrospun nanofibers have been exploited to attempt to produce an ideal TE scaffold. A multiple range of
synthetic polymers such as biodegradable aliphatic polyesters and native biopolymers, for example, collagen, silk fibroin, chitosan,
alginate, and hyaluronic acid, have been electrospun as biomimetic and temporal substrates to adapt a variety of cellular activities.
For instance, the natural collagen fiber analog generated by electrospinning served as an excellent substrate for EC growth and oste-
oblast differentiation. Electrospun hyaluronic acid also facilitate chondrocyte growth while the cell retaining typical cartilaginous
phenotypes, showing assure in the application of cartilage regeneration. The novel 3D collecting template is based on manipulation
of electric fields and forces to direct the ultimate 3D architecture; this approach generated electrospun tubes with diverse tubular
configurations.
Rapid prototyping-based microfabrication

Rapid prototyping (RP) techniques are defined as automated evidence of each tomographic layer sequence based on automatic 3D
images into the ultimate preferred architecture through additive layer-by-layer (LbL) fashion. And this RP technique is one of the
most accurate and reproducible avenues for regulating the internal pore size, porosity, pore interconnectivity, mechanical perfor-
mance, and overall dimensions of TE scaffolds. These features favor the scaffolds to be biomimetic to native tissues or organs
with regard to shape and mechano-compatibility. Though, microfabricated products frequently lack the bioactivity to stimulate
tissue regeneration. Because product fabrication is frequently implemented at the micrometer scale, integration of nanoscale
features into the microstructure is essential to finely tune cellular responses. Photolithography is a technique to replicate various
types of patterns through exposure of a photosensitive material via photomasking and vastly implemented for fabrication of micro-
electronics. However this approach is limited to photosensitive materials and only appropriate for fabrication on planar surfaces.
Soft lithography produces an optional set of techniques for microfabrication that does not have these restrictions.
Another study reported production of a dual-scale scaffold by combining the processes of RP and electrospinning. In this
process, the microfibrous layer of the scaffolds was first built via the RP process and then polymeric nanofibers were directly depos-
ited onto the microfibrous layer by electrospinning. The subsequent microfibrous layers integrated with electrospun nanofibers
were constantly laminated onto the previously integrated layers so that a 3D hybrid structure could be fabricated. The hybrid
construct consisted of a microfibrous wood pile structure and electrospun nanofibers from different biocompatible materials,
namely poly (–caprolactone) (PCL) and collagen. The dually designed scaffold showed enhanced biological function in terms
of chondrocyte adhesion and proliferation. In another study, a LbL polyelectrolyte assembly system was applied to a RP-based
microstructured scaffold for application in bone TE. Hydroxyapatite and collagen are present in the extracellular components of
natural bone tissue and show osteogenic effects on human mesenchymal stem cells. In the same study, they customized hydroxyl
apatite nanoparticles with catechol-functionalized hyaluronic acid. The personalized hydroxyapatite nanoparticles with a negative
charge and type I collagen with a positive charge were used to generate blocks for LbL assembly, which causes the formation of
a nanocomposite multilayer in RP scaffold surface. While adjusting the amounts of hydroxyapatite and collagen on the surface,
the degree of hMSC differentiation was observed by measuring alkaline phosphatase (ALP) activity and osteoblast-related genes
expression level, including BSP-II, bone morphogenetic protein-2, osteopontin (OP), and OC. ALP activity and the relevant gene
expression increased in a pattern consistent with the levels of the two surface components. The achievement of a biomimetic design
approach that combines biomimetic structures with surface chemistry opens up new frontiers in the fabrication of multifunctional
scaffolds and also offers flexibility in that the integration of other bioactive agents onto the scaffold such as bone growth factors
could additionally promote bone tissue regeneration.
Modular hierarchical assemblies

Until now, producing large engineered tissues using prefabricated scaffolds has been restricted by the nonhomogeneous allocation
of cells within the scaffold and a shortage of vascularization, which leads to cell death in the core of the scaffold. To solve this
problem, the approach of modular TE, which targets to assemble biological modules typically consisting of cell masses or cell/poly-
mer constructs, was introduced. A modular scaffold is developed to maintain flexibility and adaptability for tissue reconstruction so
that large complex tissues can be rebuilt by the connection of simple modular units in several different ways, and different modules
with diverse functionalities can be integrated into the final construct. Multicellular spheroids can be used as modular building units
for TE and were formerly self-assembled into a toroidal 3D structure using tissue liquidity mediated by cell–cell and cell–matrix
interactions. Furthermore, organ printing is a programmed system that uses bio-ink to assemble modules into 3D functional living
structures; for example, multicellular spheroids have been collected with the aid of ECM-mimicking hydrogels. Though, the forma-
tion of a stable and strong bioconstruct is a major challenge for successful implantation and functional retention of the engineered
tissue. Previous study reported that the nanofilaments mentioned earlier were altered with the RGD peptide moiety and self-
assembled with MSCs to formulate composite multicellular spheroids. And these composite spheroids presented improved
hMSC viability and increased level of adipogenic differentiation compared to blank spheroids composed only of cells. These
improved composite periods were used as prefabricated building units for adipose TE. Combinations of a suitable carboxylic acid
and n-butylstannoic acid constitute modular gelation systems, in which the creation of a well-defined “tin-drum” nanocluster subse-
quently underpins the hierarchical assembly of nanostructured fibers, which form self-supporting gel-phase networks in organic
solvents.
Molecular complementarity is crucial in the development of chemical systems and determinations a significant number of
outstanding complications in the emergence of complex systems. All physical and mathematical models of organization within
complex systems depend on nonrandom association between components. Molecular complementarity offers a naturally occurring
nonrandom linker. Most notably, the formation of hierarchically controlled stable modules massively progresses the probability of
attaining self-organization, and molecular complementarity delivers a mechanism by which hierarchically systematized stable
modules can develop. Lastly, modularity based on molecular complementarity creates a means for storing and replicating informa-
tion. Microfibrous templates formed by the RP technique were used to mechanically stabilize the assembled tissue. The microfi-
brous templates were also changed with fibroblast growth factor (bFGF), an angiogenic growth factor, to stimulate
neovascularization. These bioactive templates were consumed to support the 3D assembly of composite spheroids, yielding a biomi-
metic hierarchical assembly with solid multiscaled structures. Implantation of engineered tissue paradigm in the dorsal subcuta-
neous pockets of nude mice causes successful formation of active and vascularized adipose tissue, suggesting that retaining the
recommended tissue contours and adipose tissue function, the crucial hurdles in regeneration of soft tissue were overcome. This
assembly concept and modular design can be extended to reconstruct a variety of multifunctional tissues and organs.
Scaffold Functionalization
Advances in biological and medical sciences, materials science, and engineering have triumphed in the past few decades in
providing valuable implant materials for human tissue repair for millions of patients all over the world. Currently, several materials
were used clinically for implants in orthopedics, dentistry, etc. which include metals, polymers, ceramics, and composites. And these
materials served their roles moderately well; some of the currently used implant materials that were not initially developed for
medical applications have shortcomings in their biological environment. Though many biodegradable polymers such as PLA,
PGA, PLGA, and PCL have been used to create manageable structures for regulating the repair and regeneration of damaged tissues,
active control of cell adhesion and downstream cellular events is still challenging due to the absence of biofunctional ligand that can
instruct cells. As a result, many efforts have been made to integrate bioactive ECM molecules onto the scaffolds’ surfaces by several
modification modalities including noncovalent and covalent binding. ECM proteins can be noncovalently adsorbed onto the
surfaces of scaffolds by electrostatic interaction and van der Waals forces. To coat PLLA and PCL scaffolds fibronectin and laminin
are used and have been shown to increase the cell adhesion. Furthermore, stem cells appear to favorably differentiate into osteo-
blasts depending on the type of ECM adsorbed to the scaffolds. Recently ECM microarray study examined the effects of combination
of ECM molecules like collagen-I, III, IV, laminin, and fibronectin on selective differentiation of embryonic stem cells, and they also
emphasized the importance of ECM proteins as immobilized biological cues on scaffolds. Although, physical adsorption is
a comparatively weak force and may not be suitable for TE applications for which continued signaling are required. As a substitute,
covalent immobilization of bioactive molecules has been attained by surface etching and plasma/gamma-ray treatment, which
involves the cleavage of a degradable polymer backbone to generate carboxyl (–COOH) groups or radicals. These functional groups
can undertake additional reactions with ECM proteins and related bioactive peptides (DGEA, RGD, IKVAV, YIGSR) on the surfaces
of several scaffolds including PVDF, PLLA, PCL, and PLCL. The covalently conjugated molecules have been shown to be stable under
physiological conditions and prolonged implantation in the localized microenvironment maintains the biological activity. Origi-
nally, these bioactive ligands enhanced cell adhesion to the surface and were involved in cell-specific signaling.
Hydroxyapatite deposition technique on nanofibers using the CaP dipping method by mineralization. Unlike plasma-spraying
technique, it does not include high temperatures, thereby overcoming the disadvantages attributed to the high temperatures used
during the process, such as the possibility of fracture at the interface between the titanium and the hydroxyapatite due to the residual
stress at the interface, and variations in the porosity, composition, crystallinity, and structure of the plasma-sprayed hydroxyapatite.
So, new hydroxyapatite coating approaches have attracted great attention in recent years for substituting the plasma spraying high
temperature techniques. For increasing the process of osseointegration, it is important that the initial cell capture ratio is high.
Another work demonstrated that it is possible to capture MSCs on a substrate such as allograft bone by raising a system where it
was able to capture MSCs on allograft bone with an enrichment factor of 3–4X at best. Fibronectin conjugated to PLLA, PCL,
and silicone rubber scaffolds significantly improved the survival of osteoblasts, chondrocytes, and myoblasts, while collagen-
modified PLLA scaffolds eased the chondrocytes proliferation. RGD peptide-immobilized scaffolds enabled the differentiation of
osteoblasts and the myogenic differentiation of muscle precursor cells. Likewise, IKVAV and YIGSR have been described to regulate
neural and EC differentiation. The density and distribution of bioactive molecules presented from the surface must be optimized for
appropriate control of cell behavior. A high density of bioactive molecules on the surface is connected to greater cell spreading, focal
contact formation, and proliferation to a certain extent, although cell migration is relative to protein density on the surface in a bi-
phasic manner. Hence, a nominal amount of signaling peptide can be commonly used for surface immobilization and another
study demonstrated that as low as 1 and 10 fmol cm 2 of RGD peptide was sufficient for cell spreading and focal contact formation.
Furthermore, the peptide density and the distribution on the surface regulate integrin clustering. The size of integrins is approxi-
mately 10 nm and their clustered complex structure is < 100 nm; the distribution of peptides for cell-adhesive mimetics should
be considered on a nanometer scale. Newly, an adaptable technique to immobilize bioactive molecules based on mussel adhesive
proteins was reported. To establish the effectiveness of this technique, dopamine was polymerized onto metals, polymers, and
ceramics, and then secondary ligands, for example, ECM bioactive molecules, were covalently immobilized to the polydopamine
layer. Likewise, another study employed hybrid mussel adhesive protein attached with short ECM bioactive domains by recombi-
nant DNA technology. These methods are effective in that surface immobilization can be carried out irrespective of the chemical
composition of the materials.
Biomolecules Delivery
Existing TE approaches focus on restoring impaired tissue architectures using biologically active scaffolds. The perfect scaffold would
mimic the ECM of any tissue of interest, stimulating cell proliferation and de novo ECM deposition. A plethora of techniques have
been assessed to engineer scaffolds for the organized and targeted release of bioactive molecules to deliver a functional structure for
tissue growth and remodeling, in addition to improve recruitment and proliferation of autologous cells within the implant. The
controlled supply of growth factors and cells within biomaterial carriers can increase and accelerate functional bone formation.
The carrier system can be designed with preprogrammed release kinetics to provide bioactive molecules in a localized, spatiotem-
poral manner which is similar to the natural wound healing process. The carrier can also act as an ECM-mimicking substrate for
stimulating osteoprogenitor cellular infiltration and proliferation for integrative tissue repair. Growth factors are key molecules
that stimulate intracellular signal cascades required for tissue regeneration and also integrated into scaffolds where they can deliver
positive biochemical signals ranging from promotion of mitogenic activity to stimulation of neovascularization. The growth factors
used for creation of engineered tissues are mainly dependent on the target tissue types to be regenerated. Epidermal growth factor,
transforming growth factor-b, fibroblast growth factors, and platelet-derived growth factor are frequently used to accelerate wound
healing because of their mitogenic induction of epithelial cells and fibroblasts in addition to upregulation of matrix formation, for
example, chemical immobilization of recombinant human EGF (rhEGF) on an electrospun scaffold was effectively engaged for
wound healing. The rhEGF-adapted nanofibrous scaffold preserved the growth factor activity even under harsh conditions such
as enzymatic degradation, subsequently inducing keratinocyte differentiation. Bone morphogenic protein-2 and TGF-b are often
essential to stimulate and preserve tissue-specific properties for regeneration of hard tissues such as bone and cartilage.
Generally used methods for combining growth factors and other cues involve covalently tethering them to the scaffold material
these methods have been used before to stimulate ES cell differentiation and survival. Although such strategies are extremely effec-
tive at retaining such cues inside scaffolds, covalent linkages can impede internalization and, in order, affect the activity and func-
tion of growth factors. Affinity-based supply systems have been discovered as alternative methods of retaining growth factors inside
scaffolds via noncovalent interactions. Growth factor is a soluble signaling molecule that binds with transmembrane receptors on
target cells and controls variety of cellular functions. The ultimate biological response elicited from a growth factor depends on the
uniqueness of the growth factor and target cell, receptor type, cell number, and other signaling events. So, an important component
in designing an organized delivery system is selection of the appropriate single or combination of growth factors for maximized
tissue repair.
The TE scaffolds can be fixed with or without embedded cells. Previous study reported that cell-loaded scaffolds displayed
adverse effects leading to reduced healing processes as a result of immune responses activated by the ECM materials secreted
from the preseeded cells. The use of cell-free scaffolds may be more needed in certain cases. When using scaffold alone method,
the scaffold has to be particularly adapted with bioactive molecules to induce the infiltration of peripheral progenitor cells or multi-
potent stem cells. The regenerative conductivity or inductivity directed by the scaffold is mainly due to the composition of the mate-
rial, which can distribute biochemical cues such as growth factors. For bone lesions repair, growth factor releasing scaffolds have
been engaged to ameliorate osteogenesis and initiate the vascularization, both of which can be controlled by the dose and release
kinetics of the growth factors. Particularly, collagen sponges combining recombinant human BMP-2 (rhBMP-2), advertised as the
INFUSE Bone Graft, are clinically used for treating degenerative disc diseases. In a human clinical trial, the rhBMP-2/collagen
sponges showed more reliable osteoinduction than autogenous bone grafts.
The lack of autologous cell sources for tissue reconstruction has led to attempts to use pluripotent or multipotent stem cells.
Exactly, MSCs are most often used as another cell source for reconstructing connective tissues such as cartilage, bone, adipose,
and tendon. Some studies that have used progenitor cells or stem cells for TE have stated that scaffolds loaded with growth factors
result in a high degree of cell differentiation. Characteristically, continued release of growth factors and a short diffusion distance
between abound growth factor and a target cell are mandatory to maximize the activity of the growth factors because of their short
biological half-lives in the body, for example, gelatin microspheres loaded with TGF-b were used to stimulate the assembly of
hMSCs into cellular aggregates. Dispersion of TGF-b released from microspheres into the hMSCs of cellular aggregates induces chon-
drogenesis, as measured by the amount of DNA, GAG, and collagen type II formed. In addition, two types of electrospun nanofibers
with dissimilar bFGF release profiles and surface hydrophilicity were matched by measuring the differentiation level of hMSCs.
Stem cells grown on electrospun fibers for fast release of bFGF and a more hydrophilic surface presented better collagen production
and upregulated gene expression, consistent with fibroblastic differentiation, suggesting that the release kinetics of the growth factor
as well as the surface properties of the scaffold could change stem cell fate. Moreover, proper infiltration of blood vessels permits
long-term survival of the surrounded tissue construct by stimulating transport of indispensable nutrients, oxygen, and cell-signaling
molecules. Angiogenic growth factors including acidic or basic FGF, vascular endothelial growth factor, angiopoietins, and PDGF
can be combined into scaffolds to facilitate host tissue in growth and induce angiogenesis.
PLGA foams fabricated by using solvent casting with salt leaching or supercritical carbon dioxide have been engaged to straight
encapsulate bFGF. Protein release was fairly continuous, but an initial burst and comparatively low loading competence were
unavoidable. So, heparin and heparin sulfate have been demoralized to immobilize bFGF on the surfaces of various scaffolds
on the basis of the strong binding affinity between heparin and bFGF. The heparin facilitated supply of bFGF has separate advan-
tages in terms of protection of early degradation of growth factors and facilitation of cell recognition, eventually resulting in better
angiogenesis. Combination of chemically heparinized foams and bFGF or VEGF have showed to be an effective delivery platform
for growth factors, with steady and continued release of growth factors over a stretched period and consequently superior angiogenic
activity. Heparinized electrospun fibers also displayed a high degree of bFGF surface immobilization and more prolonged release of
the growth factor than that of bare electrospun fibers. Likewise, laminin and bFGF were simultaneously adsorbed on heparinized
nanofibers. The combination of immobilized biochemical cues, that is, laminin and bFGF, as well as nanofiber alignment, syner-
gistically induced oriented neurite outgrowth and cell migration. Furthermore to localized introduction of a single growth factor,
distribution of multiple growth factors in immediate or sequential release mode can enhance treatment efficiency. A polymeric
system for dual growth factor delivery was established for therapeutic angiogenesis. Different release kinetics was detected for
VEGF-165 and PDGFBB provided from a single PLGA scaffold; implantation of this scaffold induced the rapid formation of a mature
vascular network in a rat model.
Importance of Bioreactors
In regenerative medical therapy, TE approach has been proposed to fulfill the neotissues demand via in vitro tissue regeneration. In
this scenario, bioreactors are the key strategy to translate cells/tissue-based constructs under a controlled environment, into low-
cost, large-scale products that are clinically safe and biologically effective. Several experimentation conditions can be optimized
including temperature, pH, oxygen tension and perfusion of cells, external stimuli, etc. in order to provide physical and biochem-
ical regulatory signals desired for cellular proliferation and differentiation. For instance, in vivo cells will respond to mechanical
stimulation which bioreactors can supply, aiding cells to produce ECM within shorter time span under optimal mechanical stiff-
ness. The mechanical stimulation has also been shown to stimulate stem cells down different lineages, enabling the formation of
different cell types. The bioreactors’ designs are specific to desired tissue and application; however some of the common basics
should be followed: (i) They should be easily assembled and efficient in tissue formation in a short time span while keeping
the products sterile. (ii) The materials must be nontoxic which will be employed in designing bioreactors. This has ruled out
most metals as they have the potential of releasing ions into the media which could be highly toxic to cells in most cases. (iii)
The bioreactor should come with a sensor for accurate monitoring of culturing conditions. The principle of an in vivo bioreactor
is to implant a well-designed TE scaffold with or without cell seeding onto a well-vascularized site of a living organism and harvest
when matured. However, future trends of in vivo bioreactors lie with implantable devices which are able to monitor and control
the in vivo culturing condition and scaffolds with higher biocompatibility that is suitable for long-term implantation with
minimal rejections.
Biomimetic Scaffolds for Regenerative Engineering

Bone tissue
Bone formation involves three types of bone cells, that is osteoblasts, osteocytes, and osteoclasts that begin with sequential events
of proliferation, differentiation, matrix formation, and finally mineralization. Importantly, bones are made of remarkably strong
and flexible tissues that are capable to remodel itself perpetually. However, to cure/repair any damage/defects TE has emerged as
an extraordinary approach for the treatment with the use of designed nanostructured materials, especially osteoconductive
biomaterial scaffolds along with osteogenic cell populations and osteoinductive bioactive factors. Mainly, biomimetic scaffold
materials have been used to understand the osteoblastic differentiation of progenitor cells. These ideal biomaterial scaffolds
should act as a carrier for cells and growth factors and also should induce formation of bone with surrounding tissues after
implantation. In search of perfect biomaterial, various biomaterials including CaP ceramics, bioresorbable polymers, bioactive
glasses, functionalized hydrogels, and composites are extensively used to hold cells for bone regeneration. Recently, it has
been found that the ECM glycoproteins are expressed during new bone formation and OP, thrombospondin, and BSP as matrix
proteins were important in bone cell migration, proliferation, matrix deposition, and mineralization. Hence, biomimetic scaf-
folds’ integration into bone tissue takes place at the material–tissue interface, which results in initial cell and substrate interac-
tions. Thus, it is important to design bone matrix proteins modified biomimetic scaffolds for excellent cell–matrix interactions to
promote bone growth.
Initially, incorporation of bioactive peptides protein fragments, that is arginine–glycine–aspartic acid (RGD) is recognized by the
cell’s transmembrane integrin receptors, which is the most common adopted strategy to enhance functionality. The composite of
RGD and rosette nanotube (RNT) was prepared with a unique surface chemistry with favorable cytocompatibility properties for
bone repair. In this study, RNT’s incorporation into a biocompatible hydrogel resulted in a biologically inspired 3-D nanoscale scaf-
fold with RGD-functionalized self-assembled biomimetic features, which was more suitable for 3-D bone regeneration. Moreover,
most of the studies were reported utilizing RGD modification on traditional materials on 2-D flat substrates. RGD-containing
peptides bound to neonatal calvarial osteoblasts using membrane receptors were reported. The effect of RGD peptides was
monitored on the specific interaction with bone-derived cells in vitro, where a 15 amino acid sequence containing RGD derived
from BSP was immobilized on glass plates. The seeded osteoblasts on the modified surfaces for 2 h showed enhanced cell attach-
ment strength and promoted focal contact formation. In a study, it was reported that osteoblast adhesion for 4 h in serum-free
medium was enhanced only on an Arg-Gly-Asp-Ser (RGDS) immobilized surface; however the proliferation rate after 3 days on
peptide-modified surfaces was not significantly different than on control surfaces. They also showed the effect of the growth factor
OP-1 in conjunction with a cell-binding peptide. OP-1 exhibited synergistic enhancement of mineralization of the osteoblasts
cultured on RGD-modified surfaces. Additionally, the translation of these results in vivo remains challenging since a number of
ECM proteins are involved in wound healing and bone forming processes.
Biomimetic properties of biodegradable polymers were enhanced by chemical modification with RGD-containing peptides.
Previously, silk-based materials were used for modification of polymers with RGD-containing peptides to investigate the effect on
the long term growth of human osteoblast like cells. The modified silk-based scaffolds showed that immobilized peptides
promoted expression of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) proteins and calcified bone
nodule formation compared to nonmodified control surfaces. In another report, biodegradable polymers of PLA and PLGA
were coupled with RGD peptides using poly(L-lysine) and fabricated as scaffolds for the growth of osteoblasts. The designed poly-
mers showed enhanced initial cell attachment and also showed significant expression of osteogenic phenotype. RGD peptides
modification over biodegradable polymers is advantageous and more preferred over the other model surfaces, that is quartz,
glasses, and metals. On the other hand, synthetic biodegradable polymers are getting more attentions due to their specific inter-
actions of incorporated peptides with activated receptors and also useful to study osteoinductive abilities through in vivo
implantation.
To overcome the challenges faced during the application of biomimetic scaffolds to bone defects with an irregular shape, RGD
peptides were further tethered to unsaturated polyester and subsequently crosslinked to form solid constructs, which showed poten-
tial as injectable application. Additionally, covalently linked biomimetic hydrogels with RGD peptides synthesized using various
macromolecular structures presented good bioactive signals and also showed the capability of modulating marrow stromal osteo-
blast adhesion. Recently, heparin-binding domain in FN was used as a substitute for RGD peptide sequences, which has ability to be
recognized by polysaccharide molecules in the cell membrane and hence can be used to design biomimetic materials for bone TE. In
recent report, pectin-based injectable biomaterials were used for bone TE. They used pectin for the first time to modify RGD-
containing oligopeptide and used as an ECM alternative for bone tissue regeneration. The immobilized MC3T3-E1 preosteoblast
cells viability and differentiation were monitored through viability, metabolic activity, morphology, and osteogenic differentiation
tests. The results showed that preosteoblast cells immobilized in both types of pectin microspheres maintained a constant viability
up to 29 days and were able to differentiate. Also, grafting of the RGD peptide on pectin backbone induced improved cell adhesion
and proliferation within the microspheres. The cells were grown inside and outside of the microspheres and organized themselves in
the 3D structures that produced a mineralized ECM, suggesting the potential of pectin as an injectable cell vehicle for bone tissue
regeneration. More recently, functionalized d-form self-assembling peptide hydrogels were utilized for bone regeneration. They
demonstrated the utilization of functional motif RGD to modify peptide D-RADA16 for designing and fabricating peptide D-
RADA16-RGD. Also, they incorporated the angiogenic growth factor bFGF into these peptide hydrogel scaffolds. Finally, they
applied the self-assembling peptide D-RADA16-RGD hydrogels (with or without bFGF) to repair the bone defects of SD rat femoral
condyle and evaluated the osteogenic ability of D-RADA16-RGD hydrogels. The obtained results showed enhanced extensive bone
regeneration.
Cardiovascular tissue
The aim of cardiac TE development is the development of bioengineered biomimetic materials to facilitate and provide the phys-
ical support and help cardiac regeneration. Mainly, the removal of the damaged cardiac tissue is done by replacing certain func-
tions of the damaged ECM that avoids adverse cardiac remodeling and dysfunction after myocardial infarction. However, the
cardiac regeneration is facilitated with the help of biomimetic materials that support cardiomyocyte differentiation and expansion
both in vivo and in vitro, support and protect injured cells into the heart and repair them, and guide the formation of regenerative
heart tissues through engineered and/or native regeneration. For effective regeneration of cardiac tissues, various critical factors
(i.e., replacement of cardiomyocytes, support of electrical conduction, and mechanical force of contraction) must be maintained
or established.
Presently, nanotechnology has offered tools to design or bioengineer various biomimetic materials that mimic nature or draw
inspiration from the nature. These designed biomimetic materials are extensively explored in the field of cardiovascular science and
provided opportunities for cellular transplantation using polymeric scaffolds to repair damaged cardiac tissue. Numerous studies
were reported to explore the ability of biomimetic materials for creating tissues to support different functions. For treating patients
with valvular cardiovascular disease, mostly heart valve augmentation or replacement methods are utilized. However, such valve
should be designed using noninflammatory, biocompatible, and nonthrombogenic materials which can grow in pediatric patients.
Additionally, the mechanical and structural properties are two important factors needed for heart valve scaffold material replace-
ment. For all these problems, biomimetic materials development is necessary. To replace the damaged native tissues during cardiac
or peripheral bypass surgery, synthetic cardiovascular grafts are used when there is limited availability of healthy autologous tissues/
vessels. The lower patency rate of synthetic vascular grafts as compared to natural tissues is mainly due to thrombus formation and
intimal hyperplasia following the implantation. However, difference in the mechanical compliance of synthetic grafts compared to
the surrounding native tissues caused other complications to patients. Another important limitation is small diameter (< 6 mm) of
synthetic grafts which causes increased failure rate. Hence, attempts are made to design the vascular grafts with enhanced mechan-
ical stability and biocompatibility. The functional importance of biomimetic cardiovascular grafts relies on the anatomical structure
and the biological function of blood vessels. These blood vessels are made of three different tissues layers which play critical role in
blood circulation, maintain tensile strength and viscoelasticity of the blood vessel due to collagen and elastin structural compo-
nents, and provide smooth tissue with long-term durability. Other elements that present an ideal vascular graft need to be designed
with having important properties, that is, infection resistance, bio-compatible, suturability, and off-the-shelf availability.
Endothelial cells (ECs) seeding is another approach in which vascular grafts are engineered onto synthetic materials to render the
luminal surface antithrombogenic. In this approach foreign cells cannot be used as they may elicit an immune response. To avoid
this drawback, patient’s own cells are harvested for seeding of the graft lumen before implantation. Typically, cell culture techniques
are also introduced in case of limited supply of ECs. However, transplanted ECs have drawback of detachment from the surface
during the surface exposure to blood circulation. The biomimetic materials mimicking the fibrillar architecture of the ECM have
been used to modify the surface of cardiac specific cells (vascular grafts). The phase separation, electrospinning, and self-
assembly methods are used to develop the nano and submicron polymeric fibers. Recently, the fibronectin and RGD-containing
peptides were coated on an expanded polytetrafluoroethylene (ePTFE) in order to improve ECs attachment. In this case, the fibro-
nectin was properly adsorbed to the polymer surface and RGD-containing peptides were covalently immobilized. The saphenous
vein ECs were seeded on coated ePTFE for 30 min and exposed to the shear stress in an artificial flow circuit, which showed
enhanced cell attachment and retention by coating with fibronectin as well as immobilizing RGD-containing peptides. Saphenous
vein ECs incubated on the RGD peptides modified ePTFE surface showed excellent cell attachment and retention. In another study,
biomimetic polyurethane-based vascular grafts were designed that showed poor ECs adhesion by themselves. Hence, RGD-
containing sequences incorporated were introduced into polyurethanes using chemical and photochemical modification methods.
Improved ECs adhesion and proliferation were observed with these RGD containing sequences modified polyurethanes. However,
RGD modification might increase platelet adhesion during blood circulation in the human body due to platelets also expresses
integrin receptors recognizing RGD sequences. On the other hand, platelet adhesion to RGD-modified surface may also reduce
the patency rate of the vascular graft; thereby ultimately it will lead to the failure of the graft. The signaling peptides, that is Tyr-
Ile-Gly-Ser-Arg (YIGSR) and Arg-Glu-Asp-Val (REDV) were explored due to their specific interactions with ECs to enhance the endo-
thelialization that is needed for a nonthrombogenic vascular surfaces generation. In a study, YIGSR was immobilized on the surface
of poly(ethylene terephthalate) and glass to improve the EC attachment and spreading. The migration of ECs study showed signif-
icant increase in the persistence of cell movement that resulted in the random motility coefficient of ECs. Further, REDV peptides
derived from III-CS domain of human plasma fibronectin were used to study the interaction of a receptor present in ECs which was
due to the peptide surface modification that resulted in good attachment of ECs and spreading.
The vascular grafts were modified to modulate ECM protein production during tissue formation and control cellular interac-
tions. The ECM protein production is critical to generate robust vascular grafts, tissue viability, and provide mechanical integration
with adjacent tissues. Additionally, the produced ECM must substitute fabricated biomimetic materials for myocardial regeneration
and new tissue formation. As it is reported, ECM production by SMCs and ECs is dependent on the density and type of signaling
peptides attached onto the glass surfaces. This process can be assessed by the incorporation of 3H-glycine to the expressed proteins.
Study showed that the initial cell adhesion on modified surfaces with Val-Ala-Gly-Pro (VAGP), RGDS, Val-Ala-Val-Ala-Gly-Pro
(VAVAGP), and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) was increased. However, the total matrix production was decreased on these
surfaces after 7 days as compared to nonmodified surfaces. Further, the ECM production was increased with the addition of TGF-b in
the media. This result demonstrated that growth factors and signaling peptides are needed in order to optimize matrix production.
Growth factor such as TGF-b1 was also tethered to PEG-based hydrogels through covalent binding and hence increased hydroxypro-
line production was obtained compared to unmodified hydrogels.
Conclusions and Future Prospects
With nanoscience and nanotechnology driving the revolution in the field of materials science and engineering, enables to design
and fabricate novel biomaterial scaffolds incorporating various biomimetic features at the nanometer scales, molecular, and genetic
level. Owing to the complex and dynamic nature of cell responses toward regulatory signals, imaging of engineered biomimetic
constructs during cultivation immensely improves our understanding of cellular responses to micro-environmental parameters
with spatial and temporal specificity. Thus, with biology providing fundamental design requirements for regenerative medicine,
engineered tissues can acts as models for developmental studies, disease modeling, toxicity screening, and many more.
Acknowledgments
This research was partly supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) (grant
number: 2012M3A9C6050204) funded by the Korean government (MEST) and Technology Commercialization Support Program, Ministry for Food,
Agriculture, Forestry and Fisheries (grant number: 814005-03-3-HD020), Republic of Korea, and as well supported by a grant of the Korea Health
Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (grant number: HI15C2996), funded by Ministry of
Health&Welfare, Republic of Korea.
Further Reading
Amini, A. R., Laurencin, C. T., & Nukavarapu, S. P. (2012). Bone tissue engineering: recent advances and challenges. Critical Reviews in Biomedical Engineering, 40, 363–408.
Cordonnier, T., Sohier, J., Rosset, P., & Layrolle, P. (2011). Biomimetic materials for bone tissue engineering – state of the art and future trends. Advanced Engineering Materials,
13, 135–150.
Currie, L. J., Sharpe, J. R., & Martin, R. (2001). The use of fibrin glue in skin grafts and tissue engineered skin replacements: a review. Plastic and Reconstructive Surgery, 108,
1713–1726.
Dhivya, S., Ajita, J., & Selvamurugan, N. (2015). Metallic nanomaterials for bone tissue engineering. Journal of Biomedical Nanotechnology, 11, 1675–1700.
Dunn, D. A., Hodge, A. J., & Lipke, E. A. (2014). Biomimetic materials design for cardiac tissue regeneration. WIREs Nanomedicine and Nanobiotechnology, 6, 15–39.
Gong, T., Xie, J., Liao, J., Zhang, T., Lin, S., & Lin, Y. (2015). Nanomaterials and bone regeneration. Bone Research, 3, 15029.
Haghi, A. K., Oluwafemi, O. S., Josmin, P., & Hanna, J. M. (2013). Composites and nanocomposites. Apple Academic Press, 230.
Jennifer, P., Mikael, M., & Jeffrey, A. (2010). Biomimetic materials in tissue engineering. Materials Today, 13, 14–22.
Liu, Y., Luo, D., & Wang, T. (2016). Hierarchical structures of bone and bioinspired bone tissue engineering. Small, 12, 4611–4632.
Meyer, R. A., Sunshine, J. C., & Green, J. J. (2015). Biomimetic particles as therapeutics. Trends in Biotechnology, 33, 514–524.
Peter, X. (2008). Ma biomimetic materials for tissue engineering. Advanced Drug Delivery Reviews, 60, 184–198.
Prabhakaran, M. P., Venugopal, J., Kai, D., & Ramakrishna, S. (2011). Biomimetic material strategies for cardiac tissue engineering. Materials Science and Engineering: C, 31,
503–513.
Pradhan, S., Hassani, I., Clary, J. M., & Lipke, E. A. (2016). Polymeric biomaterials for in vitro cancer tissue engineering and drug testing applications. Tissue Engineering Part B:
Reviews, 22, 470–484.
Sears, N., Dhavalikar, P., Whitely, M., & Cosgriff-Hernandez, E. (2017). Fabrication of biomimetic bone grafts with multi-material 3D printing. Biofabrication, 9, 025020.
Shotorbani, B. B., Alizadeh, E., Salehi, R., & Barzegar, A. (2017). Adhesion of mesenchymal stem cells to biomimetic polymers: a review. Materials Science and Engineering:
Materials for Biological Applications, 71, 1192–1200.
Shoulders, M. D., & Raines, R. T. (2009). Collagen structure and stability. Annual Review of Biochemistry, 78, 929–958.
Stevens, M. M. (2008). Biomaterials for bone tissue engineering. Materials Today, 11, 18–25.
Taek, G. K., Heungsoo, S., & Dong, W. L. (2012). Biomimetic scaffolds for tissue engineering. Advanced Functional Materials, 22, 2446–2468.
Titorencu, I., Albu, M. G., Nemecz, M., & Jinga, V. V. (2017). Natural polymer-cell bioconstructs for bone tissue engineering. Current Stem Cell Research & Therapy, 12, 165–174.
Bioreactors: System Design and Application for Regenerative Engineering
Antonio Valdevit, Stevens Institute of Technology, Hoboken, NJ, United States; and SEA Limited, Columbus, OH, United States
Introduction 496
Bioreactor Considerations 497
Biological Element Selection 497
Biological Environment 497
Mechanical Environment 497
Fluid Flow 497
Gas/Fluid Pressure 498
Substrate Selection 499
Basic Description and Operation for Design of Feedback-Controlled Bioreactor Employing Direct Mechanical Loading 501
Single-Cell Loading Bioreactor 506
Optimization of Loading 508
Compressive Loading and Imaging 509
Further Reading 512
Nomenclature
a Solenoid radius (m)
b Drag Force (N)
|BZ | Magnetic field (T)
Fmag Magnetic field force (N)
I Current (A)
L Solenoid length (m)
Lw Length of wire (m)
N Solenoid turns
v Velocity (m/s)
m Mass of microsphere (kg)
rp Particle radius (m)
h Fluid viscosity ¼ 8.9 10 4 Pa s
m0 Permittivity of free space ¼ 8.85418782 10 12 m 3 kg 1 s4 A2
Introduction
The term bioreactor can be rather confusing due to the varied configurations and widespread applications. Most simply described,
a bioreactor is a self-contained entity capable of growing and/or sustaining the biological functions of living organisms. The organ-
isms may be cells, viruses, bacteria, or live tissue. Regardless of organism, the appropriate environment for growth and sustainment
of function is required and, more importantly, is specific to the organism if proliferation of the organism and sustainment of bio-
logical function are to be achieved. The environment is not solely based on the biofluidic or media conditions. The mechanical
conditions also play an important role, especially in the case of biological material comprising the musculoskeletal system. Methods
by which mechanical conditions may be generated with a reactor include fluid flow to induce shear, while gas pressure may be used
to apply compression. More recently, direct methods of compressive load application with respect to individual cells have been
realized using magnetic beads. Another aspect for consideration in bioreactor design is the substrate or layer where the biological
element will reside. With the exception of blood cells, most biological material requires a platform upon which to adhere. The
adherence permits proliferation and, when conditions are appropriate, normal biological function. The substrate may be rigid,
smooth, porous, or even flexible so as to permit application of tensile forces by stretching of the substrate. In short, the design
of a bioreactor is an attempt to mimic the normal biomechanical and physiological environment of biological material that
may be subsequently perturbed using physical and biochemical signals in an attempt to understand physiological responses and
function.

Regenerative Engineering j Bioreactors: System Design and Application for Regenerative Engineering 497
Bioreactor Considerations
In the design of any device involving biological tissue interaction and in the specific case of a bioreactor, it is vital that the design
process be parsed into less daunting engineering tasks. When each element has been addressed, one can then initiate the more diffi-
cult problem of integrating the various components. It should be noted that assuming that one can simply add the components
would be a naïve approach to a bioreactor design. The design process is iterative. One may find that some elements may require
modification in order to be integrated into the final design. Furthermore, one must be open to compromises. Bioreactor design
and application is a field that is at the forefront of basic science research. As such, components or materials to achieve an optimal
design may not be available, may require invention, or may be cost prohibitive. To minimize effort, one should do a thorough liter-
ature review to investigate what components, materials, and environmental conditions have been used with success by other inves-
tigators employing similar or identical biological material. Conducting such a task will reduce the incidence of selecting a material
or environmental condition that has been shown less efficacious or even detrimental in your specific application.
Biological Element Selection
The selection of cells and tissue for study can be as varied as the design of bioreactors. Depending on the nature of the study, appro-
priate cell or tissue selection is required in order to replicate the biomechanical or physiological function in musculoskeletal tissue.
One may consider using osteocytes or osteoblasts in the study of bone proliferation and mineralization. In the case of connective
tissue, such as cartilage or tendons and ligaments, chondrocytes or fibrocytes are an appropriate biological selection. Alternatively,
stem cells may be employed under different mechanical and environmental conditions to study the effects of the cellular environ-
ment upon differentiation. Cell use, be it specific or undifferentiated stem cells, may be either human or animal. Prior to selecting
a specific cell type, one should conduct a literature review in order to identify if a specific cell type, cell line, or species has been
associated with successful culturing and sustainment in the intended application.
Biological Environment
The biological environment may be one of the most challenging aspects in any bioreactor design and use. Regardless of cellular type
selection, consideration for nutrients, waste disposal, and maintenance of the biological environment are crucial. For example, if
one designs a reactor encompassing a scaffold where the media flow is essentially stagnant, most likely many cells residing in
the central region of the scaffold will likely be exposed to a reduced oxygen concentration due to physical barriers restricting diffu-
sion and a lack of fluid flow. Under these conditions, cells near the center of the scaffold may experience an elevated level of cellular
death as compared to those in the surface of the scaffold where nutrient and oxygen levels are significantly increased as compared to
the center of the scaffold.
Mechanical Environment
All cells respond to mechanical stimulation. Obviously, cells that constitute bone tissue are the most responsive as evidenced by
Wolff’s law. Wolff’s law is not to be taken as a physical law such as Newton’s laws of motion. That is, Wolff’s law is an observation
that states that bone will remodel so as to minimize the loading stresses upon the structure. Consequently, increased loading will
increase bone remodeling and strength. Reduced loading will reduce the bone volume and hence bone strength. However, cellular
response to the mechanical environment is not isolated to osteoblasts, osteoclasts, and osteocytes, which are located in bone. Endo-
thelial cells which constitute lumen as in blood vessels, arteries, and walls of the GI tract are also subjected to a mechanical envi-
ronment. The difficulty in applying mechanical stimulation in a bioreactor setting lies with orientation, magnitude, and frequency.
Different cell types are subjected to multiple combinations of these parameters during different activities and, as such, require
a mechanical environment to replicate the physiological state. There are several methods available to establish a mechanical envi-
ronment. These include use of fluid flow, gas pressure generation, and physical mechanical loading.
Fluid Flow
The shear stress (s) upon the cells at the base of the flow channel is given by s ¼ 6mS and/WH2, where m is the fluid viscosity, S is the
flow rate, and W and H are the width and height of the channel, respectively (Fig. 1). One must verify that the flow is laminar in
order to achieve constant fluid shear stress. This can be somewhat ensured by several considerations. The dimension of the width
should be much greater than the area where the cells are located as well as be greater than the height. The length of the channel
should be sufficiently long as compared to the region of cellular attachment so as to facilitate uniform flow by avoiding the turbu-
lent regions at the inlet and outlet of the channel. Alternatively, another configuration allows for differential shear stresses to be
displayed based on the location from a central inlet. Under this geometric configuration, the shear stress (s) is given by s ¼ 6mS/
2pDH2, where m is the fluid viscosity, S is the flow rate, D is the distance from the central inlet, and H is the height of the channel
498 Regenerative Engineering j Bioreactors: System Design and Application for Regenerative Engineering
Fig. 1 Fluid flow reactor mechanism for constant fluid stress due to laminar flow.
Fig. 2 Fluid flow reactor configuration to permit differential fluid stress based on distance from central axis of the inflow.
(Fig. 2). This fluid flow geometry permits examination of the cellular response to shear stress over a range of stress values using
a single experiment since the fluid stress varies as the reciprocal of the distance, D, from the inlet.
Gas/Fluid Pressure
While fluid flow for shear stress is common, in general they are restricted to small volumes on the order of microscope slides.
Cellular volumes may be increased through the use of gas/fluid compression geometries. In these cases, cells to be loaded are placed
at the base of a chamber that is filled part way with media. The gas phase above the media is then pressurized by depression of an
actuator which decreases the gas volume and hence pressurizes the liquid below (Fig. 3A). The advantages of such an apparatus
include ease of use and reduced variability. However, it should be noted that gas exchange due to cellular activity within the fluid
media will eventually reach equilibrium and, with continued use, result in a depletion of nutrients and required oxygen for cellular
proliferation. These configurations are suitable for short-term and acute experiments.
To reduce the complexity and retain reproducible loading, a similar configuration employs a flat compression platen placed
upon cells embedded within a matrix or scaffold. Such a static loading condition allows for controlled loading (Fig. 3B). In addi-
tion, the simplicity of the mechanism can permit multiple devices to be used with various combinations of matrix/scaffold and
platen mass in order to investigate the effects of load and 3-D cell geometry. This particular method can suffer from two disadvan-
tages. The first challenge is that cells located near the surface of the matrix/scaffold will see nutrient media at a significantly higher
level as compared to those cells embedded deep within the structure. Such a condition can lead to oxygen and nutrient depletion
within the central regions of the matrix/scaffold configuration. A second concern when using this geometry is the effect of Poisson’s
ratio. This is especially significant in the cases of soft materials. Examples include experiments involving cartilage, muscle, meniscus,
tendons, and ligaments. With these tissue and matrix configurations, compressive loading will lead to lateral expansion of the mate-
rial. When this occurs, the cells will not only be subjected to compressive loading but also experience shear due to the movement of
the matrix/scaffold perpendicular to the direction of loading. Characterization of the shear component of the overall stress will
require computational fluid dynamics simulations.
Fig. 3 (A) Compression of the gas above the fluid phase with a plunger or an actuator serves to compress the gas and in part compressive forces
through the fluid upon the cells located at the base. (B) Direct compression of the scaffold or matrix containing the cells can be performed using
a plunger or an actuator. One must be aware of the Poisson’s ratio effect due to compression of the scaffold or matrix.
These illustrations display some of the configurations possible under which cells may be subjected to stresses. When designing
a novel or traditional loading configuration, it is important to keep in mind that the resulting apparatus should attempt to replicate
the conditions in vivo with respect to applied loading, environmental fluid, and substrate. One should also keep in mind that these
techniques are generally applied over shorter durations and that long-term experiments will require mechanisms for media replen-
ishment and cellular waste removal.
Substrate Selection
Substrate selection is a vital element in bioreactor performance. The surface upon which cells are seeded can not only influence cell
migration but also elicit an effect upon physiological function of the cells. Cell types generally display a preferential affinity for
certain substrate surface composition and surface morphology. One must consider the hydrophobic/hydrophilic effects of the
surface upon the residing cells. Furthermore, surface texture plays an important role. Cells migrate across the surface by elongating
or extending the region in response to a preferred interaction with the substrate surface. At some point, the elongated region
becomes anchored or adhered to the surface of the substrate. Subsequently, the remaining material of the cell is dragged along
the surface through retraction (Fig. 4).
Fig. 4 A graphical representation of cell motion across a substrate. Extension of the cellular membrane is followed by anchoring at a specific
distance from the cell and then proceeded by retraction of the cellular body towards the anchor location.
With the advent of 3-D printing, microcontoured surfaces are possible and play a significant influence in cell migration. In
general, cells prefer a substrate for adhesion. Exceptions to this include red blood cells or other cells found in the bloodstream.
A more recent element with respect to surface effects has been demonstrated with the implementation of electric fields in various
orientations across the surface seeded with cells. Under these conditions, cells tend to align themselves with the field. In general, 2-D
considerations as outlined above are less likely to result in cellular apoptosis as waste and nutrient fluids can flow freely and can be
replenished with fresh fluids as needed. In the case of 3-D structures or scaffolds, the likelihood of cell death is increased as one
approaches the center of the construct. While the cells residing on the surface are exposed to free-flowing and/or fresh media, those
cells residing deep within the structure can only obtain nutrients and dispose of waste through diffusion within the structure and, as
such, are subjected to depleted nutrients and oxygen, as well as an elevated waste environment.
For 3-D structures, the material should provide sufficient porosities for ample diffusion to occur (Fig. 5). In addition, pulsatile
flow around the structure could be used to enhance fluid flow within the structure and hence decrease the likelihood of cell death.
However, one must keep in mind that the applied loading upon the structure may not be the resultant load upon the cellular mate-
rial contained within. Consider a 3-D scaffold capable of sustaining several thousand Newtons in compressive load. Imparting 10–
20 N of load as is commonly cited in the literature will result in virtually no loading upon the cellular material within the scaffold.
While most scaffolds are confined to basic science research or to nonload-bearing applications, with the advent of 3-D printing,
the realization of surgically sized, physiologically load-sustaining scaffolds is possible. In the case of the examples presented for
spinal fusion and segmental bone replacement applications, the scaffolds are approximately 30 mm in diameter and 10 mm in
height. The spinal scaffold consists of interwoven hyperbolic struts in concentric patterns adjusted for decreased modulus transition
as one traverses from the periphery to the central region of the scaffold (Fig. 6A). The segmental bone replacement scaffold consists
of intertwined conduits arranged to mimic the plywood configuration of bone. This dual modulus design facilitates the low
modulus cancellous bone in the center and the increased high-strength modulus of cortical bone at the periphery (Fig. 6B).
Both designs are 3-D printed from biodegradable polylactic acid (PLA). These unique geometries manifest themselves with failure
loads of 4900 and 9600 N for the spinal and bone replacement scaffold, respectively. While these static properties are essential, both
scaffolds also sustain runout loads of 800 and 1100 N to 5 million cycles, respectively, without cracking.
With respect to biological response, in the case of the spinal device, alkaline phosphatase activity (indicative of proliferating oste-
oblast cells) displayed a significant increase after only 14 days when stimulated as compared to unstimulated controls (Fig. 7A). In
the case of the segmental replacement scaffold, the calcium colorimetric assay evaluating osteoblast mineralization was manifested
significantly greater than stimulation frequencies of 2 Hz as compared to unstimulated controls with 0.5 and 5 Hz frequencies. This
2 Hz frequency continued to manifest elevated mineralization throughout 21 days of stimulation as compared to the other frequen-
cies examined (Fig. 7B). These two scaffolds represent the use of 3-D printing in the case of surgical sized implants. As such, in order
to maintain cellular viability at the center of these devices, many of the concepts presented will be applicable.
When considering 3-D materials evaluations to characterize the mechanical response of the material under static and dynamic
loading are required to ensure appropriate loading of the biological material when conducting in vitro experiments. Often, loading
a single structure is unfeasible, and as such one would like to take advantage of loading multiple 3-D structure simultaneously. To
achieve this requires fabrication of appropriate fixtures that can adjust to specified strains or stresses prior to initialization of
Fig. 5 Regardless of scaffold design or geometry, fluid flow throughout the scaffold is important if one is to avoid cellular apoptosis in the center
of the scaffold due to buildup of waste materials and lack of nutrient and oxygen flow.
pulsatile loading. This is most easily accomplished using individual plungers of known mass to reside upon each 3-D structure
(Fig. 8). A plate is fitted over all the plungers and secured to each plunger using fasteners. Such a procedure will ensure that
each 3-D structure is initialized with the plunger mass as initializing stress or strain. Subsequent loading will then ensure that
each 3-D structure is imparted with the same loading profile.
Using commercial materials, testing frames in conjunction with bioreactor vessels such as the one described here can facilitate
accurate dynamic and sinusoidal loading at controlled levels. While the bioreactor vessels may themselves be 3-D printer or fabri-
cated by traditional means, the use of commercial testing frames becomes not only impractical, but also a significant expense.
Furthermore, to employ these vessels in these testing frames requires transport from sterile incubator conditions to testing facilities
which may lead to contamination and variability during the course of a long-term experiment. The ultimate solution to such a chal-
lenge lies in the development of a bioreactor that can employ feedback control in order to reproducibly and accurately apply sinu-
soidal loading conditions. The subsequent section of this guide will provide a description for operation and design for the elements
required for such a device.
Basic Description and Operation for Design of Feedback-Controlled Bioreactor Employing Direct Mechanical
Loading
At the heart of any loading system is a transducer. Transducers can come in a variety of geometries and technologies in order for one
to evaluate physical quantities. In general, when transducers are coupled to signal conditioning circuits, an electrical output based
Fig. 6 (A) A surgical sized spinal fusion scaffold capable of sustaining physiological loading can be 3-D printed from polylactic acid (PLA). The
varying strut geometry permits a gradual modulus change from a high-strength periphery to a reduced modulus core. (B) In the segmental replace-
ment of long bone, a high-modulus periphery is required to replicate cortical bone. Centrally, a reduced modulus core permits fluid flow perpendic-
ular to a radial within the scaffold.
Fig. 6 (continued).
(A) ALP activity in stimulated hFOB cells (B) Calcium colorimetric assay
P<0.01 all other comparisons Osteoblast mineralization
0.5 Day 7
Not significant
3
Calcium content (mg/dL)
ALP activity (U/mL)
0.4
0.3 2
0.2
1
0.1
0.0 0
0 7 14 Control 0.5Hz 2Hz 5Hz
Number of stimulation days Loading frequency
P<0.05 For all comparisons
Fig. 7 (A) Stimulation of fetal osteoblasts in the spinal scaffold resulted in significant alkaline phosphatase activity after only 14 days. (B) Osteo-
blast mineralization in the segmental bone replacement device was evident and after only 7 days when using a 2 Hz frequency for stimulation.
on the physical quantity to be measured will result. In the design of the reactor, at least two transducers are required in order to
control and record the mechanical signals. A displacement transducer is required in order to measure/record deformation or if
one prefers strain. A second transducer is employed in order to measure/record applied force or alternatively the stress if one knows
or can compute the surface contact area a priori. Displacement transducers can be based upon several technologies. These include
use of linear potentiometers where the motion of the shaft changes the resistance of the potentiometer. Reluctance or Hall effect
transducers are also employed in cases where very small deformations or displacements are involved. Piezoelectric actuators can
also be used to impart known deformations or strains. In the case of force measurement and recording, one of the more dominant
methods employs strain gauges as the sensing element. More recently, piezoelectric ink that outputs a change in resistance based on
the amount of pressure applied at the ink surface has also been employed. The advantage of these thin-film ink sensors is that they
are flexible and sealed to protect against fluid/moisture penetration.
Fig. 8 The use of commercial testing machines to apply pulsatile loading, hence generating fluid flow through the 3-D structure, is possible. Under
these conditions, one must ensure that all scaffolds are subjected to equal loading conditions. Self-contained reactors that can perform continuous
loading with feedback may be a more viable option.
Most transducers require a signal conditioning circuit in order to transform the sensing element (i.e., strain gauge, potentiom-
eter, piezoelectric ink) into an electrical signal. The most simple of the circuits is the Wheatstone bridge, where one arm of the bridge
is the transducer and the remaining three arms are comprised of fixed resistors whose value is comparable to that of the sensing
element. Regardless of input voltage across the bridge, when all four arms of the bridge are at equal resistance (a balanced bridge),
no current will flow and hence a zero voltage output will result. Changes in the transducer sensing element will result in the sensing
arm to be at a different resistance with respect to the three remaining fixed arms of the bridge. This results in an unbalanced bridge
circuit and produces an output voltage comparable to the change in resistance of the unbalanced arm. The output voltage can be
subsequently calibrated to known applied displacements, forces, or other required physical properties in order to generate a curve
for transformation of output voltage to the physical quantity being applied or recorded. Displacement and force transducers are
commercially available and often incorporate the signal conditioning circuits and any additional signal amplification modules.
These commercial devices can be expensive and may be cost prohibitive if one wishes to design a series of bioreactors. What follows
is a simple technique for generation of displacement and force transducers using commercial strain gauges. The technique is not
standardized but can be customized by individual researchers to suit their needs.
A unique type of strain gauge transducer for both displacement (Fig. 9A) and force (Fig. 9B) measurements has been developed
by the author and colleagues. Both the displacement and force versions of the devices have been characterized and display excellent
repeatability and linearity. The method involves placing a piece of brass shims stock (for a displacement transducer) or brass rod
(for a force transducer) over a curved mandrel in order to produce an arc in the material. The arc only needs to be fabricated to
provide sufficient space for the attachment of the gauge. A strain gauge is then glued at the apex of the arc. Both devices work
by deformations in the arc radius, resulting in compressive or tensile deformation of the strain gauge. As one can see from the cali-
bration curves, both display excellent linearity. Furthermore, the data points associated with the deformation transducer (Fig. 9A)
represent the errors associated with four repeated calibration runs.
The rod diameter and shims stock thickness are dependent upon the loads and deformations to be applied and recorded. Both
devices can be connected to respective Wheatstone bridges utilizing fixed resistors based on the strain gauge resistance as the three
static arms of the bridge. The resulting output voltage from the transducer is then input to an amplifier for signal amplification. It
should be noted that while traditional strain gauge amplification is on the order of 10,000, these particular transducers generally
require amplification gains between 500 and 1000. This is a notable advantage as when one attempts to amplify a signal from the
transducer, the associated noise is amplified as well and can result in less than ideal signal conditioning without proper filters, either
at the input to the amplifier or within the amplifier circuit. Continuous data output from both transducers will allow for either
displacement (or strain) or force (or stress) control of the applied loading.
The next consideration in designing a bioreactor is the method as to how the applied loading will be generated. As was eluci-
dated earlier, devices such as piezoelectric actuators can be employed. Other technologies such as stepper motors, pneumatic air
cylinders, electromechanical actuators, and magnetic drives may be applicable. A simple yet easily controlled method employs
generation of the loading through acoustic speakers or voice coils. This is especially useful if one is interested in application of
continuous loading over a large number of frequencies. The loading profiles are generated electrically and are used as input to
the acoustic driver. The signals may be generated through commercial function generators or, in the case of this simple design
example, the use of a timer circuit employing a 555 timer chip. Such a circuit allows for control of the input voltage amplitude which
controls the deflection of the speaker cone. In addition, by changing the value of the control potentiometer, the frequency of the
square wave output to be used for control of the acoustic drive may be altered (Fig. 10).
Fig. 9 Typical calibration curves for the displacement (left) and force (right) transducers.
Force Acoustic drive

transducer
Actuator
Displacement
transducer
Outflow manifold
Inflow
manifold
Flexible Porous support

membrane
Fig. 10 Proposed feedback-controlled bioreactor illustrating components and overall compact geometry.
Using this method, the investigator may apply prescribed input voltages to the acoustic drive in order to achieve the desired
displacement. The resulting displacement (or strain) levels can be verified with the displacement transducer described. The resultant
loads generated due to the acoustic drive can be monitored using the force transducer described or any other load-sensing device.
This operation is known as displacement control. That is, one is driving the system based on a known or desired displacement or
strain and recording the resultant forces or stresses. Conversely, the speaker cone of the acoustic drive can apply deflections to result
in prescribed loads (or stress) upon the residing cell/scaffold configuration. Under this mode of operation, a load transducer can be
used to verify the applied forces while a displacement transducer can monitor the resulting deformations (or strains) due to the
applied forces. This operation mode is known as load control. Regardless of which mode control is employed, one must ensure
that a mechanical interface is designed to incorporate the acoustic drive and the respective transducers. It is not recommended
that a transducer directly contact the loading surface as damage to the contact interface of the transducer may result. The risk is
minimal for any given cycle but can become noteworthy as one conducts long-term cyclic loading.
An advantage of this type of design that employs both load and displacement application and monitoring is that monitoring
voltages from either the displacement or the force transducers can be employed as the feedback voltage to a comparator circuit
in order to achieve a homeostatic loading environment within the reactor. This feedback operation compares the difference in
applied voltage versus the feedback signal and subsequently adjusts the applied voltage levels so as to minimize the voltage differ-
ence. This is similar in operation to the way bone adapts its architecture in order to sustain the applied loading. The advantages of
such a method include a continuously adapting signal to maintain stress or strain, which drives the cellular remodeling mechanisms
to sustain the loading conditions. As the mechanical integrity of the cellular matrix/scaffold composite is enhanced to two cellular
proliferations, the applied loading must be increased in order to continue the cellular adaptation process similar to that described
by Wolff’s law.
In general, one would like a bioreactor operate for extended periods of time. One of the significant challenges is maintaining
a sterile environment over this time duration. In this particular design, a flexible membrane between the acoustic drive mechanism
and the loading platen connected to the acoustic drive containing the transducers serves to maintain a sterile environment within
the bioreactor well. Furthermore, this membrane provides a direct interface between the loading mechanism and the cell/scaffold
configuration residing in the bioreactor well. During the course of long-term mechanical stimulation, one must ensure that the
membrane remains intact, and if signs of deterioration are observed, replace the membrane prior to failure.
In order to reduce the fatigue burden upon the membrane, one should employ a nonbioreactive spacer between the membrane
and the cell/scaffold configuration. The requirements for the spacer include low mass, high porosity, and sufficient mechanical
integrity so as not to deform under loading. By maintaining a mechanically stiffer construct between the membrane and cell/scaf-
fold composite, the acoustic drive loading profile is directly transferred to the cell/scaffold matrix without loss due to mechanical
damping resulting in decreased effective loading. It is important that the spacer also be nonreactive with respect to the cellular envi-
ronment and surrounding media. This is required in order to ensure that only the loading and biological environments play a role in
cellular response. Environmental media conditions may be altered to elucidate effects of various therapies and are specific to the
investigation to be conducted.
The final mechanical element to be considered is the supports for the cell/scaffold composite. The interface of the cell/scaffold
composite is crucial. Nutrients and waste through the diffusion process as well as the hydrodynamic compression due to loading
must flow into and out of the cell/scaffold composite. Such a requirement will reduce cellular hypoxia at the center of the config-
uration due to reduced oxygen levels. Furthermore, the removal of cellular waste will be facilitated through these supports. The
movement of nutrients must also be permitted deep within the cell/scaffold configuration. Cells residing on the surface will demon-
strate altered response if subjected to significantly different environmental conditions as compared to those in the center of the scaf-
fold. There is a long list of porous materials that may be considered. These include ceramics porous metals and porous plastics. One
inexpensive solution is the filters contained in modern aquariums that employ ceramic cylinders as filtration units. These permit the
flow of water and oxygen while retaining sufficient mechanical integrity.
For most experiments, the media is expended over approximately 48 h. With long-term experiments, continuous exposure of the
culture within the reaction vessel will facilitate an elevated risk of contamination. To reduce this likelihood, the chamber of each
reactor is equipped with two manifolds to permit fresh media introduction and removal of expended media. While a single mani-
fold may be used in conjunction with a bidirectional valve, such a design may reintroduce expended media and cellular waste back
into the system. In this design, two manifolds are employed. One manifold consists of a one-way inlet and four one-way outlets.
This manifold is used to introduce fresh media into the bioreactor chamber. The second manifold contains four one-way inlets and
a single one-way outlet. This manifold is employed in the removal of expended media and cellular waste and can be used to obtain
media samples for monitoring of cellular function products. This setup will eliminate the likelihood of expended media and cellular
waste reintroduction. The four outlets on the first manifold allow for a reduced pressure wave when new media is introduced, hence
minimizing any currents, which would thereby disrupt the established flow pattern. The four outlets on the second manifold allow
reduced turbulence as expended media and cellular waste are drawn from the bioreactor chamber. As one can see from the design,
due to the relatively small geometry of the reactor, the device can be placed in an environmental chamber at 37 C and 5% CO2 in
order to mimic the in vivo cellular environment. Multiple reactors can be placed over multiple well plates in order to optimize the
number of cellular experiments that may be performed at any given time.
It should be stressed that while the design of a bioreactor may involve many engineering decisions, ultimately the biology of the
cellular elements under investigation takes precedence. One must establish the experimental conditions based on the cellular
loading environment that consists of mechanical and biologic cues that facilitate cellular responses. The bioreactor design proposed
here is a guide as to the processes to be considered in such an undertaking. The specific design elements required for any experiment
are dependent upon the ultimate goal of the research question. To this point, the discussion of a bioreactor design has involved the
introduction of a loading and cellular environment upon an assembly of cells. These cells can be in a 2-D planar structure or may be
comprised in a 3-D configuration involving a scaffold or matrix. While this approach is viable in assessing overall cellular response,
there are confounding signals due to cellular interconnectivity with other neighboring cells or the scaffold/matrix. The next question
then becomes this: Can a single cell in isolation be subjected to a loading profile and can the cellular response to the loading be
observed and quantified?
Single-Cell Loading Bioreactor
There is a paucity of work in this field. The basic approach involves use of paramagnetic microspheres with a bioreceptive ligand
secured to the surface. The microspheres consist of a paramagnetic core surrounded by a polystyrene layer to which the ligand or
integrated is attached. Fluid flow draws the microsphere towards a cellular network at which point the ligand binds to the cellular
surface. Application of a magnetic field will then impart a tensile load upon the paramagnetic microsphere and the associated
ligand, resulting in tensile forces on the surface of the cell. The major drawback to this approach is the resulting cellular response.
One may ask these questions: Is the cellular response due to the applied loading? Is a cellular response due to the biological ligand?
Is a cellular response due to the combination of the ligand and applied loading? In these experiments, one must assume that the
cellular response to the applied loading is orders of magnitude above the biological response to the ligand bound upon the cell
surface. However, one cannot accurately distinguish between the two aspects. To eliminate this dilemma, what if one considers
that a paramagnetic particle could be employed as an actuator upon the surface of a single cell? One can then observe if compressive
loading deforms the cellular morphology and in turn manifests a biological response to the compressive load applied.
In order to facilitate cyclic loading while applying a controlling magnetic field, implementation of a solenoid is an effective
method. A traditional solenoid involves winding wire around a hollow core. One can fabricate a solenoid that is self-contained
and designed to circumvent a microscope objective. This feature provides the ability to observe and capture images of the resulting
cellular morphological changes due to compression from magnetic microspheres directly upon cellular surfaces without the use of
ligand-binding proteins. As the number of windings increases, the magnetic field will increase accordingly (Solenoid 1 design). In
order to increase magnetic field strength, a solenoid core with flanges on the top and bottom permits layering of coils (Solenoid 2
design). However, this also increases the temperature around the solenoid. The elevation and temperature due to the solenoid pres-
ence may be detrimental in two ways. The elevated temperature surrounding the solenoid may produce thermal currents in the
atmosphere that may influence the cellular response due to elevated temperature. In addition, the elevated operating temperature
of the solenoid may, in fact, provide detrimental environmental conditions for cellular existence. In both cases, one cannot be
assured that the resulting compressive forces from microsphere compression are the sole reason for the observed cellular response.
Alternately, one may create a solenoid that is longer in length but narrower in diameter (Solenoid 3 design). In order to design an
appropriate solenoid, one can perform the following exercise. The theoretical force on a microsphere due to a solenoid can be care-
fully evaluated using a magnetic field and Newtonian kinematic equations, at least to a first approximation. The derivation for the
equation used to calculate the force exerted on spherical microsphere proceeds as follows:
Magnetic field calculated at the center of solenoid
Radius ¼ a, length ¼ L, turns ¼ N
m0 IN
jBZ j ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (1)
L2 þ 4a2
Magnetic field calculated at the edge of solenoid
Radius ¼ a, length ¼ L, turns ¼ N
m0 IN
jBZ j ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi (2)
2 L2 þ a2
Velocity of magnetic beads
Drag force ¼ b, velocity ¼ v, magnetic field force ¼ Fmag
dv
m ¼ Fmag bv (3)
dt
Fmag h i
v¼ 1 eðbt=mÞ (4)
b
.
Current ¼ I, length of wire ¼ Lw, magnetic field ¼ B

Fmag ¼ ILw, B sin 90 therefore Fmag ¼ ILw, B B 1/r where r ¼ distance to solenoid
.
ILw r B r IL m0 IN
Fmag ¼ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (5)
r r L þ 4a2
½1eðbt=mÞ
ILw m0 IN
v¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (6)
br L2 þ 4a2
Drag force ¼ b ¼ 6hprpv ¼ 6phrp
Relative velocity ¼ v ¼ 1
Particle radius ¼ rp
Fluid viscosity ¼ h ¼ 8.9 10 4 Pa s
h i
1e6phrm p t
ILw m0 IN
v¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (7)
6phrp r L2 þ 4a2
Acceleration ¼ A ¼ dv
dt

dv ILw m0 IN 6phrp 6phrp t
¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi e (8)
dt 6phrp r L2 þ 4a2 m

ILw m0 IN
¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi e6phrp t
rm L2 þ 4a2
For t 1, e6phrpt ¼ 1

dv ILw m0 IN
¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (9)
dt rm L2 þ 4a2
Final equation for force calculation comparisons:
I2 Lw m0 N I2 Lw m0 N
F ¼ mA ¼ pffiffiffiffiffiffiffiffiffi ∙m ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi (10)
rm L2 þ4a2 r L2 þ 4a2
The results of the exercise provide a graphical representation of the relative magnitude of magnetic field force generation for
different solenoid geometries upon the paramagnetic microspheres at various distances from the solenoid (Fig. 11).
It would appear from the figure that solenoid design three provides the greatest attractive force upon the paramagnetic polysty-
rene microspheres. However, such a design cannot be employed as the dimensions of the solenoid for this design cannot be incor-
porated into a traditional inverted optical microscope configuration without significant modifications to the microscope stage. The
second solenoid design can be selected as the diameter of the solenoid is able to contain the microscope objective within the coils.
In this particular design, the solenoid possesses 240 turns in six layers of 40 turns each. The solenoid design is slightly larger than the
diameter (41.3 mm) of the microscope objective, allowing for improved heat dissipation during operation. Removing solenoid
heating is crucial so as not to influence morphological changes due to thermal effects. Fig. 12 illustrates the solenoid design.
10
8
Force due to magnetic fields (N)
0
0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9
Distance from solution to solenoid edge (mm)
Solenoid 1 Solenoid 2 Solenoid 3
Fig. 11 Force exerted on magnetic particles as a function of the distance between the particle solution and the edge of solenoid from Eq. (10).
Fig. 12 Side and top views of solenoid design 2.
Optimization of Loading
To ensure the effective movement of the magnetic microspheres, different magnetic microsphere suspensions in deionized (DI)
water should be prepared to maximize microsphere movement visibility and determine optimized concentration of magnetic
microsphere suspension for the most rapid solution clearance. This exercise is important so as to determine the concentration of
the paramagnetic polystyrene microspheres required to induce loading without imparting a bombardment of the cells due to
a massive quantity of microspheres. If one employs a significantly large number of magnetic microspheres for visualization of
the field through the microscope objective that will become a difficult task, it is important to determine a minimum threshold
for observance of bead loading upon cells. Ideally, one would prefer to identify a concentration resulting in a single microsphere
residing upon a single cell. In practice, this is not feasible, and hence one should titrate the concentration of microspheres so as to
achieve a sufficient dispersion whereby a single microsphere may be observed on a cell in most cases.
An effective method to arrive at a suitable dispersion of microspheres involves the use of glass micropipette tips containing a pre-
determined volume (on the order of a milliliter) magnetic microsphere suspension at various dilution concentrations suspended at
a preset distance from the solenoid. A permanent magnet is located above the micropipette tip. The permanent magnet is required as
the combination of microsphere agglutination, adhesion to the cell surface, and presence of the earth’s magnetic field would main-
tain the magnetic microspheres in contact with the cell surface, resulting in permanent staff compression. As well, presence of these
permanent magnets serves to retard the approaching velocity of the magnetic microspheres (due to the solenoid) as they contact the
cells. Since the magnetic field strength increases as the inverse of the distance during approach to the solenoid, a high relative
velocity impact from the magnetic microsphere (due to the increasing magnetic field as a result of the decreased distance to sole-
noid) may damage the cellular surface. Furthermore, the permanent magnets draw the magnetic microspheres back into suspension
for complete unloading of the cells when power to the solenoid is terminated. This is essential during the rest component of the
duty cycle or unloading portions of dynamic loading regimens. It has been demonstrated in the literature that continuous cell
loading results in eventual cell desensitization to the loading regimen and/or cell death.
To induce a magnetic field in the hollow solenoid, a programmable DC power supply can provide preselected current profiles.
Fig. 13 illustrates the experimental configuration for determination of magnetic microsphere suspension movement.
With respect to the magnetic microsphere concentrations, suspensions with magnetic microsphere to DI water ratios of 1:2, 1:4,
1:8, 1:10, 1:16, and 1:20 using 1 mm diameter paramagnetic microspheres are a viable starting point. It is important to realize that
most paramagnetic polystyrene-coated microspheres have a surface surfactant. Depending on the nature of the cells to be examined,
one may order positive, negative, or neutral surface-charged microspheres. The surface charges are generally due to a functional
group during the processing. Depending on the experiment to be conducted, functional groups may be advantageous. In general,
one should consider the use of surfactant-free neutral paramagnetic polystyrene microspheres in order to reduce the likelihood of
electrochemical attraction between the microsphere and the cellular surface which would hinder unloading of the cell by the perma-
nent magnets and likely result in a tensile force being applied during the unloading phase of the loading cycles. The magnetic
strength required for retarding of the approach velocity is a trade-off between high-impact contact and a long-term, slow contact.
The high-impact scenario will likely damage the cell, while an exceptionally long-duration approach will require excessive solenoid
operation time and generate elevated local temperatures.
In this particular example, the programmable DC power supply applied a solenoid current between 0 and 5A at 0–15 V, resulting
in decreased time for temperature elevation. In this particular example of a single-cell bioreactor loading device, solenoid operation
consisted of a 25% duty cycle with 10 s of power and 30 s of cooling during application of 10 V and 1.85A. Such a configuration was
sufficient to keep the solenoid from overheating. This particular duty cycle was specific to this experimental example. Depending on
the specific microscope configuration and cells employed, one may require an altered duty cycle.
Fig. 13 Permanent magnets above the dish prevented the microspheres from affixing to the bottom of the dish due to microsphere agglutination,
surface adhesion, and the earth’s magnetic field.
Compressive Loading and Imaging

Fig. 14 depicts the schematic and experimental setup. This particular experiment placed the solenoid around the objective of
a microscope and searched isolated locations within the coverslip dish, where paramagnetic microspheres were located directly
on the surface of the cell. When conducting these types of studies, one must image the paramagnetic microspheres, cells, and
cell nuclei before and after loading, using the image capture system of the associated microscope. Fluorescence images of cell nuclei
before loading (red and green) and during loading (blue and green) can be overlaid and subsequently analyzed for changes in
nuclear morphology due to the applied loading. It is important to recognize that this image overlay process must be conducted
for many instances of paramagnetic microspheres residing on the surface of the cell. In order to achieve sufficient statistical power
for the experiment, one must conduct a sample size determination.
Fig. 14 Depiction of the schematic and experimental setup for application of mechanical loading to cells using paramagnetic microspheres. The
magnetic field generated by a solenoid encircling an optical microscope objective.
In order to quantify the effects of cell nucleus compression due to the solenoid force on the paramagnetic polystyrene micro-
spheres, the major and minor axes of the cell nuclei are computed by measuring the number of pixels across the perpendicular
axes of the nucleus. Fig. 15 illustrates the computation for the major and minor axes. It is critical that in image analysis quantifi-
cation for distances one obtains the relationship between pixel number and linear distance so that the pixel distances may be con-
verted to physical lengths. The margin of error for these calculated measurements is within four pixels or 2.67 mm.
In order to establish a statistically significant difference between the loaded and unloaded conditions, paired Student’s t-test can
be conducted for the major and minor axis distances at the various cell locations during active and terminated power conditions of
the solenoid. As one can see in Fig. 16, under microsphere compression (solenoid power on) the red staining of the nucleus indi-
cates a change in morphology as compared to the green staining under nonmagnetic loading conditions. No significant change in
the minor axis was noted (Fig. 17A). Interestingly, a significant difference in the major axis of the cell nucleus was observed
(Fig. 17B). This may indicate a preferential direction that a cellular nucleus may deform in an attempt to reduce the effects of
compressive loading.
Fig. 15 Measurement of major and minor axes for a given cell.
(A) (B)
Beads, cell, nuclei—solenoid ON Beads, cell, nuclei—solenoid OFF
(C)
Nuclei overlay—solenoid OFF (GREEN)
Nuclei overlay—solenoid ON (RED)
Fig. 16 Single-cell loading using paramagnetic microspheres. (A) Microspheres and cells with solenoid ON. (B) Microspheres and cells with sole-
noid OFF. (C) Magnified image of overlaid cell nuclei for solenoid OFF (GREEN) vs. solenoid ON (RED) indicative of nuclear dimensional changes.
(A) Minor axis (B) Major axis

15 15
Not statistically significant P<0.01
Microns (µm)
Microns (µm)
10 10
5 5
0 0
OFF ON OFF ON
Solenoid condition Solenoid condition
Fig. 17 Changes in cell nucleus dimensions due to magnetic microsphere loading. (A) Minor axis (P > .7) and (B) major axis (P < .01).
This single-cell bioreactor configuration has demonstrated the successful use of varying magnetic fields to induce compressive
forces via paramagnetic microspheres using a solenoid coupled to a variable power supply. A statistically significant increase in the
major axis length of the loaded cell nuclei as compared to the unloaded condition can be visualized and quantified using this partic-
ular configuration. The changes observed were the result of direct compressive loading induced by the paramagnetic microspheres
and not the result of thermal effects due to solenoid operation as no changes were observed in morphology of the cells when the
solenoid is active and paramagnetic microspheres were not present on the cell surface.
This bioreactor prototype has established the framework for a portable and economical device for application of loading condi-
tions in biological environments to study cellular response under ligand-free conditions. The device developed is applicable for
controlled single-cell loading to isolate the biological response of cells to mechanical activation. Since the microspheres are not
bound to the cell via ligands, use of superiorly positioned permanent magnets permits the possibility of unloaded conditions.
Continuous periodic, ligand-free loading represents a physiological loading regimen as compared to static, mechanical, ligand-
selective stimulation. Biological evaluations of different cells to varied loading are possible with the altered design of the solenoid,
duty cycle, and microscopic capabilities. This particular approach possesses an advantage in that since the microscope objective is
contained within the solenoid, the visualization optics are unaltered as a field within the solenoid is zero. One should be aware that
in the selection of the paramagnetic microspheres, they should be sufficiently large in diameter in order to prevent internalization by
the cells when subjected to loading. There is also a disadvantage to using very small paramagnetic microspheres. In such a case, the
magnetic field strength must be considerably larger in order to move the small-diameter microspheres through the media. Such
a condition will elevate the thermal environment due to the elevated current flow for a solenoid and extended activation time
required for microsphere migration.
In this specific configuration, cells experienced strain values of 1.3% for the minor axis of the cell nucleus while a strain level of
5.6% in the direction of the major axis. It has been reported in the literature that daily regimens of approximately 2% strain are
sufficient to manifest cellular differentiation. Therefore, one can customize the prototype design to achieve specific strain values
based on solenoid design and solenoid current flow.
Paramagnetic microspheres can be an effective method for the delivery of direct and localized forces to cellular networks or indi-
vidual cells. The advantage of using paramagnetic microspheres in such an approach has been the ability to apply force directly to
cells rather than transmitting forces through a scaffold or other intermediary mechanism, such as a biological or chemical ligand.
Forces applied directly to the cell rather than a scaffold eliminate mechanical restrictions that are commonly present.
As compared to direct compression, another standard method of load application involves the use of shear loading. Shear load
applications include cell perfusion chambers developed to simulate physiologic fluid flow. Previous studies have utilized cell perfu-
sion chambers to examine the effects of shear mechanical stimulation on cells in vitro. However, computational fluid dynamics
models and experimental fluid dynamics studies have demonstrated that perfusion chambers impart poorly controlled,
location-dependent shear stresses rather than constant shear stresses. To this end, a similar approach involving a solenoid can be
employed to generate shear loading. Using the equations previously derived, solenoid design three may be appropriate as it creates
a strong magnetic field while displaying reduced thermal effects. Furthermore, in the case of shear loading, the cells are not located
at a constant distance from the solenoid and hence are subjected to a shear force that is dependent on the distance from the solenoid
center. Under compressive loading as in the previous description, the cells were at a constant distance from the solenoid, and when
compressed by the magnetic polystyrene microspheres the distance between the microspheres and the solenoid can be considered as
constant across the cellular plane.
An advantage in conducting shear loading experiments is that one is not usually confined to the geometrical constraints of the
microscope configuration. In such a case, only a support and alignment fixture for the solenoid axis is required. It should be noted
that if one isolates specific regions at fixed distances away from the solenoid axis for observation, one can observe the cellular
response due to increasing shear forces as one approaches the solenoid due to the change of the magnetic field strength which
increases as one over the distance from the solenoid.
These two single-cell loading bioreactor examples illustrate a simple and effective mechanism for individual cell loading serving
as a starting point for investigative studies. Investigators can certainly adapt magnetic microsphere concentration and size, cellular
selection and culture, as well as other environmental conditions to suit the experiment and in vitro conditions for investigation.
Bioreactor design can employ 2-D cellular planes, 3-D structures consisting of a matrix or scaffold containing cells, or a single-
cell device. The objective of this work is to provide a starting point from which one may design a bioreactor system suitable for their
individual needs based on cellular, environmental, and mechanical requirements. There is no single universal set of design param-
eters that are suitable to all experimental investigations. Rather, one must examine the goal of the experiment and determine the
cellular species and suitable in vitro environment prior to initiating the engineering and design of a suitable loading mechanism and
criteria. The use of commercial and/or custom-fabricated transducers is a vital element in any bioreactor design. Sufficient resolu-
tion, accuracy, and reproducibility are vital in not only load application and control but also recording of cellular response due to
the applied loading.
The complexity of in vitro biomechanical studies permits a wide variety of bioreactor designs to be equally adaptable to several
or more experimental conditions. The appropriate suitability of any single reactor design universally across all experiments is unre-
alistic. More practical is that a specific bioreactor design may be suitable for several cell types and loading conditions, provided that
a sufficient operating range with respect to environmental volume, substrate, and applicable loading parameters is built into the
bioreactor design a priori. This may be accomplished with appropriate selection of materials, transducers, and environmental
media. Regardless of design, it is important to realize that all bioreactors will result in a cellular response, but it is critical that
the observed response is directly related to the operational control and loading due to the reactor and not due to other factors.
This may be achieved through repeated experiments and by sequentially altering mechanical and environmental conditions and
observing the cellular response. Bioreactor design is not an exact science, but is dependent upon in vitro cellular responses. One
may only control the mechanical and environmental conditions, but the response of the cells to both of these elements is beyond
the control of the investigator. As such, bioreactor designs should be reproducible and accurate in the establishment of the mechan-
ical and biological environment.
The future use of bioreactors will be in conjunction with surgically sized load-sustaining scaffolds. Consider a case where bone
graft is required for a surgical fusion or repair. Patients may be able to donate bone marrow or other cells suitable for bone culturing
sometime prior to surgery. Once the cells have been expanded and placed on scaffolds for a period of stimulation using a bioreactor,
the biologically active cells contained within the load-sustaining scaffold may then be implanted into the patient. This scenario
provides advantages over current methods that require traumatic bone harvesting for autograft bone or nonbiologically active
cadaveric or synthetic materials to be used as fillers. The advancement in bioreactor design may make such a scenario a reality.
Further Reading
Bancroft, G. N., Sikavitsas, V. I., & Mikos, A. G. (2003). Design of a flow perfusion bioreactor system for bone tissue-engineering applications. Tissue Engineering, 9(3), 549–554.
Boal, D. (2012). Mechanics of the cell (2nd edn.). Cambridge University Press: United Kingdon.
Chang, C.-H., Lin, C.-C., Chou, C.-H., Lin, F.-H., & Liu, H.-C. (2005). Novel bioreactors for osteochondral tissue engineering. Biomedical Engineering: Applications, Basis and
Communications, 17, 38–43.
Chung, R. (2017). 3D printed variable modulus physiological load sustaining and bioactive scaffolds for segmental bone replacement [dissertation]. Hoboken: Stevens Institute of
Technology, 175 p.
Dobson, J., Cartmell, S. H., Keramane, A., & El Haj, A. J. (2006). Principles and design of a novel magnetic force mechanical conditioning bioreactor for tissue engineering, stem
cell conditioning, and dynamic in vitro screening. IEEE Transactions on Nanobioscience, 5(3), 173–176.
Ethier, C. R., & Simons, C. A. (2007). Chapter 2: Cellular biomechanics. In Introductory biomechanics from cells to organisms. New York: Cambridge University Press.
Jin, G., Yang, G.-H., & Kim, G. H. (2015). Tissue engineering bioreactor systems for applying physical and electrical stimulations to cells. Journal of Biomedial Materials Research
Part B Applied Biomaterials, 103B, 935–948.
Korossis, S. A., Bolland, F., Kearney, J. N., Fisher, J., & Ingham, E. (2005). Topics in tissue engineering: VII. Bioreactors. In N. Ashammakhi, & R. L. Reis (Eds.), vol. 2. Bioreactors
in tissue engineering (pp. 1–23). http://www.oulu.fi/spareparts/ebook_topics_in_t_e_vol2/abstracts/korossis_0102.pdf.
López-Fagundo, C., Bar-Kochba, E., Liane, L. L., Hoffman-Kim, D., & Franck, C. (2014). Three-dimensional traction forces of Schwann cells on compliant substrates. Journal of the
Royal Society Interface, 11, 20140247. https://doi.org/10.1098/rsif.2014.0247.
Maglaras, C. (2017). Physiologic load sustaining bone scaffold for spinal fusion utilizing hyperbolic struts [dissertation]. Hoboken: Stevens Institute of Technology, 128 p.
Martin, I., Wendt, D., & Heberer, M. (2004). The role of bioreactors in tissue engineering. Trends in Biotechnology, 22(2), 80–86.
Mason, C., Hjerpe, R., Noonan, E., Leopold, P. L., & Valdevit, A. (2016). A device for ligand-free single cell loading using magnetic fields and paramagnetic microspheres. American
Journal of Biomedical Engineering, 6(6), 159–165.
Salehi-Nik, N., Amoabediny, G., Pouran, B., Tabesh, H., Shokrgozar, M. A., Haghighipour, N., Khatibi, N., Anisi, F., Mottaghy, K., & Zandieh-Doulabi, B. (2013). Engineering
parameters in bioreactor’s design: A critical aspect in tissue engineering. Biomed Research International, 762132, 15 p. https://doi.org/10.1155/2013/762132
Sladkova, M., & Maria de Peppo, G. (2014). Bioreactor systems for human bone tissue engineering. Processes, 2, 494–525.
Teo, A., Mantalaris, A., & Lim, M. (2012). Hydrodynamics and bioprocess considerations in designing bioreactors for cardiac tissue engineering. Journal of Regenerative Medicine &
Tissue Engineering. http://www.hoajonline.com/journals/pdf/2050-1218-1-4.pdf https://doi.org/10.7243/2050-1218-1-4.
Yeatts, A. B., & Fisher, J. P. (2011). Bone tissue engineering bioreactors: Dynamic culture and the influence of shear stress. Bone, 48, 171–181.
Yu, X., Botchwey, E. A., Levine, E. M., Pollack, S. R., & Laurencin, C. T. (2004). Bioreactor-based bone tissue engineering: The influence of dynamic flow on osteoblast phenotypic
expression and matrix mineralization. PNAS, 101(31), 11203–11208.
Bone Substitute Materials
M Bohner, RMS Foundation, Bettlach, Switzerland
Introduction 515
Materials 515
Ceramics 515
High Mechanical Properties 516
Bone Bonding 516
Resorbable 516
Ionic Drugs 516
Better Handling 516
Metals 517
Resorbable Metals 517
Polymers 517
Xeno- and Allografts 518
Xenografts 518
Allografts 518
Bone Substitute Formulations 519
Granules 519
Blocks/Sponges 520
Pastes (or Putties) 521
Cements 521
Membranes/Strips 522
Architecture 522
In Vivo Properties 523
Dissolution 523
Cell-Mediated Dissolution 524
Enzymatic Digestion 524
Transport 524
Hydrolysis 525
Corrosion 525
Conclusion 525
References 525
Glossary
Allograft Allotransplantation (allo- from the Greek meaning ‘other’) is the transplantation of cells, tissues, or organs, to
a recipient from a genetically nonidentical donor of the same species. The transplant is called an allograft, allogeneic
transplant, or homograft (Wikipedia, 15 August 2013).
Autologous bone (autograft) Autotransplantation is the transplantation of organs, tissues, or even proteins from one part of
the body to another in the same individual. Tissue transplanted by such ‘autologous’ procedure is referred to as an autograft or
autotransplant. It is contrasted with xenotransplantation (from other species) and allotransplantation (from other individual
of same species) (Wikipedia, 15 August 2013).
Bacteremia (also bacteraemia) is the presence of bacteria in the blood (Wikipedia, 23 August 2013).
Bioactive The spontaneous origination or communication of a material in a biological environment (Williams, 1987). Bone
substitute researchers generally consider a ‘bioactive’ bone substitute as a material that gets coated by apatite once dipped in
a ‘simulated body fluid’ and/or a material that directly bonds to bone once implanted in vivo.
Bone augmentation The action of injecting a cement into bone (generally osteoporotic) to eventually increase its strength, for
example, for ‘vertebroplasty’ procedures.
Bone wax Bone wax is a waxy substance used to help mechanically control bleeding from bone surfaces during surgical
procedures. It is generally made of beeswax with a softening agent such as paraffin or petroleum jelly and is smeared across the
bleeding edge of the bone, blocking the holes and causing immediate bone hemostasis through a tamponade effect
(Wikipedia, 15 August 2013).
Cement A cement is a binder, a substance that sets and hardens independently, and can bind other materials together

514 Regenerative Engineering j Bone Substitute Materials
Ceramic Kingery et al. (1976) define ceramics as “the art and science of making and using solid articles which have as their
essential component, and are composed in large part of, inorganic nonmetallic materials.”
Colloid A colloid is a substance microscopically dispersed throughout another substance (Wikipedia, 30 August 2013).
Cortical bone Cortical bone, synonymous with compact bone, is one of the two types of osseous tissue that form bones.
Cortical bone facilitates bone’s main functions: to support the whole body, protect organs, provide levers for movement, and
store and release chemical elements, mainly calcium. As its name implies, cortical bone forms the cortex, or outer shell, of most
bones (Wikipedia, 15 August 2013).
Ductility In material science, ductility is a solid material’s ability to deform under tensile stress (Wikipedia, 27 August 2013).
Elastic modulus An elastic modulus, or modulus of elasticity, is the mathematical description of an object or substance’s
tendency to be deformed elastically (i.e., nonpermanently) when a force is applied to it. The elastic modulus of an object is
defined as the slope of its stress–strain curve in the elastic deformation region. As such, a stiffer material will have a higher
elastic modulus (Wikipedia, 27 August 2013).
Embolism In medicine, an embolism refers to the lodging of an embolus, which may be a blood clot, a fat globule, or a gas
bubble in the bloodstream, which can cause a blockage (Wikipedia, 15 August 2013).
Hydrogel Hydrogel is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is
the dispersion medium (Wikipedia, 30 August 2013).
Micropore Even though the official definition of a ‘micropore’ is a pore smaller than 2 nm, most biomaterials researchers
consider a ‘micropore’ as a pore with a diameter comprised between 100 nm and that of a macropore (see ‘macropore’
definition). This is in analogy with the definition of a microparticle from 100 nm to 100 mm.
Macrophage Macrophages (Greek: big eaters, from makros ‘large’ þ phagein ‘eat’; abbr. MF) are cells produced by
the differentiation of monocytes in tissues. Monocytes and macrophages are phagocytes. Macrophages function in both
nonspecific defense (innate immunity) as well as help initiate specific defense mechanisms (adaptive immunity) of vertebrate
animals. Their role is to phagocytose, or engulf and then digest, cellular debris and pathogens, either as stationary or as mobile
cells (Wikipedia, 28 August 2013).
Macropore Even though the official definition of a ‘macropore’ is a pore larger than 50 nm, biomaterials researchers consider
a macropore as a pore large enough to promote vascularized bone ingrowth. A diameter of 50 or 100 mm is generally
considered to be the minimum required size.
Nanopore Pore smaller than 100 nm.
Nerve root A nerve root is the initial segment of a nerve leaving the central nervous system (Wikipedia, 15 August 2013).
Orthopedics Orthopedic surgery or orthopedics (also spelled orthopaedic surgery and orthopaedics) is the branch of surgery
concerned with conditions involving the musculoskeletal system (Wikipedia, 28 August 2013).
Osteoblast Osteoblasts (from the Greek words for ‘bone’ and ‘germ’ or embryonic) are mononucleate cells that are responsible
for bone formation (Wikipedia, 28 August 2013).
Osteochondral (¼endochondral) Endochondral ossification is one of the two essential processes during fetal development of
the mammalian skeletal system by which bone tissue is created. Unlike intramembranous ossification, which is the other
process by which bone tissue is created, cartilage is present during endochondral ossification (Wikipedia, 16 August 2013).
Osteoclast An osteoclast (from the Greek words for ‘bone’ (Ossó) and ‘broken’ (klassó2)) is a type of bone cell that removes
bone tissue by removing its mineralized matrix and breaking up the organic bone (organic dry weight is 90% collagen)
Osteoconduction The process by which bone grows on a surface (Wiktionary, 15 August 2013).
Osteogenesis Osteogenesis is the process of laying down new bone material by cells called osteoblasts (Wikipedia, 16 August
2013).
Osteoinduction A biologic response in which chemical signals induce the formation and development of bone (Wiktionary,
15 August 2013).
Osteosynthesis Osteosynthesis is the reduction and fixation of a bone fracture with implantable devices that are usually made
of metal (Wikipedia, 28 August 2013). So, it can be considered to be a sub-branch of orthopedics.
Osteotomy An osteotomy is a surgical operation whereby a bone is cut to shorten, lengthen, or change its alignment
Osteotransduction A process by which a bone defect filled with a bone substitute is transformed into new bone tissue
(Driessens et al., 1998).
Paste Paste is a term for any very thick viscous fluid (Wikipedia, 15 August 2013).
Putty Putty is a generic term for a plastic material similar in texture to clay or dough typically used in domestic construction and
repair as a sealant or a filler (Wikipedia, 15 August 2013).
Resorption There is no consensus on the terms ‘resorption’ and ‘degradation’ (with or without the prefix ‘bio’) in the
biomaterials field. Here, both terms are used as synonyms and describe the process of removal of a material in a biological
environment, either by dissolution (Williams, 1987) or by other physical or chemical processes.
Regenerative Engineering j Bone Substitute Materials 515
Sintering Sintering is a method for creating objects from powders. It is based on atomic diffusion. Diffusion occurs in any
material above absolute zero, but it occurs much faster at higher temperatures. In most sintering processes, the powdered
material is held in a mold and then heated to a temperature below the melting point. The atoms in the powder particles diffuse
across the boundaries of the particles, fusing the particles together and creating one solid piece (Wikipedia, 15 August 2013).
Spacer In the bone substitutes field, a spacer is a synonym of ‘bone substitute’ or ‘bone filler’.
Spinal cord The spinal cord is a long, thin, tubular bundle of nervous tissue and support cells that extends from the brain (the
medulla oblongata specifically) (Wikipedia, 15 August 2013).
Supersaturation The term supersaturation refers to a solution that contains more of the dissolved material than could be
dissolved by the solvent (.). It can also refer to a vapor of a compound that has a higher (partial) pressure than the vapor
pressure of that compound (Adapted from Wikipedia, 28 August 2013).
Trabecular bone (also called spongy or cancellous bone) Cancellous bone, synonymous with trabecular bone or spongy
bone, is one of two types of osseous tissue that form bones. The other osseous tissue type is cortical bone (Wikipedia, 15
August 2013).
Tissue engineering Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable
biochemical and physical–chemical factors to improve or replace biological functions (Wikipedia, 15 August 2013).
Vertebroplasty Vertebroplasty and kyphoplasty are similar medical spinal procedures in which bone cement is injected
through a small hole in the skin (percutaneously) into a fractured vertebra with the goal of relieving back pain caused by
vertebral compression fractures (Wikipedia, 15 August 2013).
Xenograft Xenotransplantation (xenos- from the Greek meaning ‘foreign’) is the transplantation of living cells, tissues, or
organs from one species to another. Such cells, tissues, or organs are called xenografts or xenotransplants (Wikipedia, 15 August
2013).
Introduction
Millions of patients suffer every year from a bone defect, for example, following diseases or accidents. Even though bone is a self-
healing organ, large bone defects may not heal. Therefore, bone defects which are considered by the clinician to be too large to spon-
taneously heal are filled with a so-called spacer. The nature of this spacer can vary. Traditionally, autologous bone is used. It is the
gold standard, but its extraction from the patient (typically at the iliac crest) has many drawbacks such as the need for a second
surgery, the risk of infection, or long-term pains. Generally, the complication rate is in the order of 20% and the amount of
bone that can be extracted is limited (Younger and Chapman, 1989; Arrington et al., 1996). Therefore, various alternatives
(‘bone substitute materials’) have been sought for, in particular using allografts and their derivatives, xenografts and their deriva-
tives, and synthetic materials. Interestingly, first efforts were triggered by the large number of wounded American soldiers during the
Vietnam War (Hench, 2006). The aim of the present article is to describe these various classes of bone substitutes, with a strong
focus on resorbable synthetic materials.
The article is divided into four sections. The first section is devoted to the various materials used for bone substitution. In the
second section, the focus is set on the various formulations that are commercially available (granules, blocks, pastes, cements,
membranes). The third part will address architectural aspects. Finally, the last part will briefly review resorption mechanisms of
bone substitutes.
Materials
Bone is a very permissive organ. As a result, most materials that have been proposed as bone substitutes have had a positive effect on
bone healing and many different materials can be found commercially. This section gives a brief overview of the materials that have
been used with their main features and some past and present research trends.
Ceramics
Considering all nonmetallic inorganic materials as ‘ceramics’ (Kingery et al., 1976), the first ceramics used for bone substitution
were inorganic nonmetallic glasses (so-called bioglass) (Hench et al., 1971); aluminum oxide (Predecki et al., 1972); calcium
aluminate (Hulbert et al., 1970); calcium sulfates (Dreesmann, 1892; Peltier, 1961); and calcium phosphates such as hydroxyap-
atite, b-tricalcium phosphate (Bhaskar et al., 1971), a-tricalcium phosphate, or a mixture thereof (Monroe et al., 1971). Since then
(¼early 1970s), the variety and number of ceramics that have been proposed has literally exploded, particularly in the field of
glasses (Jones, 2013). Several research directions can be distinguished, as described hereafter.
High Mechanical Properties

In the 1970s, efforts were devoted to the synthesis of dense ceramics with the hope of providing bone-bonding ceramics with load-
bearing properties. For example, Akao et al. (1982) described the synthesis of b-tricalcium phosphate (b-TCP) ceramics with
a compressive strength of 459 MPa and a flexural strength of 138 MPa. Jarcho et al. (1979) reported values of 687 MPa in compres-
sion and 154 MPa in tension. The same authors also worked on hydroxyapatite and produced a translucent ceramic with 917 MPa
in compression and 196 MPa in tension (Jarcho et al., 1976). These values are higher than those measured for cortical bone with
a tensile strength between 67 and 104 MPa (Evans, 1976). However, ceramics are by definition brittle whereas bone, a ceramic-
reinforced polymer composite, is fairly tough and resilient. Therefore, efforts to use ceramics in load-bearing applications have
failed due to the risk of catastrophic failures and the concomitant risk of producing knife-sharp splitters. This observation led
researchers to place efforts on the synthesis of composites, in particular polyethylene – hydroxyapatite (Bonfield, 1988; Bonfield
et al., 1981), polyhydroxybutyrate – hydroxyapatite (Doyle et al., 1991), and polylactides – calcium phosphates (Ignatius et al.,
2001a,b) (see hereafter).
Bone Bonding
Another early trend in the field of ceramic bone substitutes was to design ceramics with bone-bonding abilities to prevent instabil-
ities at the bone substitute–bone interface and hence the formation of a fibrous capsule (Hench, 1998). The most famous example is
bioglass (Hench et al., 1971). Since this ability was considered to be a central parameter for bone substitution, efforts were made to
improve it, mostly by coating the implants with a calcium phosphate, or by modifying the surface properties (chemical or physical)
(Le Guéhennec et al., 2007). Efforts were also made to find an in vitro method to predict the in vivo bone-bonding ability of a bone
substitute. These efforts led to the development of the ‘bioactivity’ test (Kokubo and Takadama, 2006), which consists in dipping
a material into a solution mimicking the ionic composition of serum for a few weeks. Materials forming an apatite coating after this
dipping time are considered to be ‘bioactive,’ i.e., able to bond to bone once implanted. Unfortunately, despite some interesting
correlations between in vitro and in vivo results, the test is not universal and present some weaknesses such as the occurrence of
false-positive and false-negative results: some materials become coated with apatite but do not bond to bone, whereas other mate-
rials do not become coated but bond to bone (Bohner and Lemaitre, 2009; Kingery et al., 1976).
Resorbable
Two main strategies can be considered to treat a bone defect with a bone substitute. The first strategy is to use a material that is
mechanically so stable (e.g., Ti–Al–Nb alloy) that the defect filled with the bone substitute materials possesses load-bearing prop-
erties throughout the whole implantation and healing period. Unfortunately, none of the strong materials used as bone substitutes
are resorbable. So, two risks exist: first, the material may eventually fail due to cyclic loading, and second, there is a risk of implant
infection if the patient suffers from bacteremia (Sendi et al., 2011). So, the second strategy for bone substitution is to fix the defect
with a strong temporary implant (e.g., osteosynthesis plates and screws) and fill the defect with a highly osteotransductive material,
i.e., a material that combines a very fast resorption and a high osteogenic potential. Once the defect is healed, the temporary fixation
can be removed. This explains why the last years have seen a surge in the study and use of highly resorbable calcium-based materials
such as bioactive glasses (Jones, 2013), calcium sulfates, b-TCP, brushite (Lemaitre et al., 1987; Munting et al., 1993), monetite
(Galea et al., 2008; Habibovic et al., 2008), or calcium carbonates (Fujita et al., 1991). Mixtures of them have also been considered,
for example, calcium sulfates – calcium phosphates (Abramo et al., 2010) or calcium carbonates – calcium phosphates (Roy and
Linnehan, 1974; Tadier et al., 2011).
Ionic Drugs
A decade ago, Hench proposed the concept of ‘third-generation biomaterials’ (Hench and Polak, 2002), i.e., biomaterials
combining resorbability and bioactivity (in the sense of a therapeutic effect). In the context of bone substitution, efforts have
been made to include specific ions in inorganic non-metallic materials and/or to control their release. One very famous and pioneer-
ing example is provided by silicon-substituted hydroxyapatite developed by Bonfield’s team (Gibson et al., 1999). Unfortunately,
this sintered material does not have a markedly different solubility than pure sintered hydroxyapatite, so Si-substituted hydroxyap-
atite is practically inert (¼non resorbable), and thereby, it is still unclear how a non-resorbable ceramic could benefit from the pres-
ence of Si in its crystallographic structure (Bohner, 2009). More recently, Habibovic and Barralet went much further in this approach
by proposing to load resorbable calcium phosphates with soluble salts containing anions and cations such as phosphates
(Habibovic et al., 2010), carbonates (Yang et al., 2010), cobalt (Patntirapong et al., 2009), copper (Barralet et al., 2009), or zinc
(Yang et al., 2010). These authors introduced the concept of ‘bioinorganics’ in bone substitution (Habibovic and Barralet,
2011). A strong trend is also seen in the bioactive glass field to incorporate specific ions in order to enhance their biological response
(Jones, 2013; Mautner et al., 2013; Hoyle et al., 2004). Using ions as ‘drugs’ is particularly attractive since ions are not considered as
drugs. So, ion-loaded ceramics can in principle be certified in the same way as a ceramic.
Better Handling
As previously mentioned, Hench proposed in 2002 the concept of ‘third-generation biomaterials,’ mentioning that efforts are now
devoted to the synthesis of resorbable biomaterials with biological function. However, some of the bone substitute materials devel-
oped in the 1970s, such as bioactive glasses and b-TCP, had already a combination of resorbability and biological function (via the
release of ions). What has definitively changed in the last 40 years is the appearance of bone substitutes with much better handling.
Figure 1 Dual paste cement after mixing in a static mixer and injection through a cannula.
In the 1970s, bone substitutes were only available as granules and blocks. In 1987, Klein et al. proposed to embed the granules in
a polymer matrix (alginate) to make them injectable. Simultaneously, first efforts were made to develop calcium phosphate cement
(CPC) formulations (Brown and Chow, 1983). Even though the first commercial formulations could be delivered from a cartridge
using a gun and injected directly through a small cannula into the treated bone defect (Constantz et al., 1995), mixing and injection
were fairly complicated. So, efforts were made to design bone substitute materials as ready-to-use pastes, either as nonsetting
(Laschke et al., 2007) or as setting pastes (Takagi et al., 2003; Lemaitre et al., 2003; Chow and Takagi, 2007). Currently, there is
one commercial CPC formulation that consists of two pastes (Lemaitre et al., 2003; Bohner, 2010). Upon application of a force
on the plunger of a syringe, the two pastes are pushed into an elongated and thin static mixer (Figure 1). Once out of the mixer
(and if required a cannula), setting occurs. Quite a few dual-paste formulations are under development, but shelf-life (storage)
and sterilization pose great problems. Such problems are much smaller for nonsetting pastes, so there is a large number of
ready-to-use nonhardening pastes that are available commercially (Bohner, 2010).
Metals
The main advantage of metals compared to ceramics and polymers is their mechanical properties, in particular their strength and
toughness. Therefore, it is not very surprising that porous metals have been used as bone substitutes. Examples include porous tita-
nium (Patil et al., 2010; Tuncer and Arslan, 2009), nitinol (Ni–Ti alloy) (Ayers et al., 1999; Bansiddhi et al., 2008), tantalum (Bobyn
et al., 1999), or magnesium and its alloys (Witte, 2010). However, bone ingrowth is often limited (in amount and distance), which
has triggered authors to work on ways to facilitate bone formation at the material surface (Miyazaki et al., 2001; Takemoto et al.,
2006).
Resorbable Metals
Lately, there has been a clear trend toward the development of resorbable metallic scaffolds, particularly those based on Mg.
However, the resorption (¼corrosion) of Mg-based scaffolds is often difficult to control, and may lead to the formation of hydrogen
bubbles or to the uncontrolled release of toxic elements, for example, Zn (Zberg et al., 2009).
Polymers
Large efforts have been made to use polymers for bone substitution. However, research has been fairly slow until the mid-1990s and
the landmark paper of Langer and Vacanti on tissue engineering (Langer and Vacanti, 1993). Since then, the number of publications
rose dramatically, with typically more than 100 articles per year. The main reason for this increase lies in the fact that polymers have
an elastic modulus and ductility very close to those of human tissues. As a result, the deformations applied on a porous polymer
scaffold can be transmitted to cells sitting on the polymer surface. In such situations, ceramics would either break or not transfer the
deformations due to their high stiffness.
Historically, first efforts to use polymers for bone applications were made in the 1970s and 1980s with synthetic polymers such
as polymethylmethacrylate (PMMA) (De Wijn, 1976; Vaandrager et al., 1983), polysulfone (Spector et al., 1978), or polyethylene
(Spector et al., 1979). At that time, the focus was set on the synthesis of porous coatings for implant fixation (Spector et al., 1988). It
is only in the late 1980s that the use of porous polymers was considered for bone substitution (Korbelar et al., 1988). Particular
emphasis was set on polylactides (Chen et al., 2013) but numerous other polymers such as polyurethanes (Guelcher, 2008), poly-
phosphazene (Syed et al.), and polycaprolactones (Kim et al., 2004; Williams et al., 2005) were also tested.
Polymer scaffolds have in general two problems in bone regeneration: low mechanical properties and limited biological
response (Zürz et al., 1991), possibly due to the uncontrolled release of toxic residues (Ignatius et al., 2001b). In fact, Hubbell
mentioned in 1995 (Hubbell, 1995) that the main challenge in tissue engineering is “the development of resorbable biomaterial
with more favorable tissue interactions during degradation.” To decrease this problem, polymers releasing acid residues during
degradation (such as polylactides) were associated with calcium phosphates (Agrawal and Ray, 2001; Hutmacher, 2000; Rezwan
et al., 2006; Wei and Ma, 2004; Zhang and Ma, 1999). Another approach was the use of hydrogels (Gutowska et al., 2001; Lee
and Mooney, 2001), either natural or synthetic, since the volume fraction of solid material, which may potentially create negative
biological reactions, is very small. Typical natural hydrogels include chitosan (Imahashi, 1994; Snyder and Bish, 1989), alginates
(Lee and Mooney, 2012), or hyaluronan (Bish and Post, 1989; Collins and Birkinshaw, 2013; Burdick and Prestwich, 2011). Among
synthetic hydrogels, Hubbell and coworkers proposed to design hydrogels in such a way that they would react to biological stimuli,
for example, to metalloproteinases (Lutolf and Hubbell, 2005; Hubbell, 1999). Unfortunately, the development of a new polymer
as bone substitute is very expensive and the market is very small, which hinders innovation. As a result, the importance of polymers
for bone substitution is fairly limited. Currently, polymers are mostly used as vehicle for the delivery of ceramic bone substitutes
(D’Este and Eglin, 2013). Before ending this section on polymers, it is important to mention that mussel-inspired synthetic hydro-
gels have been used as glue with the hope (among others) to mend pieces of broken bones (Lee et al., 2007; Waite, 2008).
Xeno- and Allografts

Xeno- and allografts play a very important role in bone substitution. For example, the number one product used in the dental field
(‘BioOss’) is derived from bovine bone. Similarly, the market share of allografts in the US is close to that of autografts and way ahead
of that of synthetic materials. In both cases, there are risks related to disease transmission and immune responses. For instance,
several patients who received an allograft became HIV positive (Hofmann et al., 1995). The potential presence of prions in
bovine-derived xenografts was also discussed (Wenz et al., 2001). In 2005, Albert et al. (2006) mentioned a risk of 1:1 000 000
for the transmission of HIV and 1:200 000 for the transmission of hepatitis C virus. Immunologically, negative results obtained
from the injection of animal-derived hyaluronan were attributed later on to a bad purification of the material (Goldberg and Coutts,
2004; Madhavan and Roy, 1998).
Xenografts
Many xenografts have been used as bone substitutes. The first material that was used on a large scale was ‘Pro Osteon,’ a coral-
derived calcium phosphate ceramic (Figure 2(e)). This material is obtained by the hydrothermal conversion of coralline calcium
carbonate into calcium phosphate in the presence of ammonium phosphate (Roy and Linnehan, 1974). ‘BioOss,’ the most wide-
spread bone substitute in the dental field, is cleaned “through a combination of chemical processing and heat that result in dena-
turation and removal of proteins and other organic substances” (Wenz et al., 2001). Since the temperature of the thermal treatment
is fairly low (close to 300 C), the mineral nanostructure is preserved and the specific surface area remains very high, close to
80 m2 g1 (Weibrich et al., 2000). The product called ‘Endobone’ (Figure 2(d)) is also obtained from bovine bone, but organics
are removed by high-temperature sintering. Compared to ‘BioOss,’ the risk of having organic immunologically relevant residues is
eliminated, but the resulting material has a low-specific surface area, below 1 m2 g1 (Weibrich et al., 2000), and a much higher
crystallinity, which makes the produce fairly inert in vivo.
In recent years, several commercial xenograft products have disappeared, possibly because the requirements set for the certifica-
tion of bone substitutes have increased and/or because commercial products may contain immunogenic remnants (Ghanaati et al.,
2014). When xenografts can be copied synthetically, a shift occurs from a natural to a synthetic production. This is, for example, the
case for hyaluronan which used to be extracted from root comb but which is increasingly produced by bacterial fermentation.
Allografts
Surprisingly, whereas the importance of xenografts is rather decreasing, that of allografts is increasing (Ghanaati et al., 2014). Some-
times, this trend is promoted by the authorities. For example, in France, the reimbursement prices of allografts are much higher than
those of synthetic bone substitutes, possibly because many hospitals possess ‘tissue banks.’
In general, two types of bone substitute can be distinguished. The first type, which could be denominated ‘nonprocessed
allografts,’ is basically a piece of bone that has been retrieved from a patient or a corpse, checked for diseases, and finally cleaned
to remove cells and marrow (Albert et al., 2006). Even though it sounds as a straightforward process, it is long and demanding
(Vangsness Jr. et al., 2006). Such nonprocessed allografts can be machined to be used as spinal fusion cage or to repair segmental
defects, for example, in hip revision. The second type of allograft can be called ‘processed allograft’ because it implies the removal of
the mineral component of bone. Such products are then called ‘demineralized bone’ (Tiedeman et al., 1991; Chakkalakal et al.,
1994; Gruskin et al., 2012), and are often combined with other materials (e.g., hyaluronan) to improve their handling (e.g.,
‘DBX’ product). Demineralized bone is generally found to be osteoinductive (Eid et al., 2001).
As a side note, it must be mentioned that there have been a few scandals in the past related to the use and sale of allografts.
Indeed, the commercial value of bones retrieved from one corpse can reach 100 000 US$. So, the incentive to use human bone
Figure 2 Examples of commercial bone substitute blocks: (a) chronOS (synthetic, b-TCP), (b) Synthacer (synthetic, hydroxyapatite (HA)),
(c) Lubboc (xenograft, bovine HA), (d) Endobon (xenograft, bovine HA), (e) Pro Osteon (xenograft, coralline calcium carbonate converted into
hydroxyapatite), and (f) Vitoss (synthetic, b-TCP).
illegally is high. The most famous case involved a British journalist, Alistair Cooke, and led to the conviction (among others) of an
oral surgeon.
Bone Substitute Formulations
Bone substitutes have been designed in various formulations: granules, blocks/sponges, pastes (or ‘putties’), cements, and
membranes/strips. The decision to take one specific formulation depends on many factors, such as the application, the clinicians
preferences, or simply their availability.
Granules
The granule formulation is the most used among bone substitutes. This is probably related to its relatively low cost and good bio-
logical properties (fast bone ingrowth). Also, granules can fit in any defect form.
Granules are generally sold as angular or spherical particles with a size varying between 0.1 and 5 mm. Too large particles cannot
be inserted into narrow bone defects, whereas too small particles are difficult to handle, particularly if they fall beside the defect.
Therefore, dental products tend to consist of small package volumes (mostly 0.5–1.0 ml) and small granules (<1 mm), whereas
larger package volumes (typically 5–10 ml) and larger granules (>1 mm) are sold in orthopedics.
Among the various design parameters, not only granule size but also granule shape is an important aspect (Figure 3). Indeed,
spherical particles roll well compared to angular granules, which may be advantageous when trying to place or inject such particles
into a small defect. Contrarily, spherical particles present high packing densities, which may be detrimental for bone ingrowth
(limited gap size between granules) and resorption (much material to resorb). Currently, the majority of commercial granules
are angular.
Figure 3 Examples of commercial granular products: (a) Biosorb, (b) Triosite, (c) Cerasorb, (d) Biphasic calcium phosphate (BCP; mixture of b-
tricalcium phosphate and hydroxyapatite), (e) Bioglass, and (f) Ceros/chronOS. Most granules possess a fairly extensive internal porosity. All gran-
ules shown here are synthetic.
As all bone substitutes, granules have to fulfill their ‘spacer’ function, i.e., fill the defect, provide fast bone ingrowth, and possibly
fast turnover into new bone. These requirements have led researchers to optimize the bone substitute architecture, typically porosity,
pore size, and pore interconnection size. Whereas large efforts have been made in the field of scaffolds/blocks (see Section Archi-
tecture), little has been done to determine the best granule porosity, size, and shape. Specifically, there are only very few studies
looking at the size of pores present between piled-up granules and ways to control the intergranular space by optimizing the granule
shape (Choi et al., 2012; Field et al., 2009; Ylä-Soininmäki et al., 2013). Nevertheless, all granules tend to be highly porous
(Figure 3).
Blocks/Sponges
Blocks are generally used to fill large bone defects or defects with very well-defined geometries. For example, wedges are often
applied in knee osteotomies (van Hemert et al., 2004). Also, cages used for intervertebral body fusion can be prefilled with blocks
(Steffen et al., 2001). The size of blocks varies depending on the applications and the producers, but their size (apparent volume) is
typically in the range of 0.1–5 ml. As for granules, smaller blocks are used more intensively in dentistry than in orthopedics.
To provide fast bone ingrowth and possibly fast osteotransduction, the blocks are generally highly porous (Figure 2), typically
between 60 and 90% porosity, and the porosity consists of macropores (Figure 2) and micropores (Figure 4(a)). Occasionally, they
may also contain nanopores (Figure 4(b)), for example, when the scaffolds are produced with CPCs without subsequent sintering.
Bone ingrowth is more difficult with blocks than with granules. Contrarily, due to their architecture, blocks have a higher
mechanical stability than a pile of granules. Another advantage of blocks is their easy handling and the possibility to use them
for tissue engineering purposes.
Commercial blocks are stiff, which has some advantages (mechanical stability), but also some disadvantages, such as the
inability to fit in complex defect shapes or to sustain deformations during loading, which is very important for mechanically stim-
ulating cells in tissue engineering applications.
Figure 4 (a) Microporosity of a synthetic b-TCP bone substitute (courtesy of G. Richards, A.O. Davos); (b) nanoporous calcium-deficient hydroxy-
apatite (CDHA) scaffold.
Pastes (or Putties)

As previously mentioned, handling of granules might be quite cumbersome. For example, filling a narrow bone defect takes much
time and patience, even after mixing the granules with blood or bone marrow to obtain a liquid paste. Also, removing granules that
have fallen beside the bone defect is not easy, particularly when the granules have submillimeter sizes. Therefore, efforts have been
made to provide injectable and/or moldable bone substitute formulations. Most approaches have been based on the use of a poly-
mer matrix and ceramic powders or granules (Bohner, 2010). For example, Klein et al. (1987) proposed in 1987 to mix b-TCP gran-
ules with a sodium alginate solution to obtain an injectable bone substitute paste that would harden after implantation. A similar
approach was used in 1988 by Gerhart et al. who combined b-TCP granules with gelatin, sodium salicylate, paraformaldehyde, and
water. As a third example, Ito (1991) used chitosan to bond hydroxyapatite, Zn oxide, and Ca oxide. However, either purely organic
or purely inorganic approaches have also been proposed. Organic approaches include PMMA and demineralized bone. Interest-
ingly, the latter product was already in use in the nineteenth century and became very popular after the landmark paper of Urist
who demonstrated that demineralized bone induced bone formation after implantation in a muscle (Gruskin et al., 2012; Urist,
1965). It is perhaps of interest to mention here that pastes have been commonly used in bone to control bleeding in bone
(‘sealant’), for example, using fibrin/fibrin clot (Matras, 1982) and beeswax (the so-called bone wax) (Achneck et al., 2010).
Pure ceramic approaches to design bone substitute pastes are mostly based on hydraulic cements, such as calcium sulfate cements
(already in use in the late nineteenth century) (Dreesmann, 1892), and CPCs (Brown and Chow, 1983). However, there is a rapid
growth in the use of nonhardening ceramic pastes consisting of nano- or microsized calcium phosphate particles and an aqueous
solution (Getter et al., 1972; Bohner, 2010; Laschke et al., 2007).
Since many pastes consist of a mixture of a binding phase and granules, their clinical use may potentially lead to leakage and/or
granule release. This might have particularly dramatic consequences, for example, close to blood vessels (risk of embolism (Krebs
et al., 2007; Urlings and Van Der Linden, 2013)) or to the spinal cord (risk of nerve root compression, possibly leading to paralysis
(Ratliff et al., 2001; Kelekis et al., 2003)). Less dramatic but still annoying consequences can be the aseptic leakage of the resorbing
paste through the skin after several weeks of implantation. This has been mentioned by several clinicians in association with non-
setting calcium phosphate pastes.
Cements
As suggested in the last paragraph, it is difficult to categorize pastes and cements because cements can have a very thick (‘putty’)
consistency prior to hardening, whereas nonhardening injectable pastes that do not harden might have a fairly liquid consistency.
Here, pastes that harden are considered to be ‘cements.’
Many cements have been used as bone substitute. The most famous is PMMA because it is widely used to anchor joint replace-
ment prosthesis and to augment bone (Heini and Berlemann, 2001; Lewis, 2006). However, it is not resorbable and has certain
drawbacks such as monomer toxicity leading to hypotension (Karlsson et al., 1995) and high exothermicity causing bone and tissue
necrosis (Belkoff and Molloy, 2003). As a result, alternative polymeric cements have been looked for. Unfortunately, product devel-
opment is hindered by tight regulations (high investment costs until product approval) so the only real innovation in this field over
the last decades has been the launch of a bis-GMA/bis-EMA/TEGDMA cement (‘Cortoss’) a decade ago (Heini and Berlemann, 2001;
Lewis, 2006). Innovation is currently limited to incremental changes of PMMA cements, for example, to change the radiopacity or
the viscosity profile during setting.
Ceramic-based cements include plaster of Paris (PP) (Dreesmann, 1892; Peltier, 1961) and CPCs (Bohner, 2010; Brown and
Chow, 1983). There are also combinations of calcium sulfates and phosphates (Bohner, 2010), calcium aluminates (Axen et al.,
2004), and calcium silicates (Engqvist et al., 2006). Currently, there are close to 50 ceramic-based cement formulations (Bohner,
2010).
In ceramic-based cements, hardening follows a sequence of events, consisting first in the dissolution of the reagent in the aqueous
phase and second in the nucleation (¼formation) and growth of crystals. For PP, the end product is gypsum (¼calcium sulfate dihy-
drate), whereas for CPCs, it is generally either an apatite or brushite (¼calcium sulfate dihydrate). This explains why it is often
referred to ‘apatite CPCs’ and ‘brushite CPCs.’ Recently, various authors have shown that monetite CPCs can be obtained in
nonaqueous hydrophilic liquids (Takemoto et al., 2006; Engstrand et al., 2013).
Since mineral-based cements have transient rheological properties and are meant to be injected in situ, several handling param-
eters are of paramount importance: (1) the setting time (Bohner, 2007), which should be close to 10 min; (2) the setting rate (Boh-
ner, 2007), which should be such that the overall setting reaction is finished shortly after the setting time; (3) the injectability
(Bohner and Baroud, 2005), which is the ability of the cement paste to be injected without a solid–liquid phase separation leading
to syringe plugging; and (4) the cohesion (Bohner et al., 2006), which is the ability of the cement paste to keep its physical integrity
during setting (no wash-out).
Generally, a large excess of water has to be used during the hardening reaction, which leads to the formation of a high porosity
(typically 40–60%). The size of pores present in the cements is controlled by the size of the entangled crystals. Since apatite crystals
are generally smaller than 100 nm (Figure 4(b)), apatite CPCs tend to contain nanopores. Contrarily, brushite CPCs and PP harden
via the entanglement of micrometer-sized crystals, so these cements contain micropores.
Due to the absence of macropores in mineral cements, blood vessel and tissue ingrowth is only possible once the cement is
resorbed, which leads to a fairly slow osteotransduction (Frankenburg et al., 1998; Kobayashi et al., 2007), particularly when
the amount of injected cement is large (Bohner and Baumgart, 2004) and when the reaction product is an apatite. For example,
Kobayashi et al. (2009) reported that only 14% of a vertebral defect filled with an apatite CPC was replaced by bone or marrow
after 3 years of implantation. Thus, apatite CPCs are often considered to present a too slow resorption rate.
The mechanical properties of mineral cements result from the physical entanglement of crystals (Figure 4(b)). As a result,
ceramic-based cements are brittle and weak, particularly under tension and shear. For example, Norian SRS was reported to have
a compressive strength of 55 MPa and a tensile strength of 2.1 MPa (Choi et al., 2012). The mechanical properties of mineral-
based cements are similar to those of a bag full of sand: a high stability under compression (e.g., while sitting on it) and a low
stability under shear or tension (e.g., while pulling sand grains out of the bag).
Membranes/Strips
Besides granules, blocks/sponges, pastes, and cements, bone substitutes are also available as membranes. Membranes are used to
contain bone substitutes in a given location and/or to protect a bone cavity from soft tissue ingrowth (Pineda et al., 1996).
Membranes are particularly used in the dental field for the so-called guided bone regeneration (Schmid et al., 1997; Zitzmann
et al., 1997). Whereas (thin) membranes have generally a rather passive role, mm-thick membranes, also called ‘strips,’ have
a more active role because bone is expected to form within the membrane over time.
Architecture
Very early, researchers wondered what architecture bone substitute materials should have in order to optimize their effect on bone
healing (Hulbert et al., 1970; Klawitter and Hulbert, 1971; Hulbert et al., 1972). It appeared that porous bone substitutes fared
better than nonporous ones, and that results were particularly good when the pores were interconnected and larger than about
100 mm. In the meantime, hundreds of studies have been performed. In 2005, Karageorgiou and Kaplan summarized in a compre-
hensive review the results of these studies by stating that: “the minimum requirement for pore size is considered to be z 100 mm
due to cell size, migration requirements and transport. However, pore sizes >300 mm are recommended, due to enhanced new bone
formation and the formation of capillaries. Because of vascularization, pore size has been shown to affect the progression of osteo-
genesis. Small pores favored hypoxic conditions and induced osteochondral formation before osteogenesis, while large pores, that
are well-vascularized, lead to direct osteogenesis.” In other words, the conclusions that can be drawn from the literature remain
vague apart from the fact that the porous network should be larger than roughly 100 mm.
In 2011, Bohner et al. proposed that one problem in defining the most adequate bone substitute architecture is related to the fact
that the interaction between a bone substitute and the surrounding tissues is multifactorial, and that, as a result, each clinical appli-
cation requires a different bone substitute architecture. These authors also mentioned that the link between bone substitute archi-
tecture and in vivo behavior could be better understood by more adequately monitoring the in vivo response and by relating the
results to finite element models. Specifically, most studies look at a bone substitute globally even though advanced imaging tech-
niques allow local assessments (Komlev et al., 2009, 2010; Langer et al., 2010). Via such means, each and every pore in a bone
substitute could be looked at separately. For example, it should be possible to relate the in vivo evolution of the geometry of a given
pore to the amount of bone formed within this pore. So, instead of getting a few results from the implantation of a scaffold con-
taining, for example, 1000 pores, it should be possible to generate the same information for all 1000 pores of the scaffold.
During many years, the research focus was set on macroporosity and macropore interconnections. Lately, the focus has shifted
toward micropores and microporosity. For example, Lan Levengood et al. (2010) wrote in 2010 that micropores are essential to
achieve ‘multiscale osteointegration’ and that “multiscale osteointegration has the potential to greatly improve overall perfor-
mance of these scaffolds through an improvement of mechanical properties, load transfer, and stability in the long and short
term, and represents a new paradigm for scaffold design.” Some authors are even proposing to use macropore-free bone substi-
tutes provided they are resorbable and contain a high microporosity (Mayr et al., 2009). Indeed, microporous bone substitutes
have been shown to degrade faster than nonmicroporous materials (Klein et al., 1985; Wei et al., 2010; Yokozeki et al., 1998).
Klein et al. (1985) even wrote that changes in microporosity played a more important role in the resorption process than changes
in macroporosity.
In the last two decades, bone substitutes, and in particular calcium phophates, have been shown to promote bone formation in
ectopic sites (e.g., in muscles, subcutaneous). As the formation of bone in an ectopic location has been used as criteria to demon-
strate the osteoinductive potential of an implant or organic substances/molecules, the community has started talking about the
osteoinductive potential of calcium phosphates. Various studies have suggested a link between the architecture and the osteoinduc-
tivity of bone substitutes. For example, Ripamonti et al. (1999) underlined the importance of concavities compared to convexities.
More recently, Yuan et al. (1999) showed that microporous concavities were much more osteoinductive than smooth ones. This
result was confirmed by various researchers, for example, by Coathup et al. (2012) and Chan et al. (2012). The mechanism by which
micropores induce bone formation is still unclear. Le Nihouannen et al. (2005) suggested that the resorption of microporous
samples led to the release of microparticles, hence activating macrophages and osteoinduction (Sage et al., 2010; Brown et al.,
2012). In that respect, it is of interest to mention that the group of Fratzl showed an increase in in vitro bone formation rate
with a decrease in pore curvature (Rumpler et al., 2008; Dunlop et al., 2010).
As explained herein, Lan Levengood et al. (2010) talked about a ‘new paradigm’ when assessing the importance of micropores.
Indeed, Karageorgiou and Kaplan (2005) wrote that pore sizes of 100 mm are required for bone formation even though there is
abundant evidence that bone can form in much smaller pores. Also, cells have been found to invade micropores despite the absence
of blood vessels within the micropores (Lan Levengood et al., 2010; Gaasbeek et al., 2005; Zerbo et al., 2005). In fact, it is known
that cells (like osteocytes) can survive in very confined environment provided there are no further than a few 100 mm from a blood
vessel. Nutrient transport occurs then via diffusion.
So far, the bone substitute architecture has been discussed in terms of porosity. However, it is likely that solid features, such as
grain size, are also important. In fact, various authors mentioned in the past that grain and neck size were important for ceramic
resorption (Van der Meulen and Koerten, 1994; Klein et al., 1984). Moreover, Klein et al. (1986) suggested that the composition
and dimensions of grain boundaries should also be taken into account. Figure 5 shows an example of four different microstructures,
which are likely to affect ceramic resorption.
In Vivo Properties
As indicated herein, most materials that have been tested as bone substitute have been successful, i.e., have promoted bone forma-
tion. This observation is particularly surprising for resorbable bone substitutes because various resorption mechanisms have been
described. Some details are given hereafter.
Dissolution
Most materials sustain a certain degree of dissolution once implanted provided they are soluble in vivo. When solubility is high, for
example, for calcium sulfate dihydrate (gypsum) and in a smaller extend for calcium phosphate dihydrate (brushite), dissolution
Figure 5 Four microstructures of b-TCP bone substitutes. The samples were embedded in a resin, polished, coated, and looked under scanning
electron microscopy (SEM). Pores are in dark color. The scale bar corresponds to a length of 10 mm.
occurs within a few days or weeks (Stubbs et al., 2004; Ikenaga et al., 1998; Bohner et al., 2003). When solubility is very low but not
nil, for example, for metals, dissolution may occur during the whole implantation time, but at such a small rate that detection might
be difficult. Elevated metal ion levels are generally found in patients having orthophedic or osteosynthesis implants (Yan et al.,
2007; Cadosch et al., 2009; Siddiqi et al., 2011). Some materials are not soluble in vivo because physiological fluids are supersat-
urated toward these materials. A typical example is hydroxyapatite. In that case, dissolution can only occur via cell mediation
(see under Cell-Mediated Dissolution).
Cell-Mediated Dissolution
During loading, our bones may fail locally, hence leading to the formation of small cracks. Since an accumulation of these cracks
would eventually lead to fatigue failure, human bodies have a mechanism to regenerate bone. The two main actors of this process
are osteoclasts and osteoblasts (Ducy et al., 2000; Teitelbaum, 2000). Osteoclasts remove bone around cracks, and osteoblasts form
new bone in these resorbed areas. Since some calcium phosphate materials have a composition and solubility similar to those of
bone mineral, they can be resorbed by osteoclasts and replaced by new bone via the action of osteoblasts. The best examples are
probably b-TCP and apatites produced at or close to room temperature (Figure 6). Calcium phosphates that are thermodynamically
more stable than these two calcium phosphates, for example, sintered apatites, can also be resorbed by osteoclasts, but their resorp-
tion rate is so slow that the materials can be considered to be inert. In some cases, bone fractures have been associated with the
presence of sintered apatite bone substitutes (Linhart et al., 2004).
The second cells that play an important role in bone substitute resorption are macrophages. Whereas osteoclasts act on surfaces,
macrophages ingest loose particles or debris (Brown et al., 2012). As previously mentioned, various authors have proposed that the
activation of macrophages by the release of loose calcium phosphate microparticles might trigger osteoinduction (Le Nihouannen
et al., 2005; Yuan et al., 1999). Even though this mechanism is only one of various mechanisms proposed to explain osteoinduc-
tion, it is of great interest to better understand what parameters lead to particle release, and how these particles stimulate macro-
phages, eventually leading to osteoinduction. Despite efforts in this direction (and a lot of activities with intellectual property), our
current knowledge is very limited.
Enzymatic Digestion
Polymers that are naturally present in the human body are degraded by enzymes. For example, collagen is degraded by collagenase
(West and Hubbell, 1999), hyaluronan by hyaluronase (Fraser et al., 1984), and fibrin by plasmin (West and Hubbell, 1999).
Transport
Some polymers such as cellulose derivatives are soluble in vivo, but cannot be degraded by the human metabolism. As a result,
resorption proceeds by a combination of dissolution and transport, first to the lymph nodes, and then to the kidneys.
Figure 6 Osteoclasts (in red) resorbing a b-TCP porous ceramic.

Hydrolysis
Many polymers, like polylactides, polyglycolides, and polycaprolactones, are resorbed by hydrolysis (Woodruff and Hutmacher,
2010). This process is generally fairly independent of the biological environment, which means that the in vivo resorption time
can be fairly well predicted with in vitro tests. This is very interesting for drug delivery purposes, because drug release can be timed,
but somewhat disadvantageous for bone regeneration because the latter may vary a lot depending on the patient and the clinical
indication. As a result, a mismatch between bone substitute degradation and new bone formation might occur.
The hydrolytically driven resorption pattern is polymer dependent. Some polymers like polyglycolides present an autocatalytic
degradation (Woodruff and Hutmacher, 2010). This may lead to “an outer layer of higher molecular weight skin with a lower
molecular weight, degraded, interior” (Woodruff and Hutmacher, 2010). It may also lead to inflammatory reactions if the latter
skin breaks (Böstman and Pihlajamäki, 2000). The occurrence of this autocatalytic reaction is the reason why polylactides and poly-
glycolides are often combined with calcium phosphate powders to buffer the pH (Ignatius et al., 2001a,b). Chemical modifications
can obviously be used to control resorption rate. This is widely used in pharmaceutics, by varying, for example, the fraction of poly-
glycolic acid in polylactic acid. However, this approach may bear some risks related to product certification, particularly with new
copolymer compositions.
Corrosion
The last mechanism leading to resorption is corrosion. A typical example is Mg. The reaction is:
Mg þ 2H2O ¼ Mg(OH)2 þ H2
This reaction leads to the formation of a fairly basic compound and the release of gas. If Mg corrosion is fast, the release of a basic
compound can trigger the precipitation of a calcium phosphate (Witte et al., 2005) and lead to the formation of gas bubbles. One
gram of Mg can produce one liter of hydrogen (Witte et al., 2009). Another example of corrodible material is iron (Peuster et al.,
2006).
Conclusion
Many materials have been proposed as bone substitute, and most of them have proved to work well. Generally, inorganic nonme-
tallic materials (zceramic) present the best biological performance. Metals have to be favored when high loads are at play. Finally,
polymers and hydrogels are of particular interest for tissue engineering and to improve the handling of ceramic-based bone graft
substitutes.
The performance of bone substitute materials is a function of many physicochemical parameters, such as composition, size,
surface area, or architecture. However, they also depend on their handling because the materials will eventually have to be used
and implanted by a clinician. In that respect, ready-to-use injectable pastes are of particular interest.
Many new approaches have been proposed over the past decades to improve the performance of bone substitutes. Many involve
the use of drugs, cells, and new materials. However, increasingly stringent regulations and pressure on healthcare expenditures are
hindering innovation. So, current commercial innovations are mainly focused on incremental improvements, far from academic
research. The gap between academic research and commercial reality is expected to widen even more.
References
Abramo, A., Geijer, M., Kopylov, P., & Tägil, M. (2010). Clinical device-related article osteotomy of distal radius fracture malunion using a fast remodeling bone substitute consisting
of calcium sulphate and calcium phosphate. J.Biomed. Mater. Res. – Part B Appl. Biomater., 92, 281–286.
Achneck, H. E., Sileshi, B., Jamiolkowski, R. M., Albala, D. M., Shapiro, M. L., & Lawson, J. H. (2010). A comprehensive review of topical hemostatic agents: efficacy and
recommendations for use. Ann. Surg., 251, 217–228.
Agrawal, C. M., & Ray, R. B. (2001). Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J. Biomed. Mater. Res., 55, 141–150.
Akao, M., Aoki, H., Kato, K., & Sato, A. (1982). Dense polycrystalline b-tricalcium phosphate for prosthetic applications. J. Mater. Sci., 17, 343–346.
Albert, A., Leemrijse, T., Druez, V., Delloye, C., & Cornu, O. (2006). Are bone autografts still necessary in 2006? A three-year retrospective study of bone grafting. Acta Orthop.
Belg., 72, 734–740.
Arrington, E. D., Smith, W. J., Chambers, H. G., Bucknell, A. L., & Davino, N. A. (1996). Complications of iliac crest bone graft harvesting. Clin. Orthop., 300–309.
Axen, N., Persson, T., Bjorklund, K., Engqvist, H., & Hermansson, L. (2004). An injectable bone void filler cement based on Ca-aluminate. Bioceramics, 16, 254–2, 265–268.
Ayers, R. A., Simske, S. J., Bateman, T. A., Petkus, A., Sachdeva, R. L., & Gyunter, V. E. (1999). Effect of nitinol implant porosity on cranial bone ingrowth and apposition after
6 weeks. J. Biomed. Mater. Res., 45, 42–47.
Bansiddhi, A., Sargeant, T. D., Stupp, S. I., & Dunand, D. C. (2008). Porous NiTi for bone implants: a review. Acta Biomater., 4, 773–782.
Barralet, J., Gbureck, U., Habibovic, P., Vorndran, E., Gerard, C., & Doillon, C. J. (2009). Angiogenesis in calcium phosphate scaffolds by inorganic copper ion release. Tissue Eng.
Part A, 15, 1601–1609.
Belkoff, S. M., & Molloy, S. (2003). Temperature measurement during polymerization of polymethylmethacrylate cement used for vertebroplasty. Spine, 28, 1555–1559.
Bhaskar, S. N., Brady, J. M., Getter, L., Grower, M. F., & Driskell, T. (1971). Biodegradable ceramic implants in bone. Electron and light microscopic analysis. Oral Surg. Oral Med.
Oral Pathol., 32, 336–346.
Bish, D. L., & Post, J. E. (1989). Modern Powder Diffraction. Washington, DC: The Mineralogical Society of America.
Bobyn, J. D., Stackpool, G. J., Hacking, S. A., Tanzer, M., & Krygier, J. J. (1999). Characteristics of bone ingrowth and interface mechanics of a new porous tantalum biomaterial.
J. Bone Joint Surg. Br., 81, 907–914.
Bohner, M. (2007). Reactivity of calcium phosphate cements. J. Mater. Chem., 17, 3980–3986.
Bohner, M. (2009). Silicon-substituted calcium phosphates - a critical view. Biomaterials, 30, 6403–6406.
Bohner, M. (2010). Design of ceramic-based cements and putties for bone graft substitution. Eur. Cells and Mater., 20, 1–12.
Bohner, M., & Baroud, G. (2005). Injectability of calcium phosphate pastes. Biomaterials, 26, 1553–1563.
Bohner, M., & Baumgart, F. (2004). Theoretical model to determine the effects of geometrical factors on the resorption of calcium phosphate bone substitutes. Biomaterials, 25,
3569–3582.
Bohner, M., & Lemaitre, J. (2009). Can bioactivity be tested in vitro with SBF solution? Biomaterials, 30, 2175–2179.
Bohner, M., Theiss, F., Apelt, D., Hirsiger, W., Houriet, R., Rizzoli, G., Gnos, E., Frei, C., Auer, J. A., & Von Rechenberg, B. (2003). Compositional changes of a dicalcium phosphate
dihydrate cement after implantation in sheep. Biomaterials, 24, 3463–3474.
Bohner, M., Doebelin, N., & Baroud, G. (2006). Theoretical and experimental approach to test the cohesion of calcium phosphate pastes. Eur. Cell Mater., 12, 26–35.
Bohner, M., Loosli, Y., Baroud, G., & Lacroix, D. (2011). Deciphering the link between architecture and biological response of a bone graft substitute. Acta Biomater., 7, 478–484.
Bonfield, W. (1988). Composites for bone replacement. J. Biomed. Eng., 10, 522–526.
Bonfield, W., Grynpas, M., Tully, A. E., Bowman, J., & Abram, J. (1981). Hydroxyapatite reinforced polyethylene–a mechanically compatible implant material for bone replacement.
Böstman, O. M., & Pihlajamäki, H. K. (2000). Adverse tissue reactions to bioabsorbable fixation devices. Clin. Orthop. Relat. Res., 216–227.
Brown, W. E., & Chow, L. C. (1983). A new calcium phosphate setting cement. J. Dent. Res., 62, 672.
Brown, B. N., Ratner, B. D., Goodman, S. B., Amar, S., & Badylak, S. F. (2012). Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative
medicine. Biomaterials, 33, 3792–3802.
Burdick, J. A., & Prestwich, G. D. (2011). Hyaluronic acid hydrogels for biomedical applications. Adv. Mater., 23, H41–H56.
Cadosch, D., Chan, E., Gautschi, O. P., & Filgueira, L. (2009). Metal is not inert: role of metal ions released by biocorrosion in aseptic loosening – current concepts. J. Biomed.
Mater. Res. – Part A, 91, 1252–1262.
Chakkalakal, D. A., Mashoof, A. A., Novak, J., Strates, B. S., & Mcguire, M. H. (1994). Mineralization and pH relationships in healing skeletal defects grafted with demineralized bone
matrix. J. Biomed. Mater. Res., 28, 1439–1443.
Chan, O., Coathup, M. J., Nesbitt, A., Ho, C. Y., Hing, K. A., Buckland, T., Campion, C., & blunn, G. W. (2012). The effects of microporosity on osteoinduction of calcium phosphate
bone graft substitute biomaterials. Acta Biomater., 8, 2788–2794.
Chen, Z., Liu, H., Liu, X., Lian, X., Guo, Z., Jiang, H. J., & Cui, F. Z. (2013). Improved workability of injectable calcium sulfate bone cement by regulation of self-setting properties.
Mater. Sci. Eng. C, 33, 1048–1053.
Choi, S., Liu, I. L., Yamamoto, K., Igawa, K., Mochizuki, M., Sakai, T., Echigo, R., Honnami, M., Suzuki, S., Chung, U. I., & Sasaki, N. (2012). Development and evaluation of
tetrapod-shaped granular artificial bones. Acta Biomater., 8, 2340–2347.
Chow, L.C., Takagi, S., 2007. Dual-phase cement precursor system for bone repair. WO Patent Application PCT/US2006/401034.
Coathup, M. J., Hing, K. A., Samizadeh, S., Chan, O., Fang, Y. S., Campion, C., Buckland, T., & blunn, G. W. (2012). Effect of increased strut porosity of calcium phosphate bone
graft substitute biomaterials on osteoinduction. J. Biomed. Mater. Res. – Part A, 100 A, 1550–1555.
Collins, M. N., & Birkinshaw, C. (2013). Hyaluronic acid based scaffolds for tissue engineering – a review. Carbohydr. Polym., 92, 1262–1279.
Constantz, B. R., Ison, I. C., Fulmer, M. T., Poser, R. D., Smith, S. T., Vanwagoner, M., Ross, J., Goldstein, S. A., Jupiter, J. B., & Rosenthal, D. I. (1995). Skeletal repair by in situ
formation of the mineral phase of bone. Science, 267, 1796–1799.
D’Este, M., & Eglin, D. (2013). Hydrogels in calcium phosphate moldable and injectable bone substitutes: sticky excipients or advanced 3-D carriers? Acta Biomater., 9, 5421–5430.
De Wijn, J. R. (1976). Poly(methyl methacrylate)–aqueous phase blends: in situ curing porous materials. J. Biomed. Mater. Res., 10, 625–635.
Doyle, C., Tanner, E. T., & Bonfield, W. (1991). In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite. Biomaterials, 12,
841–847.
Dreesmann, H. (1892). Ueber Knochenplombierung. Beitr. Klin. Chir., 9, 804–810.
Driessens, F. C., Planell, J. A., Boltong, M. G., Khairoun, I., & Ginebra, M. P. (1998). Osteotransductive bone cements. Proc. Inst. Mech. Eng. H, 212, 427–435.
Ducy, P., Schinke, T., & Karsenty, G. (2000). The osteoblast: a sophisticated fibroblast under central surveillance. Science, 289, 1501–1504.
Dunlop, J. W. C., Fischer, F. D., Gamsjã¤ger, E., & Fratzl, P. (2010). A theoretical model for tissue growth in confined geometries. J. Mech. Phys. Solids, 58, 1073–1087.
Eid, K., Zelicof, S., Perona, B. P., Sledge, C. B., & Glowacki, J. (2001). Tissue reactions to particles of bone-substitute materials in intraosseous and heterotopic sites in rats:
discrimination of osteoinduction, osteocompatibility, and inflammation. J. Orthop. Res., 19, 962–969.
Engqvist, H., Edlund, S., Gomez-Ortega, G., Loof, J., & Hermansson, L. (2006). In vitro mechanical properties of a calcium silicate based bone void filler. Key Eng. Mater., 309–
311(II), 829–832.
Engstrand, J., Aberg, J., & Engqvist, H. (2013). Influence of water content on hardening and handling of a premixed calcium phosphate cement. Mater. Sci. Eng. C, 33, 527–531.
Evans, F. G. (1976). Mechanical properties and histology of cortical bone from younger and older men. Anat. Rec., 185, 1–11.
Field, J. R., Mcgee, M., Wildenauer, C., Kurmis, A., & Margerrison, E. (2009). The utilization of a synthetic bone void filler (JAX) in the repair of a femoral segmental defect. Vet.
Comp. Orthop. Traumatol., 22, 87–95.
Frankenburg, E. P., Goldstein, S. A., Bauer, T. W., Harris, S. A., & Poser, R. D. (1998). Biomechanical and histological evaluation of a calcium phosphate cement. J. Bone Joint
Surg. Am., 80, 1112–1124.
Fraser, J. R., Laurent, T. C., Engstrom-Laurent, A., & Laurent, U. G. (1984). Elimination of hyaluronic acid from the blood stream in the human. Clin. Exp. Pharmacol. Physiol., 11,
17–25.
Fujita, Y., Yamamuro, T., Nakamura, T., Kotani, S., Ohtsuki, C., & Kokubo, T. (1991). The bonding behavior of calcite to bone. J. Biomed. Mater. Res., 25, 991–1003.
Gaasbeek, R. D., Toonen, H. G., Van Heerwaarden, R. J., & Buma, P. (2005). Mechanism of bone incorporation of beta-TCP bone substitute in open wedge tibial osteotomy in
patients. Biomaterials, 26, 6713–6719.
Galea, L. G., Bohner, M., Lemaitre, J., Kohler, T., & Muller, R. (2008). Bone substitute: transforming b-tricalcium phosphate porous scaffolds into monetite. Biomaterials, 29,
3400–3407.
Gerhart, T. N., Miller, R. L., Kleshinski, S. J., & Hayes, W. C. (1988). In vitro characterization and biomechanical optimization of a biodegradable particulate composite bone cement.
J. Biomed. Mater. Res., 22, 1071–1082.
Getter, L., Bhaskar, N. S., Cutright, D. E., Perez, B., Brady, J. M., Driskell, T. D., & O’hara, M. J. (1972). Three biodegradable calcium phosphate slurry implants in bone. J. Oral
Surg., 30, 263–268.
Ghanaati, S., Barbeck, M., Booms, P., Lorenz, J., Kirkpatrick, C. J., & Sader, R. (2014). Potential lack of “standardized” processing techniques for production of allogeneic and
xenogeneic bone blocks for application in humans. Acta Biomater. doi: https://doi.org/10.1016/j.actbio.2014.04.017.
Gibson, I. R., Best, S. M., & Bonfield, W. (1999). Chemical characterization of silicon-substituted hydroxyapatite. J. Biomed. Mater. Res., 44, 422–428.
Goldberg, V. M., & Coutts, R. D. (2004). Pseudoseptic reactions to hylan viscosupplementation: diagnosis and treatment. Clin. Orthop. Relat. Res., 130–137.
Gruskin, E., Doll, B. A., Futrell, F. W., Schmitz, J. P., & Hollinger, J. O. (2012). Demineralized bone matrix in bone repair: history and use. Adv. Drug Deliv. Rev., 64, 1063–1077.
Guelcher, S. A. (2008). Biodegradable polyurethanes: synthesis and applications in regenerative medicine. Tissue Eng. – Part B: Rev., 14, 3–17.
Gutowska, A., Jeong, B., & Jasionowski, M. (2001). Injectable gels for tissue engineering. Anat. Rec., 263, 342–349.
Habibovic, P., & Barralet, J. E. (2011). Bioinorganics and biomaterials: bone repair. Acta Biomater., 7, 3013–3026.
Habibovic, P., Gbureck, U., Doillon, C. J., Bassett, D. C., Van Blitterswijk, C. A., & Barralet, J. E. (2008). Osteoconduction and osteoinduction of low-temperature 3D printed
bioceramic implants. Biomaterials, 29, 944–953.
Habibovic, P., Bassett, D. C., Doillon, C. J., Gerard, C., Mckee, M. D., & Barralet, J. E. (2010). Collagen biomineralization in vivo by sustained release of inorganic phosphate ions.
Adv. Mater., 22, 1858–1862.
Heini, P. F., & Berlemann, U. (2001). Bone substitutes in vertebroplasty. Eur. Spine J., 10(Suppl. 2), S205–S213.
Hench, L. L. (1998). Bioceramics. J. Am. Ceram. Soc., 81, 1705–1727.
Hench, L. L. (2006). The story of bioglass. J. Mater. Sci. Mater. Med., 17, 967–978.
Hench, L. L., & Polak, J. M. (2002). Third-generation biomedical materials. Science, 295, 1014–1017.
Hench, L. L., Splinter, R. J., Allen, W. C., & Greenlee, T. K. (1971). Bonding mechanisms at the interface of ceramic prosthetic materials. J. Biomed. Mater. Res., 5(6), 117–141.
Hofmann, G. O., Kirschner, M. H., Wangemann, T., Falk, C., Mempel, W., & Hammer, C. (1995). Infections and immunological hazards of allogeneic bone transplantation. Arch.
Orthop. Trauma Surg., 114, 159–166.
Hoyle, C. E., Lee, T. Y., & Roper, T. (2004). Thiol-enes: chemistry of the past with promise for the future. J. Polym. Sci., Part A: Polym. Chem., 42, 5301–5338.
Hubbell, J. A. (1995). Biomaterials in tissue engineering. Biotechnology (N.Y.), 13, 565–576.
Hubbell, J. A. (1999). Bioactive biomaterials. Curr. Opin. Biotechnol., 10, 123–129.
Hulbert, S. F., Young, F. A., Mathews, R. S., Klawitter, J. J., Talbert, C. D., & Stelling, F. H. (1970). Potential of ceramic materials as permanently implantable skeletal prostheses.
Hulbert, S. F., Morrison, S. J., & Klawitter, J. J. (1972). Tissue reaction to three ceramics of porous and non-porous structures. J. Biomed. Mater. Res., 6, 347–374.
Hutmacher, D. W. (2000). Scaffolds in tissue engineering bone and cartilage. Biomaterials, 21, 2529–2543.
van Hemert, W. L., Willems, K., Anderson, P. G., Van Heerwaarden, R. J., & Wymenga, A. B. (2004). Tricalcium phosphate granules or rigid wedge preforms in open wedge high
tibial osteotomy: a radiological study with a new evaluation system. Knee, 11, 451–456.
Ignatius, A. A., Augat, P., & Claes, L. E. (2001a). Degradation behavior of composite pins made of tricalcium phosphate and poly(L,DL-lactide). J. Biomater. Sci. Polym. Ed., 12,
185–194.
Ignatius, A. A., Betz, O., Augat, P., & Claes, L. E. (2001b). In vivo investigations on composites made of resorbable ceramics and poly(lactide) used as bone graft substitutes.
Ikenaga, M., Hardouin, P., Lemaitre, J., Andrianjatovo, H., & Flautre, B. (1998). Biomechanical characterization of a biodegradable calcium phosphate hydraulic cement:
a comparison with porous biphasic calcium phosphate ceramics. J. Biomed. Mater. Res., 40, 139–144.
Ito, M. (1991). In vitro properties of a chitosan-bonded hydroxyapatite bone-filling paste. Biomaterials, 12, 41–45.
Jarcho, M., Bolen, C. H., Thomas, M. B., Bobick, J., Kay, J. F., & Doremus, R. H. (1976). Hydroxylapatite synthesis and characterization in dense polycrystalline form. J. Mater. Sci.
Lett., 11, 2027–2035.
Jarcho, M., Salsbury, R. L., Thomas, M. B., & Doremus, R. H. (1979). Synthesis and fabrication of b-tricalcium phosphate (whitlockite) ceramics for potential prosthetic applications.
J. Mater. Sci., 14, 142–150.
Jones, J. R. (2013). Review of bioactive glass: from hench to hybrids. Acta Biomater., 9, 4457–4486.
Karageorgiou, V., & Kaplan, D. (2005). Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials, 26, 5474–5491.
Karlsson, J., Wendling, W., Chen, D., Zelinsky, J., Jeevanandam, V., Hellman, S., & Carlsson, C. (1995). Methylmethacrylate monomer produces direct relaxation of vascular smooth
muscle in vitro. Acta Anaesthesiol. Scand., 39, 685–689.
Kelekis, A. D., Martin, J. B., Somon, T., Wetzel, S. G., Dietrich, P. Y., & Ruefenacht, D. A. (2003). Radicular pain after vertebroplasty: compression or irritation of the nerve root?
Initial experience with the “cooling system”. Spine, 28, E265–E269.
Kim, H. W., Knowles, J. C., & Kim, H. E. (2004). Hydroxyapatite/poly(ε-caprolactone) composite coatings on hydroxyapatite porous bone scaffold for drug delivery. Biomaterials, 25,
1279–1287.
Kingery, W. D., Bowen, H. K., & Uhlmann, D. R. (1976). Introduction to Ceramics. USA: John Wiley and Sons.
Klawitter, J. J., & Hulbert, S. F. (1971). Application of porous ceramics for the attachment of load bearing internal orthopedic applications. J. Biomed. Mater. Res. Symp., 2,
161–229.
Klein, C. P. A. T., Driessen, A. A., & De Groot, K. (1984). Relationship between the degradation behaviour of calcium phosphate ceramics and their physical/chemical characteristics
and ultrastructural geometry. Biomaterials, 5, 157–160.
Klein, C. P., De Groot, K., Driessen, A. A., & Van Der Lubbe, H. B. (1985). Interaction of biodegradable beta-whitlockite ceramics with bone tissue: an in vivo study. Biomaterials, 6,
189–192.
Klein, C. P. A. T., De Groot, K., Driessen, A. A., & Van Der Lubbe, H. B. M. (1986). A comparative study of different b-whitlockite ceramics in rabbit cortical bone with regard to their
biodegradation behaviour. Biomaterials, 7, 144–146.
Klein, C. P., Van Der Lubbe, H. B., & De Groot, K. (1987). A plastic composite of alginate with calcium phosphate granulate as implant material: an in vivo study. Biomaterials, 8,
308–310.
Kobayashi, N., Ong, K., Villarraga, M., Schwardt, J., Wenz, R., Togawa, D., Fujishiro, T., Turner, A. S., Seim, H. B., III, & Bauer, T. W. (2007). Histological and mechanical evaluation
of self-setting calcium phosphate cements in a sheep vertebral bone void model. J. Biomed. Mater. Res. – Part A, 81, 838–846.
Kobayashi, H., Fujishiro, T., Belkoff, S. M., Kobayashi, N., Turner, A. S., Seim, H. B., III, Zitelli, J., Hawkins, M., & Bauer, T. W. (2009). Long-term evaluation of a calcium phosphate
bone cement with carboxymethyl cellulose in a vertebral defect model. J. Biomed. Mater. Res. – Part A, 88, 880–888.
Kokubo, T., & Takadama, H. (2006). How useful is SBF in predicting in vivo bone bioactivity? Biomaterials, 27, 2907–2915.
Komlev, V. S., Mastrogiacomo, M., Peyrin, F., Cancedda, R., & Rustichelli, F. (2009). X-ray synchrotron radiation pseudo-holotomography as a new imaging technique to investigate
angio- and microvasculogenesis with no usage of contrast agents. Tissue Eng. – Part C: Methods, 15, 425–430.
Komlev, V. S., Mastrogiacomo, M., Pereira, R. C., Peyrin, F., Rustichelli, F., & Cancedda, R. (2010). Biodegradation of porous calcium phosphate scaffolds in an ectopic bone
formation model studied by X-ray computed microtomography. Eur. Cells Mater., 19, 136–146.
Korbelar, P., Vacik, J., & Dylevsky, I. (1988). Experimental implantation of hydrogel into the bone. J. Biomed. Mater. Res., 22, 751–762.
Krebs, J., Aebli, N., Goss, B. G., Sugiyama, S., Bardyn, T., Boecken, I., Leamy, P. J., & Ferguson, S. J. (2007). Cardiovascular changes after pulmonary embolism from injecting
calcium phosphate cement. J. Biomed. Mater. Res. B Appl. Biomater., 82, 526–532.
Lan Levengood, S. K., Polak, S. J., Wheeler, M. B., Maki, A. J., Clark, S. G., Jamison, R. D., & Wagoner Johnson, A. J. (2010). Multiscale osteointegration as a new paradigm for
the design of calcium phosphate scaffolds for bone regeneration. Biomaterials, 31, 3552–3563.
Langer, R., & Vacanti, J. P. (1993). Tissue engineering. Science, 260, 920–926.
Langer, M., Liu, Y., Tortelli, F., Cloetens, P., Cancedda, R., & Peyrin, F. (2010). Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone
scaffold. J. Microsc., 238, 230–239.
Laschke, M. W., Witt, K., Pohlemann, T., & Menger, M. D. (2007). Injectable nanocrystalline hydroxyapatite paste for bone substitution: in vivo analysis of biocompatibility and
vascularization. J. Biomed. Mater. Res. B Appl. Biomater., 82, 494–505.
Le Guéhennec, L., Soueidan, A., Layrolle, P., & Amouriq, Y. (2007). Surface treatments of titanium dental implants for rapid osseointegration. Dent. Mater., 23, 844–854.
Le Nihouannen, D., Daculsi, G., Saffarzadeh, A., Gauthier, O., Delplace, S., Pilet, P., & Layrolle, P. (2005). Ectopic bone formation by microporous calcium phosphate ceramic
particles in sheep muscles. Bone, 36, 1086–1093.
Lee, K. Y., & Mooney, D. J. (2001). Hydrogels for tissue engineering. Chem. Rev., 101, 1869–1879.
Lee, K. Y., & Mooney, D. J. (2012). Alginate: properties and biomedical applications. Prog. Polym. Sci. (Oxford), 37, 106–126.
Lee, H., Dellatore, S. M., Miller, W. M., & Messersmith, P. B. (2007). Mussel-inspired surface chemistry for multifunctional coatings. Science, 318, 426–430.
Lemaitre, J., Mirtchi, A., & Mortier, A. (1987). Calcium phosphate cements for medical use: state of the art and perspectives of development. Silic. Ind., 9-10, 141–146.
Lemaitre, J., Pittet, C., Brendlen, D., 2003. Pasty or liquid multiple constituent Patentfor injectable calcium phosphate cements. Source: WO 03/041753 A1.
Lewis, G. (2006). Injectable bone cements for use in vertebroplasty and kyphoplasty: state-of-the-art review. J. Biomed. Mater. Res. B Appl. Biomater., 76, 456–468.
Linhart, W., Briem, D., Amling, M., Rueger, J. M., & Windolf, J. (2004). Mechanical failure of a porous hydroxyapatite ceramic 7.5 years after treatment of a fracture of the proximal
tibia. Unfallchirurg, 107, 154–157.
Lutolf, M. P., & Hubbell, J. A. (2005). Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol., 23, 47–55.
Imahashi, T. M. (1994). Transformations of gypsum to calcium sulfate hemihydrate and hemihydrate to gypsum in NaCl solutions. Bull. Chem. Soc. Jpn., 67, 1961–1964.
Madhavan, H. N., & Roy, S. (1998). Effects of viscoelastic ophthalmic solutions on cell cultures. Indian J. Ophthalmol., 46, 37–40.
Matras, H. (1982). The use of fibrin sealant in oral and maxillofacial surgery. J. Oral Maxillofac. Surg., 40, 617–622.
Mautner, A., Qin, X., Wutzel, H., Ligon, S. C., Kapeller, B., Moser, D., Russmueller, G., Stampfl, J., & Liska, R. (2013). Thiol-ene photopolymerization for efficient curing of vinyl
esters. J. Polym. Sci., Part A: Polym. Chem., 51, 203–212.
Mayr, H. O., Dietrich, M., Fraedrich, F., Hube, R., Nerlich, A., Von Eisenhart-Rothe, R., Hein, W., & Bernstein, A. (2009). Microporous pure beta-tricalcium phosphate implants for
press-fit fixation of anterior cruciate ligament grafts: strength and healing in a sheep model. Arthroscopy, 25, 996–1005.
Miyazaki, T., Kim, H. M., Kokubo, T., Kato, H., & Nakamura, T. (2001). Induction and acceleration of bonelike apatite formation on tantalum oxide gel in simulated body fluid. J. Sol-
gel Sci. and Technol., 21, 83–88.
Monroe, E. A., Votava, W., Bass, D. B., & Mcmullen, J. (1971). New calcium phosphate ceramic material for bone and tooth implants. J. Dent. Res., 50, 860–861.
Munting, E., Mirtchi, A., & Lemaître, J. (1993). Bone repair of defects filled with a phosphocalcic hydraulic cement: and in vivo study. J. Mater. Sci. Mater. Med., 4, 337–344.
Patil, J. S., Kamalapur, M. V., Marapur, S. C., & Kadam, D. V. (2010). Ionotropic gelation and polyelectrolyte complexation: the novel techniques to design hydrogel particulate
sustained, modulated drug delivery system: a review. Dig. J. Nanomater. Bios., 5, 241–248.
Patntirapong, S., Habibovic, P., & Hauschka, P. V. (2009). Effects of soluble cobalt and cobalt incorporated into calcium phosphate layers on osteoclast differentiation and activation.
Peltier, L. F. (1961). The use of plaster of Paris to fill defects in bone. Clin. Orthop., 21, 1–31.
Peuster, M., Hesse, C., Schloo, T., Fink, C., Beerbaum, P., & Von Schnakenburg, C. (2006). Long-term biocompatibility of a corrodible peripheral iron stent in the porcine
descending aorta. Biomaterials, 27, 4955–4962.
Pineda, L. M., Busing, M., Meinig, R. P., & Gogolewski, S. (1996). Bone regeneration with resorbable polymeric membranes. III. Effect of poly(L-lactide) membrane pore size on the
bone healing process in large defects. J. Biomed. Mater. Res., 31, 385–394.
Predecki, P., Stephan, J. E., Auslaender, B. A., Mooney, V. L., & Kirkland, K. (1972). Kinetics of bone growth into cylindrical channels in aluminum oxide and titanium. J. Biomed.
Mater. Res., 6, 375–400.
Ratliff, J., Nguyen, T., & Heiss, J. (2001). Root and spinal cord compression from methylmethacrylate vertebroplasty. Spine, 26, E300–E302.
Ripamonti, U., Crooks, J., & Kirkbride, A. N. (1999). Sintered porous hydroxyapatites with intrinsic osteoinductive activity: geometric induction of bone formation. S. Afr. J. Sci., 95,
335–343.
Roy, D. M., & Linnehan, S. K. (1974). Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange. Nature, 247, 220–222.
Rumpler, M., Woesz, A., Dunlop, J. W., Van Dongen, J. T., & Fratzl, P. (2008). The effect of geometry on three-dimensional tissue growth. J. R. Soc. Interface., 5, 1173–1180.
Sage, A. P., Tintut, Y., & Demer, L. L. (2010). Regulatory mechanisms in vascular calcification. Nat. Rev. Cardiol., 7, 528–536.
Schmid, J., Hammerle, C. H., Fluckiger, L., Winkler, J. R., Olah, A. J., Gogolewski, S., & Lang, N. P. (1997). Blood-filled spaces with and without filler materials in guided bone
regeneration. A comparative experimental study in the rabbit using bioresorbable membranes. Clin. Oral Implants Res., 8, 75–81.
Sendi, P., Banderet, F., Graber, P., & Zimmerli, W. (2011). Periprosthetic joint infection following Staphylococcus aureus bacteremia. J. Infect., 63, 17–22.
Siddiqi, A., Payne, A. G. T., De Silva, R. K., & Duncan, W. J. (2011). Titanium allergy: could it affect dental implant integration? Clin. Oral Implan. Res., 22, 673–680.
Snyder, R. L., & Bish, D. L. (1989). Quantitative analysis. In D. L. Bish, & J. E. Post (Eds.), Modern Powder Diffraction. Washington, DC: The Mineralogical Society of America.
Spector, M., Michno, M. J., Smarook, W. H., & Kwiatkowski, G. T. (1978). A high-modulus polymer for porous orthopedic implants: biomechanical compatibility of porous implants.
Spector, M., Harmon, S. L., & Kreutner, A. (1979). Characteristics of tissue growth into proplast and porous polyethylene implants in bone. J. Biomed. Mater. Res., 13, 677–692.
Spector, M., Heyligers, I., & Roberson, J. R. (1988). Porous polymers for biological fixation. Clin. Orthop. Relat. Res., 207–219.
Steffen, T., Stoll, T., Arvinte, T., & Schenk, R. K. (2001). Porous tricalcium phosphate and transforming growth factor used for anterior spine surgery. Eur. Spine J., 10(Suppl. 2),
S132–S140.
Stubbs, D., Deakin, M., Chapman-Sheath, P., Bruce, W., Debes, J., Gillies, R. M., & Walsh, W. R. (2004). In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial
defect model. Biomaterials, 25, 5037–5044.
Syed, K. H., Gulrez, S. A.-A., & Phillips, G. O. (2011). Hydrogels: methods of preparation, characterisation and applications. In A. Prof. Carpi (Ed.), Progress in Molecular and
Environmental Bioengineering – From Analysis and Modeling to Technology Applications. ISBN: 978-953-307-268-5, InTech, Available from http://www.intechopen.com/
books/progress-in-molecular-and-environmental-bioengineeringfrom-analysis-and-modeling-to-technology-applications/hydrogels-methods-of-preparation-characterisationand-
applications.
Tadier, S., Le Bolay, N., Rey, C., & Combes, C. (2011). Co-grinding significance for calcium carbonate-calcium phosphate mixed cement. Part I: effect of particle size and mixing on
solid phase reactivity. Acta Biomater., 7, 1817–1826.
Takagi, S., Chow, L. C., Hirayama, S., & Sugawara, A. (2003). Premixed calcium-phosphate cement pastes. J. Biomed. Mater. Res. – Part B Appl. Biomater., 67, 689–696.
Takemoto, M., Fujibayashi, S., Neo, M., Suzuki, J., Matsushita, T., Kokubo, T., & Nakamura, T. (2006). Osteoinductive porous titanium implants: effect of sodium removal by dilute
HCl treatment. Biomaterials, 27, 2682–2691.
Teitelbaum, S. L. (2000). Bone resorption by osteoclasts. Science, 289, 1504–1508.
Tiedeman, J. J., Connolly, J. F., Strates, B. S., & Lippiello, L. (1991). Treatment of nonunion by percutaneous injection of bone marrow and demineralized bone matrix. An
experimental study in dogs. Clin. Orthop., 294–302.
Tuncer, N., & Arslan, G. (2009). Designing compressive properties of titanium foams. J. Mater Sci., 44(6), 1477–1484.
Urist, M. R. (1965). Bone: formation by autoinduction. Science, 150, 893–899.
Urlings, T. A. J., & Van Der Linden, E. (2013). Elastoplasty: first experience in 12 patients. Cardiovasc. Intervent. Radiol., 36, 479–483.
Vaandrager, J. M., Van Mullem, P. J., & De Wijn, J. R. (1983). Porous acrylic cement for the correction of craniofacial deformities and repair of defects, animal experimentation and
two years of clinical application. Biomaterials, 4, 128–130.
Van der Meulen, J., & Koerten, H. K. (1994). Inflammatory response and degradation of three types of calcium phosphate ceramic in a non-osseous environment. J. Biomed. Mater.
Res., 28, 1455–1463.
Vangsness, C. T., Jr., Wagner, P. P., Moore, T. M., & Roberts, M. R. (2006). Overview of safety issues concerning the preparation and processing of soft-tissue allografts.
Arthroscopy, 22, 1351–1358.
Waite, J. H. (2008). Surface chemistry: mussel power. Nat. Mater., 7, 8–9.

Wei, G., & Ma, P. X. (2004). Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials, 25, 4749–4757.
Wei, J., Jia, J., Wu, F., Wei, S., Zhou, H., Zhang, H., Shin, J. W., & Liu, C. (2010). Hierarchically microporous/macroporous scaffold of magnesium-calcium phosphate for bone
tissue regeneration. Biomaterials, 31, 1260–1269.
Weibrich, G., Trettin, R., Gnoth, S. H., Gotz, H., Duschner, H., & Wagner, W. (2000). Determining the size of the specific surface of bone substitutes with gas adsorption. Mund
Kiefer Gesichtschir, 4, 148–152.
Wenz, B., Oesch, B., & Horst, M. (2001). Analysis of the risk of transmitting bovine spongiform encephalopathy through bone grafts derived from bovine bone. Biomaterials, 22,
1599–1606.
West, J. L., & Hubbell, J. A. (1999). Polymeric biomaterials with degradation sites for proteases involved in cell migration. Macromolecules, 32, 241–244.
Williams, D. F. (Ed.). (1987). Definitions in Biomaterials. Amsterdam, Oxford, New York, Tokyo: Elsevier.
Williams, J. M., Adewunmi, A., Schek, R. M., Flanagan, C. L., Krebsbach, P. H., Feinberg, S. E., Hollister, S. J., & Das, S. (2005). Bone tissue engineering using polycaprolactone
scaffolds fabricated via selective laser sintering. Biomaterials, 26, 4817–4827.
Witte, F. (2010). The history of biodegradable magnesium implants: a review. Acta Biomater., 6, 1680–1692.
Witte, F., Kaese, V., Haferkamp, H., Switzer, E., Meyer-Lindenberg, A., Wirth, C. J., & Windhagen, H. (2005). In vivo corrosion of four magnesium alloys and the associated bone
response. Biomaterials, 26, 3557–3563.
Witte, F., Fischer, J., Nellesen, J., Vogt, C., Vogt, J., Donath, T., & Beckmann, F. (2009). In vivo corrosion and corrosion protection of magnesium alloy LAE442. Acta Biomater., 6(5),
1792–1799.
Woodruff, M. A., & Hutmacher, D. W. (2010). The return of a forgotten polymer – polycaprolactone in the 21st century. Prog. Polym. Sci. (Oxford), 35, 1217–1256.
Yan, Y., Neville, A., & Dowson, D. (2007). Biotribocorrosion of CoCrMo orthopaedic implant materials-Assessing the formation and effect of the biofilm. Tribol. Int., 40, 1492–1499.
Yang, L., Perez-Amodio, S., Barrère-De Groot, F. Y. F., Everts, V., Van Blitterswijk, C. A., & Habibovic, P. (2010). The effects of inorganic additives to calcium phosphate on in vitro
behavior of osteoblasts and osteoclasts. Biomaterials, 31, 2976–2989.
Ylä-Soininmäki, A., Moritz, N., Turco, G., Paoletti, S., & Aro, H. T. (2013). Quantitative characterization of porous commercial and experimental bone graft substitutes
with microcomputed tomography. J. Biomed. Mater. Res. – Part B Appl. Biomater., 101(8), 1538–1548.
Yokozeki, H., Hayashi, T., Nakagawa, T., Kurosawa, H., Shibuya, K., & Ioku, K. (1998). Influence of surface microstructure on the reaction of the active ceramics in vivo. J. Mater.
Sci. Mater. Med., 9, 381–384.
Younger, E. M., & Chapman, M. W. (1989). Morbidity at bone graft donor sites. J. Orthop. Trauma., 3, 192–195.
Yuan, H., Kurashina, K., De Bruijn, J. D., Li, Y., De Groot, K., & Zhang, X. (1999). A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics. Biomaterials,
20, 1799–1806.
Zberg, B., Uggowitzer, P. J., & Loffler, J. F. (2009). MgZnCa glasses without clinically observable hydrogen evolution for biodegradable implants. Nature Mater., 8, 887–891.
Zerbo, I. R., Bronckers, A. L., De Lange, G., & Burger, E. H. (2005). Localisation of osteogenic and osteoclastic cells in porous beta-tricalcium phosphate particles used for human
maxillary sinus floor elevation. Biomaterials, 26, 1445–1451.
Zhang, R., & Ma, P. X. (1999). Poly(a-hydroxyl acids)/hydroxyapatite porous composites for bone- tissue engineering. I. Preparation and morphology. J. Biomed. Mater. Res., 44,
446–455.
Zitzmann, N. U., Naef, R., & Scharer, P. (1997). Resorbable versus nonresorbable membranes in combination with Bio-Oss for guided bone regeneration. Int. J. Oral Maxillofac.
Implants, 12, 844–852.
Zürz, A., Odler, I., Thiemann, F., & Berghöfer, K. (1991). Autoclave-Free Formation of a-Hemihydrate Gypsum. J. Am. Ceram. Soc., 74, 1117–1124.
Case Studies for Soft Tissue Regenerative Engineering
Jorge Luis Escobar Ivirico and Cato T Laurencin, University of Connecticut, Storrs, CT, United States; and University of
Connecticut Health Center, Farmington, CT, United States
Case Studies for Soft Tissue Injuries and Treatment Strategies 530
Tendons 530
Skeletal Muscle 531
Ligaments 532
Regenerative Engineering Approaches for Soft Tissue Regeneration 533
Adipose Tissue Regenerative Engineering 533
Skeletal Muscle Regenerative Engineering 534
Tendon Regenerative Engineering 534
Ligament Regenerative Engineering 535
Further Reading 536
Glossary
Allografts A graft transplanted between genetically nonidentical individuals of the same species.
Arthroscopy Minimally invasive surgical procedure on a joint in which an examination and sometimes treatment of damage
are performed using an arthroscope or an endoscope that is inserted into the joint through a small incision.
Autograft A tissue or an organ grafted into a new position in or on the body of the same individual.
Lymphadenopathy Disease of the lymph nodes, in which they are abnormal in size, number, or consistency.
Semiinterpenetrating networks Polymer comprising two or more networks that are at least partially interlaced on a polymer
scale but not covalently bonded to each other.
Tenoblast An immature tendon cell.
Soft tissue is a broad term used to describe tissue-related functions of connecting and supporting organs and specific tissues. The soft
tissue is divided into connective (including tendon, ligament, fibrous tissue, synovial membrane, skin, fat, fascia, among others)
and not connective tissue, which include muscle, blood vessels, and nerves. Acquired and congenital conditions like trauma, infec-
tions, diseases, and aging can cause damage to the soft tissues that often lead to nonself-healing defects. Allografts and xenografts
have been widely used for soft tissue repair, but the success associated with these treatments is low due to some drawbacks such as
the implanted tissue rejection, disease transmission, and cost. Autologous transplantation is the primary treatment so far. However,
donor-site morbidity and loss of tissue volume over time are its principal challenges. In recent decades, new knowledge about stem
cell therapy has been acquired as an alternative treatment for soft tissue repair; however, the limitations associated with cell
numbers and their location in the target tissue make this treatment ineffective most of the time. To address these limitations,
the field of tissue engineering emerged as a promising strategy for tissue regeneration. The development of living tissue constructs
as a combination of cells with biodegradable biomaterials, which mimic the functions of native tissue, and growth factors allow cell
survival, proliferation and differentiation, cell localization, and cell-biomaterial integration in the host tissue. Recently, a field of
regenerative engineering has been established to facilitate the convergence of the fields of tissue engineering, advanced material
science, stem cell science, physics, developmental biology, and clinical translation toward the main goal of regeneration of complex
tissues and organs.
Case Studies for Soft Tissue Injuries and Treatment Strategies

Tendons
Tendons are fibrous connective tissues that bind and transmit forces from muscle to bone. They are bright white in color that varies
in shapes and sizes depending on the role of muscle. Tendons have the highest tensile strength and are remarkably strong, compared
with other soft tissues. Histologically, tendons consist of bundles of collagenous fibers densely packed and arranged in parallel and
proteoglycan. Collagen fibers are responsible for the tensile strength whereas the proteoglycans govern the viscoelastic nature of the
tendon. The fibroelastic nature of tendon is responsible for providing strength and transmitting large mechanical forces. Tenoblast
and tenocytes are tendon-specific cell types and comprise around 95% of the cellular content. Tendons are populated with tenoblast
(immature, spindle-shaped tendon cells that give rise to tenocytes), and tenocytes are mature tendon cells that are found anchored

Regenerative Engineering j Case Studies for Soft Tissue Regenerative Engineering 531
to collagen fibers throughout the tendon structure. Other cell types include vascular cells, chondrocytes, and synovial cells of the
tendon sheaths.
Tendon disorders are a common clinical problem due to the poor natural healing response and long-lasting periods of rehabil-
itation. Rotator cuff tears are a common pathology causing shoulder pain in the adult. Traditionally, conservative treatments such as
extracorporeal shockwave therapy, NSAIDs, and corticosteroid injections usually show short-term pain relief but lack long-term effi-
cacy. Severe tendon injury is mainly treated surgically using autografts, allografts, xenografts, or medical devices to repair or replace
the damaged tendon. However, none of these therapies have provided a complete solution for recovery of a functional tendon. A
meta-analysis of three randomized controlled trials (RCTs) involving 252 patients on treatment of rotator cuff tear showed no clin-
ically significant difference between surgery and physiotherapy treatment in 1-year follow-up. Since physiotherapy is less expensive
and prone to complications than surgery, a conservative approach is recommended as the initial treatment modality. However, it is
still unknown if surgery and conservative treatment have the same impact on patients’ quality of life, speed of recovery, return to
work, and cost-effectiveness. In addition, with improvements in arthroscopic repair of the rotator cuff, the mini-open technique is
becoming less popular. A recent study evaluated repair integrity, shoulder function, and satisfaction postoperation in patients aged
> 50 years. In comparison with the arthroscopic repair, mini-open rotator cuff repair resulted in superior repair integrity and
shoulder function. However, the correlation between the rotator cuff integrity and patient satisfaction and function remained
controversial after repair. Moreover, arthroscopic surgery was twice as expensive as open surgery initially, but no significant differ-
ence in overall cost-effectiveness was found after 2 years. Despite the advances in arthroscopy, miniopen rotator cuff repair remains
a useful technique.
Furthermore, a systematic review aimed to determine the effectiveness of early or conservative rehabilitation for patients after
surgical repair of rotator cuff tears. The study showed no difference in function, pain, range of motion, or retears ratio between early
and conservative rehabilitation. However, early mobilization may be beneficial for small and medium tears. Moreover, a meta-
analysis of RCTs and non-RCTs compared the clinical outcomes of autograft with allograft tendons in patients with posterior
cruciate ligament (PCL) regeneration. PCL plays an important role in maintaining the stability of the knee joint. PCL rupture
can lead to meniscal and articular cartilage damage, which can accelerate degeneration of the knee joint. The results obtained in
the meta-analysis data suggest that there were equivalent effects on the Lysholm knee function score in patients who underwent
autograft versus allograft PCL reconstruction. In addition, the patients with autograft tendons had a higher Tegner activity scale
than those with allograft tendons. Altogether, the most important finding of this study is that the application of an autograft
does not bring about more successful outcomes than an allograft.
Achilles tendon rupture is another tendinopathy associated with physical exercise and age-related degeneration that affect both
athletic and general population. Multiple RCTs have shown conflicting results while comparing surgical with nonsurgical treatment
for acute Achilles tendon rupture. Based on a systematic review of overlapping metaanalyses, the current best evidence shows that
surgical treatment may be preferred at centers that do not have functional rehabilitation, and nonsurgical intervention may be the
best choice at centers offering functional rehabilitation. Moreover, four RCTs involving 169 patients were conducted to determine
whether augmented repair provides better clinical benefits compared with nonaugmented repair. This metaanalysis suggested that
no statistical difference was found between augmented and nonaugmented repair groups in terms of infection rate, patient satis-
faction, and rerupture rate. Further, prospective randomized studies need to be performed to assess clinical outcome of these
two techniques for acute Achilles tendon rupture.
Skeletal Muscle
Skeletal muscle is one of three types of muscle that comprises approximately 45% of human body mass and is essential for gener-
ating forces that are involved in movement and locomotion. It is a highly organized structure encompassing nerves, blood vessels,
myofibers, and extracellular connective tissue. In a minor injury situation such as muscle strain, the skeletal muscle can self-repair
through the activation of muscle satellite cells (MuSCs, muscle stem cells). The formation of myofibers and their integration into
muscle tissue results because of the proliferation and differentiation of satellite cells into myoblast, in response to a muscle injury
(Fig. 1). The stem cell niche (interaction between the satellite cells and their environment) plays a key role in the muscle regener-
ation process.
The current treatments include applying ice and resting the strained tissue for a few days. Also, nonsteroidal anti-inflammatory
drugs (NSAIDs) and acetaminophen help in reducing pain and swelling. A systematic review and metaanalysis of 41 studies per-
formed between 1985 and 2015 reported that the use of NSAIDs is more effective in the treatment of lower body muscle injuries
when compared with upper body injuries. Additionally, after a short-term acute muscle injury, NSAIDs reduced pain, blood creatine
kinase level, and strength loss.
Severe damage to skeletal muscle such as traumatic injuries, myopathies, and tumors can lead to an irreversible loss of muscle
mass. So, the formation of fibrotic scar tissue in the affected tissue after injury prevents the muscle regeneration. When severe trauma
occurs, the current treatment includes the implantation of healthy, vascularized, and innervated autologous tissue (well known as
muscle flaps, isolated from the vicinity of the injury) in the damaged area. However, the limitations of this treatment are the donor-
site morbidity and long periods of rehabilitation, leading in most of cases to muscle regeneration with poor functionality and
strength.
532 Regenerative Engineering j Case Studies for Soft Tissue Regenerative Engineering
Fig. 1 (A) Immediate response to muscle injury. Upon injury, MuSCs activate, self-renew, proliferate, and differentiate into myoblasts. Fibroadipo-
genic progenitors (FAPs) are also activated and secrete transient extracellular matrix. Debris is removed by the infiltration of neutrophils in the injury
tissue. (B) Regeneration in young niche. Recruitment of neutrophils and FAP apoptosis is induced by the action of pro-inflammatory M1 macro-
phages. Also, angiogenic and anti-inflammatory factors are secreted by anti-inflammatory M2 macrophages. The process of differentiation and fusion
of myoblasts is essential in the new muscle fibers formation. (C) Regeneration in aged/pathologic niche. Stiffening is the result of changes in the
MuSC niche composition. The inflammation process persists due to the dysregularization of the inflammatory response. Fibrosis is produced by the
FAPs’ oversecretion of ECM. Reproduced from Han, W. M., Jang, Y. C. and García, A. J. (2017). Engineered matrices for skeletal muscle satellite cell
engraftment and function. Matrix Biology 60–61, 96–109.
Ligaments
Ligaments are connective tissue comprising fibrous bands that connect the bones together at the joints and support some internal
organs. Dense bundles of collagenous fibers and fibrocytes (bone marrow-derived mesenchymal progenitor cells that are involved
in the vascularization and healing processes) are the main components of ligaments. Ligaments can be classified in two types: (1)
white ligament composed by collagenous fibers that are not stretchy, very strong, and allow subjective freedom movement and (2)
yellow ligaments rich in elastin fibers that are very tough and elastic. The anterior cruciate ligament (ACL) is one of the most impor-
tant ligaments that play an important role in the knee stability. The ACL does not self-repair due to its avascular nature, that’s why
some treatments like surgical reconstruction and rehabilitation are needed for ACL restoration. In the United States alone,
> 200,000 people suffer ACL rupture each year, so conventional treatments are being practiced using surgical reconstruction
with/without allograft or autograft.
Two clinical studies were done to compare outcome between patients treated with rehabilitation alone with early ACL recon-
struction and patients with rehabilitation and optional delay ACL reconstruction for 5 years. One hundred twenty-one cases
were studied, 62 patients with early ACL reconstruction and 59 with delay ACL reconstruction. In all cases, patient received similar
rehabilitation. The studies showed that patients who opted for delayed ACL reconstruction had greater instability in the knee joint
compared with the early ACL reconstruction group. However, the study also concluded that both groups (early and delay ACL
reconstruction) did not show any radiographic differences and thus did not provide better results after 5 years. Another study of
3–5 years clinical trial compared autograft with allograft ACL reconstruction, including 64 patients. Symptoms like activity level,
physical examination, and knee testing were recorded. Overall, the International Knee Documentation Committee ratings and Cin-
cinnati knee score showed no clinical statistical difference between groups. This study provided evidences for using allograft as an
alternative reconstruction treatment. Recently, a 10 years clinical trial compare allograft with autograft for primary ACL reconstruc-
tion, involving 99 patients. There were 4 autograft (8.3%) and 13 allograft (26.5%) failures that required further revision and
reconstruction. The results concluded that there was no significant difference in the scoring (Tegner or International Knee Documen-
tation Committee ratings), but patients with allograft implant showed three times higher failure rate compared with those who
received the autograft implants.
In order to overcome the limitation of allograft and autograft, a different approach using synthetic and natural materials was
explored for ACL reconstruction patients. Clinical trial using the artificial ligament, GORE-TEX (made by polytetrafluoroethylene
(PTFE)) showed only one prosthetic breakage in 30 patients after 2 years. Further, in another study, the implantation of PTFE liga-
ments resulted in improvements of objective and parameters after 18 months. However, after a certain period, GORE-TEX graft was
withdrawn from the market because the implantation of these materials resulted in complication in patients such as synovial reac-
tion and inguinal lymphadenopathy. On the other hand, polyester like polyethylene terephthalate (PET) was employed as synthetic
ligament and upon implantation in 130 sportsman showed good results in terms of joint stability, and complete integration in the
host tissue was observed after 2 years. Besides, in another study, the implantation of PET in 47 patients improved subjective param-
eters, Tegner activity level, and higher stability.
Regenerative Engineering Approaches for Soft Tissue Regeneration

Adipose Tissue Regenerative Engineering
The regenerative engineering of adipose tissue is the field that addresses the pathologies of adipose tissue and the clinical needs. The
use of new mechanisms of tissue regeneration based on specific cell precursors and new technologies for biomaterials production
may address the limitations of conventional treatments (Fig. 2).
Some studies have described the development of different cell-laden artificial extracellular matrices (ECMs) for adipose tissue
regenerative engineering applications with promising results. In this sense, synthetic and natural polymers have been proposed
to manufacture 3-D biodegradable structures. Key factors such as pore size, total porosity, and pore interconnectivity are essential
properties necessary to produce 3-D structures that determine the transport of nutrients and metabolites that in the end modulate
cell colonization and extracellular matrix production. Among synthetic polymers, poly(lactic acid) (PLA), poly(glycolic acid) (PGA),
and poly(lactic-co-glycolic acid) (PLGA) have been used as cell delivery platforms. Specifically, insulin, insulin-like growth factor-1
(IGF-1), basic fibroblast growth factor (bFGF)-loaded PLGA-polyethylene glycol (PLGA-PEG) microparticles, and insulin and IGF-
1-laden PLGA-PEG microparticles/PLGA scaffold constructs were implanted in a subcutaneous rat model, enhancing the free fat
graft volume and weight. In another case, after 35 days of in vitro culture, 3T3-L1 cells (adipogenic cell line) seeded within PGA
scaffolds showed a lipid accumulation with mature and unilocular morphology. Finally, the formation of mature fat pads was
observed after 35 days of subcutaneous implantation in nude mice.
Moreover, the natural polymers offer a good alternative for tissue regeneration due to their similarity with some
biomacromolecules present in the in vivo environment and with the ECM. In addition, natural polymers possess good biocompat-
ibility, and they may prevent foreign body reactions, frequently detected with synthetic polymers. Collagen, hyaluronic acid (HA),
Fig. 2 Strategy for adipose tissue regenerative engineering combining adipose-derived cells with engineered scaffolds.
gelatin, and fibrin have been widely used. For instance, subcutaneous implantation of preadipocytes/collagen porous constructs in
immunodeficient mice showed new vessels and adipose tissue formation.
Decellularized adipose tissue (DAT) is another natural substrate widely used for adipose regenerative engineering. DAT matrices
are obtained through a decellularization process of adipose tissue into acellular 3-D porous substrates. Recently, with the develop-
ment of new technologies, engineered adipose tissue constructs have been produced using 3-D bioprinting technique. Well-defined
3-D printed tissue constructs have been obtained by a combination of DAT (as bioink) and human adipose tissue-derived mesen-
chymal stem cells (hASCs). Good cell penetration, tissue remodeling, and adipose tissue formation were observed after 2 weeks of
in vivo subcutaneous implantation of the DAT/hASC constructs in nude mice.
Skeletal Muscle Regenerative Engineering

New muscle regeneration strategies have emerged to overcome the limitation of the current treatments. The design and develop-
ment engineered muscle constructs based on cell seeding into 3-D scaffolds until the tissue becomes functional and their subse-
quent transplantation into the affected tissue in patients is the main concept for muscle regenerative strategies.
Recently, some studies have shown promising results. For instance, mesenchymal stem cell (MSC)-laden alginate cryogel (with
porous size between 70 and 150 mm) has been investigated as multifunctional substrate. The engineered material was used as MSC
artificial niche and, at the same time, as IGF-1 and vascular endothelial growth factor (VEGF) release platform to enhance the MSC
paracrine effect on muscle progenitor’s cells. 7, 28, and 56 days after the implantation of the artificial niches in a muscle trauma
model in rats, the muscle strength improved, reducing considerably the fibrosis, and muscle fiber density increased. Another study
reported the fabrication of mouse mesoangioblast-laden PEG-fibrinogen hydrogels. After the implantation of the constructs under
the skin on the surface of the anterior tibial muscle, the attraction of host vessels and nerves induced by the mesoangioblasts was
observed, as well as the formation and maturation of aligned myofibers. Finally, an artificial muscle like normal tibialis anterior
resulted after replacing the ablated tibialis anterior with a mouse mesoangioblast-laden PEG-fibrinogen construct. Keratin-based
hydrogels have been widely used as cell/growth factor delivery platform for functional muscle regeneration. Skeletal muscle progen-
itor cells, IGF-1, and/or bFGF-laden keratin hydrogels were implanted in a murine model of volumetric muscle loss injury to the
latissimus dorsi (LD) muscle. The influence of topographical cues like groove on the surface of different materials including HA and
poly(L-lysine) polyelectrolyte complex, poly(dimethylsiloxane) (PDMS), engineered gelatin methacrylate, and polystyrene
substrates on muscle derived cells has been investigated. For instance, myoblast alignment and myotube formation were observed
on grooved micropatterned PDMS substrates. Interestingly, additional cells added on top of the previous myotube layer followed
the same pattern, so the cells attached, aligned, and fused formed a 3-D tissue multilayer patch.
Tendon Regenerative Engineering

The use of cell niche matrices is the new regenerative engineering approach to speed the healing process of rotator cuff tendon
injuries. The main concept is to use them as tissue bridge between the tendon and the bone facilitating cell growth and collagen
deposition.
Oriented electrospun nanostructure scaffolds have been produced to mimic: (1) the tendon extracellular matrix (scaffolds
composed by fibers with diameter in a range of 50–500 nm, like collagen structure) and (2) the biomechanical properties of the
rotator cuff. Scaffolds of poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone functionalized with poly [(ethyl alanato)1
(p-methyl phenoxy)1] phosphazene (PNEA-nPh) fabricated by electrospinning technique are promising nanofiber matrices for
tendon repair. Specifically, when the polycaprolactone/PNEA-mPh hybrid scaffolds were implanted in a rat model of rotator
cuff augmentation, the morphology of regenerated tendon was like the suture repair group (see Fig. 3). However, the implantation
of rat MSC/polycaprolactone/PNEA-mPh constructs resulted in a tissue morphology like the intact tendon indicating a faster tendon
Fig. 3 (A) Randomly oriented PLGA scaffolds (nanofibers diameter between 400 and 800 nm). (B) Repair augmentation using PLGA scaffolds.
Reproduced from Taylor, E. D., Nair, L. S., Nukavarapu, S. P., McLaughlin, S. and Laurencin, C. T. (2010). Novel nanostructured scaffolds as thera-
peutic replacement options for rotator cuff disease. Journal of Bone and Joint Surgery 92, 170–179.
remodeling process. Only traces of donor rat MSC were observed after 6 weeks suggesting that the observed therapeutic effect is due
to the delivery of growth factors, immunomodulators, etc.
MSC-laden PLGA scaffold implanted in rabbit model of an infraspinatus tear resulted in the formation of Sharpey fibers and
fibrocartilage. In groups with cell-material constructs, the resultant enthesis had better tensile strength and greater collagen fiber
formation compared with controls (cell-free scaffolds). On the other hand, several growth factors can augment rotator cuff healing
process. bFGF-loaded PLGA electrospun scaffolds were employed in a rat model of a chronic rotator cuff tear. During the initial stage
of the healing process, local delivery of bFGF induced high fibrocartilage formation and better collagen fiber organization compared
with controls (bFGF-free scaffolds).
Ligament Regenerative Engineering

The strategy of ligament regenerative engineering involves the use of biocompatible and biodegradable biomaterials with the neces-
sary mechanical properties and the cell source, preferably primary cells isolated from autologous healthy ligament tissue. Ideally,
the development of scaffolds as cell support, cell maturation in vitro followed by the implantation of the engineered constructs in
patients, is the principal aim of this concept. From the clinical point of view, the principal advantages are the simple surgical proce-
dures, minimal patient morbidity, good mechanical stability, and minimal risk of disease transmissions or infections. Ligament–
bone interface is another important aspect to consider. This is a multilayered transition zone involving tissues with different
mechanical properties that’s why the fabrication of constructs, able to mimic the ligaments in vivo, is a challenge for regenerative
engineering field.
Natural and synthetic materials have been extensively used for ligament reconstruction. For example, collagen and collagen-
platelet sponges were used to regenerate the anterior cruciate ligament (ACL). The scaffolds were implanted in a minipig model
of lateral ACL transection (ACLT). Normal suture repair procedure was used as control (see Fig. 4). Thirteen weeks after the implan-
tation of collagen sponges, mature fibroblast and vascular tissue were observed in the healing area, and no inflammatory cells and
Fig. 4 Gross appearance of control group (suture repair procedure) (A) and scaffold groups (B). In both cases, the repair tissue happens from the
anterior slope of the tibial spines to the intercondylar lateral wall. Change in cartilage structure was not observed. Individual synovialized band was
observed in the repair tissue. Reproduced from Fleming, B. C., Magarian, E. M., Harrison, S. L., Paller, D. J. and Murray, M. M. (2010). Collagen
scaffold supplementation does not improve the functional properties of the repaired anterior cruciate ligament. Journal of Orthopaedic Research 28,
703–709.
residues of collagen sponges were detected. Collagen sponges alone were not sufficient to enhance the healing process; however, the
combination of collagen and platelet improve the tissue repair.
Silk is another interesting material that has widely been used as ligament replacement substitute. Tensile strength and toughness
are its main advantages compared with other biomaterials from natural sources. MSC-loaded braided silk scaffold constructs were
used to repair the ACL using a porcine model. Twenty-four weeks after implantation, the new regenerated ligament was visually like
the ACL of the control group. Implanted materials were almost degraded, and a layer of fibrous tissue was formed due to cell infil-
tration and ECM production. Besides, positive type I collagen and almost negative type III collagen and tenascin-C markers were
found in the newly regenerated tissue.
Furthermore, poly(hydroxyesters) like PLLA and PGA have been employed to reconstruct the ACL due to their biocompatibility,
degradation rates, and good mechanical properties. In an interesting study, a million of primary rabbit ACL cells were seeded on
PLLA braided scaffolds and implanted in a rabbit ACL model. Twelve weeks after implantation, the constructs showed a dense
connective tissue and vascularization throughout the replacement, production, and deposition of collagen fibers surrounding
the PLLA fibers. The tensile mechanical properties of rabbit ACL and ligament replacements were measured. Significant differences
between the strength retention of construct and cell-free scaffolds were observed at 12-week time point. In conclusion, the combi-
nation of cells and biomaterials yielded better results than cell-free materials because of better cell infiltration along the scaffold and
more mature collagen remodeling during the transfer of load to the neoligament.
The challenging regeneration of soft tissues can thus be taken to a next level by making advancement in tissue engineering by
integrating it with areas of advanced materials sciences, physics, stem cell science, and developmental biology. The emergence of
new regenerative approaches would bring a paradigm shift in the soft tissue regeneration field.
Further Reading
Benjamin, T., Corona, S., & Greising, M. (2016). Challenges to acellular biological scaffold mediated skeletal muscle tissue regeneration. Biomaterials, 104, 238–246.
Choi, J. H., Gimble, J. M., Lee, K., Marra, K. G., Rubin, J. P., Yoo, J. J., Vunjak-Novakovic, G., & Kaplan, D. L. (2010). Adipose tissue engineering for soft tissue regeneration.
Tissue Engineering. Part B, Reviews, 16, 413–426.
Escobar Ivirico, J. L., Bhattacharjee, M., Kuyinu, E., Nair, L. S., & Laurencin, C. T. (2017). Regenerative engineering for knee osteoarthritis treatment: Biomaterials and cell-based
technologies. Engineering, 3, 16–27.
Han, W. M., Jang, Y. C., & García, A. J. (2017). Engineered matrices for skeletal muscle satellite cell engraftment and function. Matrix Biology, 60–61, 96–109.
Harris, K., Driban, J. B., Sitler, M. R., Cattano, N. M., & Hootman, J. M. (2015). Five-year clinical outcomes of a randomized trial of anterior cruciate ligament treatment strategies:
An evidence-based practice paper. Journal of Athletic Training, 50, 110–112.
James, R., & Laurencin, C. T. (2014). Musculoskeletal regenerative engineering: Biomaterials, structures and small molecules. Advances in Biomaterials, 123070, 12 p.
Kwee, B. J., & Mooney, D. J. (2017). Biomaterials for skeletal muscle tissue engineering. Current Opinion in Biotechnology, 47, 16–22.
Peach, M. S., Ramos, D. M., James, R., Morozowich, N. L., Mazzocca, A. D., Doty, S. B., Allcock, H. R., Kumbar, S. G., & Laurencin, C. T. (2017). Engineered stem cell niche
matrices for rotator cuff tendon regenerative engineering. PLoS ONE, 12, e0174789.
Pei, B., Wang, W., Fan, Y., Wang, X., Watari, F., & Li, X. (2017). Fiber-reinforced scaffolds in soft tissue engineering. Regenerative Biomaterials, 4, 257–268.
Taylor, E. D., Nair, L. S., Nukavarapu, S. P., McLaughlin, S., & Laurencin, C. T. (2010). Novel nanostructured scaffolds as therapeutic replacement options for rotator cuff disease.
Journal of Bone and Joint Surgery, 92, 170–179.
Yang, G., Rothrauff, B. B., & Tuan, R. S. (2013). Tendon and ligament regeneration and repair: Clinical relevance and developmental paradigm. Birth Defects Research Part C:
Embryo Today, 99, 203–222.
Characterizing the Properties of Tissue Constructs for Regenerative Engineering
Yusuf Khan, University of Connecticut Health Center, Farmington, CT, United States
Introduction 537
Evaluating the Mechanical Properties of Tissues and Tissue Constructs 537
Mechanics of Materials 538
Anisotropy 538
Viscoelasticity 538
Methods for Testing Mechanical Properties of Biological Tissues and Synthetic Materials 539
Compression and tensile testing 540
3-Point and 4-point bending 541
Torsion testing 541
Indentation testing 541
Evaluating Tissue Construct Biocompatibility and Cell-Construct Interactions 542
Toxicity 542
Cell viability and proliferation 542
Differentiation 543
In Vivo Models for Tissue Construct Evaluation 543
Hard Tissue Defect Models 543
Soft Tissue Defect Models 543
Conclusion 544
References 544
Further Reading 545
Introduction
At the intersection of tissue engineering and regenerative medicine lies regenerative engineering. Defined as “the integration of tissue
engineering with advanced material science, stem cell science, biophysical stimulation, and areas of developmental biology,” regen-
erative engineering describes the newest strategies for replacing biological tissues. It leans heavily on the latest developments in
materials science, stem cell biology, and clinical translation and as places importance on understanding how these materials,
cellular processes, and their clinical translation potential can be measured in terms of mechanical properties, cellular biocompat-
ibility, cell-material interactions, and preclinical in vivo testing. Later, we discuss methods of evaluating hard and soft tissues, mate-
rials, cell behavior and cell-material interactions, and preclinical animal models that can help pave the way from benchtop to
bedside.
Evaluating the Mechanical Properties of Tissues and Tissue Constructs
Properly matching the mechanical properties of the tissue to be regenerated has long been a fundamental design criterion for tissue-
engineered constructs. Whether replacing hard tissues like bone or soft tissues like ligament, tendon, muscle, cartilage, or blood
vessel, properly matching the mechanical properties is essential. In hard tissues like bone, a failure to match the compressive
modulus of the construct to that of the surrounding bone can lead to failure of either the construct or the tissue around it. Engi-
neering a construct with inferior mechanical properties can jeopardize the mechanical integrity of the injury site by chancing cata-
strophic failure or simply eliminate that construct from consideration of such load-bearing defect sites. Over-engineering a construct
such that the mechanical properties are considerably higher than those of the surrounding tissue can lead to a phenomenon called
stress shielding in which the mechanical stresses applied to the defect area are absorbed and solely supported by the construct that is
mechanically overmatched to the surrounding bone tissue, shielding the surrounding tissue from physical loading and stimulating
the body to resorb the bone tissue from those unloaded areas. Since the body responds to physical loading by stimulating the
production of more bone, it contrarily responds to a lack of physical loading, as would be experienced in stress shielding, by resorb-
ing the unloaded bone. So regardless of whether the mismatch under- or over-compensates for the mechanical strength of the
surrounding bone, it leads to hindrances in healing. The same mismatch of mechanical properties can be seen in soft tissues
like ligament, which has a very specific s-shaped stress–strain curve that has to be mimicked in an engineered construct, and blood
vessel, where the compliance of the synthetic vessel should best approximate that of a native vessel to minimize the disruption of
blood flow as it passes from native vessel to synthetic conduit and back to native vessel. Disruptions in this flow regime can lead to
changes in the forces applied to the vessel wall stimulating overproliferation of cells near the vessel-construct anastomosis,

538 Regenerative Engineering j Characterizing the Properties of Tissue Constructs for Regenerative Engineering
a phenomenon called neointimal hyperplasia. This can cause the lumen of the vessel and/or construct to become partially or
completely obscured. Below is a summary of the mechanical properties of some hard and soft tissues and the mechanical properties
of some of the more common materials used to repair or restore these tissues. Of note is the dramatic mismatch in these properties
of many of these materials, clarifying the importance of novel approaches to designing constructs. It is important to note that for
some materials there is a very wide range of mechanical properties. This is the case for several reasons. For the human tissue, the
mechanical properties of a particular tissue, skin for instance, can vary considerably depending on where it was harvested prior
to testing. The skin on the plantar surface of the foot, for instance, is considerably different than that of the face, for instance.
The wide range of properties seen in the synthetic materials may be less intuitively obvious, but these properties can vary based
on material testing protocols, processing techniques, synthesis techniques, modifications to materials, and the inherent variability
that exists within materials of similar type. While this variability in material properties may initially appear as a challenge to the
regenerative engineer when making choices for the design of a construct, it is actually an advantage that a material with very similar
characteristics can be formed to have such a wide spectrum of mechanical strength (Table 1).
Mechanics of Materials
Anisotropy
Beyond compression and tension are a number of other mechanical characterizations that are relevant to tissues and should guide
the development of tissue replacement constructs. Many tissues, for instance, exhibit anisotropic mechanical properties. Anisotropy
describes the property of a material that exhibits different properties in the x, y, and/or z direction. Mechanical anisotropy suggests
that a material compressed or placed in tension in one axis would not necessarily display the same mechanical response in
a different axis. For example, a natural ligament that is tested in tension along its longitudinal axis would prove to have dramatically
stronger mechanical properties than that same ligament tested in tension across its transverse axis. Some biological materials exhibit
anisotropic mechanical properties like ligament, tendon, and cortical bone, while others exhibit more of an isotropic mechanical
profile like cartilage (to some degree) and trabecular bone. A microscopic examination of the hierarchical structure of these tissues
often reveals a structural basis for either isotropy or anisotropy.
Viscoelasticity
Traditionally materials under mechanical loading express some amount of deformation in response to applied load, or strain in
response to stress. The material is loaded until failure and can be represented by a simple stress–strain curve (Fig. 1). Many soft
tissues within the human body, however, respond to mechanical loading in a way that resembles both a fluid and a solid, or as
having a combination of viscous and elastic mechanical properties, respectively. These viscoelastic tissues respond differently in
traditional tensile testing depending on how parameters like strain rate are varied in the testing. Strain rate, the rate at which the
crosshead moves in a tensile testing machine, can result in different mechanical properties for viscoelastic tissues and therefore
should be chosen carefully depending on the tissue, or engineered construct, being tested. Other testing method variations can
reveal tissue behaviors under mechanical loading such as stress relaxation, creep, and hysteresis. Stress relaxation occurs when
Table 1 Mechanical properties of biological tissues and synthetic materials used to regenerate those tissues
Ultimate tensile strength Elastic modulus Ultimate compressive Elastic modulus in

Tissue/material type (MPa) in tension (MPa) strength (MPa) compression (MPa)
Cortical bone 124–174 (Pal, 2014) 17,000–19,000 90–167 (Athanasiou 5000–15000 (Athanasiou
(Pal, 2014) et al., 2000) et al., 2000)
Trabecular bone 2.5 (Athanasiou et al., 483 (Athanasiou et al., 0.15–14 (Athanasiou 12–900 (Athanasiou
2000) 2000) et al., 2000) et al., 2000)
Ligament 50–100 (Holzapfel, 2001) 345 – –
Tendon 50–100 (Holzapfel, 2001) 800–2000 – –
Cartilage 9–40 (Holzapfel, 2001) 1–10 (Pal, 2014) – 1–20 (Barker and
Seedhom, 2001)
Aorta 0.3–0.8 (Holzapfel, 2001) 0.05–0.1 (Isnard et al., – –
1989)
Skin 1–20 (Holzapfel, 2001) 0.02–0.1 (Liang, 2010) – –
Hyaluronic acid – – – 0.02–0.15 (Levett et al., 2014)
Chitosan 0.5–45 (Foster et al., 2015; 23–68 (Jana et al., 2012) 0.3–1.7 (Jana et al., 2012) 5.5–18 (Jana et al., 2012)
Jana et al., 2012)
Poly(lactide) 37 – – 600–7000 (Bergström and
Hayman, 2016)
Poly(caprolactone) 1.1–16 (Eshraghi and Das, 35–350 (Eshraghi and Das, 0.6–38 (Eshraghi and Das, 12–317 (Eshraghi and
2010) 2010) 2010) Das, 2010)
It is important to note that the ranges of properties given for tissues and materials reflect the effect of testing protocols, processing techniques, material synthesis techniques,
modifications to materials, and the inherent variability that exists within tissues and materials of similar type.
Regenerative Engineering j Characterizing the Properties of Tissue Constructs for Regenerative Engineering 539
Fig. 1 Stress–strain curve. Stress (y-axis) is a measure of the load placed on an object and strain (x-axis) is the material deformation in response
to the stress. The linear portion of the (solid) curve indicates the elastic region, in which the material will revert to its original form if the load was
removed. The curved portion of the (solid) curve is the plastic region, beyond which the material is permanently disfigured. Once the material breaks
(point of material failure) the stress drops to 0.
a tissue is placed under tension and the crossheads are moved a fixed distance apart and held in that position such that the tissue is
held in tension with a constant load (see Fig. 2). Over time as the tissue is held in tension at a fixed distance the stress exerted on the
load cell by the tissue relaxes or decreases, indicating that the tissue is no longer under the same tension as it was at the onset of
testing. This is a phenomenon of viscoelastic tissues, as a purely elastic tissue would not undergo this stress relaxation. Creep occurs
when a soft tissue or material is placed in tension with a constant strain rate and the tissue or material constantly deforms as the
crosshead continues to move, rather than resist the crosshead movement and ultimately fail (see Fig. 3). When the crosshead stops
moving and returns to its original position, removing the load from the tissue, the stress–strain curve follows a different path than
when initially loaded, unlike a purely elastic material that would follow the same path. This discrepancy between the initial and
recovery path of the stress–strain curve of viscoelastic tissues or materials is termed hysteresis (see Fig. 4) and gives insight into
how a tissue, or a material, will recover after the loading is removed. The tissue or material may or may not fully regain its original
shape but the rate at which it recovers may be different than a purely elastic material.
Methods for Testing Mechanical Properties of Biological Tissues and Synthetic Materials
Evaluating the mechanical properties of tissue or materials is most effective when following specific protocols such that the results of
testing from one laboratory can be accurately compared to another laboratory. This is critical given the varied results that can be
obtained depending on the specific testing protocol as described earlier. Consistency in mechanical testing can be achieved by
observing published protocols or using testing standards like those published by the American Society for Testing and Materials
(now simply known as ASTM) or the International Organization for Standards (ISO). These standard organizations publish detailed
protocols for a full range of materials evaluation, including mechanical testing. For instance, ASTM standard D695 provides
a protocol for testing the compressive strength of solid plastic materials, specifying the dimensions of the sample (aspect ratio),
the crosshead speed of the mechanical testing machine, and the collection of data. Typically force and displacement data are
recorded by the mechanical testing machine and are readily converted into stress and strain data, either by the testing machine’s
Fig. 2 Stress relaxation occurs when a material is loaded initially and held under constant strain for a period of time, during which the load experi-
enced by the tissue decreases without a change in strain.
Fig. 3 Creep occurs when a material is loaded with a constant fixed strain rate but the applied stress does not increase.
Fig. 4 The hysteresis curve shows the stress–strain relationship as a material is loaded (blue line) and unloaded (red line). After the material is
loaded it recovers its initial shape differently than a perfectly elastic material.
accompanying software or by the user from the raw force/displacement data. Plotting the stress versus strain curve yields informa-
tion such as the elastic modulus, yield stress, fracture stress, and toughness.
Compression and tensile testing

As described earlier, compression testing of a material involves a sample of the material of dimensions indicated by the appropriate
standard by bringing two compression platens together to compress the sample. Specific protocols call for the compression of
a cylinder of material but the specific shape of the material can vary by standard protocol. Applied force and crosshead displacement
data are collected in real time and the test continues until the sample fails (as indicated by the shape of the force-displacement
curve) or the tester chooses to end the test. As reported earlier, compression testing can reveal the ultimate compressive strength,
yield strength, elastic modulus, and toughness of a sample. Tensile testing is done in a similar manner but rather than being
compressed between two platens the sample is connected on either end to tensile testing grips. Typically when samples are tested
in tension, the sample is formed into a dogbone shape such that the ends of the sample are wider than the middle which serves two
purposes; (1) it increases the surface area available for the tensile grips to hold onto the sample to provide more surface area for
gripping and (2) it biases the sample to more likely fail along the mid-region rather than at the tensile grip since the mid-region
is thinner and therefore mechanically less robust. This strategy allows for stress concentrations that may exist at the tensile grips
to have less of an effect on the failure of the material, allowing for a more accurate test. Tensile testing can reveal the ultimate tensile
strength, young’s modulus, and yield strength. Hard tissues like bone are often tested in compression, as are implant materials. One
may question the validity of pure compression in testing tissues like bone since it is rarely ever in pure compression, but having
a uniform testing strategy to compare bone between anatomical sites or between subjects can serve to eliminate other testing vari-
ables and permit proper comparison.
3-Point and 4-point bending

Bending tests are conducted by placing a length of material across a span and pushing down along the span to bend the
material until failure. Bending tests reveal the elastic modulus of bending, flexural stress, and flexural strain of a material.
3-Point bending involves placing the material across a span supported on either ends of the material and bringing down
a point source to the center of the span and bending the material until failure while recording applied force and crosshead
displacement (see Fig. 5). The 3-point description comes from the two points of support at the ends of the material and the
one point of deflection brought down to the middle of the material. 4-point bending tests are conducted similarly to 3-point
bending tests except that rather than one point source being brought down to the center of the span of material two points
slightly separated from the center of the material are brought down in contact with the material. This separation of the two
point sources spreads the region of bending out from the center such that a larger portion of the material is tested than with
only one point of deflection. Hard materials like bone and implants can be tested using bending tests as a measure of the
tissue or material in both tension (the bottom of the sample as it is tested) and compression (the top of the material as it is
tested).
Torsion testing
Torsion testing involves the twisting of a sample along an axis and is a useful test for acquiring information like torsional shear
stress, maximum torque, shear modulus, and breaking angle of a material or the interface between two materials. Typically
a longitudinal sample is placed in a torsion tester and one end of the sample is twisted around the long axis until failure, during
which the force, or in the case of rotation the torque, and the displacement, or in the case of rotation the angular displacement,
are recorded. Torsion testing is appropriate for materials that may experience a torsional load like a metallic bone screw, an intra-
medullary rod, rubber tubing that may become twisted, or to measure the shear strength of a bond between an implant and
native tissue like bone.
Indentation testing
In contrast to traditional mechanical testing that provides one mechanical assessment of an entire material, indentation
methods can mechanically characterize a material at the macro-, micro-, and nano-scale level. This becomes a valuable tool
when looking at tissues and complex tissue constructs in which several tissue types or materials may be integrated together.
Doing traditional tensile, compressive, or torsion testing of heterogeneous tissues or tissue constructs would provide one
numerical value for each test performed, but that one value may not accurately characterize the various tissue or material types.
Indentation studies at both the micro and nano level allow for the precise mechanical testing of either small volumes of mate-
rial or across very small, specific regions of a tissue or construct. Whether nanoindentation or microindentation is performed
depends on the specific questions being asked but is ultimately dictated by the physical size of the indenter, which are typically
either spherical or pyramidal in shape. The choice of tip shape depends on the information to be collected and the brittleness
or softness of the material itself.
Nanoindentation, or depth-sensing indentation, requires a small load from a point source to be applied to a tissue or material
surface to yield a small, localized deformation. The loading and resulting displacement of the probe are monitored and recorded
and from this data the material hardness and modulus can be calculated. Loads used in nanoindentation can range from micro-
newtons to millinewtons and displacements can range from nanometers to micrometers. Advantages of this mechanical testing
technique include the ability to evaluate complex tissues and constructs with precision and accuracy with relatively little destruction
of the sample. Tissues like cartilage that can have regional differences in mechanical properties within the same tissue can benefit
from nanoindentation. Mechanical properties like elastic modulus, hardness, storage and loss modulus, and compliance can be
calculated with indentation tests.
Fig. 5 3-Point (left) and 4-point (right) bending test apparatus. 3-point bending provides three points of contact; two supports and one center point
where the loading is applied. 4-point bending provides four points of contact; two supports and two points where loading is applied. The two points
spread the loading region along the specimen so a larger portion of the material is tested in bending.
Evaluating Tissue Construct Biocompatibility and Cell-Construct Interactions
While characterizing the mechanical properties of a tissue construct is critical to its overall success, it is equally critical to characterize
the construct in terms of its interactions with the building blocks of regenerated tissues: the cells. Cellular evaluation is a vast area of
study and fills textbooks with theory, technique, and outcome measurements and an exhaustive evaluation is beyond the scope of
this article. There are, however, some fundamental considerations and techniques that provide an important foundation to future
studies. Here, we discuss these fundamental analyses, the theory behind their utility, and some suggested methodologies that help
determine the overall biocompatibility of a tissue construct. The term biocompatibility refers to the degree of response a material
may invoke after being implanted. Generally speaking, any foreign material that is implanted into the human body will invoke
a response but that response can vary widely from toxic resulting in serious injury or death to benign that raises no concerns
but should be understood, to a response that is predicted and desired because it may lead to enhanced healing.
Toxicity
The toxicity of a material or construct refers to how toxic it is, or to how much damage it can do to an organism in general. When
considering regenerative engineering constructs under development, one may choose to focus on the toxicity of a construct specif-
ically to cells, or its cytotoxicity. A cytotoxic material would be one that caused considerable harm or death to the cells on, in, or near
the construct, but it is reasonable to assume that a material or construct that is toxic to cells would be toxic to larger organs or tissues
made up of similar cells. While the toxicity of a material is less of a concern for biomaterials that have been evaluated previously and
in use currently new materials would certainly need to undergo toxicity testing. These materials can be evaluated by placing cells
directly on or in proximity to the material to determine whether any compounds or eluents that may leach from the material
are toxic. This latter case is especially important in the development of resorbable materials since an intact material may be cyto-
compatible but its degradation products may not be. For instance, polyesters such as polylactide, polyglycolide, and poly(lactide-co-
glycolide) are well-studied and widely used degradable biomaterials and are inherently nontoxic to cells that attach, migrate, and
proliferate while in contact with the material. These polymers, however, degrade when in contact with an aqueous medium and
their degradation products are lactic and glycolic acid, both highly acidic and potentially damaging to cells on or near the material.
Despite these acidic degradation products, these polymers are widely and successfully used but serve as a good example of why the
degradation products are as important as the material itself when it comes to cytotoxicity studies.
Testing for cytotoxicity generally involves determining whether the cell membrane is intact. An intact cell membrane indicates
a live cell and a ruptured cell membrane indicates a dead cell. Assays have been developed to evaluate the integrity of the cell
membrane in which solutions are added to a cellularized environment, be it a tissue construct or cells in culture, that either stain
the intracellular components of a dead cell or react with extracellular components that have been released from a cell because of the
damaged cell membrane. For instance, trypan blue is used to visually determine a live or dead cell. Since trypan blue does not pass
through the cell membrane, a live cell with an intact cell membrane will not stain blue while a dead one with a damaged and there-
fore permeable membrane will. Another common method is a live/dead assay, often coupled with fluorescent markers. The fluo-
rescent markers are tagged to either a molecule that passes through the cell membrane or one that does not, the former fluorescing at
a different wavelength than the latter and therefore distinguishing between live and dead cells. Other assays interact with
compounds normally maintained within the cell membrane in live cells but are released into the extracellular milieu if the
membrane is broken, indicating a dead cell. Some cytotoxicity assays provide an overall level of cell death based on the entire pop-
ulation of cells without the specificity of which cells are live or dead, while other methods allow the determination of which cells are
alive and which are dead. The choice of assay depends on the information needed; an overall picture of cytotoxicity of a material or
construct or a regional measure of cell survival within a construct or material.
Cell viability and proliferation

Cell viability and proliferation are important metrics of a material or construct for tissue repair. They can provide important feed-
back on the suitability of surface modifications, three-dimensional architecture, oxygen transport, degradation product compati-
bility, and many other aspects of the material or structure. While often used interchangeably cell viability and proliferation are
similar to each other in that both are measures of live cells but distinct in that viability is a measure of cellular activity and overall
health, while proliferation is a measure of the rate of growth and production of daughter cells of a cell population. Either may be
suitable as a tool to measure the health of a cell population and the suitability of a construct or material for cell survival but the
assays and techniques used to assess each can vary and care should be taken to ensure that the method of analysis or choice of assay
is appropriate. Generally speaking, proliferation is required to have a construct fully populated by cells which in turn mandates
a fully interconnected pore structure or other means of adequate nutrient and waste exchange to allow the cells to remain viable
within the depths of the construct. If the construct has insufficient nutrient transport cells will not proliferate to the center of the
construct and new tissue will not be deposited. Measuring proliferation is one measure of how well a construct can support the
residence of cells throughout its structure. Common assays used to test cellular viability include the MTT and MTS assays, which
measure the viability of a cell through its metabolic activity, DNA synthesis assays which can translate to actual cell numbers,
and manual cell counts using a dye such as trypan blue, which, as discussed earlier, will permeate a damaged cell membrane as
an indicator of a nonviable, or dead, cell.
Differentiation
For a tissue construct to facilitate the regeneration of tissues, it must be able to sustain not only the proliferation of cells but also
their differentiation as well. Cell differentiation involves the maturation and subsequent specialization of a cell. As cells mature in
their life cycle they become more specialized, or more focused in their function. For instance a mesenchymal stem cell can
contribute to the regeneration of multiple tissue types depending on its gene expression and subsequent maturation, or differen-
tiation. Once it differentiates its overall potential to become multiple tissue types is reduced to one specific tissue type. Differenti-
ation is not limited to stem cell populations though. An osteoblast, for instance, will differentiate into an osteocyte and its function
will become more specialized. Understanding how cells differentiate, what causes them to differentiate, and how to control their
differentiation are powerful tools available to the regenerative engineer through the design of tissue constructs. Materials scientists
and engineers have developed methods to influence or control cellular differentiation by modulating material aspects like compo-
sition, topography, architecture, delivery of payload, and mechanical properties to name a few. Evaluating the differentiation of
a cell is one way of measuring cellular function on a construct but also its overall health. As cells differentiate they undergo a number
of changes that can be measured or tracked to assess the nature and extent of differentiation. Surface receptors, protein expression,
and genetic expression are a few of the ways the regenerative engineer can assess the extent of differentiation of a cell and gather
a picture of the functionality of those cells on the construct. Whether as a passive measure of cellular function on a cell or as
a measure of the intended influence on a particular cell, differentiation is an important tool in assessing the efficacy of a tissue
construct.
In Vivo Models for Tissue Construct Evaluation
In vitro cellular analysis provides important insight into the potential utility of a tissue construct as a tool for regenerating tissue and
offers the advantage of an environment that is easy to control and while it can act as an important first approximation of efficacy it is
only an estimate of the in vivo environment. To truly evaluate a tissue construct an in vivo model is necessary. Also unlike human
studies where tissue is evaluated after injury or onset of disease animal models can chronicle the progression of injuries. Many
different in vivo models exist to test toxicity, biocompatibility, mechanical stability, and tissue repair potential. An exhaustive
list of in vivo models is beyond the scope of this article, but here we provide some defect models as examples of the different
approaches that can be taken. Within musculoskeletal regenerative engineering there are several models of hard and soft tissue
injuries and defects. Below are descriptions of each, the type of injury they model, why they are chosen, and how they inform
the clinical implementation of the tissue construct.
Hard Tissue Defect Models

The in vivo models used in hard tissue construct evaluation can focus on critical size or noncritical size defects. A critical size defect,
typically made in long bones, is one that will not heal on its own. Unlike a noncritical size defect that, if left alone, would eventually
heal, a critical size defect is of sufficient size to prevent spontaneous repair. In regenerative engineering we mostly focus on those
tissues that cannot heal themselves and therefore use critical size defects. The presumption is if the engineered construct can heal
a critical size defect in an animal model it can support large-scale defect healing in humans. To this end several long bone models
have been used in mice, rats, and rabbits, where different animals have different physical sizes and healing times. Mice tend to heal
faster than rats and rabbits while the larger size of the rat and rabbit make for easier defect formation and, depending on the
construct, more realistic preclinical models. Long bone defects allow for the evaluation of cortical bone repair while calvarial defects,
circular defects in the skulls of animals, allow for the evaluation of trabecular bone. While long bone defects in these animals would
provide a good assessment of long bone repair in humans, a calvarial defect in mice may provide a good assessment tool for trabec-
ular bone found in the facial bones while providing a bony area large enough to create a defect and assess healing using a construct.
Trying to create a jaw or facial bone defect in a mouse may prove to be too challenging and certain types of constructs may not be
able to be manufactured on such a small scale. Some of the long bones that have been evaluated in these animals are rat tibial
segmental defects, rat femoral segmental defects, rabbit ulnar segmental defects, and rabbit radial segmental defects.
Soft Tissue Defect Models

The in vivo models used for soft tissue construct evaluation focus on the repair and/or regeneration of tissues like muscle, ligament,
tendon, and cartilage. Soft tissue defect models can assess the construct’s ability to provide mechanical stability to the defect site, to
encourage cellular migration and infiltration, and over time to evaluate the overall healing of the defect. As with hard tissue models,
many soft tissue models focus on defects that do not fully heal on their own such as full ligament and tendon tears, but can also
evaluate methods of increasing the rate and extent of tissue repair for injuries that may heal on their own. The specific animals
chosen for soft tissue defects depend on the animal anatomy, physical size, and the biomechanics of how the tissue in question
is loaded. For instance, the rat is physically small and can be challenging to work with when considering the repair or regeneration
of tissues like the anterior cruciate ligament (ACL) or rotator cuff, but studies have demonstrated that due to anatomic similarities
between the rat and human rotator cuff the rat is uniquely suited as a model for human rotator cuff tears and subsequent surgical
repairs. In this instance, the challenge of the small size is outweighed by the benefits of having a model that more accurately matches
the biomechanical loading and anatomical structure of a human rotator cuff. Rotator cuff defect models typically involve the tran-
section of one or more tendons in the shoulder or chronic overuse of the tendon leading to injury or a combination of both. Repair
typically involves the resuturing of the transected tendon back to the bone of origin, but depending on the construct that has been
developed to facilitate repair the strategy may vary.
For anterior cruciate ligament repair small animals are used as well but larger animals offer fewer challenges surgically, which
may result in less surgical error or variability between animals. Small animals have been used for cell-based ligament repair where
a surgical implantation of a construct is not required, but larger animals like rabbits have found favor when a construct is being
surgically implanted and sutured to existing tissues. In these instances the knee cavity is opened and the intact ACL is transected.
Tunnels are drilled through the tibia and femur (proximal and distal to the transected ACL) and the construct is fed through
each of the tunnels such that the two bones are connected by the construct, much like the native ACL. Once the construct is
adequately secured within each bone (which can be done by a variety of methods), the soft tissue around the knee is sutured closed
and the animal is free to ambulate. This model allows the construct to be mechanically loaded just as the native ACL was prior to
transection and is exposed to the same extracellular milieu as the native ACL, providing an excellent recapitulation of the native ACL
environment for testing.
For cartilage repair, there are a few different animal models. Some models involve the creation of a focal defect, which is a defect
in the cartilage or the cartilage and underlying trabecular bone, with well-defined borders. Other models involve the creation of
osteoarthritis, a degenerative disease of the cartilage that results in more diffuse, less-localized, cartilage damage across an articulat-
ing surface. Each of these models of injury is created in different ways and is designed to measure different treatment strategies. The
focal injuries can be created by impact or surgical removal of cartilage with or without the underlying bone while the less-localized
injuries like osteoarthritis can be created surgically by creating grooves in the cartilage, surgically manipulating the meniscus to
induce osteoarthritis, or surgically transecting the ACL to induce osteoarthritis. The choice of model depends on the treatment
strategy to be tested. The choice of animal may depend on the questions being asked or the budget available. Small animal cartilage
defects are used quite commonly where one of the above methods is applied to mice, rats, rabbits, and guinea pigs, but the anatomy
of small animal cartilage and underlying bone is somewhat different from humans so larger animals have been used as well. The
anatomical similarity of the cartilage and underlying bone in animals like dogs, goats, sheeps, and pigs to humans is better but the
cost associated with these studies is considerably higher. Each approach, small or large animal, has its benefits and drawbacks so the
selection of a model is often dictated by the questions being asked or the stage of development of the construct.
Conclusion
The field of regenerative engineering presents an exciting new paradigm for tissue construct design and implementation that seeks
to take advantage of the newest discoveries in materials development, stem cell biology, developmental biology, and clinical
translation. Here, we have discussed some of the fundamental aspects of tissue construct evaluation from mechanical testing
to cellular evaluation and preclinical testing. While not an exhaustive analysis of the area it presents a starting point for the
budding regenerative engineer and provides a scope from which to build a thorough testing programs for novel tissue constructs.
Each distinct area is constantly under development and therefore constantly changing and being updated, but a foundational
knowledge of these areas will serve the regenerative engineer well as they move forward with their novel approaches to complex
tissue repair.
References
Athanasiou, K. A., Zhu, C. F., Lanctot, D. R., Agrawal, C. M., & Wang, X. (2000). Fundamentals of biomechanics in tissue engineering of bone. Tissue Engineering, 6(4), 361–381.
Barker, M. K., & Seedhom, B. B. (2001). The relationship of the compressive modulus of articular cartilage with its deformation response to cyclic loading: Does cartilage optimize its
modulus so as to minimize the strains arising in it due to the prevalent loading regime? Rheumatology (Oxford), 40(3), 274–284.
Bergström, J. S., & Hayman, D. (2016). An overview of mechanical properties and material modeling of polylactide (PLA) for medical applications. Annals of Biomedical Engineering,
44(2), 330–340.
Eshraghi, S., & Das, S. (2010). Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two-dimensional, and three-dimensional orthogonally
oriented porous architectures produced by selective laser sintering. Acta Biomaterialia, 6, 2467–2476.
Foster, L. J., Ho, S., Hook, J., Basuki, M., & Marçal, H. (2015). Chitosan as a biomaterial: Influence of degree of deacetylation on its physiochemical, material and biological
properties. PLoS One, 25, 10(8).
Holzapfel, G. A. (2001). Biomechanics of soft tissue. In J. Lemaitre (Ed.), Handbook of material behavior: Nonlinear models and properties (pp. 1057–1075). Cachan: LMT-Cachan.
Isnard, R. N., Pannier, B. M., Laurent, S., London, G. M., Diebold, B., & Safar, M. E. (1989). Pulsatile diameter and elastic modulus of the aortic arch in essential hypertension: A
noninvasive study. Journal of the American College of Cardiology, 13(2), 399–405.
Jana, S., Florczyk, S. J., Leung, M., & Zhang, M. (2012). High-strength pristine porous chitosan scaffolds for tissue engineering. Journal of Materials Chemistry, 22,
6291–6299.
Levett, P. A., Hutmacher, D. W., Malda, J., & Klein, T. J. (2014). Hyaluronic acid enhances the mechanical properties of tissue-engineered cartilage constructs. PLoS
One, 9(12).
Liang, X. (2010). Boppart SA. Biomechanical properties of in vivo human skin from dynamic optical coherence elastography. IEEE Transactions on Biomedical Engineering, 57(4),
953–959.
Pal, S. (2014). Mechanical properties of biological materials. In Design of artificial human joints and organs (pp. 23–40). New York, NY: Springer.
Further Reading
Athanasiou, K. A., Zhu, C. F., Lanctot, D. R., Agrawal, C. M., & Wang, X. (2000). Fundamentals of biomechanics in tissue engineering of bone. Tissue Engineering, 6(4), 361–381.
Buffinton, C. M., Tong, K. J., Blaho, R. A., Buffinton, E. M., & Ebenstein, D. M. (2015). Comparison of mechanical testing methods for biomaterials: Pipette aspiration,
nanoindentation, and macroscale testing. Journal of the Mechanical Behavior of Biomedical Materials, 51, 367–379.
Sah, R. L., & Ratcliffe, A. (2010). Translational models for musculoskeletal tissue engineering and regenerative medicine. Tissue Engineering. Part B, Reviews, 16(1), 1–3.
Clinical and Laboratory Aspects of Hematopoietic Stem Cell Transplantation
ST Avecilla and MM Cushing, New York Presbyterian Hospital, Weill Cornell Medical College, New York, NY, USA
Hematopoiesis: A Brief Introduction to How Blood Cells Are Created and Formed 547
The Utility of Hematopoietic Stem Cell Transplantation 547
HPC Collection 548
HPC-M (Bone Marrow) 548
HPC-A (Peripheral Blood Stem Cells) 548
HPC-C (Umbilical Cord) 548
HPC Characterization 550
HPC Manipulation 550
Cryopreservation 550
HPC Infusion 551
Adverse Reactions to HPC Infusions 551
Complications after HSCT 552
Advances in HSCT 553
Further Reading 553
Glossary
Allogeneic transplant Stem cells utilized are from a donor.
Apheresis Medical procedure during which a patient or donor has their blood separated and a specific fraction collected via
a continuous flow centrifuge.
Autologous transplant Stem cells utilized are from the patient’s own cells (rather than from a donor).
Conditioning regimen Chemotherapy or irradiation given immediately prior to a transplant to eradicate the patient’s disease
and suppress immune reactions, which could lead to rejection.
Cryoprotectant Liquid substance capable of inhibiting intracellular ice formation in the process of freezing cells for long-term
storage.
Cytokine Signaling protein, which stimulates the proliferation of cells via specific receptors.
Differentiation The process whereby an unspecialized stem cell acquires the features of a specialized cell such as a mature red
blood cell. Differentiation is controlled by the interaction of a cell’s genes with the physical and chemical conditions outside
the cell, usually through signaling pathways involving proteins in the cell surface.
Engraftment A state where a hematopoietic progenitor or stem cell has homed to the bone marrow and reconstituted
hematopoiesis for the recipient of the transplant.
Flow cytometer A device that measures various optical parameters of single cells in suspension.
Graft-vs-host disease (GvHD) Common complication of allogeneic stem cell transplant where immunocompetent T cells in
the donor graft recognize and attack host tissue as a foreign target.
Hematologic malignancy Cancer of the blood or bone marrow.
Hematopoiesis Process of production, multiplication, and specialization of blood cells in the bone marrow beginning with
a hematopoeitc stem cell.
Hematopoietic progenitor cell (HPC) A cell with the capability to proliferate and generate differentiated blood cells, but
which is already at a further stage of differentiation than a stem cell and may only differentiate into its designated target
hematopoietic cells. In addition, HPCs only can divide a limited number of times, unlike stem cells. For the purpose of this
article, the term HPC will include HPCs and hematopoietic stem cells.
Hemostasis Arrest of bleeding by vasoconstriction and coagulation.
HPC mobilization Process by which drugs are used to cause release of stem cells from the bone marrow into the peripheral
blood.
Peripheral blood stem cells (PBSCs) HPCs circulating in peripheral blood that are collected by apheresis from an autologous
or allogeneic donor.
Stem cell An undifferentiated, omnipotent cell with an unlimited capability of self-renewal.

Regenerative Engineering j Clinical and Laboratory Aspects of Hematopoietic Stem Cell Transplantation 547
Hematopoiesis: A Brief Introduction to How Blood Cells Are Created and Formed
Blood is a liquid tissue that has many important functions in the human body. In addition to serving a critical role in transporting
oxygen and nutrients, blood and its constituents are essential in host defenses, hemostasis, and many other functions. Hematopoi-
esis is a process whereby stem and progenitor cells, through a process of proliferation and differentiation, result in the generation of
the cellular elements of blood. This differentiation process is influenced by lineage-specific cytokine signaling such as granulocyte-
colony stimulating factor (G-CSF), erythropoietin, thrombopoietin, in addition to microenvironmental stimuli such as interaction
with bone marrow stroma and blood vessels. Due to the ephemeral nature of the components of blood, hematopoiesis is crucial in
the continuous replenishment of functional cells. There are several instances where clinical treatment of disease results in the irre-
versible destruction of the bone marrow, which can be fatal if a compatible hematopoietic progenitor cell transplant is not
performed.
The Utility of Hematopoietic Stem Cell Transplantation
Hematopoietic stem cell transplantation (HSCT) refers to the intravenous infusion of autologous or allogeneic hematopoietic
progenitor cells (HPCs), collected from bone marrow, peripheral blood, or umbilical cord blood (CB), to replace damaged or aber-
rant stem cells in a patient. A list of common indications for HSCT is given in Table 1.
In the currently practiced methods of treating hematologic malignancies, HSCT is a frequently chosen modality in conjunction
with chemotherapy and/or total body irradiation. Nonmalignant conditions that are treated with HSCT include severe sickle cell
disease, severe combined immunodeficiency, in addition to marrow failure states such as aplastic anemia. Most chemotherapy regi-
mens employ cytotoxic agents which are intended to kill the neoplastic cells. The nonneoplastic bone marrow, which has a high
proliferative baseline, is also severely and adversely affected and may be irrevocably damaged. In order to rescue the patient
from a bone marrow depleted state, an HSCT must be done. Due to the fact that the immune cells are primarily derived from
the bone marrow and associated tissues, it is highly important that the donor HPCs come from a human leukocyte antigen
(HLA)-matched party. The requirement for HLA matching even outweighs the need for A/B/O blood group matching. Sources
for HLA-compatible HPCs can vary from autologous HPCs to those harvested from related or unrelated donors. Transplant-
related mortality and morbidity may be affected by numerous aspects of the transplant (Table 2).
Prior to collecting an allogeneic donor candidate, several health criteria must be assessed. The donor must pass a physical exam-
ination in addition to answering an HPC donation questionnaire, which assesses infectious disease risk. Infectious disease marker
assays are performed on the donor’s blood to ensure that no infectious agents are transmitted unknowingly.
Table 1 Common indications for HSCT
Autologous Allogeneic
Multiple myeloma/amyloidosis Acute myeloid leukemia

Non-Hodgkin lymphoma Acute lymphoblastic leukemia
Hodgkin lymphoma Myeloproliferative disorders
Acute myeloid leukemia Multiple myeloma
Neuroblastoma Non-Hodgkin lymphoma
Germ cell tumors Hemoglobinopathies (thalassemia/sickle cell)
Inborn errors of metabolisms
Autoimmune disorders
Severe congenital immune deficiencies
Table 2 Aspects of HSCT, which may affect morbidity/

mortality
Toxicity and effectiveness of conditioning regimen

Degree of HLA matching
Infection prevention and treatment
Autologous vs allogeneic transplant
Source of transplant (bone marrow, peripheral blood, or
umbilical cord)
Time from transplant until engraftment
Incidence and severity of graft-vs-host disease
548 Regenerative Engineering j Clinical and Laboratory Aspects of Hematopoietic Stem Cell Transplantation
HPC Collection
HPC-M (Bone Marrow)
The first recognized source of HPCs was bone marrow. It was discovered that a small population of the harvested cells had the prop-
erty of giving rise to the full repertoire of hematopoietic cell lineages and most importantly they could reconstitute a depleted bone
marrow for the lifetime of the recipient. While no longer the most popular method of extracting HPCs for transplant, hematopoietic
progenitor cell-bone marrow (HPC-M) has some advantages to the methods developed in later years to harvest HPCs for transplant
(Table 3).
First and foremost, the prospective donor need not take any medication or cytokine which may have both short- and long-term
negative health effects. The extraction process is a surgical procedure during which the donor is placed in a prone position under
local or general anesthesia for pain management. Once the donor is properly prepared, an 11–14 gauge needle is inserted into the
posterior iliac crest until it is in the marrow space. Marrow is then aspirated from the donor until multiple locations accessible from
the single insertion site have been exhausted. Typically, the marrow is collected with heparin as the anticoagulant and a biocompat-
ible solution for cell suspension such as Plasma-Lyte A (Baxter Healthcare, Deerfield, IL). Additional anticoagulant such as acid
citrate dextrose (ACD) may be added to further prevent sample clotting.
Due to the relative rarity of the HPCs within the marrow space, a large volume of material is usually extracted which often
includes a significant contamination by peripheral blood. The harvest is monitored by total nucleated cell (TNC) yield with a usual
target of >3.0 108 nucleated cells/kg of recipient weight. Drawbacks of harvesting HPC-M include the donor’s exposure to anes-
thesia, blood loss, postcollection pain, bruising, and other less common symptoms. Due to the blood loss, a great majority of
donors require at least one red blood cell transfusion (both autologous and less commonly allogeneic), which is also a risk to
the donor. Because T-lymphocytes are not produced in the marrow, the product tends to have much less CD3 T-cell content which
can result in a lower incidence of graft-vs-host disease (GvHD).
HPC-A (Peripheral Blood Stem Cells)

In the initial experience with HSCT and patients with hematologic malignancies, it was found that in some chemotherapy regimens,
patients demonstrated a low but substantially increased number of HPCs circulating in the peripheral blood. With this observation,
HPC mobilization regimens were developed which utilized chemotherapy as a mobilization agent. This technique could ethically
only be used on patients with malignancies and not with allogeneic healthy donors since the risk of chemotherapy is considered
unacceptable. Once cytokines such as G-CSF were discovered and developed for clinical use, mobilization protocols utilizing G-CSF
became available. Typically allogeneic and/or autologous donors will receive a subcutaneous injection of G-CSF daily for 5 days
with HPC collection via apheresis (Figure 1) occurring on the fifth day. Of key importance is the donor vascular access due to
the fact that peripheral vein access is the preferred and least invasive route to obtaining peripheral blood. Donors with poor periph-
eral venous access can still undergo HPC mobilization and apheresis harvest of HPCs, however, a central line venous catheter or
comparable vascular access will first have to be placed. While central vascular access allows for higher apheresis flow rates and typi-
cally shorter collection cycles, it comes with the risks of having a central line placed which include infection, air embolus, and
bleeding. In addition to cytokine and chemotherapy mobilization regimens, newer agents have been developed to further enhance
HPC mobilization. An agent that is FDA approved for HPC mobilization in specific patient populations (lymphoma and multiple
myeloma) is plerixafor. Plerixafor (formally known as AMD3100) is a small antagonist molecule of the chemokine receptor CXCR-
4, which is found on HPCs in addition to other cells. It is hypothesized that disruption of CXCR-4 chemotactic signaling of HPCs
while in the bone marrow niche allows for the peripheral mobilization of HPCs into the bloodstream. The apheresis machine is
anticoagulated with ACD-A and therefore products collected via this modality have ACD-A as the primary anticoagulant. Collection
is monitored by enumerating CD34/CD45 positive cells and collection is done until a goal dose is reached e.g., >2–5 106 CD34þ
cells/kg. Unlike HPC-M, apheresis collected HPCs have a substantial number of T cells which has implications in both graft-vs-host
and graft-vs-leukemia (GvL) effects of the transplant. An additional benefit of HPC-A is that engraftment times are usually shorter in
comparison with HPC-M and HPCs from human umbilical vein CB. See Figure 2 for an example of a HPC-A.
HPC-C (Umbilical Cord)

The most recently described, readily available source of hematopoietic progenitor cells for clinical use is human umbilical vein CB,
HPC-C. An insightful observation was made upon the microscopic examination of CB smear preparations that there was an
Table 3 Hematopoietic progenitor comparison
HPC source Cell dose (per kg recipient weight) Speed of engraftment GvHD risk HLA match stringency
8
HPC-M 2–4 10 nucleated cells Moderate Moderate High
HPC-A 5 106 CD34þ cells Fast Highest High
HPC-C >1.5 107 nucleated cells Slow/Delayed Lowest Moderate-high
Figure 1 Apheresis collection on a cobe spectra.
Figure 2 HPC-A.
immature appearing white cell population that resembled blast cells. Further research into this cell population revealed that this
blastlike population was in fact a benign hematopoietic progenitor cell population, which could be used successfully in HSCTs.
Immediately prior to giving birth, prospective donor mothers are screened with a donor questionnaire and upon giving birth,
maternal and CB samples are obtained for appropriate infectious disease marker testing. Once the child is born, the umbilical
cord is cross-clamped and the attached placenta is placed in a sterile container and transferred to a workroom where the CB is har-
vested under as sterile conditions as possible. The harvested CB is then processed to isolate the mononuclear cell fraction and
analyzed for transplant appropriateness, which include TNC count, CD34þ/CD45þ cell count, microbiological cultures, and
HLA typing. Once the testing samples have been obtained, the product is cryopreserved and banked for future use in HSCT treat-
ments. Due to the immature, immune state of the product, HPC-C is associated with lower rates of GvHD in addition to successful
transplants having an increased tolerance for HLA mismatches. Because HPC-C are collected, characterized, and stored before
a specific recipient is identified, they represent a rapidly obtainable source of HPCs that can be used without a long lead-time.
Conversely, HPC-A and HPC-M both require identification of a suitably HLA-matched donor with genotyping in addition to the
performance of a medical procedure to harvest the HPCs (apheresis or surgical marrow collection), which may be accompanied
with possible HPC mobilization in the case of HPC-A. Due to the small nature of the umbilical cord, there is an intrinsic limit of the
quantity of HPCs that can be harvested per cord. Because HPC dose is one of the most important predictors of HSCT outcome (the
higher the dose, the more likely engraftment will occur), HPC-C as the only source of HPCs from single donors for HSCT has limited
success in larger patients (mostly adults).
HPC Characterization
In order to properly prepare an HPC product, several parameters are measured. First a sample of the HPC product is taken and
analyzed with a hematology analyzer in order to obtain a TNC count. In the case of HPC-M, the TNC count is used to measure
harvest adequacy with the assumption that the typical CD34þ cell content is approximately 1–3% of the total TNC count. For
a more accurate assessment of CD34þ cell content, the sample is subjected to flow cytometric analysis. Antibodies to CD34
(HPC marker) and CD45 (pan-leukocyte marker) antigens are used in conjunction with a viability stain to accurately quantify
the number of live CD34þ cells that have the proper cellular characteristics to qualify as HPCs (small cells with low granularity,
similar to lymphocytes). Viability can also be assessed using a vital stain such as trypan blue in which viable cells exclude the
dye; however, trypan blue is considered to be less sensitive than flow cytometric viability using 7-AAD. To test the product for
contamination risk, which may occur at any stage between collection and processing, a sample of product is inoculated in the auto-
mated microbial culture media at several points in processing. Finally, some laboratories perform HPC potency testing. The most
common example of potency testing is the colony forming unit (CFU) growth assay. Briefly, a sample of HPC product is plated in
a defined, cytokine-rich medium and allowed to incubate for 14 days, after which the cell culture is examined for the growth of
colonies characteristic for different classes of progenitors (see an example in Figure 3). While CFU growth assays are helpful, at
this time they cannot assess the presence of hematopoietic stem cells but rather the committed progenitor content (HPC).
HPC Manipulation
ABO mismatched grafts are acceptable for HSCT, but there may be related complications, such as acute or delayed hemolysis, or
delayed or lack of red cell engraftment. The cells may be red blood cell depleted, but this is often at the expense of the loss of
some HPCs.
Cryopreservation
While minimally manipulated HPC products are preferred due to higher levels of viability and lower chances that processing
adversely affects the ‘stemness’ and viability of the HPC products, there are clinical situations where cryopreservation is an essential
component of HPC processing. First and foremost, when autologous HSCT therapy is indicated such as in multiple myeloma and
some forms of lymphoma, chemotherapy induction and conditioning regimens cannot occur concurrently with HPC collection due
to the cytotoxic effects of the regimens. In order to harvest viable HPCs, the patient is first mobilized and his or her HPCs are
collected, characterized, processed, and cryopreserved for later use after ablative chemotherapy and radiation have been adminis-
tered. Another example where cryopreservation is widely used is in HPC-C. Situations may also occur where potential donors are
Figure 3 CFU assay.

Figure 4 Cryofacility.
unavailable at the time the patient needs an HSCT and, if available beforehand, the donor may preemptively donate HPC-A with
subsequent cryopreservation for use in the HSCT when appropriate.
Due to the fact that water expands when it transitions from liquid to solid phase, and the subsequent expansion results in cellular
damage, cells cannot be stored in a frozen state without proper cryoprotection. Industry-wide, dimethyl sulfoxide (DMSO) is used
in various concentrations as an agent that crosses the plasma membrane of cells and displaces water such that the cells become rela-
tively dehydrated. DMSO is also thought to inhibit cell-damaging ice formation and promote water solidification in an amorphous,
vitreous state that results in reduced amounts of cellular damage upon thawing. Once infused, DMSO is converted to dimethyl
sulfone and dimethyl sulfide and eliminated by urinary excretion. Renal excretion accounts for almost half of the administered
DMSO dose. Serum half-life of DMSO is about 20 h; the half-life of dimethyl sulfone, a renally excreted metabolite, is about
72 h. A small amount of DMSO is converted to dimethyl sulfide, which is excreted through the lungs for about 24 h and is respon-
sible for the characteristic odor of the patient’s breath following a stem cell infusion.
Cryopreserved HPCs are stored in liquid nitrogen tanks, most often at vapor phase (150 C) until they are needed for trans-
plant (Figure 4 for an example of a cryofacility).
HPC Infusion
The infusion of an HPC product (i.e., the transplant) is usually an anticlimactic event that resembles the intravenous infusion of any
blood product. The product may be infused fresh, as is most often the case for allogeneic transplants, or thawed if it was cryopre-
served after collection, as is most often the case for autologous transplants or HPC-C infusions. Usually the bags are thawed near the
patient’s room with a 37 C water bath to shorten the exposure of the HPCs to DMSO. Cryopreserved HPCs should always be
administered through a central venous line. A 10% DMSO solution has a high molality that can cause pain when infused through
a peripheral line.
The thawed HPC product may have a volume of 2–25 ml kg1 of the recipient. The product can be administered via a syringe or
directly from the bag. Direct infusion from the bag can avoid the risk of contamination and cell loss during transfer from bag to
syringe. At the end of the infusion, the bag and line should be flushed with normal saline to minimize cell loss. The line can
also be flushed during the infusion to increase the infusion rate. The stem cell product should never be irradiated or transfused
through a leukoreduction filter. The dose of DMSO given during the infusion of thawed stem cells varies, but most centers set
an upper limit of 1 g of DMSO per kg of patient weight over a 24-h period.
To minimize the time of contact between the thawed stem cells and DMSO, HPC infusions should occur as rapidly as possible.
The infusion speed of DMSO cryopreserved products reported in the literature varies between 5 and 20 ml/min. A slower infusion
rate may avoid some of the adverse reactions associated with DMSO infusion and volume overload. Patients should have their
symptoms and vital signs carefully monitored during the infusion and for several hours afterward. Noncardiogenic side effects
usually occur during the infusion and often resolve after the infusion is stopped. Bradycardia, however, has been reported to occur
several hours after the infusion.
Adverse Reactions to HPC Infusions
The incidence of adverse reactions to HPC infusion varies depending on the type of product infused (bone marrow, peripheral
blood stem cells, or umbilical cord and autologous vs allogeneic), whether the product is fresh or thawed, and whether the product
was washed after thawing vs bedside thawing. Reported rates of reactions generally range from 20 to 30% of infusions. Little data
exist on the incidence of each individual type of reaction.
As with all blood components, there is a risk of microbial contamination with HPCs. Contamination of HPCs can occur during
any step in the transplant process, including collection, processing, and thawing. Many transplantation centers perform HPC
product cultures before and after processing, but unlike other products, HPCs are not readily replaceable making the management
of positive culture results far more complex. Many factors pertaining to the infectious organism, the product, and the patient must
be taken into account when evaluating the culture result. The virulence of the identified organism is of great importance, with gram-
negative rods raising particular concern. The possibility of replacing the contaminated product should be investigated. If, for
instance, only a fraction of the vials for a transplant are culture positive and an adequate number of CD34 positive cells are present
in the remaining vials, then the contaminated vials can simply be discarded. Recollection is another option to be evaluated. The
stage of the patient’s treatment is critical because it affects how long the transplant can be delayed, particularly if the patient has
already undergone a conditioning regimen. If proceeding with the infusion of a culture positive HPC product, prophylactic antibi-
otic therapy may be administered. When time allows pretransplant, the identity and antibiotic susceptibilty of the contaminant
organism can be determined and targeted preemptive antibiotic therapy can be done.
Allergic reactions are common in a stem cell recipient and manifest in a similar way to allergic transfusion reactions, with
common features including pruritus, urticaria, and orbital swelling. Upper and lower airway obstruction and anaphylaxis may occur
during more severe reactions. The allergen may be a protein in an allogeneic donor’s plasma or a product added during processing,
such as dextran or DNAse. Allergic reactions can often be prevented by premedication with antihistamines, steroids, or a combina-
tion of both.
Febrile reactions most commonly occur due to the accumulation of platelet- or leukocyte-derived cytokines in the product or to
leukocyte antibodies in the recipient that react with the leukocytes in the product. These reactions may also occur as a result of bacte-
rial contamination of the product or hemolysis due to plasma or red cell antigen incompatibility in allogeneic transplants.
Thawed products have additional, associated adverse events. The thawed HPC product contains many components besides the
HPCs themselves, including DMSO, plasma, white blood cells, granulocyte debris, and red blood cell debris (including free hemo-
globin). Most reactions associated with thawed products are related to DMSO and include nausea, vomiting, abdominal cramping,
hyper- or hypotension, bradycardia, chest tightness, fever, chills, and headache. DMSO gives off an odor that has been compared to
creamed corn or garlic. The odor may last for 1–2 days and may cause nausea; sucking on hard candy may help eliminate the nausea.
HPC products that are washed to reduce the DMSO content have less infusion-related toxicity.
Complications after HSCT
There is a high mortality associated with hematopoietic stem cell transplant. Complications may occur that are related to the
patient’s underlying disease, previous treatments, conditioning regimen, transfusions, and medications. In addition, the patient’s
immune system may become dysregulated. Early complications are often related to the time to engraftment. An engraftment delay
leaves the patient susceptible to infections and bleeding. HPC donor source, chemotherapy regimen, and glucocorticoid treatment
can influence the type and propensity for infections, which include bacterial, fungal, and viral etiologies. Mucositis and gastroin-
testinal toxicity are frequent early treatment complications, especially in the first 14 days after transplant. Hepatic veno-occlusive
disease (sinusoidal obstruction syndrome) is a serious complication that is more common in allogeneic transplant vs autologous
with a mortality of 20–50%. Late complications of stem cell transplant include avascular necrosis of bone, cataract formation, bron-
chiolitis obliterans, and gonadal failure. The development of a secondary malignancy after transplant, often myelodysplastic
syndrome or leukemia, infrequently occurs.
Graft failure results when the recipient’s immune system rejects the infused donor cells and engraftment does not occur. It is less
likely to occur after fully myeloablative conditions, but it is more common after reduced intensity conditioning or after the infusion
of products with lower numbers of stem cells (such as umbilical CB). Relapse of the underlying disease is the most frequent cause of
graft failure. Risk factors for relapse include transplant for advanced disease, autologous transplant, T-cell depleted graft, and
reduced intensity conditioning regimens. Other risk factors for graft failure include HLA mismatch between donor and recipient
and damage to the stem cell product during processing (rare). Donor lymphocyte infusion, collected from donors with or without
G-CSF mobilization, can also be helpful in the treatment of patients with progressive disease after transplant.
Immunologic events after allogeneic transplant can be both beneficial and deleterious. GvHD is a cause of morbidity, mortality,
and decreased quality of life after allogeneic stem cell transplant. GvHD is caused by donor T cells that recognize antigenic dispar-
ities between the donor and recipient and cause a reaction. GvHD causes selective epithelial damage of target organs including the
skin, liver, and the gastrointestinal tract. Acute GvHD occurs within the first 100 days after transplant. GvHD is a clinical diagnosis
with laboratory (biopsy) confirmation. Risks for GvHD include HLA and gender disparity, increased age of the donor or recipient,
and higher quantity of T cells in the product. T cells are the main mediators of GvHD; however, although T-cell depleted grafts
decrease the risk of GvHD, they increase the risk of engraftment failure. Glucocorticoids are the main treatment for GvHD; antith-
ymocyte globulin, mesenchymal stem cell, and photophoresis have also been used to treat steroid-refractory GvHD with variable
responses. The severity of GvHD is inversely related to the risk of relapse. Patients with GvHD have a lower risk of relapse (due to
GvL effect).
GvL is a term that refers to the adoptive immunotherapeutic effect of transplanted allogeneic hematopoietic cells against
leukemia cells. This condition parallels GvHD and measures that decrease GvHD also decrease GvL.
Monitoring for engraftment and GvHD is important for a transplant program and is required by accrediting bodies such as Foun-
dation for the Accreditation of Cellular Therapy and AABB (formerly American Association of Blood Banks). It is crucial that
programs perform outcome analysis (i.e., time to neutrophil and platelet engraftment following product administration) and relate
this to product efficacy. Overall and treatment-related morbidity and mortality must be assessed at 100 days and 1 year after
transplantation.
Advances in HSCT
Although HSCT has made remarkable progress since its inception in the 1950s, there is still room for significant advancement.
Efforts to use reduced intensity conditioning regimens, improve stem cell mobilization and homing after transplant, and develop
procedures to enhance ex vivo expansion of stem cells are all under way. In addition, manipulation of the stem cell product to
include the sorting of cells into subsets (natural killer cells, T cells, regulatory T cells, etc.) may allow the selective infusion of
only the desired components, thus avoiding any unwanted complications.
Further Reading
Copelan, E. A. (2006). Hematopoietic stem-cell transplantation. N. Engl. J. Med., 354, 1813–1826.

Delaney, M., & Haspel, R. L. (2009). Thawing and infusing cellular therapy products. In E. M. Areman, & K. Loper (Eds.), Cellular Therapy: Principles, Methods, and Regulations (pp.
375–382). Bethesda, MD: AABB.
Donmez, A., Aydemir, S., Arik, B., et al. (2012). Risk factors for microbial contamination of peripheral blood stem cell products. Transfusion, 52, 777–781.
Gratwohl, A., Baldomero, H., Aljurf, M., et al. (2010). Hematopoietic stem cell transplantation: a global perspective. JAMA, 363, 2091–2101.
Kumar, S., DeLeve, L. D., Kamath, P. S., & Tefferi, A. (2003). Hepatic veno-occlusive disease (sinusoidal obstruction syndrome) after hematopoietic stem cell transplantation. Mayo
Clin. Proc., 78, 589–598.
Linenberger, M. L. (2009). Collection of cellular therapy products by apheresis. In E. M. Areman, & K. Loper (Eds.), Cellular Therapy: Principles, Methods, and Regulations (pp.
251–260). Bethesda, MD: AABB.
Oran, B., & Shpall, E. (2012). Umbilical cord blood transplantation: a maturing technology. Hematol. Am. Soc. Hematol. Educ. Program, 2012, 215–222.
Sauer-Heilborn, A., Kadidlo, D., & McCullough, J. (2004). Patient care during infusion of hematopoietic progenitor cells. Transfusion, 44, 907–916.
Spitzer, T. R. (2009). Bone marrow collection. In E. M. Areman, & K. Loper (Eds.), Cellular Therapy: Principles, Methods, and Regulations (pp. 236–250). Bethesda, MD: AABB.
Relevant Websites
http://www.aabb.org.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm.
http://www.celltherapysociety.org/.
http://www.factwebsite.org/.
http://marrow.org.
Dental Stem Cells
M Nakashima and Y Hayashi, Department of Dental Regenerative Medicine, Center of Advanced Medicine for Dental and Oral
Diseases, National Center for Geriatrics and Gerontology, Research Institute, Obu, Japan
Introduction: Mesenchymal Stem Cells 554

Dental Pulp Stem Cells 555
Stem Cells from Human Exfoliated Deciduous Teeth 555
Stem Cells from Apical Papilla 556
Dental Follicle Progenitor Cells 556
Periodontal Ligament Stem Cells 556
Isolation of Dental Stem Cells 557
Characterization of Dental Stem Cells 557
Surface Markers 558
Dentin/Pulp 558
Periodontal Ligament 558
Bone 560
Neuron 560
Blood Vessel 560
Trophic Factors 561
Clinical Application 561
Conclusions 561
References 561
Glossary
Adult stem cells Undifferentiated cells, found throughout the body after embryonic development, that multiply by cell
division to replenish dying cells and regenerate damaged tissues. Also known as somatic stem cells, they can be found in
juvenile as well as adult animals and humans.
Mesenchyme A type of undifferentiated loose connective tissue that is derived mostly from mesoderm, although some are
derived from other germ layers, e.g., some mesenchymes are derived from neural crest cells and thus originate from the
ectoderm.
Neural crest cells A transient, multipotent, migratory cell population unique to vertebrates that gives rise to a diverse cell
lineage including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia.
Regeneration Growth anew of lost tissue or destroyed parts or organs.
Repair Improve or restore damaged or injured parts or organs.
Introduction: Mesenchymal Stem Cells
Since the term stromal stem cells and mesenchymal stem cells (MSCs) have been introduced in the 1980s, MSCs have been isolated
from almost all adult tissues (e.g., bone marrow, adipose tissue, synovium, dermis, periosteum, peripheral blood, solid organs (e.g.,
liver, spleen, and lung), and teeth). MSCs are rare and quiescent populations in their niche within fully specialized tissues, having
self-renewal properties and multilineage differentiation potential in vitro (Dominici et al., 2006). MSCs possess potential immuno-
modulatory, antiinflammatory, and trophic effects. MSCs are heterogeneous, comprising a subset of stem cells (or different subsets
of stem cells) and more committed progenitor cells. According to the International Society for Cellular Therapy, the term ‘multi-
potent mesenchymal stromal cells’ is used for the plastic adherent cells independent of their origins, and the MSCs should be
termed only for the subset. Due to lack of standardization in isolation and culture techniques and definitive and specific markers,
the three minimal criteria are proposed for MSCs to be defined: (1) plastic adherence, (2) expression of CD73, CD90, and CD105,
and lack of CD11b, CD14, CD19, CD79a, CD45, and HLA-DR expression, and (3) their trilineage (ecto-, meso-, endodermal line-
ages) differentiation potential (Dominici et al., 2006). Regenerative medicine has especially been paying attention to clinical appli-
cations of MSCs-based therapy to repair or regenerate damaged tissues. Advantages of harnessing MSCs are their easy availability,
high proliferative activity, fewer of the ethical concerns associated with embryonic stem cells and low immunogenicity. Several clin-
ical studies have already demonstrated therapeutic utility of MSCs in diseases such as graft-versus-host disease (GVHD), myocardial

Regenerative Engineering j Dental Stem Cells 555
infarcts, diabetes, and different types of neurological disorders (Sensebé et al., 2010). The therapeutic effects of MSCs are not
restricted to their differentiation ability but depend on their potency to modulate local environment, activate endogenous stem/
progenitor cells, and secrete various trophic factors (Phinney and Prockop, 2007). The regenerative mechanisms, however, are
not completely understood. This section will address the current knowledge on MSCs from dental tissues, especially their charac-
terization and regenerative potential.
Dental Stem Cells
All stem cells involved in odontogenesis originate in mesenchyme except ameloblast progenitor cells. The mesenchyme of the oral
and facial region originates almost exclusively from the paraxial mesoderm up to the third week of development. Neural crest cells
of the ectoderm migrate into the hyoid arches in the fourth week, and most of the mesenchyme is ultimately of neuroectodermal
origin. These cells are known to have an ectomesenchymal origin (Ulmer et al., 2010). The various MSC populations are isolated
from teeth, and include cells from the pulp of both adult teeth (dental pulp stem cells (DPSCs)) and exfoliated deciduous teeth
(stem cells from human exfoliated deciduous teeth (SHEDs)) and, from the root apical part of the papilla (stem cells from apical
papilla (SCAPs)), from the tissue (dental follicle) that surrounds the unerupted tooth (dental follicle progenitor cells (DFPCs)), and
from the periodontal ligament (PDL) that links the tooth root with the bone (periodontal ligament stem cells (PDLSCs)). These
distinct populations differ in terms of self-renewal capability, proliferation rate, stem cell marker expression and differentiation
potential, etc. Their biological properties are described in more detail in the following.
Dental Pulp Stem Cells

The postnatal pulp contains several niches of potential progenitor/stem cells, which is important for mediating reparative dentin
formation. There is the ‘true’ or ‘mother’ adult stem cell central to the niche. This subset of undifferentiated cells displays an infre-
quent, yet unlimited self-renewal, and gives rise to a renewed mother stem cell and a daughter transit amplifying progenitor cell at
mitosis. These daughter progenitor cells have more limited self-renewal capacity, but are highly proliferative. Cell–cell and cell–
matrix communication is critical to maintain the stem cell status within the niche (Sloan and Waddington, 2009). The niche usually
maintains a quiescent state and specific signals derived from precise area of niche permit stem cells to stay alive, and change their
number and fate (Scadden, 2006; Chen et al., 2011). The niche resides predominantly in the perivascular and perineural sheath
regions of the pulpal cavity (Shi and Gronthos, 2003), from where they migrate to the site of injury (Téclès et al., 2005). Additional
niche in the odontoblast–subodontoblast layers and the pulpal stroma, however, is also suggested by elevated expression of Notch
signals (Løvschall et al., 2005).
DPSCs can be isolated by a colony isolation method from the adult dental pulp (Gronthos et al., 2000), which can be acquired
from third molars, pulpectomized teeth or unneeded teeth for the purpose of orthodontic treatment. DPSCs exhibit a self-renewal
property, a high proliferative capacity, and a multilineage differentiation potential to give rise to a variety of cell types, such as oste-
oblasts, chondrocytes, adipocytes, myoblasts, endotheliocytes, and melanocytes, as well as neurons and glia, being of neural crest
origin (Ulmer et al., 2010; Kawashima, 2012). In vivo, DPSCs can differentiate into odontoblasts and induce host cells to participate
in regeneration by generating dentin–pulp-like complex after subcutaneous transplantation in conjunction with hydroxyapatite/tri-
calcium phosphate (HA/TCP) into immunocompromised mice (Gronthos et al., 2000, 2002; Batouli et al., 2003). A dentin–pulp-
like complex with well-established vascularization can be regenerated de novo in emptied root canal by DPSCs ectopically (Cordeiro
et al., 2008; Prescott et al., 2008; Huang, 2009a, 2010). DPSCs can also differentiate into osteoblasts and endothelial cells to
produce adult bonelike tissue with an integral blood supply (d’Aquino et al., 2007). Differentiation of DPSCs into mature oligo-
dendrocytes or functionally active neurons is demonstrated after transplantation into the embryonic mesencephalon (Arthur et al.,
2008). Preincubated DPSCs toward odontogenic, adipogenic, and myogenic lineages differentiate along distinct pathways after
transplantation into immunocompromised mice (Kerkis et al., 2006; Zhang et al., 2008). The multilineage differentiation potential
of DPSCs strongly suggests their possible applications in regenerative medicine.
The colony-derived populations of DPSC are heterogenous and contain more than one stem cell population. Therefore, various
strategies have been tried to isolate a more defined clonal subset of stem/progenitor cells using immunoselection of some cell
surface antigen markers such as STRO-1 (Shi and Gronthos, 2003; Laino et al., 2006; Yang et al., 2007), and CD105 (Nakashima
et al., 2009; Iohara et al., 2011) by flow cytometry or magnetic-activated cell sorting. Isolation of the ‘true’ or ‘mother’ adult stem
cells, which exhibited lower level of the DNA-binding fluorescent dye, Hoechst 33342, than that of the rest of the pulp cells (Iohara
et al., 2006), is an alternative method. So far, at least two different stem cell populations are identified. One is originating from the
neural crest (ectomesenchyme) and the other is of mesenchymal origin. However, the isolation results of different stem populations
are still inconclusive due to lack of specific cell surface markers. Thus, a novel isolation method is necessary to be developed before
subsequent characterization of distinct DPSC populations.
Stem Cells from Human Exfoliated Deciduous Teeth

SHEDs are isolated from dental pulp derived from exfoliated deciduous teeth. Compared to DPSCs, SHEDs exhibit higher growth
rates, and differentiate into a variety of cell types to a greater extent, including neural cells, chondrocytes, adipocytes, osteoblasts, and
556 Regenerative Engineering j Dental Stem Cells
myocytes (Miura et al., 2003; Kerkis et al., 2006; Wang et al., 2010, 2012a; Nourbakhsh et al., 2011). Higher expression of pluripotent
markers such as Oct4, Sox2, Nanog, and Rex1, lower neurosphere formation, and lower expression of neuronal markers after
neuronal induction are found in SHEDs compared with DPSCs (Govindasamy et al., 2010). The use of embryonic stem cells is asso-
ciated with ethical concerns, and autologous postnatal stem cells with multipotency have the limitations of readily available sources.
On the other hand, deciduous teeth are disposable and readily accessible and SHED is an attractive alternative. When SHEDs are
injected into tooth slice with scaffold and transplanted subcutaneously into immunocompromised mice, SHEDs are able to differ-
entiate into odontoblasts and endothelial cells to generate dentin–pulp-like tissue (Sakai et al., 2010). SHEDs also induce recipient
murine cells to differentiate into bone-forming cells to repair critical-size bone defects (Miura et al., 2003; Seo et al., 2008). SHEDs
are capable of differentiating into blood vessels that anastomosed with the host vasculature (Cordeiro et al., 2008), neuronal devel-
opmental potential is demonstrated by injecting SHEDs into the dentate gyrus of the hippocampus of immunocompromised mice
(Miura et al., 2003). Transplantation of SHED spheres into the rat striatum of Parkinson disease partially improves the apomorphine-
evoked rotation of behavioral disorders (Wang et al., 2010). These features and accessibility of tissue resource make SHEDs a poten-
tial source of stem cells to repair and regenerate vasculogenic and neurogenic diseases and damaged tooth structures.
Stem Cells from Apical Papilla

A unique population of dental stem cells known as stem cells from the root apical papilla is residing in the tips of growing tooth
roots of the permanent immature teeth (Sonoyama et al., 2008). The apical papilla tissue is only present during root development
before or during the tooth erupts into the oral cavity. In developing teeth, root formation starts as the epithelial cells from the
cervical loop proliferate apically, and influence the differentiation of odontoblasts from undifferentiated mesenchymal cells and
cementoblasts from follicle mesenchymal cells (Linde and Goldberg, 1993). There is an apical cell-rich zone lying between the
dental pulp and the apical papilla. The distinction between the dental pulp and the apical papilla is that the apical papilla
represents a precursor tissue for the radicular pulp. SCAPs proliferate faster with greater population doubling than DPSCs
(Sonoyama et al., 2006). SCAP have osteo/dentinogenic, neurogenic, and adipogenic differentiation potential, but not myogenic
and chondrogenic differentiation potential (Sonoyama et al., 2006; Abe et al., 2011). SCAPs, similar to DPSCs, are more committed
to osteo/dentinogenicity. SCAPs express osteo/dentinogenic markers and growth factor receptors similar to DPSCs, but express
lower level of dentin sialophosphoprotein, matrix extracellular phosphoglycoprotein, transforming growth factor (TGF) bRII, fibro-
blast growth factor receptor (FGFR) 3, and Flt-1 (VEGFR-1) than do DPSCs (Sonoyama et al., 2008). In clinical case of apexogenesis,
SCAPs give rise to odontoblasts to induce root formation since SCAP residing in the apical papilla survive in an infected immature
tooth with periapical periodontitis (Chueh and Huang, 2006). When SCAPs are transplanted into immunocompromised mice with
HA/TCP as a scaffold, they differentiate into odontoblasts to form the dentin structure (Sonoyama et al., 2006). Most human tissues
from early in their development are not clinically available for stem cell isolation. On the other hand, the root apical papilla is acces-
sible postnatally from extracted wisdom teeth. The embryonic-like properties (i.e., in the process of development) and the higher
proliferative potential of SCAP make this population suitable for cell-based regeneration and preferentially for forming root.
Dental Follicle Progenitor Cells

The dental follicle is a loose ectomesencyme-derived connective tissue sac surrounding the enamel organ and the dental papilla of
the developing tooth germ before eruption. The dental follicle plays a crucial role in tooth development and contains progenitors
for cementoblasts, PDL fibroblasts, and osteoblasts. DFPCs are isolated from the dental follicle of impacted third molars or during
their tooth eruption usually between 15 and 28 years of age (Morsczeck et al., 2005; Yao et al., 2008). DFPCs are fibroblastlike,
colony-forming, and plastic-adherent cells, and have the ability to form compact mineralized nodules in vitro. Putative stem cell
markers, Notch-1, nestin, STRO-1, and FGFRI-IIIC are expressed (Morsczeck et al., 2005). DFPCs have the ability to differentiate
toward cementoblasts, PDL fibroblasts, osteoblasts, adipocytes, and neurons, showing greater plasticity than other dental stem cells
(Morsczeck et al., 2005, 2008, 2009; Kémoun et al., 2007; Yao et al., 2008; Coura et al., 2008). DFPCs differentiate into PDL fibro-
blasts that secrete collagen and interact with fibers on the surfaces of adjacent bone and cementum to form PDL. DFPCs can form
PDL after transplantation into severe combined immune deficiency (SCID) mice (Yokoi et al., 2007), but hard tissues such as
dentin, cementum, or bone are not identified (Yagyuu et al., 2010). Dentin noncollagenous proteins extracted from dentin can stim-
ulate DFPCs to differentiate into cementoblast lineages. Col-I facilitates the differentiation of DFPCs along the mineralization
process. Enamel matrix derivatives or bone morphogenetic protein (BMP)-2/-7 activate DFPCs toward the cementoblastic pheno-
type (Kémoun et al., 2007). DFPCs are heterogenous populations containing highly undifferentiated state of PDL-type lineage and
cementoblastic or alveolar bone osteoblastic lineage, suggesting they play a role in tissue regeneration as much as the individual
lineages might do. DFPCs transplanted into the alveolar fossa in conjunction with a dentin matrix-treated scaffold contribute to
the formation of rootlike tissues with a pulp–dentin complex and a PDL connecting a cementum-like layer to host alveolar
bone (Guo et al., 2012b). Further research is necessary for potential uses of DFPCs.
Periodontal Ligament Stem Cells

The PDL is a fibrous connective tissue derived from the dental follicle and originates from neural crest cells. PDL connects the ce-
mentum to alveolar bone and maintains the root of the tooth in the alveolar socket by acting as a ‘shock absorber’ during
mastication. PDL contains progenitor cells that migrate and differentiate into either cementoblasts or osteoblasts in response to
lesions (McCulloch, 1985). A population of stem cells from PDL (PDLSCs) has been identified (Seo et al., 2004). PDLSCs can
differentiate into cementoblasts, adipocytes, odontoblasts, chondrocytes, myotubes, neurons, astrocytes, and oligodendrocytes
under defined culture conditions (Seo et al., 2004; Sonoyama et al., 2006; Gronthos et al., 2006). In vivo, they generate
cementum/PDL-like structures similar to native PDL as a thin layer of cementum after transplantation with HA/TCP into immu-
nocompromised mice (Seo et al., 2004). After transplantation into the sockets of the mandible in minipig models, autologous
PDLSCs with HA/TCP and gelform scaffolds form a bioroot encircled with PDL connecting to the surrounding bone (Sonoyama
et al., 2006). After transplantation into apical involvement defects of a canine advanced periodontitis model, PDLSCs show better
regenerating capacity of PDL, alveolar bone, and cementum as well as peripheral nerves and blood vessels in the regenerated PDL
space compared with DPSCs (Park et al., 2011). These findings suggest that PDLSCs have a potential function of repair and regen-
eration of PDL.
Isolation of Dental Stem Cells
Dental stem cells can be isolated by using the explant outgrowth technique or the enzymatic digestion technique. The former tech-
nique is that the tissues cut into 2-mm3 pieces are directly incubated in culture dishes and stem cells migrating out from the tissues
are collected after 2–3 weeks (Stanislawski et al., 1997; Spath et al., 2010). The latter technique is that the cell suspensions after
digestion with collagenase or a combination of collagenase and dispase are seeded in culture dishes to be colonized (Gronthos
et al., 2000). These populations are highly proliferative but heterogenous in cellular differentiation and multipotentiality. There-
fore, a more defined clonal subset of stem/progenitor cells are isolated using immunoselection of some cell surface antigen
markers by flow cytometry or magnetic-activated cell sorting. Those cell surface markers include STRO-1 (the perivascular cell
marker), CD34 (the hematopoietic/endothelial marker), c-kit/CD117 (the putative stem/progenitor cell protooncogene marker),
b1 integrin (preferentially expressed in primitive cells), low-affinity nerve growth factor receptor (LANGFR/p75/CD271) (the
embryonic neural crest cell marker), CD105/endoglin, and stage-specific embryonic antigen (SSEA)-4. Furthermore, the ‘true’
or ‘mother’ adult stem cells, side population cells, are isolated from dental pulp and PDL by flowcytometry using the DNA-
binding fluorescent dye Hoechst 33342 exclusion (Nakashima et al., 2009; Hynes et al., 2012; Kawanabe et al., 2012). Those
fractionated subpopulations show higher proliferation and migration activities, a longer proliferative life span, and greater multi-
differentiation potential including angiogenesis and neurogenesis compared with unfractionated colony-derived populations
(Nakashima et al., 2009; Iohara et al., 2011). There are two different origins in dental stem cells: mesenchymal stem/progenitor
cells and more embryonic neural crest stem cells (Coura et al., 2008; Degistirici et al., 2008; Waddington et al., 2009; d’Aquino
et al., 2011; Dangaria et al., 2011; Janebodin et al., 2011; Abe et al., 2012; Achilleos and Trainor, 2012; Kaku et al., 2012).
SSEA-4þ cells isolated from dental follicle have all the characteristics of neural crest cells and share some features with embryonic
stem (ES) cells: high level of embryonic stem markers (TRA-1-60, TRA-1-81, OCT-4, SSEA-4), mRNA expression of Nanog and Rex-
1, pluripotency in vitro and in vivo, integration into the inner cell mass of blastocytes, high level of telomerase activity, and ability to
form embryoid bodies (d’Aquino et al., 2011). Fractionated stem cells might be more suitable for some tissue regeneration since
some regenerated tissues are much higher in volume compared with unfractionated colony-derived populations (Iohara et al.,
2011). A novel simple method should be developed prior to clinical trials using cost-effective and potential disposable apparatus,
by which dental stem cells can be isolated from a small amount of tissue with efficiency and in safety. Potential utility of differences
in migratory activity among the different cell populations in place of cell surface antigen marker expressions is recently demon-
strated (Murakami et al., 2013).
Characterization of Dental Stem Cells

Surface Markers (Table 1)
Dental stem cells are composed of more than 95% of CD29þ, CD44þ, CD73þ, CD90þ, and CD105þ cells. Less than 2% of dental
stem cells express the panleukocyte marker CD45, the hemotopoietic/endothelial cell marker CD34, the monocyte and macrophage
markers CD11 and CD14, the B-cell marker CD79 and CD19, or HLA class II. Different types of dental stem cells share common
stem cell antigen marker expressions (Huang et al., 2009b; Kerkis and Caplan, 2012). Nevertheless, the data currently available on
other markers such as CD34, CD117, and CD146, are inconsistent, and the clear definition of an exclusive population of dental
stem cells is difficult. This is based on the following facts: (1) Each type of dental stem cells contains several stem cell subpopula-
tions. (2) The cells are related to the plastic adherent and cultured population, which changes the phenotype easily during cell
culture. (3) The heterogeneity also depends on isolation and culture methods, which are varied in different laboratories. There
are many variables that affect the final outcome in the composition of subpopulations: initial plating density, coating of culture
dishes, composition of cell culture media (glucose content, calcium concentration, supplementation with antioxidants), supple-
ment (bovine serum, human serum, platelet lysate, or growth factors), oxygen supply (hypoxia), and method of subculturing
and cryopreservation. Therefore, standardization of the isolation and culture procedure is needed for good reproducibility of results
from different laboratories and studies.
Table 1 Surface markers of dental stem cells
DPSC ( Lindroos et al., SHED ( Govindasamy

2008; Nakashima et al., et al., 2010; Rodrı´guez- SCAP ( Huang et al., DFPC ( Guo et al., PDLSC ( Huang et al.,
2009; Rodrı´guez-Lozano Lozano et al., 2011; 2009b; Rodrı´guez- 2012a; Rodrı´guez- 2009b; Rodrı´guez-Lozano
et al., 2011) Suchánek et al., 2010) Lozano et al., 2011) Lozano et al., 2011) et al., 2011)
CD(þ) CD10 B B
CD13 B B B B B
CD24 B
CD29 B B B B B
CD44 B B B B B
CD59 B B B
CD71 B
CD73 B B B B B
CD90 B B B B B
CD105 B B B B B
CD106 B B
CD117 B
CD146a B B B
CD166 B
CD271 B B
STRO- B (5–10%) B (9%) B (>18%) B B
1
CD() CD3 B
CD14 B B B B
CD18 B
CD31 B B
CD34 B B B B B
CD45 B B B B B
CD150 B
a
CD146 is reported in Nakashima, M., Iohara, K., Sugiyama, M., 2009. Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp
regeneration. Cytokine Growth Factor Rev. 20, 435–440.
Regenerative Potential (Table 2)

Dental stem cells offer potential for regeneration of damaged tooth tissues such as dentin, pulp, and PDL. Bone, nerve, and blood
vessel are also repaired or regenerated by dental stem cells.
Dentin/Pulp
DPSCs, SHEDs, and SCAPs are derived from pulp tissue or the precursor of pulp, and have dentin/pulp repair/regeneration poten-
tial (Huang et al., 2009b; Volponi et al., 2010; Estrela et al., 2011). When DPSCs are transplanted alone or together with BMP2 on
the exposed pulp in the cavity, the dentin–pulp complex is induced (Nakashima and Akamine, 2005; Iohara et al., 2006, 2009).
DPSCs and SCAPs form the dentin–pulp complex after subcutaneous transplantation into immunocompromised mice, whereas
SHEDs form mineralized tissue without the distinct pulp–dentin complex (Huang, et al., 2009b). In ectopic tooth transplantation
models, dentin–pulp complex with well-established vascularity can be regenerated de novo in emptied root canal space of tooth slice
or tooth root by either DPSCs or SHEDs (Huang, 2011). In orthotropic pulpectomized tooth model as a regenerative endodontic
therapy, pulp tissue with good vasculature and innervation is completely regenerated after transplantation of DPSCs with cell-
derived factor-1 (SDF-1) (Iohara et al., 2011). Dentin formation is also induced in the coronal and the apical parts of the regener-
ated pulp tissue to prevent microleakage as well as along the dentinal wall. Transplantation of DPSCs with SDF-1 yields
a significantly larger amount of the regenerated pulp tissue compared with transplantations of bone marrow or adipose-derived
stem cells (Ishizaka et al., 2012).
Periodontal Ligament (Hynes et al., 2012)

Transplantation of PDLSCs into immunocompromised mice validates the ability of PDLSCs to form functional cementoblast-like
cells and cementum/PDL-like tissue, including Sharpey’s fibers. The regenerative capacity of PDLSCs is presented in a range of dental
defects in various animal models with scaffold including HA/TCP and collagen, demonstrating the regeneration of normal peri-
odontal tissues, containing organized cementum, alveolar bone, and the PDL attachment apparatus. Multilayered cell sheets of
PDLSCs supported by scaffold including polyglycolic acid (PLGA), hyaluronic acid, HA/TCP, and collagen enhance periodontal
regeneration with the formation of new cementum and well-oriented PDL fibers. The autologous DFPCs generate new cementum,
alveolar bone, and Sharpey’s fibers of PDL into the apical involvement defect. However, PDLSCs have the best regenerative capacity
compared with DFPCs and DPSCs, showing good innervation and vascularization in addition to regeneration of PDL, alveolar
Table 2 Biological properties of dental stem cells
DPSC ( Bakopoulou et al., 2011;

Demarco et al., 2011; Gronthos SHED ( Demarco et al., 2011;
et al., 2000; Iohara et al., 2011, Morsczeck et al., 2010; Nishino
2009, 2008; Nakashima et al., et al., 2011; Nourbakhsh et al., SCAP ( Bakopoulou et al., 2011; DFPC ( Demarco et al., 2011; PDLSC ( Feng et al., 2010;
2009; Sugiyama et al., 2011; 2011; Wang et al., 2012a; Seo Demarco et al., 2011; Ding et al., Morsczeck et al., 2010; Tomic Kémoun et al., 2011; Liu et al.,
Properties Waddington et al., 2009) et al., 2008) 2010; Sonoyama et al., 2008) et al., 2011) 2008)
Location Permanent tooth pulp Exfoliated deciduous tooth Apical papilla of developing root Dental follicle of developing Periodontal ligament
tooth
Proliferation rate þ þþ þþ þþ þþ
Heterogeneity þ þ þ þ þ
Multipotency Odontoblast, osteoblast, Odontoblast, osteoblast, Odontoblast, osteoblast, Odontoblast, osteoblast, Odontoblast, osteoblast,
chondrocyte, myocyte, chondrocyte, myocyte, neurocyte, adipocyte, iPS neurocyte, adipocyte, chodrocyte, neurocyte,
neurocyte, adipocyte, neurocyte, adipocyte, iPS cementoblast
choroneal epithelial cell,
melanoma cell, iPS
Migration þ þ þ þ þ
Regenerative potential Bone regeneration, neural Bone regeneration, neural Bone regeneration, neural Bone regeneration, PDL Bone regeneration, PDL
regeneration, myogenic regeneration, tubular dentin, regeneration, dentin–pulp regeneration regeneration, root formation
regeneration, dentin–pulp wound healing regeneration, root formation
regeneration
Regenerative Engineering j Dental Stem Cells

Trophic effect
Antiapoptosis (Iohara et al., 2008; B B
Jewett et al., 2010; Sugiyama
et al., 2011; Wanachottrakul et al.,
2011; Zhao et al., 2012)
Immunosuppressive (Ding et al., B B B B
2010; Ishizaka et al., 2013; Jewett
et al., 2010; Morsczeck et al.,
2010; Tomic et al., 2011;
Wanachottrakul et al., 2011; Zhao
et al., 2012)
Proliferation B B B B B
Migration B B B B
559
bone, and cementum. DPSCs transplanted into various periodontal defects result in inconsistencies among different research
groups. A root/periodontal complex with significantly better compression strength is formed when a root-shaped HA/TCP block
containing SCAP coated with PDLSC-seeded gel foam is implanted in tooth socket.
Bone (Ulmer et al., 2010; Hynes et al., 2012; Kawashima, 2012; Kerkis and Caplan, 2012)
DPSCs transplanted with HA/TCP, PLGA, collagen, nanofiber hydrogel, or HA nanohydroxyapatite/collagen/poly (L-lactide) exhibit
bonelike structure rather than dentin. Pretreatment of DPSCs with BMP2 promotes osteogenesis. SHEDs with a proper scaffold such
as platelet-rich plasma have also the ability to form mature bone. PDLSCs show better osteogenic properties than SHEDs when
treated with retinoic acid in combination with insulin. Critical size bone defect is repaired by DPSCs and SHEDs.
Neuron
Dental stem cells differentiate into neural lineage in vitro. SHEDs injected into the hippocampus of immunocompromised mice
after 1 week cultivation in the neuronal differentiation medium express neural markers, indicating neuronal differentiation
in vivo (Miura et al., 2003). DPSCs transplanted into the mesencephalon of day-2 chicken embryo acquire a neuronal morphology
and function, suggesting DPSCs exposed to the appropriate environmental cues differentiate into active neuron (Arthur et al.,
2008). DPSCs coordinate axon guidance via CXCR-4 and stromal SDF-1/CXCL12 axis to induce neuroplasticity within a receptive
host nervous system (Arthur et al., 2009). Transplantation of DPSCs into the hippocampus promotes proliferation, cell recruitment,
and maturation of endogenous neural stem/progenitor cells by secreting neurotrophic factors, suggesting their therapeutic potential
as a stimulator and modulator of the local repair response in the central nervous system (Huang et al., 2008). Significant recovery
from neurological dysfunction in cerebral ischemia and spinal cord injury models are reported after transplantation of DPSCs or
SHEDs, suggesting their neural regenerative potential in the central nervous system (Yang et al., 2009; de Almeida et al., 2011;
Sugiyama et al., 2011, Sakai et al., 2012). In a peripheral nerve injury model, artificial nerve conduits containing DPSCs promote
nerve regeneration with myelinated fibers (Sasaki et al., 2008, 2011).
Blood Vessel
DPSCs and SHEDs accelerate blood flow and vasculogenesis/angiogenesis in an ischemic hind-limb model and a peripheral nerve
injury model (Iohara et al., 2008; Nakashima et al., 2009; Sasaki et al., 2008). DPSCs differentiate into osteoblasts and endothe-
liocytes synergically after transplantation into immunocompromised rats and lead to generation of adult bone structure with an
integral blood supply, suggesting that angiogenesis may be regulated by distinct mechanisms (d’Aquino, 2007). When SHEDs
are seeded in biodegradable scaffolds within tooth slices and are transplanted into immunodeficient mice, they also differentiate
Table 3 Soluble factors secreted by dental stem cells
Soluble trophic factors and cytokines DPSC SHED SCAP DFPC PDLSC
BDNF (Iohara et al., 2011; Ishizaka et al., 2012, 2013; Brain-derived neurotrophic factor þ þ þ þ þ
Sakai et al., 2012; Sugiyama et al., 2011)
EGF (Kim et al., 2012) Epidermal growth factor þ þ þ þ
FGF2 (Kim et al., 2010, 2012; Nakamura et al., 2009) Fibroblast growth factor 2 þ þ þ
GDNF (Gale et al., 2011; Howard et al., 2010; Ishizaka Glial cell-line derived neurotrophic factor þ þ
et al., 2012, 2013; Sakai et al., 2012; Sugiyama et al.,
2011)
GM-CSF (Iohara et al., 2008, 2009, 2011; Ishizaka et al., Granulocyte macrophage colony-stimulating þ
2012, 2013) factor
HGF (Iohara et al., 2008; Su et al., 2012) Hepatocyte growth factor þ þ
IGF1 (Kim et al., 2012; Ochiai et al., 2012) Insulin-like growth factor 1 þ þ þ þ þ
MMP3 (Iohara et al., 2008, 2009, 2011; Ishizaka et al., Matrix metalloproteinase-3 þ þ
2012, 2013)
NPY (Iohara et al., 2008; Iohara et al., 2011; Ishizaka et al., Neuropeptide Y þ
2012, 2013)
NGF (Iohara et al., 2011; Ishizaka et al., 2012, 2013; Kim Nerve growth factor þ þ þ þ þ
et al., 2010, 2012; Nakamura et al., 2009; Sakai et al.,
2012; Sugiyama et al., 2011)
PDGF (Iohara et al., 2008; Kim et al., 2010, 2012) Platelet-derived growth factor beta þ þ
TGF-beta (Derringer and Linden, 2007; Gale et al., 2011; Transforming growth factor beta þ þ þ þ þ
Howard et al., 2010; Kim et al., 2012; Nakamura et al.,
2009; Ochiai et al., 2012)
VEGF (Derringer and Linden, 2007; Dissanayaka et al., Vascular endothelial growth factor þ þ þ þ þ
2012; Iohara et al., 2008, 2009, 2011; Ishizaka et al.,
2012, 2013; Kim et al., 2010, 2012; Zhou et al., 2004)
into endothelial cells in addition to odontoblast-like cells in the regenerated pulp tissue within the slices (Cordeiro et al., 2008;
Sakai et al., 2010).
Trophic Factors (Table 3)

Among the various types of cell-to-cell signaling, paracrine signaling transmits over short distances between different cell types.
Dental stem cells secrete a broad panel of growth factors and cytokines and provide instructive cues via paracrine signaling. The
factors secreted by dental stem cells suppress the local immune system, inhibit apoptosis, enhance mobilization of endogenous
stem/progenitor cells, and promote regeneration including angiogenesis and neurogenesis. These immunomodulatory, antiapop-
totic, migratory, angiogenic, and neuroprotective/neuroregenerative effects, which are referred to as trophic effects (Nakashima
et al., 2009; Nakashima and Iohara, 2011, 2014; Nakashima and Huang, 2013), are distinct from the direct differentiation of dental
stem cells into repair/regenerated tissue. Studies in vivo in animal models of pulpectomy, ischemic hind limb, ischemic cerebrum,
and spinal cord injury indicate these trophic effects of dental stem cells (Iohara et al., 2011; Sugiyama, 2011; Ishizaka et al., 2012,
2013; Inoue et al., 2012). This understanding may lead to the rational design of new therapies utilizing dental stem cells for
damaged tissue and degenerative disorders.
Immunomodulatory Roles (Table 3) (Kerkis and Caplan, 2012; Wang et al., 2012b)
The two outstanding hallmarks of dental stem cells (DSCs) and MSCs are the homing to sites of injury and ischemia and immu-
nomodulation. The immunomodulatory role of DSCs and MSCs is due to (1) activated T-cell apoptosis via the FAS ligand/FAS-
mediated death pathway, (2) upregulation of T regulatory cells (Tregs), which results in immune tolerance, and (3) suppression
of natural killer cells, as these are effector cells of innate immunity and play a key role in cytotoxic potential and secretion of prein-
flammatory cytokines such as tumor necrosis factor a. While the suppressive effects of DSCs and MSCs on T lymphocytes are known,
their effect on B-lymphocytes is not clear. In some studies, MSCs inhibit B-cell proliferation, whereas in other investigations they
have a stimulatory role on proliferation of B lymphocytes. Allogeneic MSCs have been used in immunotherapy in systemic lupus
erythematosus, rheumatoid arthritis, and multiple sclerosis. It is noteworthy that allogeneic MSCs have been used to treat GVHD.
Although the immunosuppressive and immunomodulatory roles of DSCs and MSCs are known, it is important to point out that
major histocompatibility complex (MHC) class I is low and MHC class II antigens are absent in MSCs. The candidate immunomod-
ulators secreted by DSCs and MSCs include TGF-b, hepatic growth factors, interleukins 6 and 10. In conclusion, the immunomod-
ulatory roles of DSCs have a profound effect on the clinical effectiveness by inhibition of T-lymphocyte function, and upregulation
of Tregs stimulating immune tolerance.
Clinical Application
Clinical examination has demonstrated the potential efficacy and safety of transplanting autologous PDL progenitor cells with HA/
TCP in the treatment of human periodontitis with deep intrabony defect. All three patients show no inflammation in the treatment
area and no systemic disorder associated with transplantation (Feng et al., 2010). Another clinical trial has demonstrated that
implantation of a biocomplex composed of DPSCs and a collagen sponge scaffold resulted in optimal bone repair and complete
bone regeneration in oromaxillofacial bone defect (d’Aquino et al., 2009).
Conclusions
Apart from all the basic scientific evidence on dental stem cells accumulated during the last decade, we are at the heralding of a new
era of stem cell therapy. Dental stem cells are one of the most potential adult stem cells. Preclinical studies have started autologous
or allogenic cell therapies harnessing dental stem cells. Further studies using in vivo models are needed to augment the under-
standing of the regulation of the migration, growth, and differentiation of dental stem cells or trophic effects of dental stem cells
by interactions with resident cells and immune cells, growth factors, and cytokines during regeneration/repair. The lack of standard-
ized isolation methods and culture protocols needs to be also overcome to guarantee high cell quality. A safe and efficacious
method to isolate good manufacturing practice-grade dental stem cells should be developed prior to clinical trials. The
cost consuming manufacturing needs to be evaluated and improved before regenerative dentistry/medicine can replace the conven-
tional treatment. One possibility might be off-the-shelf allogeneic stem/progenitor cells and banking for every day usage. Dental
stem cells are on the direct path to their clinical usage for the regenerative treatment of a multitude of diseases and injuries.
References
Abe, S., Hamada, K., Yamaguchi, S., Amagasa, T., & Miura, M. (2011). Characterization of the radioresponse of human apical papilla-derived cells. Stem Cell Res. Ther., 20, 2.
Abe, S., Hamada, K., Miura, M., & Yamaguchi, S. (2012). Neural crest stem cell property of apical pulp cells derived from human developing tooth. Cell Biol. Int., 36, 927–936.
Achilleos, A., & Trainor, P. A. (2012). Neural crest stem cells: discovery, properties and potential for therapy. Cell Res., 22, 288–304.
Arthur, A., Rychkov, G., Shi, S., Koblar, S. A., & Gronthos, S. (2008). Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate
environmental cues. Stem Cells, 26, 1787–1795.
Arthur, A., Shi, S., Zannettino, A. C., Fujii, N., Gronthos, S., et al. (2009). Implanted adult human dental pulp stem cells induce endogenous axon guidance. Stem Cells, 27,
2229–2237.
d’Aquino, R., Graziano, A., Sampaolesi, M., Laino, G., Pirozzi, G., et al. (2007). Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal
synergy leading to adult bone tissue formation. Cell Death and Differ., 14, 1162–1171.
d’Aquino, R., De Rosa, A., Lanza, V., Tirino, V., Laino, L., et al. (2009). Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge
biocomplexes. Eur. Cell. Mater., 18, 75–83.
d’Aquino, R., Tirino, V., Desiderio, V., Studer, M., De Angelis, G. C., et al. (2011). Human neural crest-derived postnatal cells exhibit remarkable embryonic attributes either in vitro
or in vivo. Eur. Cell. Mater, 21, 304–316.
de Almeida, F. M., Marques, S. A., Ramalho Bdos, S., Rodrigues, R. F., Cadilhe, D. V., et al. (2011). Human dental pulp cells: a new source of cell therapy in a mouse model of
compressive spinal cord injury. J. Neurotrauma, 28, 1939–1949.
Bakopoulou, A., Leyhausen, G., Volk, J., Tsiftsoglou, A., Garefis, P., et al. (2011). Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp
stem cells (DPSCs) and stem cells from the apical papilla (SCAP). Arch. Oral Biol., 56, 709–721.
Batouli, S., Miura, M., Brahim, J., Tsutsui, T. W., Fisher, L. W., et al. (2003). Comparison of stem-cell-mediated osteogenesis and dentinogenesis. J. Dent. Res., 82,
976–981.
Chen, S., Wang, S., & Xie, T. (2011). Restricting self-renewal signals within the stem cell niche: multiple levels of control. Curr. Opin. Genet. Dev., 21, 684–689.
Chueh, L. H., & Huang, G. T. (2006). Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. J. Endod., 32, 1205–1213.
Cordeiro, M. M., Dong, Z., Kaneko, T., Zhang, Z., Miyazawa, M., et al. (2008). Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J. Endod., 34,
962–969.
Coura, G. S., Garcez, R. C., de Aguiar, C. B., Alvarez-Silva, M., Magini, R. S., et al. (2008). Human periodontal ligament: a niche of neural crest stem cells. J. Periodontal Res., 43,
531–536.
Dangaria, S. J., Ito, Y., Luan, X., & Diekwisch, T. G. (2011). Differentiation of neural-crest-derived intermediate pluripotent progenitors into committed periodontal populations
involves unique molecular signature changes, cohort shifts, and epigenetic modifications. Stem Cell. Dev., 20, 39–52.
Degistirici, O., Jaquiery, C., Schönebeck, B., Siemonsmeier, J., Götz, W., et al. (2008). Defining properties of neural crest-derived progenitor cells from the apex of human
developing tooth. Tissue Eng. Part A, 14, 317–330.
Demarco, F. F., Conde, M. C., Cavalcanti, B. N., Casagrande, L., Sakai, V. T., et al. (2011). Dental pulp tissue engineering. Braz. Dent. J., 22, 3–13.
Derringer, K., & Linden, R. (2007). Epidermal growth factor released in human dental pulp following orthodontic force. Eur. J. Orthod., 29, 67–71.
Ding, G., Liu, Y., An, Y., Zhang, C., Shi, S., et al. (2010). Suppression of T cell proliferation by root apical papilla stem cells in vitro. Cells Tissues Organs, 191, 357–364.
Dissanayaka, W. L., Zhan, X., Zhang, C., Hargreaves, K. M., Jin, L., et al. (2012). Coculture of dental pulp stem cells with endothelial cells enhances osteo-/odontogenic and
angiogenic potential in vitro. J. Endod., 38, 454–463.
Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., et al. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The international society
for cellular therapy position statement. Cytotherapy, 8, 315–317.
Estrela, C., Alencar, A. H., Kitten, G. T., Vencio, E. F., & Gava, E. (2011). Mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration. Braz. Dent. J., 22,
91–98.
Feng, F., Akiyama, K., Liu, Y., Yamaza, T., Wang, T. M., et al. (2010). Utility of PDL progenitors for in vivo tissue regeneration: a report of 3 cases. Oral Dis., 16, 20–28.
Gale, Z., Cooper, P. R., & Scheven, B. A. (2011). Effects of glial cell line-derived neurotrophic factor on dental pulp cells. J. Dent. Res., 90, 1240–1245.
Govindasamy, V., Abdullah, A. N., Ronald, V. S., Musa, S., Ab Aziz, Z. A., et al. (2010). Inherent differential propensity of dental pulp stem cells derived from human deciduous and
permanent teeth. J. Endod., 36, 1504–1515.
Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., & Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A., 97,
13625–13630.
Gronthos, S., Brahim, J., Li, W., Fisher, L. W., Cherman, N., et al. (2002). Stem cell properties of human dental pulp stem cells. J. Dent. Res., 81, 531–535.
Gronthos, S., Mrozik, K., Shi, S., & Bartold, P. M. (2006). Ovine periodontal ligament stem cells: isolation, characterization, and differentiation potential. Calcif. Tissue Int., 79,
310–317.
Guo, W., Chen, L., Gong, K., Ding, B., Duan, Y., et al. (2012a). Heterogeneous dental follicle cells and the regeneration of complex periodontal tissues. Tissue Eng. Part A, 18,
459–475.
Guo, W., Gong, K., Shi, H., Zhu, G., He, Y., et al. (2012b). Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root. Biomaterials, 33, 1291–1302.
Howard, C., Murray, P. E., & Namerow, K. N. (2010). Dental pulp stem cell migration. J. Endod., 36, 1963–1966.
Huang, A. H., Snyder, B. R., Cheng, P. H., & Chan, A. W. (2008). Putative dental pulp-derived stem/stromal cells promote proliferation and differentiation of endogenous neural cells
in the hippocampus of mice. Stem Cells, 26, 2654–2663.
Huang, G. T. (2009a). Pulp and dentin tissue engineering and regeneration: current progress. Reg. Med., 4, 697–707.
Huang, G. T., Gronthos, S., & Shi, S. (2009b). Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.
J. Dent. Res., 88, 792–806.
Huang, G. T., Yamaza, T., Shea, L. D., Djouad, F., Kuhn, N. Z., et al. (2010). Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous
layer of dentin in an in vivo model. Tissue Eng. Part A, 16, 605–615.
Huang, G. T. (2011). Dental pulp and dentin tissue engineering and regeneration: advancement and challenge. Front Biosci. (Elite Ed.), 3, 788–800.
Hynes, K., Menicanin, D., Gronthos, S., & Bartold, P. M. (2012). Clinical utility of stem cells for periodontal regeneration. Periodontology 2000, 59, 203–227.
Inoue, T., Sugiyama, M., Hattori, H., Wakita, H., Wakabayashi, T., et al. (2012). Stem cells from human exfoliated deciduous tooth-derived conditioned medium enhance recovery of
focal cerebral ischemia in rats. Tissue Eng. Part A, 19, 24–29.
Iohara, K., Zheng, L., Ito, M., Tomokiyo, A., Matsushitam, K., et al. (2006). Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for
dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells, 24, 2493–2503.
Iohara, K., Zheng, L., Wake, H., Ito, M., Nabekura, J., et al. (2008). A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp.
Stem Cells, 26, 2408–2418.
Iohara, K., Zheng, L., Ito, M., Ishizaka, R., Nakamura, H., et al. (2009). Regeneration of dental pulp after pulpotomy by transplantation of CD31(-)/CD146(-) side population cells from
a canine tooth. Reg. Med., 4, 377–385.
Iohara, K., Imabayashi, K., Ishizaka, R., Watanabe, A., Nabekura, J., et al. (2011). Complete pulp regeneration after pulpectomy by transplantation of CD105þ stem cells with
stromal cell-derived factor-1. Tissue Eng. Part A, 17, 1911–1920.
Ishizaka, R., Iohara, K., Murakami, M., Fukuta, O., & Nakashima, M. (2012). Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone
marrow and adipose tissue. Biomaterials, 33, 2109–2118.
Ishizaka, R., Hayashi, Y., Iohara, K., Sugiyama, M., Murakami, M., et al. (2013). Stimulation of angiogenesis, neurogenesis and regeneration by side population cells from dental
pulp. Biomaterials, 34(8), 1888–1897.
Janebodin, K., Horst, O. V., Ieronimakis, N., Balasundaram, G., Reesukumal, K., et al. (2011). Isolation and characterization of neural crest-derived stem cells from dental pulp of
neonatal mice. PLoS One, 6, e27526.
Jewett, A., Arasteh, A., Tseng, H. C., Behel, A., Arasteh, H., et al. (2010). Strategies to rescue mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) from NK cell
mediated cytotoxicity. PLoS One, 5, e9874.
Kaku, M., Komatsu, Y., Mochida, Y., Yamauchi, M., Mishina, Y., et al. (2012). Identification and characterization of neural crest-derived cells in adult periodontal ligament of mice.
Arch. Oral Biol., 57, 1668–1675.
Kawanabe, N., Murata, S., Fukushima, H., Ishihara, Y., Yanagita, T., et al. (2012). Stage-specific embryonic antigen-4 identifies human dental pulp stem cells. Exp. Cell Res., 318,
453–463.
Kawashima, N. (2012). Characterisation of dental pulp stem cells: a new horizon for tissue regeneration? Arch. Oral Biol., 57, 1439–1458.
Kémoun, P., Laurencin-Dalicieux, S., Rue, J., Farges, J. C., Gennero, I., et al. (2007). Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and
enamel matrix derivatives (EMD) in vitro. Cell Tissue Res., 329, 283–294.
Kémoun, P., Gronthos, S., Snead, M. L., Rue, J., Courtois, B., et al. (2011). The role of cell surface markers and enamel matrix derivatives on human periodontal ligament
mesenchymal progenitor responses in vitro. Biomaterials, 32, 7375–7388.
Kerkis, I., & Caplan, A. I. (2012). Stem cells in dental pulp of deciduous teeth. Tissue Eng. Part B Rev., 18, 129–138.
Kerkis, I., Kerkis, A., Dozortsev, D., Stukart-Parsons, G. C., Gomes Massironi, S. M., et al. (2006). Isolation and characterization of a population of immature dental pulp stem cells
expressing OCT-4 and other embryonic stem cell markers. Cells Tissues Organs, 184, 105–116.
Kim, J. Y., Xin, X., Moioli, E. K., Chung, J., Lee, C. H., et al. (2010). Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng. Part A, 16, 3023–3031.
Kim, S. G., Zhou, J., Solomon, C., Zheng, Y., Suzuki, T., et al. (2012). Effects of growth factors on dental stem/progenitor cells. Dent. Clin. North Am., 56, 563–575.
Laino, G., Graziano, A., d’Aquino, R., Pirozzi, G., Lanza, V., et al. (2006). An approachable human adult stem cell source for hard-tissue engineering. J. Cell. Physiol., 206,
693–701.
Linde, A., & Goldberg, M. (1993). Dentinogenesis. Cri. Rev. Oral Biol. Med., 4, 679–728.
Lindroos, B., Mäenpää, K., Ylikomi, T., Oja, H., Suuronen, R., et al. (2008). Characterisation of human dental stem cells and buccal mucosa fibroblasts. Biochem. Biophys. Res.
Com., 368, 329–335.
Liu, Y., Zheng, Y., Ding, G., Fang, D., Zhang, C., et al. (2008). Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells, 26, 1065–1073.
Løvschall, H., Tummers, M., Thesleff, I., Füchtbauer, E. M., & Poulsen, K. (2005). Activation of the notch signaling pathway in response to pulp capping of rat molars. Eur. J. Oral
Sci., 113, 312–317.
McCulloch, C. A. (1985). Progenitor cell populations in the periodontal ligament of mice. Anat. Rec., 211, 258–262.
Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, L. W., et al. (2003). SHED: stem cells from human exfoliated deciduous teeth. Proc. Natl. Acad. Sci. U.S.A., 13, 5807–5812.
Morsczeck, C., Götz, W., Schierholz, J., Zeilhofer, F., Kühn, U., et al. (2005). Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol., 24, 155–165.
Morsczeck, C., Ernst, W., Florian, C., Reichert, T. E., Proff, P., et al. (2008). Gene expression of nestin, collagen type I and type III in human dental follicle cells after cultivation in
serum-free medium. Oral Maxillofac. Surg., 12, 89–92.
Morsczeck, C., Petersen, J., Völlner, F., Driemel, O., Reichert, T., et al. (2009). Proteomic analysis of osteogenic differentiation of dental follicle precursor cells. Electrophoresis, 30,
1175–1184.
Morsczeck, C., Völlner, F., Saugspier, M., Brandl, C., Reichert, T. E., Driemel, O., & Schmalz, G. (2010). Comparison of human dental follicle cells (DFCs) and stem cells from human
exfoliated deciduous teeth (SHED) after neural differentiation in vitro. Clin. Oral Investig., 14, 433–440.
Murakami, M., Horibe, H., Iohara, K., Hayashi, Y., Osako, Y., et al. (2013). The use of granulocyte-colony stimulating factor induced mobilization for isolation of dental pulp stem cells
with high regenerative potential. Biomaterials, 34, 9036–9047.
Nakamura, S., Yamada, Y., Katagiri, W., Sugito, T., Ito, K., et al. (2009). Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp
stem cells by gene expression profile from promising dental pulp. J. Endod., 35, 1536–1542.
Nakashima, M., & Akamine, A. (2005). The application of tissue engineering to regeneration of pulp and dentin in endodontics. J. Endod., 31, 711–718.
Nakashima, M., & Huang, G. T. J. (2013). Pulp and dentin regeneration. In I. Thesleff, & G. T. Huang (Eds.), Stem Cells and Craniofacial Tissue Bioengineering. Stem Cells in
Craniofacial Development, Regeneration and Repair (pp. 461–484). Hoboken: Wiley-Blackwell.
Nakashima, M., & Iohara, K. (2011). Regeneration of dental pulp by stem cells. Adv. Dent. Res., 23, 313–319.
Nakashima, M., & Iohara, K. (2014). Mobilized dental pulp stem cells (MDPSCs) for pulp regeneration: Initiation of clinical trial. J. Endod., 40, S26–S32.
Nakashima, M., Iohara, K., & Sugiyama, M. (2009). Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration. Cytokine
Growth Factor Rev., 20, 435–440.
Nishino, Y., Yamada, Y., Ebisawa, K., Nakamura, S., Okabe, K., et al. (2011). Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of
novel cell therapy. Cytotherapy, 13, 598–605.
Nourbakhsh, N., Soleimani, M., Taghipour, Z., Karbalaie, K., Mousavi, S. B., et al. (2011). Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-
derived stem cells. Int. J. Dev. Biol., 55, 189–195.
Ochiai, H., Okada, S., Saito, A., Hoshi, K., Yamashita, H., et al. (2012). Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-b1 (TGF-
b1) administration suppresses osteoblast differentiation. J. Biol. Chem., 287, 22654–22661.
Park, J. Y., Jeon, S. H., & Choung, P. H. (2011). Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis. Cell Transplant., 20, 271–285.
Phinney, D. G., & Prockop, D. J. (2007). Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair–current views.
Stem Cells, 25, 2896–2902.
Prescott, R. S., Alsanea, R., Fayad, M. I., Johnson, B. R., Wenckus, C. S., Hao, J., John, A. S., & George, A. (2008). In vivo generation of dental pulp-like tissue by using dental pulp
stem cells, a collagen scaffold, and dentin matrix protein 1 after subcutaneous transplantation in mice. J. Endod., 34, 421–426.
Rodríguez-Lozano, F. J., Bueno, C., Insausti, C. L., Meseguer, L., Ramírez, M. C., et al. (2011). Mesenchymal stem cells derived from dental tissues. Int. Endod. J., 44, 800–806.
Sakai, V. T., Zhang, Z., Dong, Z., Neiva, K. G., Machado, M. A., et al. (2010). SHED differentiate into functional odontoblasts and endothelium. J. Dent. Res., 89, 791–796.
Sakai, K., Yamamoto, A., Matsubara, K., Nakamura, S., Naruse, M., et al. (2012). Human dental pulp-derived stem cells promote locomotor recovery after complete transection of
the rat spinal cord by multiple neuro-regenerative mechanisms. J. Clin. Invest., 122, 80–90.
Sasaki, R., Aoki, S., Yamato, M., Uchiyama, H., Wada, K., et al. (2008). Tubulation with dental pulp cells promotes facial nerve regeneration in rats. Tissue Eng. Part A, 14,
1141–1147.
Sasaki, R., Aoki, S., Yamato, M., Uchiyama, H., Wada, K., et al. (2011). PLGA artificial nerve conduits with dental pulp cells promote facial nerve regeneration. J. Tissue Eng. Regen.
Med., 5, 823–830.
Scadden, D. T. (2006). The stem-cell niche as an entity of action. Nature, 441, 1075–1079.
Sensebé, L., Krampera, M., Schrezenmeier, H., Bourin, P., & Giordano, R. (2010). Mesenchymal stem cells for clinical application. Vox Sanguinis, 98, 93–107.
Seo, B. M., Miura, M., Gronthos, S., Bartold, P. M., Batouli, S., et al. (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 364,
149–155.
Seo, B. M., Sonoyama, W., Yamaza, T., Coppe, C., Kikuiri, T., et al. (2008). SHED repair critical-size calvarial defects in mice. Oral Dis., 14, 428–434.
Shi, S., & Gronthos, S. (2003). Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J. Bone Miner. Res., 18, 696–704.
Sloan, A. J., & Waddington, R. J. (2009). Dental pulp stem cells: what, where, how? Int. J. Paediatr. Dent., 19, 61–70.
Sonoyama, W., Liu, Y., Fang, D., Yamaza, T., Seo, B. M., et al. (2006). Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One, 1, e79.
Sonoyama, W., Liu, Y., Yamaza, T., Tuan, R. S., Wang, S., Shi, S., & Huang, G. T. (2008). Characterization of the apical papilla and its residing stem cells from human immature
permanent teeth: a pilot study. J. Endod., 34, 166–171.
Spath, L., Rotilio, V., Alessandrini, M., Gambara, G., De Angelis, L., et al. (2010). Explant-derived human dental pulp stem cells enhance differentiation and proliferation potentials.
J. Cell. Mol. Med., 14, 1635–1644.
Stanislawski, L., Carreau, J. P., Pouchelet, M., Chen, Z. H., & Goldberg, M. (1997). In vitro culture of human dental pulp cells: some aspects of cells emerging early from the
explant. Clin. Oral Invest., 1, 131–140.
Su, Y., Xie, W., Wang, C., Peng, L., Zhou, X., et al. (2012). JNK/P38 mitogen-activated protein kinase used for hepatocyte growth factor-induced proliferation, differentiation, and
migration in human dental papilla cells. J. Endod., 38, 1207–1213.
Suchánek, J., Visek, B., Soukup, T., El-Din, Mohamed SK., et al. (2010). Stem cells from human exfoliated deciduous teeth–isolation, long term cultivation and phenotypical
analysis. Acta Medica (Heradec Kra´love´), 53, 93–99.
Sugiyama, M., Iohara, K., Wakita, H., Hattori, H., Ueda, M., et al. (2011). Dental pulp-derived CD31-/CD146- side population stem/progenitor cells enhance recovery of focal cerebral
ischemia in rats. Tissue Eng. Part A, 17, 1303–1311.
Téclès, O., Laurent, P., Zygouritsas, S., Burger, A. S., Camps, J., et al. (2005). Activation of human dental pulp progenitor/stem cells in response to odontoblast injury. Arch. Oral
Biol., 50, 103–108.
Tomic, S., Djokic, J., Vasilijic, S., Vucevic, D., Todorovic, V., et al. (2011). Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are
susceptible to activation by toll-like receptor agonists. Stem Cell. Dev., 20, 695–708.
Ulmer, F. L., Winkel, A., Kohorst, P., & Stiesch, M. (2010). Stem cells–prospects in dentistry. Schweiz. Monatsschr. Zahnmed., 120, 860–883.
Volponi, A. A., Pang, Y., & Sharpe, P. T. (2010). Stem cell-based biological tooth repair and regeneration. Trends Cell Biol., 20, 715–722.
Waddington, R. J., Youde, S. J., Lee, C. P., & Sloan, A. J. (2009). Isolation of distinct progenitor stem cell populations from dental pulp. Cells Tissues Organs, 189, 268–274.
Wanachottrakul, N., Chotigeat, W., & Kedjarune-Leggat, U. (2011). Translationally controlled tumor protein against apoptosis from 2-hydroxy-ethyl methacrylate in human dental
pulp cells. J. Mater. Sci. Mater. Med., 22, 1479–1487.
Wang, J., Wang, X., Sun, Z., Wang, X., Yang, H., et al. (2010). Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells. Stem Cell.
Dev., 19, 1375–1383.
Wang, X., Sha, X. J., Li, G. H., Yang, F. S., Ji, K., et al. (2012a). Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch.
Oral Biol., 57, 1231–1240.
Wang, L., Zhao, Y., & Shi, S. (2012b). Interplay between mesenchymal stem cells and Lymphocytes: Implications for immunotherapy and tissue regeneration. J. Dent. Res., 91,
1003–1010.
Yagyuu, T., Ikeda, E., Ohgushi, H., Tadokoro, M., Hirose, M., et al. (2010). Hard tissue-forming potential of stem/progenitor cells in human dental follicle and dental papilla. Arch.
Oral Biol., 55, 68–76.
Yang, X., van der Kraan, P. M., van den Dolder, J., Walboomers, X. F., Bian, Z., et al. (2007). STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated
human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation. Tissue Eng., 13, 2803–2812.
Yang, K. L., Chen, M. F., Liao, C. H., Pang, C. Y., & Lin, P. Y. (2009). A simple and efficient method for generating nurr1-positive neuronal stem cells from human wisdom teeth
(tNSC) and the potential of tNSC for stroke therapy. Cytotherapy, 11, 606–617.
Yao, S., Pan, F., Prpic, V., & Wise, G. E. (2008). Differentiation of stem cells in the dental follicle. J. Dent. Res., 87, 767–771.
Yokoi, T., Saito, M., Kiyono, T., Iseki, S., Kosaka, K., et al. (2007). Establishment of immortalized dental follicle cells for generating periodontal ligament in vivo. Cell Tissue Res.,
327, 301–311.
Zhang, W., Walboomers, X. F., Van Kuppevelt, T. H., Daamen, W. F., Van Damme, P. A., et al. (2008). In vivo evaluation of human dental pulp stem cells differentiated towards
multiple lineages. J. Tissue Eng. Regen. Med., 2, 117–125.
Zhao, Y., Wang, L., Jin, Y., & Shi, S. (2012). Fas ligand regulates the immunomodulatory properties of dental pulp stem cells. J. Dent. Res., 91, 948–954.
Zhou, R. H., Yao, M., Lee, T. S., Zhu, Y., Martins-Green, M., et al. (2004). Vascular endothelial growth factor activation of sterol regulatory element binding protein: a potential role in
angiogenesis. Circ. Res., 95, 471–478.
Drug and Gene Delivery for Regenerative Engineering
Morgan A Urello, Tianzhi Luo, Bing Fang, Kristi L Kiick, and Millicent O Sullivan, University of Delaware, Newark, DE, United
States
Introduction 566
Drug- and Gene-Delivery Mechanisms 567
Diffusion-, Degradation-, and Swelling-Controlled Drug Release 567
Microenvironment-Stimulated Drug Release 568
Hydration and swelling 568
pH-Responsive 569
Thermoresponsive 569
Externally Stimulated Drug Release 569
Thermoresponsive systems 569
Light-responsive 570
Magnetic or electric field-responsive 570
Cell-Triggered Drug Release 570
Cellular interactions 570
Cellular by-product-responsive 570
Drug- and Gene-Delivery Systems 571
Ceramics 571
Hydrogels 572
Electrospun Fibrous Scaffolds 573
Micro and Nanocarriers 574
Types of Therapeutic Molecules in Drug and Gene Delivery 575
Small-Molecule Drugs 575
Protein-Based Therapeutics 576
Growth factors 576
Monoclonal antibodies 577
Nucleic Acids 577
Genes 577
RNAi 577
Delivery System Selection/Comparison 577
Therapeutic Delivery in Chronic Wound Care 578
Delivery System 578
Small-Molecule Drug Delivery 579
Protein-Based Therapeutic Delivery 580
Gene-Based Therapeutic Delivery 580
Current Obstacles and Future Applications 580
Further Reading 582
Glossary
Controlled therapeutic delivery Spatiotemporally controlled administration of therapeutics to avoid off-target effects and
achieve localized drug delivery within the desired ranges of concentration and duration.
Gene Primary sequence of nucleic acids that encodes a protein.
Growth factor A class of signaling proteins that stimulate cell proliferation, differentiation, survival, inflammation, and/or
tissue repair.
Monoclonal antibody Antibodies produced from a single cell clone with monovalent affinity to a single epitope on a targeted
antigen.
Regenerative engineering Restoration or regeneration of living tissue through integration of materials science and biochemical
signaling.
“Smart” polymers Polymers purposefully engineered to change chemically and/or physically in response to changes in
temperature, pH, light, or other physicochemical stimuli (e.g., “stimuli-responsive polymers”).
siRNA Small RNA with the capacity to bind and catalyze degradation of target mRNA sequences through assembly of the RNA-
induced silencing complex (RISC).

566 Regenerative Engineering j Drug and Gene Delivery for Regenerative Engineering
Substrate-mediated delivery The administration of a drug or biologic within a scaffold to promote localized, controlled
delivery.
Systemic delivery The administration of medications, drugs, biologics, or drug-loaded nanocarriers into the circulatory system.
Introduction
The delivery of drugs, such as small molecules and growth factors, represents a vital part of regenerative engineering approaches
aimed at restoring or regenerating living tissue through integration of materials science and biochemical signaling. Efficient drug
administration aids in the activation of specific reparative pathways that promote cellular recruitment, proliferation, and differen-
tiation within the injured or diseased site. Moreover, in more recent years, gene delivery has garnered increasing interest in regen-
erative engineering by providing a means to reprogram cells and propagate/promote desired cellular lineages and behaviors.
Systemic administration of therapeutic agents often results in rapid clearance from circulation and/or degradation before activity
is manifested. To overcome these obstacles, therapeutics are generally administered using extraphysiological and repetitive dosing
regimens; however, such routines may cause serious side effects including immune activation and carcinogenic responses. For
instance, the only FDA-approved recombinant platelet-derived growth factor (PDGF) therapy for diabetic neuropathic ulcers has
been demonstrated to reduce ulcer healing time by 32%, yet PDGF application has also been linked to a fivefold increase in
cancer-related deaths in diabetic patients. Advances in biomaterials science have provided more elegant solutions for combating
the aforementioned issues by providing platforms for targeted, sustained delivery of therapeutics with tight spatial and temporal
control.
Traditional regenerative engineering approaches employ a wide variety of biomaterials, ranging from ceramics to polymers, that
form 3-D structures to guide repair. Ideally, implanted biomaterials provide a scaffold for cellular attachment, proliferation, and
differentiation while also serving as a platform for controlled therapeutic delivery. Over the past 25 years, advances in biomaterials
science have greatly expanded the choice of biomaterials, such that polymeric and inorganic materials can be engineered with
a range of mechanical properties, degradation rates, and chemical functionalities. Increased biomaterial diversity has allowed for
selective control of cell behaviors and also has permitted therapeutic delivery with greater spatial and temporal control. Biomaterials
can regulate delivery kinetics via a multitude of mechanisms including diffusion-based, environmentally responsive, and cell-
triggered release.
Within therapeutic delivery systems, bioactive molecules may be physically entrapped or immobilized though a variety of mech-
anisms, depending upon both the material design and the therapeutic agent’s properties. Nonspecific electrostatic, van der Waals,
and hydrophobic interactions have been shown to facilitate the immobilization of various proteins and DNA vectors onto different
biomaterials and to successfully facilitate sustained therapeutic delivery. Alternatively, biomaterials and therapeutics have been
engineered to contain complementary functional groups to mediate binding, including biotin–avidin pairs, antibody–epitope
pairs, and extracellular matrix (ECM)-specific targeting. Such approaches may enable substrate specific binding, reduce off-target
effects, and improve control over release kinetics. The goal is to design delivery approaches able to provide the required duration
and dosage of therapeutic delivery while also maintaining and/or enhancing the specific biological activities of the therapeutic. For
example, the biological activity of growth factors is dependent not only on local concentration but also upon the context of growth
factor presentation within the cellular microenvironment. While some growth factors are active only when tethered to a biomaterial,
others are active only after release and cellular internalization. These differences must be accommodated when choosing or
designing an optimized delivery system.
Additionally, drug- and gene-delivery systems have employed a wide range of micro and nanocarriers. The administration of
smaller devices is typically less invasive, and nanocarriers additionally benefit from unique pharmacokinetic properties that can
prolong circulation. Delivery via nanocarriers can also be used to improve hydrophobic drug solubility and bioactivity, which
increases the quantity and efficacy of deliverable drug. Furthermore, polymeric and metal-based micro and nanocarriers have
been utilized for site-specific therapeutic delivery and/or controlled release from scaffold-mediated delivery systems. The inclusion
of cell- and tissue-targeting moieties on these carriers has enabled targeted delivery to multiple organs, diseased tissues, and tumors,
while their inclusion in bulk delivery systems has enabled enhanced control over therapeutic delivery kinetics, multitherapeutic
delivery, improved therapeutic stability, and cellular uptake. For instance, polyplex-based DNA nanocarriers, formed via electro-
static interactions between DNA and cationic polymers, have been demonstrated in numerous studies to enhance nuclease resis-
tance and improve cellular uptake, resulting in improved gene-transfer efficiency.
This review will provide an overview of the diverse array of drug- and gene-delivery systems in regenerative engineering, with
a focus on material-mediated delivery systems. Micro/nanocarriers will be discussed in the context of scaffold-mediated delivery.
For more information on their micro/nanocarrier design following systemic administration, the suggested reading, including
a review of stimuli-responsive nanocarriers by Ganta, S. et al., can be referenced. Different delivery mechanisms and smart material
designs will be discussed broadly, followed by an overview of delivery system selection based on the objective of the application and
the properties of the therapeutic. The article will conclude with an application-focused section detailing delivery system design and
application in chronic wound repair, an area of great interest in regenerative engineering.
Regenerative Engineering j Drug and Gene Delivery for Regenerative Engineering 567
Drug- and Gene-Delivery Mechanisms
The development of an appropriate drug delivery system plays a vital role in determining the rate and efficacy of tissue regeneration.
The release kinetics and bioactivity of the therapeutics, as well as how the delivery systems interact and integrate into the
surrounding host environment need to be considered. In most cases, release is controlled at least in part by diffusion, where net
molecular transport is driven by a concentration gradient, and delivery kinetics are largely controlled by the fluid properties and
physical characteristics within the scaffold. Scaffold degradation and swelling properties largely influence release. The advent of
“smart” or stimuli-responsive polymers, which display significant physiochemical changes in response to changes in their environ-
ment, has provided a means for triggering release using either external stimuli, such as heat, light, or a magnetic field, or microen-
vironmental stimuli, such as pH changes, presence/absence of enzymes, or temperature changes. Furthermore, some delivery
systems have been designed to respond to cell-mediated stimuli, including protease release and generation of reactive oxygen
species (ROS). The types of stimuli-responsive systems are summarized and compared in Fig. 1.
Diffusion-, Degradation-, and Swelling-Controlled Drug Release

Diffusion regulates drug release from most delivery systems. Therapeutics must pass through the water-insoluble material (e.g.,
a ceramic or polymer-based matrix) that forms the delivery device for release to occur. Diffusion can be controlled at a macromo-
lecular level, such as through pores in a matrix, or on a molecular scale, by passing between polymeric chains. Diffusion-controlled
delivery systems are typically categorized as either matrix-based or reservoir diffusion systems. Within matrix-based systems, the
therapeutic is distributed throughout the delivery scaffold. Water permeation promotes uniform swelling, leading to a volume
expansion in the bulk polymeric matrix and a subsequent increase in matrix pore size. Alternatively, within reservoir diffusion
systems, the therapeutic is encapsulated within a permeable polymer membrane. Swelling is observed as a nonuniform volume
expansion, wherein only the membrane region allows water permeation and swelling. Within both systems, effective diffusion
only occurs when the pore size of the swelled matrix is considerably larger than the dimensions of the therapeutic molecule.
In many diffusion-based systems, release is characterized by an initial burst release phase followed by a steady, slower release
phase. Within hydrophilic or porous matrices, this two-phase release is the result of an initial period of rapid water uptake that
triggers the burst release, followed by uniform degradation throughout the matrix structure leading to steady drug release. Alterna-
tively, within hydrophobic and inert matrices, the materials behave as reservoir systems in which zero-order release kinetics are ob-
tained without an initial burst release phase due to the surface-only wetting phenomenon. However, bioactive, biodegradable
materials are typically employed in regenerative engineering. To improve control over therapeutic release kinetics while avoiding
an initial burst release phase, various approaches have been developed. One approach is to modify the microstructure of the
delivery scaffold. A wide range of cross-linking techniques, including UV photopolymerization and various physical and chemical
cross-linking approaches, have been used to improve both scaffold stability and therapeutic retention. These cross-linked structures
are better able to achieve sustained, tailorable delivery through physical entrapment of drug within tailorable scaffolds. For
example, Y. Tabata and coworkers regulated hepatocyte growth factor (HGF) release from chemically cross-linked gelatin hydrogels
by varying the amount of the cross-linking reagent glutaraldehyde and thereby altering the resulting hydrogel degradability. More-
over, when implanted subcutaneously, cross-linked gelatin-mediated delivery of HGF induced significantly enhanced angiogenic
activity as compared with free HGF codelivered with a gelatin scaffold lacking any growth factors. Polymer–polymer and poly-
mer–peptide cross-linking strategies are also commonly employed. For instance, polyethylene glycol diacrylate (PEGDA) is
Fig. 1 A summary of the stimuli-responsive drug and gene delivery release mechanisms and the responsive components incorporated into the
delivery system.
commonly cross-linked with thiol-modified natural polymers including gelatin, hyaluronic acid, and chondroitin sulfate. This
approach has been implemented by R. A. Peattie and coworkers to mediate the codelivery of bioactive vascular endothelial growth
factor (VEGF) and basic fibroblast growth factor (bFGF) to promote enhanced angiogenic activity within murine models.
Alternatively, both physical and chemical methods have been employed to modulate the affinity between the therapeutic mole-
cule and the matrix and have achieved better controlled delivery from polymeric networks and porous scaffolds. Nonspecific elec-
trostatic interactions between ionic polymers and charged drugs are commonly used to improve drug retention and delay release.
Phosphate- and amino-functionalized polymers have achieved sustained, tailorable release of cationic and anionic drugs, respec-
tively. For instance, A. Concheiro and coworkers demonstrated that the copolymerization of 4-vinylpyridine and poly(hydroxyethyl
methacrylate) increased the loading and retention of nonsteroidal anti-inflammatory drugs (NSAIDS) by up to 20-fold and
expanded retention from hours to a week without altering the mechanical properties of the hydrogel. In another study, W. Jiskoot
and coworkers showed that model protein release from methacrylated-gelatin hydrogels was directly controlled by varying protein–
gelatin electrostatic interactions. Alternatively, natural growth-factor binding sites within ECM proteins such as fibronectin, fibrin-
ogen, and collagen have been incorporated into delivery systems to improve therapeutic retention/release profiles. For instance, the
growth factor-binding capacity of fibrinogen has been utilized to efficiently deliver low dosages of fibroblast growth factor-2 (FGF-
2) and placenta growth factor-1 (PIGF-1) from fibrin matrices to facilitate wound healing in diabetic mice (db/db). Furthermore, J. A.
Hubbell and coworkers prepared PEG-based matrices functionalized with growth-factor binding sequences isolated from fibrinogen
and discovered that these synthetic matrices could sequester multiple growth factors and fully recapitulate the effect of fibrin within
a diabetic mouse impaired healing model. In another study, R. J. Levy and coworkers prepared collagen implants functionalized
with antiadenoviral antibodies, and they showed that these implants had the capacity to achieve highly localized and sustained
gene delivery when applied to stents in pig coronary arteries.
Others have achieved sustained delivery through incorporation of particulate systems within polymeric matrices. Within these
composite systems, the matrix contains therapeutic molecules that are preloaded in secondary controlled release carriers such as
micelles, liposomes, or other particle-based delivery vehicles capable of sustained release. Secondary delivery systems have been
fabricated using both synthetic (e.g., polyvinyl alcohol (PVA), poly(lactide-co-glycolide) (PLGA), and polycaprolactone (PCL))
and natural polymers (e.g., gelatin, alginate, and chitosan). These nanocarrier constructs have been successfully used to preserve
the bioactivity of the therapeutic, sustain its release, and even enable tailorable, multitherapeutic release. For example, K. T. Nguyen
and coworkers demonstrated the codelivery of vascular endothelial growth factor (VEGF) and PDGF-BB from electrospun chitosan/
poly(ethylene oxide) (PEO) fibers, and they showed that codelivery accelerated wound healing in full-thickness rat skin wounds.
Specifically, the encapsulation of VEGF and PLGA nanoparticles preloaded with PDGF-BB within the nanofibers facilitated a fast
delivery of VEGF and a sustained delivery of PDGF-BB that resulted in significantly enhanced angiogenesis and reepithelization.
Additionally, secondary carriers have been functionalized to enable specific, tailorable affinities for both natural and synthetic
delivery substrates and thereby enhance control over delivery. For instance, polyethylenimine-DNA (PEI-DNA) polyplexes have
been incorporated into collagen by functionalizing the PEI with either biotin or collagen mimetic peptide (CMP), such that
collagen-based substrates can be modified with polyplexes through biotin–avidin binding or CMP–collagen hybridization, respec-
tively. The application of functionalized micro and nanocarriers has many additional benefits in regard to preserving therapeutic
bioactivity and targeted delivery as discussed later in this article.
Microenvironment-Stimulated Drug Release

Advances in materials science over the past three decades have enabled the development of environmentally responsive polymers
and, in turn, better integration of therapeutic delivery with delivery site processes. These biomaterials are preengineered to release
therapeutic molecules in response to physiologically relevant stimuli, including aqueous solutions and changes in pH and/or
temperature. Stimuli may induce simple one-time release or on–off delivery profiles. The response within the material typically
occurs through an induction of swelling or degradation or by stimulation of a reversible phase transition that facilitates rapid diffu-
sion or degradation-mediated release of an immobilized therapeutic.
Hydration and swelling

Delivery systems are often preengineered to swell upon application in aqueous biological fluids or media and thereby trigger drug
delivery. The application of hydrophilic or porous materials facilitates rapid water uptake, and system parameters such as cross-
linking density and charge density determine the degree of equilibrium swelling and the therapeutic diffusion rates. For example,
increasing the number of ionic groups within a hydrogel is known to increase its swelling capacity. The increase in swelling is caused
by an increase in counterions within the gel, which produces an increase in osmotic pressure. To utilize this delivery mechanism,
delivery systems are often composed of polymers like hydroxyethylcellulose, whose ionic pendant groups can release loaded ther-
apeutics upon swelling in aqueous biological fluids or media.
Other delivery systems have been engineered with hydrolyzable chemical linkages that can be used to regulate scaffold degra-
dation rates or directly immobilize therapeutics via a biodegradable linkage. In hydrolytic degradation, polymer bonds react with
water molecules and break apart. Commonly employed hydrolytic polymer types include polyanhydrides, polyamides, and poly-
esters. The primary factor affecting the rate of hydrolysis is the partial charge of the reactive carbon atom; therefore, different chem-
ical groups have inherently different reactivities and can be chosen based on the desired degradation rate for a drug-delivery
application. Furthermore, the hydrolysis rate of ester and amide groups in hydrophilic polymer networks can be altered through
the incorporation of adjacent charged molecules. For instance, M. P. Lutolf and coworkers reported that varying the charge of hydro-
lytic cross-links within a polymeric network could promote as much as a 12-fold difference in degradation, leading to significant
changes in model protein retention periods ranging from 6 to over 70 days. Additionally, E. Alsberg and coworkers demonstrated
the capacity to achieve tailorable and sustained delivery of siRNA by covalent incorporation of cationic linear PEI into photocross-
linked dextran hydrogels through hydrolyzable ester linkages.
pH-Responsive
Swelling and collapsing behavior within polymer networks may also be regulated by pH. A local decrease in pH is associated with
multiple conditions including inflammation, wound healing, and myocardial ischemia due to the overproduction of lactic acid,
increased concentration of acidic by-products from bacterial metabolism, and/or glycolytic activity of infiltrated neutrophils. For
this reason, pH is one of the most well-studied stimuli in drug delivery, and a plethora of pH-responsive materials have been docu-
mented. Ionic pH-responsive polymers have the capacity to accept or release protons due to changes in pH because their structures
consist of either acidic groups, like carboxylic or sulfonic acids, or basic groups, like amines. Thus, the electric charge of the polymer
changes in response to pH, and these changes in charge can alter solubility and/or morphology. For instance, polyanions (e.g.,
albumin, poly(methacrylic) acid (PMAA), and polyacrylic acid (PAA)) contain a large number of ionizable acid groups, which
accept protons at low pH and release protons at high pH. Thus, when pH increases, the polymer swells due to electrostatic repulsion
of the negatively charged groups, and these pH-driven changes in swelling increase the diffusivity of the therapeutic. A similar effect
occurs in cationic polymers (e.g., polylysine, chitosan, and PEI) when the pH decreases. The charge of a polymer at a given pH is
based upon its pKa, which in turn is determined by the polymer composition and molecular weight.
pH-responsive polymers have been incorporated into bulk delivery systems like hydrogels and electrospun scaffolds, and they
have also been incorporated into micro and nanocarriers. Additionally, polycationic materials, such as PEI, polyamidoamine
(PAMAM), and other dendrimers, are commonly utilized in nonviral gene delivery. Polycationic polymers are commonly used
to complex and protect nucleic acids through electrostatic interactions. The pH-sensitive nature of these polymers can also enable
endosomal escape through the “endosomal buffering” mechanism. This mechanism occurs in response to decreases in pH
following cellular uptake, when the polymer becomes increasingly charged, and correspondingly, the increased counterion concen-
tration within the endosome increases the osmotic pressure and results in endosomal lysis. Moreover, pH-triggered release has been
achieved using acid-sensitive bonds like hydrazones or acetal groups or derivatives of N-ethoxybenzylimidazole that undergo accel-
erated hydrolysis under mildly acidic conditions. These bonds are used within scaffold cross-linkers or as direct tethers between
therapeutics and scaffolds.
Thermoresponsive
Thermoresponsive materials are another useful tool in therapeutic delivery. The human body has a narrow range of temperatures,
and significant deviations are often indicative of medical problems including infection. Moreover, external sources may be used to
heat or cool tissues, and therefore, external heating or cooling can be used to trigger release as discussed in the next section.
Thermoresponsive systems are composed of polymers that undergo significant changes in solubility due to the inclusion and
interactions of hydrophobic and hydrophilic moieties in the polymer chain. The balance point temperature is referred to as the
lower critical solution temperature (LCST), at which point the polymer favors neither hydrogen bonding with the polymer nor
hydrogen bonding with water. The most commonly employed temperature-responsive materials include those with LCSTs close
to body temperature such as poly(N-isopropylacrylamide) (PNIPAAm), poly(methyl vinyl ether) (PMVE), and elastin-like polypep-
tides. These materials exhibit sol-to-gel transformationsdfor example, transitions from liquid to solid phasednear body temper-
ature. In turn, these materials have been utilized to achieve noninvasive therapeutic delivery of drugs, proteins, and cells, which can
be mixed with the polymer in its soluble state at temperatures below the LCST and subsequently delivered by topical application or
injection. Exposure to physiological temperatures induces the phase separation of the polymer and the concomitant formation of
a therapeutic gel reservoir, enabling improved control over delivery. This approach is often coupled with additional stimuli-
responsive delivery systems to achieve multitriggered therapeutic release as discussed later in the article. Thermoresponsive mate-
rials also have been used to achieve oscillatory drug release from hydrogels, electrospun porous scaffolds, gel-based microparticles,
and nanoparticles.
Externally Stimulated Drug Release

Advances in material design have enabled the release of therapeutic in response to external stimuli such as changes in temperature,
light, and electric fields. These responsive systems provide additional spatiotemporal control over delivery and, in some cases, an
“on–off” switch for delivery that enables marked improvements in delivery localization and safety.
Thermoresponsive systems
Thermoresponsive systems triggered by external stimuli typically respond to a wider range of temperatures than those engineered for
responses to microenvironmental stimuli (e.g., body temperature). As previously described, these systems employ polymers such as
poly N-isopropylacrylamide, poly(ethylene glycol), and elastin-like polypeptides, engineered to undergo temperature-dependent
and reversible sol-gel transitions through the inclusion of hydrophilic and lipophilic moieties within the polymer chain. Polymers
with an LCST near but below body temperature are ideal for therapeutic delivery. Therapeutic agents can be mixed with the polymer
solution in liquid form below the LCST, and the polymer will rapidly form a gel-like therapeutic reservoir following application in
response to body temperatures. Moreover, external heat may be applied to achieve oscillatory therapeutic delivery. For instance, S.
K. Sia and coworkers demonstrated on-demand remote release within implantable N-isopropylacrylamide-co-acrylamide pellets via
ultrasound-induced temperature changes. Other groups have similarly demonstrated remote release from thermoresponsive poly-
mers via topical application of heat or hyperthermia or through inducing localized temperature changes using light or magnetic and
electric fields. These noninvasive approaches will be discussed in greater depth in the next sections.
Light-responsive
Alternatively, light-sensitive systems have also been developed for on-demand release in response to illumination of a specific wave-
length in the ultraviolet, visible, or near-infrared regions. A common strategy is to incorporate chromophores such as o-nitrobenzyl,
trisodium salt of copper chlorophyllin, coumarin, or metals. Within these systems, the chromophore absorbs light of the appro-
priate wavelength. In some cases, the absorbed light dissipates as heat, increasing the local temperature and inducing cargo release
from a secondary thermoresponsive component in the materials (e.g., PNIPAAm, PMVE, and elastin-like polypeptides). Alterna-
tively, some light-responsive systems rely upon photosensitive polymers that demonstrate reversible photoisomerization behaviors
including open-ring transitions and cis–trans conversions including spiropyran, coumarin, and azobenzene derivative-based
systems. These materials can exhibit reversible swelling behaviors and/or “on–off” affinities for drugs in response to light
stimulation.
Magnetic or electric field-responsive

“On–off” therapeutic release has also been achieved through the reversible application of magnetic fields by incorporating magnetic
nanoparticles within polymeric networks. Magnetic field application causes the network to vibrate, which can result in increased
diffusion rate of encapsulated therapeutics. Network vibrations can also trigger sol-gel transitions when the magnetic nanoparticles
are incorporated into thermoresponsive systems composed of polymers such as PNIPAAm, as the thermoresponsive component
will respond secondarily to magnetic field-induced heating. On the other hand, the application of external electric fields has simi-
larly been used to trigger release through incorporation of pH-sensitive polymers with high concentrations of ionizable groups.
Upon application of an electric field, the subsequent change in pH disrupts hydrogen bonding between the polymer chains, which
in turn can cause polymer degradation or deformation (i.e., swelling or deswelling) and subsequent drug release.
Cell-Triggered Drug Release

Within the field of regenerative engineering, many biologically inspired materials utilize native regenerative processes to mediate
delivery. Synthetic biomaterials have been designed to contain biological cues, including domains of ECM molecules, growth
factors, and protease substrates, to promote cell behaviors, therapeutic retention, or cell-triggered therapeutic release.
Cellular interactions
To promote cellular interactions, natural biomaterials like collagen, laminin, and fibronectin are commonly incorporated into the
delivery systems. In some applications, cell-adhesive peptide motifs are incorporated into delivery systems and have several advan-
tages over the use of whole proteins. These advantages include enhanced stability against conformational change, control over
ligand density and location, minimized immune response, and cost-effectiveness. Common cell-adhesion motifs include RGD
(fibronectin, laminin, vitronectin, and various collagen), YIGSR and IKLLI (laminin), GFOGER and DGEA (type-I collagen), and
REDV and RGD/PHSRN (fibronectin). In addition to cell adhesion, the incorporation of ligands can affect canonical cell behaviors
including proliferation, viability, migration, and therapeutic responsiveness. For instance, synergistic signaling between certain
integrins and growth-factor receptors has been linked to significant alterations in cellular adhesion and/or growth-factor signaling.
Furthermore, in cases where a therapeutic is stably immobilized within a scaffold-based delivery system, cellular invasion is the key
factor in determining delivery kinetics.
Cellular by-product-responsive
Cell-triggered therapeutic delivery can also be achieved through the application of materials that are responsive to cellular by-
products, which include proteases, ROS, and reductants. In contrast to materials that respond through chemically induced mech-
anisms such as hydrolysis, which often produce bulk degradation, proteolytic materials enable localized, cell-triggered degradation
and drug delivery. Proteolytic delivery systems often are fabricated from natural materials such as collagen, laminin, or fibronectin
that innately contain specific proteolytic domains. Alternatively, such delivery systems can be formed from synthetic materials in
which protease-labile peptide sequences (i.e., collagenase- and plasmin-sensitive peptides) are incorporated as cross-linkers. For
example, cell-mediated release of VEGF has been reported from PEG-based hydrogels conjugated to the cell-adhesive peptide
RGD through the incorporation of matrix metalloproteinase 2 (MMP 2)-labile peptidic cross-linkers. Additionally, altering the
density of proteolytic sequences has been shown to control cell-mediated degradation and subsequent therapeutic delivery; as
the density of these sites increases, degradation and release also increase.
Because many pathological conditions are associated with elevated, cell-produced ROS, delivery systems composed of hydrogels,
polymeric nanoparticles, and inorganic nanoparticles have utilized a series of ROS-labile linkers and materials to achieve spatio-
temporally controlled release in both the extra- and intracellular environment. For example, C. L. Duvall and coworkers developed
a thermoresponsive hydrogel with the capacity to release a model drug in an H2O2 dose-dependent manner. Moreover, the potential
for producing materials for cell-mediated, multidrug release was achieved by K. Kiick and coworkers using a liposome-PEG hybrid
hydrogel whose thioether succinimide cross-links degrade upon exposure to thiols such as glutathione, which is found at elevated
concentrations in intracellular compartments.
Drug- and Gene-Delivery Systems
The scaffolds employed in regenerative engineering have been synthesized from a diverse array of organic and inorganic polymers,
ceramics, and composites. Ideal scaffolds mimic the 3-D structure of the native tissue and have the ability to perform as a delivery
platform for therapeutic drugs, proteins, and genes. In this section, recent developments in scaffold-mediated delivery and carrier
selection will be discussed.
Ceramics
Inorganic 3-D scaffolds and substrates, particularly calcium-based substrates for bone repair, constitute a major thrust in regener-
ative engineering. Hydroxyapatite (HAp) is the calcium apatite species in bone, making it an especially important naturally occur-
ring mineral in the inorganic scaffold/substrate group. HAp also is an excellent biomaterial in bone tissue engineering due to a series
of unique and appealing properties such as biocompatibility, bioresorbability, and osteogenicity. With rational design approaches
in scaffolds, controllable delivery and directed differentiation of stem cells can be achieved. HA-based materials can be applied
either as implants or implant coatings that enhance local osteogenesis. HA also is an excellent candidate material for the controlled
delivery of specific drugs and/or genes at localized sites, and it effectively promotes the differentiation of nearby stem cells into tar-
geted lineages (e.g., chondrogenic and/or osteoblastic cells). HAp colloids also have been developed, as these materials can be used
to form coatings or construct the inorganic phase of hybrid materials/composites.
Tricalcium phosphate (TCP) is another important member of the calcium-based inorganic scaffold/substrate family. TCP has
been used clinically as a bone substitute in grafting operations. It is also considered advantageous as a drug/gene-delivery material
due to its excellent biocompatibility and lack of significant inflammatory responses. TCP can transform into HAp through hydro-
lysis, such that many of the materials in this class are actually a mixture of TCP and/or HAp and other organic materials. Several
investigations have shown that incorporating TCP into an organic gel scaffold demonstrates a better wound-healing effect. In
many cases, polymer coatings are used in conjunction with TCP-based materials to reduce the brittleness of the pure inorganic phase
and to enhance the controllable drug release profile, and the applied polymers also can provide additional drug carrier functions.
For example, M. Zhang and coworkers have shown that TCP scaffolds with PCL coatings enable sustained and steady bone morpho-
genetic protein-2 (BMP-2) delivery over a 2-week period that promoted enhanced osteogenic activity within rat models. Further-
more, to enable controlled deformation of the material to accommodate the geometry of different defect sites, efforts have been
made to develop injectable composites containing TCP beads. Calcium ions and inorganic phosphate together have been shown
to have a boosting effect on osteogenesis. Reports have also documented the functions of TCP scaffolds when directly used as
growth-factor carriers including BMP-2 and VEGF.
Biphasic calcium phosphate (BCP) scaffolds contain both hydroxyapatite and beta-tricalcium phosphate. As a mixed material,
BCP exhibits many superior properties that combine the benefits of its two components, such as highly hierarchical porosities,
biocompatibility, bioresorbability, osteoconductivity, and mechanical strength appropriate for bone grafts. However, in order to
enable BCP-based materials to conform to the geometries necessary in various applications, polymer/BCP ceramic composites
are often prepared for scaffold use in bone tissue engineering. Furthermore, the FDA-approved bioactive protein, bone morphoge-
netic protein-2 (BMP-2), has been loaded into BCP-based scaffolds to form a biomaterial with improved osteoinductivity. Micro/
macroporous BCP (MBCP) scaffolds also have been used as carriers for mRNA encoding for hBMP-2, enabling sustained release,
enhanced osteogenic stem-cell differentiation, and mineralization. As a bioactive coating, BCP is also functional in drug delivery,
triggering early osseointegration in titanium implants.
Amorphous calcium phosphate (ACP) is the first solid phase precipitated after the rapid mixing of aqueous solutions containing
Ca2 þ and PO3 ions, and ACP also is an essential mineral phase formed in mineralized tissues. It does not have a periodic order of
crystalline structures, making it distinct from other forms of calcium phosphates. ACP has high drug adsorption efficiency, and the
amorphous surface facilitates release of the bound drug molecules. A variety of interesting and promising studies have been per-
formed with ACP-based scaffolds or composites for tissue engineering. For example, in situ precipitation of ACP and ciprofloxacin
crystals in chitosan hydrogels produces a composite scaffold suitable for drug delivery and controlled release of therapeutics like
BMP-2. ACP porous microspheres have a high capacity for drug loading and can provide pH-responsive drug release, demonstrating
promise for applications in drug delivery. Strontium has been doped with amorphous calcium phosphate to form porous micro-
spheres (SrAPMs). SrAPMs were incorporated into collagen, and the SrAPM–collagen scaffolds could effectively stimulate osteogen-
esis and promote bone regeneration.
In all cases, therapeutics (either drugs or genes) can be incorporated into ceramics/composites in multiple different ways,
including physical adsorption, chemical conjugation, and encapsulation. The most straightforward loading procedure is physical
adsorption, in which drugs/genes or drug/gene-containing nanocarriers are loaded and retained on ceramic/composite substrates
by electrostatic or van der Waals interactions. For example, in aqueous media, hydroxyapatite crystal surfaces gain a positive charge
due to release of OH– ions. As the majority of proteins present negative charges at physiological pH range, electrostatic interactions
between hydroxyapatite and proteins can be readily established in aqueous condition. The roughness of the hydroxyapatite helps
protein adsorption. Moreover, with appropriate modification/functionalization, the inorganic carriers also are able to chemically
bond to the cargoes. For instance, L. Bachas and coworkers demonstrated biphosphonate linkers enable oriented immobilization
of proteins on to HAp through hydrazine bonding. In other examples, the HAp surface is modified with amino acid, which can
chemically connect to growth factors through carboxyl–amine chemistry.
Hydrogels
Hydrogels are three-dimensional hydrophilic networks capable of absorbing a large amount of water. These networks are
composed of polymeric chains that are cross-linked via chemical conjugation or alternatively by physical interactions such as
entanglements, crystallites, van der Waals interactions, and/or hydrogen bonding. Because of their excellent biocompatibility,
biodegradability, nontoxicity, and critical role in the ECM, native macromolecules such as sugars and proteins are widely
used as the building blocks for hydrogels. Polysaccharides including alginate, chitosan, cellulose, dextran, and hyaluronic
acid have been widely studied, as have proteins such as collagen and gelatin. However, although natural materials exhibit a variety
of well-defined hierarchical structures and important biological functions, such as cell recognition and adhesion, their use in
therapeutic applications can be limited by immune responses and susceptibility to enzymatic degradation. To overcome these
limitations, synthetic polymers also have been used to fabricate hydrogels for drug and gene delivery. Among the various types
of polymeric hydrogels, hydrogels synthesized from PEG, an FDA-approved polymer, have been extensively studied with prom-
ising preclinical and clinical results. Biomimetic peptides also have been widely studied in hydrogen applications, where they
serve either as biological stimuli or as cross-linking sites. For example, CMP, synthetic peptides that mimic the triple-helical
conformation of native collagens, have been conjugated to four-arm PEG. When equipped with a CMP domain, the four-arm
PEGs form a hydrogel with these peptides serving as physical cross-linkers. Moreover, at temperatures above the melting temper-
ature (TM) of the CMP, the unfolding of the triple helices induces hydrogel disassembly, allowing a convenient mechanism to
thermally trigger the release of encapsulated drugs.
Due to their biocompatibility, high swelling in aqueous media, and responsiveness to pH, temperature, and other stimuli,
hydrogels have been extensively investigated as drug-delivery systems for molecules ranging from nonsteroidal anti-
inflammatory drugs (NSAIDs) to proteins. Compared with other drug-delivery systems, hydrogels closely resemble living tissues,
with high water content, well-designed mechanical properties, and minimal tendency to adsorb proteins from bodily fluids. Addi-
tionally, the pore size of hydrogels can be easily manipulated via changing the chemical composition and cross-linking ratio of the
polymeric network, which can in turn be utilized as a method to control the loading and releasing of encapsulated drugs.
Due to their high water content, drug release from hydrogels is typically relatively fast and occurs over a period of hours to days.
For applications in which a slower release rate is desirable, a wide range of strategies have been employed, including physical entrap-
ment and covalent conjugation to the hydrogel network. One of the most widely used methods to reduce the release rate is to intro-
duce electrostatic interactions between ionic polymer networks and oppositely charged drugs. To improve the strength of the
pairwise charge–charge interactions, multivalently charged polymers, such as phosphate-functionalized polymers, are often used
to form the hydrogel. For example, functionalizing a PNIPAAm-based hydrogel with polyoxyethyl phosphate-containing como-
nomer drastically improved the encapsulation of cationic lysozyme into the hydrogel. In another example, adding positively
charged N-(3-aminopropyl)methacrylamide or 4-vinylpyridine monomer into poly(hydroxyethyl methacrylate) networks
increased the amount of encapsulated NSAIDs by more than one order of magnitude and extended the period of sustained release
up to approximately 1 week. Chemical conjugation is widely used to load drugs into hydrogel matrices, and these methods can
enable stimuli-responsive release depending on the nature of the covalent bonds linking the drug to the gel. The details on envi-
ronmentally triggered release applications are discussed later in the article.
Thermoresponsive hydrogels are commonly developed using the principles previously discussed employing a wide range of
polymers that exhibit temperature-responsive phase transitions. The most common characteristic of these polymers is the presence
of hydrophobic groups, such as methyl, ethyl, and/or propyl groups. Hydrogels with desired thermoresponsiveness can be injected
into the body in a liquid state with encapsulated drug, followed by gelation in the body at physiological temperature to form a cross-
linked hydrogel. Additionally, the transition temperature of the hydrogel can be easily tuned by making copolymers of hydrophobic
(e.g., NIPAAm) and hydrophilic (e.g., acrylic acid) monomers and adjusting the ratio of the hydrophilic and hydrophobic segments
of the polymer to tailor therapeutic release. Generally, a higher content of hydrophobic polymers in the hydrogels, a lower transi-
tion temperature is obtained.
The pH-sensitive hydrogels are another commonly studied class of stimuli-responsive hydrogels. Hydrogels with pH-sensitivity
are primarily used in delivering therapeutics to tumor cells due to the existence of an acidic pH within the tumor stroma. The pH-
sensitive hydrogels are usually constructed using polymers with acid-sensitive bonds that can be easily cleaved in acidic conditions.
For example, hydrogels cross-linked via Schiff’s base reactions are generally stable at physiological pH yet can be degraded under
mildly acidic conditions due to the cleavage of the imine bond. The pH responsiveness also can be obtained by using polymers with
ionizable chemical groups whose chemical properties such as swelling ratio and water solubility are altered based upon the charge
state of such groups. For instance, S. A. Hegazy and coworkers designed pH-responsive hydrogels that were copolymerized from
PEG/acrylic acid. The diffusion coefficient of the encapsulated model drug ketoprofen was highly dependent upon both the pH
and the ionic strength of the medium, as the degree of ionization of the acrylic acid increases with increases in pH resulting in greater
numbers of fixed charges, chain repulsion, and thus hydrogel swelling, while increases in solution ionic strength result in increases
in the number of counterions and thus a decrease in repulsive forces and swelling.
In tissues and cells, enzymes are secreted to change the structure and properties of ECM, facilitating cell proliferation, differen-
tiation, and tissue regeneration. The incorporation of enzyme substrates as a biologically responsive component in hydrogel
networks is a useful strategy to mimic the proteolytic sensitivity of ECM and enable the triggered release of encapsulated drugs.
For example, by introducing the matrix metalloproteinase (MMP)-responsive peptide thymosin b4 (Tb4), Langer and coworkers
designed a PEG-based hydrogel that was conducive to human umbilical vein endothelial cell (HUVEC) adhesion, survival, migra-
tion, and organization. Incorporation of Tb4 significantly increased the secretion of MMP-2 and MMP-9 from encapsulated
HUVECs, which subsequently triggered the degradation of the hydrogel itself and the release of Tb4. The enzymatically controlled
release of the peptide induced vascular-like network formation within the PEG hydrogels. These results indicated that the Tb4-
encapsulating, enzymatically degradable hydrogels may be useful as scaffolds for in situ regeneration of ischemic tissues.
In addition to temperature-, pH-, and enzyme-responsive hydrogels, smart hydrogels that respond to other stimuli also have
shown promise in regenerative medicine. For example, electric current has been applied as an external trigger to induce cargo release.
Polyelectrolyte hydrogels are used for this purpose due to their capacity to undergo swelling or shrinkage when an electric field is
applied, allowing the delivery of encapsulated therapeutics such as edrophonium chloride and hydrocortisone in an “on–off”
manner when the applied electric field is switched. Light also has been used as a trigger to induce responses within hydrogels.
Light-sensitive hydrogels are advantageous in terms of their control mechanism since light stimuli can be applied instantly with
precisely controlled location and intensity. For example, the addition of bis(4-di-methylamino)phenylmethyl leucocyanide into
a PNIPAAm-based hydrogel produced a network that swelled in response to UV irradiation but shrank when the light was shut
off. This property was conferred by the bis(4-di-methylamino)phenylmethyl leucocyanide, which is usually a neutral molecule
but can dissociate into ion pairs under UV irradiation. Such a property may have potential for light-sensitive drug-delivery
applications.
In addition to the widely used environmental triggers discussed earlier, other types of stimuli responses also have been intro-
duced into hydrogels for drug delivery purposes, including sensitivity to magnetic fields, pressure, specific ions, thrombin, and
various antigens. Readers interested in details of these types of hydrogels are redirected to previous published reviews in the sug-
gested reading.
Electrospun Fibrous Scaffolds

Fibrous scaffolds produced by electrospinning have become increasingly popular in regenerative engineering due to their high
surface area-to-volume ratio and porosity, which together simulate the structure of protein fibers within the ECM. Moreover, the
versatility of electrospinning in terms of polymer content, fiber structure, and functionalization has made it possible to fabricate
scaffolds with a broad range of mechanical properties, bioactivities, and mass transport properties. For instance, drug diffusion rates
within these porous scaffolds can be tailored by alteration of polymer degradation kinetics, matrix crystallinity, porosity, geometry,
and other chemical phenomena. The diversity and biomimicry of electrospun fibrous scaffolds have led to their use in numerous
regenerative medicine applications including wound healing and nerve and spinal muscle regeneration.
Several methods have been developed for incorporation of therapeutic molecules into electrospun scaffolds, including surface
modulation, blending, and coaxial processing. In most cases, therapeutic delivery from electrospun scaffolds is either achieved by
binding the therapeutic onto the fiber surface or by encapsulating it within the fiber. While localized delivery has been achieved
using both methods, encapsulation usually offers greater control over therapeutic diffusion rates and overall release kinetics. For
example, blending approaches require the therapeutic to be codissolved or dispersed with the polymer in the organic solvent
that evaporates during the electrospinning process. This one-step fabrication technique has been successfully used to achieve sus-
tained release of hydrophobic drugs such as rifampicin and paclitaxel from hydrophobic polyester polymers and hydrophilic drugs
such as doxorubicin from hydrophilic polymers including gelatin, PEG, and PVA. One challenge is that insufficient solubility and/or
heterogenous distribution of drugs within the polymer solution can limit drug and polymer choices and often produces materials
that exhibit an inconsistent burst release of encapsulated therapeutic.
Core–shell electrospinning techniques, such as coaxial and emulsion electrospinning, overcome solubility complications and
have a proven capacity to enhance control over release while maintaining the bioavailability of unstable biologics. Unlike blended
electrospun fibers, which are produced from a single phase, core–shell fibers are fabricated using dual-solvent approaches that mini-
mize contact between the biologic compound and organic solvents. Furthermore, the core–shell structures generated have a poly-
meric shell that deters direct contact between biomolecules and the external environment. Core–shell fibers are commonly
fabricated either through coaxial electrospinning, which employs an inner jet and outer jet to generate fibers with a drug core
and a protective polymeric shell, or through emulsion electrospinning, in which two immiscible solutions (typically an apolar poly-
mer solution and an aqueous therapeutic solution) are spun simultaneously to generate the core-sheath fiber morphology. Both
approaches have been used to obtain the sustained release of active therapeutics. For instance, C. H. Wang and coworkers con-
structed PLGA/hydroxyapatite (HA) composite scaffolds for the delivery of BMP-2 plasmid DNA. These investigators demonstrated
improved therapeutic loading efficiency and extended release periods when chitosan BMP-2 plasmid DNA complexes were encap-
sulated through emulsion electrospinning versus scaffold dipping following fiber fabrication. The same group also demonstrated
BMP-2 protein delivery via PLGA/HAp composite fibrous scaffolds that were generated using emulsion electrospinning. These struc-
tures maintained native conformations of the BMP-2, and the BMP-2 release profile could be controlled via varying HAp content.
Furthermore, the versatile nature of electrospinning has provided an ideal platform for tailored, multitherapeutic delivery. For
instance, T. W. Wang and coworkers demonstrated the capacity to tailor the release of four angiogenic growth factors by incorpo-
rating the therapeutics either directly or preloaded into gelatin nanoparticles into electrospun collagen/hyaluronic acid nanofibers.
These multicomponent structures produced accelerated wound closure and elevated collagen deposition and enhanced blood-
vessel maturation when applied in a diabetic rat wound model.
Electrospun scaffolds also have been developed using stimuli-responsive polymers and biologically inspired strategies. For
instance, the thermoresponsive polymer PNIPAAm was incorporated into an electrospun fiber mat and shown to retain its LCST
behavior after electrospinning. Fibers fabricated from PNIPAAm copolymers or alternatively cospun with PNIPAAm and polymers
such as polystyrene and/or PEO have shown reversible deswelling/swelling behaviors that are conducive to thermally triggered drug
delivery. Additionally, H. Q. Liu and coworkers described the preparation of pH-responsive, cross-linked poly(styrene-co-(maleic
sodium anhydride)) (SMA) and SMA-cellulose acetate composite nanofibers that exhibited improved mechanical strength relative
to classically cast hydrogels and also exhibited pH-dependent swelling in aqueous solution. Alternatively, J. Weng and coworkers
demonstrated pH-triggered release of the drug paracetamol from electrospun scaffolds though the incorporation of acid-labile
acetal groups into the backbone of poly(D,L-lactide)-poly(ethylene glycol). Fibrous scaffolds responsive to additional types of
external stimuli, including light, electric fields, and magnetic fields, also have been demonstrated. For instance, S. Ramakrishna
and coworkers prepared skin grafts in which the photosensitive polymer poly(3-hexylthiophene) (P3HT) and epidermal growth
factor were encapsulated into core–shell-structured gelatin/poly(L-lactic acid)-co-poly(ε-caprolactone) nanofibers (Gel/PLLCL/
P3GF(cs)) by coaxial spinning. Light stimulation in these scaffolds enhanced healing within in vitro wound-healing models.
Furthermore, E. T. Kang and coworkers achieved externally controlled, UV-triggered release of the prodrug a-cyclodextrin-5-
fluorouracil through the incorporation of photosensitive azo groups into electrospun poly(vinylbenzyl chloride-glycidyl methacry-
late) nanofibers. UV light application triggered the release of bound drug via UV-induced isomerization of the azo groups from trans
to cis confirmation.
Similarly, biologically inspired strategies have been employed to achieve controlled therapeutic delivery. For instance, one of the
most common bioinspired approaches is to coat electrospun scaffolds with ECM components like collagen to promote enhanced
cellular invasion and growth-factor binding and release. Alternatively, biomacromolecules with growth-factor affinities have been
directly incorporated or covalently tethered to the scaffold surface to prolong release. B. S. Kim and coworkers improved BMP-2
retention on PLG scaffolds through covalently modifying its surface with heparin. The functionalized scaffold promoted enhanced
bone formation relative to scaffolds directly loaded with BMP-2 through sustained BMP-2 release. In other cases, growth factor has
been covalently bound to electrospun scaffolds, mimicking native ECM GF sequestering and release. Furthermore, protease-
degradable electrospun scaffolds have been developed through the incorporation of protease-labile sequences. These proteolytically
sensitive scaffolds offer a promising platform for cell-triggered delivery in a variety of regenerative engineering applications.
Micro and Nanocarriers

A large variety of micro and nanocarriers have been utilized in the delivery of therapeutic small-molecule drugs, proteins, and DNA.
Particles have been prepared from a multitude of organic and inorganic materials including nondegradable and biodegradable
polymers, lipids, self-assembling amphiphilic molecules, dendrimers, and metals. Material selection is largely dependent on the
type and administration route of the drug, as well as the therapeutic objective. For instance, liposomes are lipid-based carriers
that consist of an outer lipid bilayer and an inner aqueous space that is ideal for the delivery of soluble factors. Alternatively,
micelles are composed of a self-assembling lipid monolayer with a hydrophobic core that is ideal for the delivery of hydrophobic
drugs. Additionally, a number of carriers have been specifically engineered for nucleic acid delivery through electrostatic interaction
with the negatively charged nucleic acid phosphate backbone. These carriers are typically composed of cationic polymers or cationic
lipids that form nucleic acid complexes known as polyplexes and lipoplexes, respectively. Alternatively, lipopolyplexes, composed
of polymer–nucleic acid cores and a lipid shell, also are employed, benefiting from the unique properties of both materials. Nucleic
acid carriers must protect DNA from enzymatic degradation, promote cellular uptake, and stimulate intracellular unpackaging.
Parameters such as polymer hydrophobicity/hydrophilicity, charge density, biodegradability, and the molecular weight can be
adjusted to optimize the carrier.
In most applications, micro and nanocarriers are delivered via bolus injection, and controlled delivery is achieved by either
passive or active targeting. Passive targeting is commonly reliant on carrier size. For instance, microparticles are unable to cross
the majority of biological barriers, and therefore, microparticles are most effective when applied at the delivery site where they avoid
clearance and can remain present for weeks. In contrast, nanoparticles have the capacity to pass through biological barriers such as
the cellular membrane but are typically cleared from the body within days and commonly accumulate in the liver, spleen, and/or
kidney. Prolonged circulation and evasion of the mononuclear phagocyte system have been achieved through the incorporation of
hydrophilic polymers or glycolipids with flexible chains, such as PEG or GM1, which occupy the immediate area adjacent to the
carrier and sterically block interactions with serum proteins and cell-surface receptors.
Multiple active targeting strategies also have been employed in regenerative engineering to target specific tissue components and/
or cell types. Active targeting is typically achieved through biologically inspired strategies including the display of cell-specific
ligands, antibodies, and/or ECM-binding peptides. For instance, multiple peptides with the capacity to bind exclusively to intact
or remodeled collagen, a primary ECM component whose state is indicative of many regenerative processes, have been identified.
Sequences have been derived from sources such as collagen-I platelet receptors, collagenase, and decorin or found through phage
display. Alternatively, CMPs have been engineered to hybridize with remodeled collagen through integration into the natural
collagen triple helix. CMP tethers have been utilized to create self-assembling nanocarriers with potential collagen-binding affini-
ties, and these tethers also can retain cytoactive factors and fluorophores within areas of excessive collagen remodeling including
joints and wounds. The control over cellular uptake also has been achieved through the inclusion of receptor-specific ligands
and cationic cell penetrating peptides. In the case of DNA delivery, where nuclear delivery is essential, additional components
such as nuclear localization signal sequences, nuclear proteins such as histones, and histone-mimetic peptides have been incorpo-
rated into carriers.
Once retained in the delivery site, micro and nanocarriers may provide an additional mechanism to modulate release via diffu-
sion or stimuli-responsive methods. Moreover, scaffold-mediated delivery systems composed of micro or nanocarriers encapsulated
increased capacity to regulate release and overcome pharmacological limitations while preserving the bioactivity or stability of the
therapeutic. For instance, therapeutic often is loaded into microgels whose diffusion-based delivery may be modulated by cross-link
density. Growth factor-loaded gelatin microcarriers have been utilized to modulate the delivery of bioactive GF including insulin-
like growth factor (IGF), BMP-2, and transforming growth factor-beta 1 (TGF-beta 1) from PEG-based hydrogels. These structures
have been shown in several examples to support cartilage repair.
“Smart” or stimuli-responsive materials, coupled with microenvironmental differences in pH and/or temperature that are typical
in diseased or healing tissues, can be utilized to trigger therapeutic delivery via increased drug diffusivity or carrier destabilization.
Therapeutic molecules have been loaded into biocompatible micro and nanocarriers constructed from stimuli-responsive materials
as previously discussed. For instance, T. G. Park and coworkers prepared thermoresponsive, super-expandable pluronic/PEI poly-
plex nanocarriers with the capacity to efficiently deliver small interfering RNA (siRNA) into the cytosol and subsequently silence
the targeted mRNA. To trigger release following cellular uptake, the temperature was lowered to 20 C, and the decreased temper-
ature caused the system to undergo a phase transition with dramatic swelling that led to an 800-fold size increase and a subsequent
increase in therapeutic diffusivity. In most cases, thermoreponsive micro and nanocarriers are liposomes, polymeric micelles, or
nanoparticles typically composed of components that exhibit LCST behavior such as PNIPAAm; however, metal-based carriers
may be secondarily incorporated to provide a mechanism to raise the system temperature in response to light or magnetic and elec-
tric fields.
Additionally, pH-sensitive nanocarrier delivery systems often have advantageous features such as enhanced cellular uptake,
endosomal/lysosomal escape induced by the proton sponge effect (osmotic swelling), and the capacity for surface charge reversion.
For instance, the natural pH-responsiveness and biocompatibility of chitosan and chitosan-based nanocarriers make them ideal
candidates for controlled release in naturally acidic environments such as wounds or intracellular environments. H. Q. Zhao
and coworkers demonstrated that the grafting of chitosan onto PEI polyplexes preserved the buffering capacity of the PEI while
achieving transfection efficiencies comparable with the standard transfection agent Lipofectamine and improvements of over
40% in cell viabilities in chondrocyte and synoviocyte cell lines.
Other stimuli responses also have been demonstrated. M. O. Sullivan, T. H. Epps, III and coworkers have fabricated siRNA
mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polyplexes with the capacity to silence
targeted mRNA sequences in response to UV-stimulated siRNA release. Alternatively, M. F. Mieler and coworkers reported light-
responsive release of a model drug from gold’silver nanorods coated with DNA-cross-linked polymeric shells. Within these systems,
the nanorods acted as photothermal convertors absorbing light near infrared that was then converted to heat and thus elevated
temperatures and membrane permeability. In other examples, cell-triggered release of polyplex has been achieved through integra-
tion into protease-labile scaffolds or the use of proteolytic linkages. For instance, M. O Sullivan, K. L. Kiick, and coworkers fabricated
DNA–PEI polyplex collagens in which release/retention of polyplex was tailored through variation of CMP display on the polyplex.
The transfection levels in the murine NIH/3T3 fibroblast cell line were approximately an order of magnitude greater when protease
expression was stimulated via TNF-alpha treatments.
Types of Therapeutic Molecules in Drug and Gene Delivery
Within the field of regenerative engineering, therapeutics can be categorized as small molecular drugs, proteins, or nucleic acids
(Fig. 2). The primary objective is to augment or enable the complex reparative process. Each therapeutic type has its own distinct
benefits and delivery obstacles.
Small-Molecule Drugs
The majority of small-molecule drugs have been identified based on their capacity to modulate key signaling pathways involved in
tissue repair. For instance, pyrvinium is an FDA-approved drug used to treat infection by inhibiting the Wnt pathway, which drives
the expression of several inflammatory molecules that are upregulated during bacterial infections. P. P. Young and coworkers
demonstrated that daily administration of pyrvinium into subcutaneous, PVA sponges in mice generated better-organized and
better-vascularized granulation tissue and an increased tissue proliferative index. The same group also evaluated the therapeutic
value of pyrvinium in murine myocardial repair and demonstrated that the administration of a single dose via coronary artery liga-
tion reduced adverse cardiac remodeling based on the decrease in the left ventricular internal diameter in diastole relative to the
control. Alternatively, deferoxamine is an FDA-approved iron chelator that has been in clinical use for decades. Specifically, it is
Fig. 2 A summary of the therapeutic types in regenerative engineering and potential obstacles to efficient delivery.
known to aid in wound healing by inhibiting HIF-1alpha degradation, and in so doing, it decreases oxidative stress in the wound
bed and stimulates subsequent decreases in necrotic tissue and enhanced wound healing in murine wounds. In addition to anti-
inflammatory drugs such as pyrvinium and NSAIDs and chelators for reducing oxidative stress, another class of drugs commonly
used in regenerative engineering is statins, which are broadly characterized as lipid-lowering agents. The application of statins
demonstrated promising results in multiple regenerative engineering applications including wound healing, cardiovascular repair,
and bone growth. For instance, the anabolic effect of both simvastatin and lovastatin increased the expression of BMP-2 mRNA. In
general, small-molecule drugs have lower manufacturing costs and higher stability relative to biologics, which enables longer shelf
life, increased half-lives within the body, and more flexibility in regards to chemical modification/conjugation. Furthermore, the
treatments are less complicated and already well understood by the FDA and the pharmaceutical industry. However, small-
molecule drugs are typically nonspecific and have been documented in many cases to have adverse systemic effects and rapid
clearance.
Protein-Based Therapeutics
The application of protein-based therapeutics has been enabled by the advent of cost-effective recombinant DNA technology, which
is used to clone, express, and purify proteins of interest such as growth factors and antibodies. Growth factors play vital roles in the
pathways underlying healing within all tissues, and the delivery of growth factors can significantly augment the reparative process.
On the other hand, numerous monoclonal antibodies with the capacity to target specific proteins and cells have been identified.
This targeting capacity has been utilized for controlled therapeutic delivery and pathway inhibition.
Growth factors
Growth factors are signaling proteins capable of triggering specific cellular behaviors including migration, proliferation, and differ-
entiation, through interaction with transmembrane receptors. Most preclinical trials to date have been conducted with angiopoie-
tins, which promote blood-vessel maturation and stability; BMP-2, which stimulates osteogenic differentiation and cell migration;
fibroblast growth factor (FGF), which encourages migration, proliferation, and survival of endothelial cells; PDGF, which regulates
endothelial cell proliferation and migration; and VEGF, which promotes angiogenesis. Due to the innate role of growth factors in
the healing cascade, growth-factor administration has been demonstrated to reestablish endogenous healing responses through
coordination of multiple regenerative cascades.
Despite their promise, growth factors typically have incredibly short half-lives within the body, generally on the order of hours
(i.e., for FGF-2 7.6 h, for VEGF165 1.5 h, and for PDGF-BB < 4 h). The instability of these protein-based therapeutics is exacer-
bated in injured areas where protease activity is elevated, and in the case of chronic wounds, cells in the wound bed often exhibit
alterations in behavior leading to limited growth-factor responses. To overcome growth-factor instability in regenerative medicine
applications, extraphysiological and repetitive dosage regimens often are applied; however, these types of dosing approaches
increase the danger of growth-factor toxicity, off-target responses, and oncogenicity. Protein engineering can be used to enhance
growth-factor stability, but preserving engineered growth-factor bioactivity is not trivial. Moreover, the delivery of multiple growth
factors at different times and concentrations is required to fully recapitulate the regenerative process and account for the synergistic
nature of growth factors.
Monoclonal antibodies
Therapeutic monoclonal antibodies have the capacity to specifically bind to cells or proteins. Accordingly, this targeting capacity has
been used in a variety of contexts in drug delivery. In regenerative engineering applications, a common application of monoclonal
antibodies is to bind to growth factors involved in pathological pathways and thereby inhibit their activity. For instance, the appli-
cation of the FDA-approved anti-VEGF agents bevacizumab, ranibizumab, pegaptanib, and aflibercept have revolutionized the
treatment of neovascularization retinal disorders by inhibiting angiogenesis. The application of anti-TGF-beta 1, 2, and 3 in wound
healing and prevention of hypertrophic scar formation has been widely explored, and findings in a rabbit ear impaired healing
model strongly suggested that TGF-beta 1, 2, and 3 concentrations are important during distinct periods during both early and
late stages of repair. Additionally, the balance of TGF-beta 1, 2, and 3 plays a distinct role in hypertrophic scar formation. Mono-
clonal antibodies generally have longer half-lives than growth factors, with typical half-lives on the order of days as opposed to
hours; however, the large size of the antibodies typically causes poor perfusion and complications such as cytokine release
syndrome and immunogenicity. Many of these obstacles can be overcome though delivery using smart delivery systems, as previ-
ously discussed.
Nucleic Acids
Nucleic acid-based strategies in regenerative engineering can generally be categorized as gene or RNA interference (RNAi) therapies.
Both types of therapies can be used to reprogram cells in vivo or ex vivo, and nucleic acid delivery approaches can also be used to
increase or reduce expression of proteins controlling wound-healing cascades. Nucleic acid-based strategies can be especially bene-
ficial in regenerative engineering because they can induce cell-mediated expression/delivery of signaling molecules such as growth
factors, and the cellular production/secretion processes mimic those involved during endogenous delivery of these molecules.
Genes
Gene delivery can be used to overcome some of the shortcomings of protein-mediated delivery. The successful delivery of thera-
peutic plasmid DNA or viral vectors into target cells facilitates the expression of fresh, bioactive proteins with appropriate posttrans-
lational modifications, and these delivery methods also microlocalize delivery. Accordingly, the delivery of growth-factor genes, as
opposed to the direct delivery of growth-factor proteins, has been shown to have significant therapeutic benefits within the wound
environment.
Viral vectors are the most common tool for delivering genetic material into a cell, owing to the innate ability of viruses to trans-
fect cells. Numerous viruses have been used as vectors, and viral vectors can be purposefully chosen to facilitate permanent or
temporal transfection and to a certain degree enable targeting of a specific cell host. To date, most gene-therapy clinical trials utilize
retroviruses to achieve long-lasting transgene expression and adenoviruses to obtain transient transfection. While viral vectors
obtain high-efficiency transfection, off-target delivery and immunogenic and oncogenic risk factors have greatly limited their clinical
success. Plasmid DNAs are attractive tools in gene delivery due to their ease of modification and the ability of plasmids to self-
replicate in bacterial hosts. Moreover, tissue-specific promoters may be incorporated to achieve localized delivery; however, plasmid
DNA requires a carrier to protect it from nucleases and enable efficient transfection.
RNAi
Alternatively, nucleic acids with the capacity to silence protein expression, such as siRNA or microRNA, are often explored in regen-
erative engineering. These approaches exploit the ability of noncoding small RNAs to knock down gene expression through binding
to specific mRNAs and tagging them for nuclease destruction (siRNA) or to reduce gene expression through physically blocking
translation into proteins (microRNA). Like plasmid DNA, small RNAs require a delivery vehicle to protect them from degradation
and induce cellular uptake. Unlike plasmid DNA, the active compartment for small RNAs is the cytosol; therefore, nuclear transport
is not required. In multiple regenerative engineering applications, excessive proteolytic activity makes MMPs an attractive target for
small-RNA delivery. For example, H. S. Yoo and coworkers recently demonstrated that the MMP-responsive delivery of MMP-2
siRNA in diabetic ulcer models has the capacity to reduce wound closure time, decrease MMP-2 expression, and increase cytokeratin
levels as compared with nontreated controls. Similar to anti-VEGF monoclonal antibodies, VEGF and its receptor are also intuitive
targets for knockdown, and small-RNA approaches also have used to reduce excessive angiogenesis in ocular disorders to prevent
macular degeneration. Additionally, microRNAs have been demonstrated to play a prominent role in the survival of cardiac progen-
itor cells, and thus, their delivery shows potential in cardiac regeneration. Major obstacles for small-RNA delivery include its high
cost, potential off-target effects, inefficient uptake into cells, and lack of effective release within the delivery site.
Delivery System Selection/Comparison
Within this review, several types of delivery systems used in regenerative engineering have been discussed. To determine the correct
type of delivery system for a given application, several factors must be simultaneously considered. These include the physicochem-
ical properties of the therapeutic, the pathology of the delivery site, and the potential systemic versus localized effects of the delivery
system itself. Typically, a therapeutic for use in regenerative engineering is chosen based on its impact on healing cascades. For
instance, NSAIDs such as ibuprofen often are administered due to their well-recognized ability to reduce pain and inflammation
through blocking cyclooxygenase (COX) enzymes, which trigger the formation of prostaglandin-inflammatory signaling molecules.
In many applications, growth factors or the genes that encode them are delivered due to their roles in cellular differentiation, prolif-
eration, migration, and other behaviors essential for tissue repair. Alternatively, siRNA can be delivered to silence or reduce the
expression of proteins inhibiting regeneration such as overexpressed proteases in chronic wounds.
Once a therapeutic is selected, a delivery system must be chosen based on drug properties such as molecular weight, aqueous
solubility, charge, serum stability, and the characteristics of the targeted delivery site. For instance, hydrophobic drugs may need
to be loaded into liposomes, micelles, or the hydrophobic cores of electrospun fibers to enhance their solubility within aqueous
biological systems. Similarly, the half-life of the therapeutic should be considered, and precautions should be taken, as appropriate,
to preserve bioactivity. Precautions may include direct alteration of the therapeutic to enhance its stability, immobilization to
a substrate to reduce aggregation, or loading into polymeric- or lipid-based particles (or the cores of electrospun scaffolds) to reduce
drug contact with biological fluids. On the other hand, charged drugs may be adsorbed or immobilized onto polymeric or ceramic
scaffolds to promote localized delivery, and in the specific case of nucleic acid delivery, charge can be used to form a complex that
preserves integrity and promotes cellular uptake.
Considerations of the delivery site and disease pathology are also of great value in therapeutic delivery. Extracellular delivery is
commonly achieved utilizing bulk or microcarrier delivery systems, whereas intracellular delivery is commonly reliant on nanocar-
riers or viral vectors. However, in tissue engineering, the application of implantable or injectable 3-D scaffolds may provide structure
for cellular adhesion and ECM deposition and a platform for controlled bioactive cue delivery. On the other hand, systemic appli-
cations of targeted micro or nanocarriers can provide a less invasive approach for reaching less accessible tissues or cell types. Addi-
tionally, specific pathologies often are associated with characteristic microenvironmental changes that can be used to modulate
release. For instance, acute wounds progress from an alkaline state to an acidic state when healing begins; however, chronic wounds
beds continue to have an elevated pH over a prolonged duration. The pH of stage 1 pressure ulcers has a pH similar to that of intact
skin (pH 5.4–5.6), while the pH values of stage 2 and 3 ulcers have been reported as 6.9 and 7.6, respectively. The microenviron-
mental differences in pH can be used as a logical trigger for therapeutic release. Moreover, the enhanced proteolytic activity, ECM
degradation, and ROS production during the early stages of acute healing and within chronic wounds can be harnessed to trigger
release in responsive systems.
In applications in which “on–off” delivery is of value, the accessibility of the delivery site to external stimuli should also be
considered. For instance, the application of UV light, electric current, and heat/cold induction are sensible means for modulating
responsive delivery for topical applications; however, for certain applications, exogenous stimuli with enhanced penetration depth
are required, such as near infrared radiation (NIR), radiofrequency electromagnetic fields (EMFs), and ultrasound. For instance,
high-power NIR was reported to penetrate at least 3 cm into the brain when wavelengths of 810 and 980 nm with 10–15 W power
were applied, which demonstrates the potential to pass through skin and bone and remotely trigger therapeutic release in traumatic
brain injury. Furthermore, at 400 kHz, 99% of EMF radiation penetrates into 15 cm of tissue, and background heating of the tissue
is insignificant.
The localized and systemic impacts of the delivery system itself must also be taken into account. For instance, biocompatibility is
vital for avoiding immune responses. Moreover, delivery systems may be engineered to have direct therapeutic value on their own.
In many applications, delivery systems are purposefully designed to mimic the ECM and subsequently perform its native roles in
facilitating cellular attachment, proliferation, and phenotypic commitment. Biomimicry is typically achieved through incorporation
of native ECM components, such as collagen, or addition of mimetic peptides that contain either ECM-derived cell binding sites or
ECM structures such as the collagen triple helix. For instance, collagen-based materials are commonly employed as both gels and
sponges in regenerative medicine, where they can enhance healing by serving as both sacrificial substrates for proteolytic activity and
scaffolds for cellular adhesion. Other studies have highlighted important physical characteristics that determine the ability of
a delivery system to trigger cellular responses. For instance, the diameter and orientation of electrospun fibers within tubular nerve
guides and wound dressings were determined to impact nerve regrowth and wound closure, respectively.
Therapeutic Delivery in Chronic Wound Care

Delivery System
Impaired healing is the result of alterations in normal physiological processes typically caused by aging or diabetes. Acute wound
repair proceeds through four distinct but overlapping phasesdhemostasis, inflammation, proliferation, and remodeling; however,
chronic wounds become stalled in prolonged, exaggerated inflammatory phases. As a result, the wound bed is characterized by
excessive oxidative stress, proteolytic activity, and bacterial infections. Given the localized nature of this application coupled
with the inherent lack of blood flow within chronic wounds, bulk, localized therapeutic strategies are highly beneficial. The appli-
cation of a 3-D scaffold such as a hydrogel or electrospun fiber mat has been used to achieve spatiotemporal control over delivery
while also providing additional therapeutic benefit by serving as an analog for the ECM though facilitating cellular adhesion and
phenotypic commitment. For instance, the ECM component collagen is a primary component in many wound dressing approved
by the FDA for diabetic foot ulcer care, including Allograft, Dermagraft, and Promogran. These collagen-based products have been
demonstrated to increase fibroblast proliferation and decrease protease activity. For instance, wound closure was documented in
91% of DFU patients treated with Dermagraft within 12 weeks, whereas only 78% of patients exhibited the same outcome in
a control group treated with the conventional wet-to-dry dressing approach.
Small-Molecule Drug Delivery

The primary goal of any therapeutic approach in chronic wound healing is to replicate or enhance normal healing processes. A
plethora of small-molecule drugs and biologics have shown potential in restoring coordination between the intracellular, inter-
cellular, and extracellular pathways vital for repair. For instance, as previously discussed, FDA-approved small-molecule drugs
such as pyrvinium are used to treat wound infection through inhibiting the expression of several inflammatory molecules,
and deferoxamine (DFO) is used to reduce oxidative stress in the wound bed through inhibiting hypoxia-inducible factor 1-
alpha degradation. However, systemic delivery of these agents is not a viable option for treating chronic wounds due to the
toxicity of these agents and their short plasma half-life exhibited in in vitro and in vivo preclinical studies. Accounting for the
delivery site and the properties of the drug, G. C. Gurtner and coworkers developed a local transdermal drug-delivery system
of potential clinical value as pictured in Fig. 3. To enable the relatively large and hydrophobic DFO to penetrate the stratum cor-
neum, the lipophilic outermost layer of skin, DFO/polyvinylpyrrolidone (PVP) complex-loaded reverse micelles were fabricated
and encapsulated within a slow-releasing ethyl cellulose matrix. Upon topical application of the DFO patch to the skin, the
reverse micelles were released from the degradable polymeric matrix, and they subsequently penetrated through the stratum cor-
neum into the hydrophilic, aqueous environment of the dermis; this process caused the reverse micelle to disintegrate, enabled
the DFO/PVP complex to disassociate, and ultimately freed DFO for transdermal delivery. This system was successfully used in
diabetic murine models to prevent ulcer formation and promote diabetic wound healing through reduction of hyperglycemia-
induced oxidative stress. As our understanding of the fundamental and intricate pathways underlying repair improves, additional
drugs of value in chronic wound repair continue to be uncovered.
Fig. 3 (A) Development of a transdermal drug delivery system for DFO. DFO aggregates with PVP and surfactants to form reverse micelles (RMs).
RMs are dispersed in the polymer ethyl cellulose. After release from the polymer matrix the RMs enter the stratum corneum and disintegrate. PVP
dissolves and DFO is delivered to the dermis. DFO TDDS improves healing of diabetic ulcers. (B) Full-thickness ulcer wounds of diabetic mice treated
with a transdermal DFO TDDS formulation or vehicle control (n ¼ 10). TDDS were replaced every 48 h. Duscher, D., Neofytou, E., Wong, V. W.,
Maan, Z. N., Rennert, R. C., Inayathullah, M., Januszyk, M., Rodrigues, M., Malkovskiy, A. V., Whitmore, A. J., Walmsley, G. G., Galvez, M. G., Whit-
tam, A. J., Brownlee, M., Rajadas, J. and Gurtner, G. C. (2015). Transdermal deferoxamine prevents pressure-induced diabetic ulcers. Proceedings of
the National Academy of Sciences of the United States of America 112, 94–99.
Protein-Based Therapeutic Delivery

Protein-based therapeutics such as growth factors and monoclonal antibodies have been extensively studied in chronic wound clin-
ical trials. The delivery of various growth factors from polymeric hydrogels and electrospun scaffolds has been demonstrated to
promote faster wound closure, angiogenesis, and collagen deposition within various impaired healing animal models. The objective
of the therapy is to replace the degraded growth factors in the hostile wound bed in order to recapitulate normal repair; therefore,
delivery systems must promote sustained growth-factor delivery and bioactivity by overcoming the inherently short half-lives of
most of these factors in the protease-rich wound environment. In some cases, delivery systems are engineered to codeliver multiple
growth factors, typically through encapsulating one therapeutic directly into a polymeric scaffold and preloading the second ther-
apeutic into micro or nanocarriers that can act as a secondary barriers to regulate diffusion or facilitate stimuli-triggered release. Dual
growth-factor delivery often enhances healing in animal wound models as compared with delivery of only one growth factor;
however, in clinical delivery, synergistic interactions between different growth factors and the ECM often are not accounted for.
As shown in Fig. 4, J. A. Hubbell and coworkers presented a multifaceted approach for engineering the cellular microenvironment
to greatly enhance VEGF-A and PDGF-BB levels in wound healing or alternatively BMP-2 and PDGF-BB levels in bone repair. Specif-
ically, a multifunctional recombinant fibronectin fragment engineered to contain (i) a factor XIIIa substrate fibrin-binding
sequence; (ii) the 9th–10th type III FN repeat (FN III9–10) containing a major integrin-binding domain; and (iii) the 12th–
14th type III FN repeat (FN III12–14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2
was covalently cross-linked into a fibrin matrix. The modified fibrin was loaded with VEGF-A and PDGF-BB, and this loaded scaffold
was shown to significantly enhance angiogenesis leading to healing in a diabetic murine wound model as compared with fibrin
alone, modified fibrin without growth factors, or growth factors that were individually applied without fibrin. These findings high-
light the potent synergistic signaling between certain integrins and growth-factor receptors and show the importance of harnessing
these biomimetic interactions when designing biomaterials and delivery systems. Dual regulation through integrin signaling and
growth-factor signaling also has been shown to present a viable strategy to attenuate growth-factor potency in other regenerative
engineering applications such as osteodifferentiation. Alternatively, therapeutic monoclonal antibodies are utilized to neutralize
proteins that are elevated in chronic wound repair versus acute wound repair, including TGF-beta, flightless, or interleukin-6.
For instance, N. H. Voelcker and coworkers demonstrated significantly enhanced wound healing when flightless I neutralizing anti-
bodies were delivered from silicon nanoparticles, as compared with a no-treatment control or wounds in which the antibody alone
was administered.
Gene-Based Therapeutic Delivery

The capacity of nucleic acid delivery to reprogram cells is of particular value in chronic wound repair. Dr. David Margolis reported
the results of the first clinical trial in humans for gene therapy in wound healing in 2000. Chronic wound gene therapies have
become increasingly clinically relevant over the past few decades due to their potential ability to enhance or inhibit protein expres-
sion and restore wound-bed homeostasis. For instance, bioactive scaffold-mediated delivery of genes encoding for PDGF, VEGF,
FGF, and/or EGF has been used to upregulate protein expression and facilitate enhanced angiogenesis, epithelization, and overall
faster wound closure times within impaired animal wound models; however, vector escape and immune responses have greatly
inhibited the clinical translation of these therapies. RNA interference therapies have encountered similar obstacles. Biologically
inspired approaches that not only account for the wound environment but also harness pathways known to occur in excess in
chronic versus acute wounds have great potential in restoring wound-bed homeostasis. For instance, MMPs are expressed in excess
in chronic wounds. Under normal wound-healing conditions, these proteases facilitate ECM remodeling and promote wound
repair, yet in chronic wounds, the overexpression of these factors contributes to the pathology of the wound bed. M. O. Sullivan,
K. L. Kiick, and coworkers harnessed ECM remodeling to achieve efficient gene delivery by stably integrating DNA polyplexes into
collagens using adjustable CMP tethers, such that gene release and expression were dependent upon MMP protease expression and
subsequent collagen turnover. Furthermore, mechanistic studies from this group suggested that CMP–collagen hybridization per-
sisted after scaffold release, such that natural endocytic clearance mechanisms for collagen facilitated higher levels of CMP-polyplex
uptake and expression in cells. Alternatively, H. S. Yoo and coworkers developed an MMP-inspired treatment (Fig. 5) in which anti-
MMP siRNA polyplexes were displayed on fibrous electrospun scaffolds using MMP-labile peptide linkers. These structures were
shown to silence MMP expression and encourage wound recovery in a diabetic murine model. The continued development of
controlled, biologically inspired nucleic acid therapies has great promise in chronic wound repair.
Current Obstacles and Future Applications
Within the field of regenerative engineering, an ever-growing understanding of the processes underlying repair is rapidly expanding
the library of possible therapeutics; however, delivery remains a major concern. While beneficial under the right conditions, the
therapeutics required to promote regeneration commonly cause serious adverse side effects ranging from blindness to cancer
when delivered to off-target locations. To overcome these obstacles, better control over therapeutic delivery is a necessity. The
continued development of “smart” polymers and inorganic particles has great potential in overcoming many of these dangers.
Moreover, there is a great need for new delivery strategies that better replicate the complex, multicomponent presentation of healing
factors during the normal course of tissue healing. Strategies for mediating multitherapeutic delivery and better recapitulating
Regenerative Engineering j Drug and Gene Delivery for Regenerative Engineering
Fig. 4 (A) A multifunctional recombinant FN fragment is engineered to display the integrin-binding domain (FN III9–10) linked to the GF-binding domain (FN III12–14) and to comprise the substrate sequence a2PI1–8
for factor XIIIa. The fragment is covalently cross-linked into a fibrin matrix during the natural polymerization process of fibrin via the transglutaminase activity of factor XIIIa. Delivering VEGF-A165 and PDGF-BB within
functionalized fibrin matrices enhances skin wound healing in diabetic mice compared to treatment with fibrin only, fibrin functionalized with FN III9–10/12–14 only, fibrin containing GFs only, and fibrin functionalized
with FN III9–10/12–14 containing GFs. (B) After 7, 10, and 15 days, wound closure and granulation tissue area were evaluated by histology. (C) Wound histology (hematoxylin and eosin staining) at 10 days. Black
arrows indicate wound edges; red arrows indicate tips of epithelium tongue. The granulation tissue (pink-violet) is characterized by a large number of granulocytes with nuclei that stain in dark-violet or black. Muscle
under the wounds is stained in red. Fat tissue appears as transparent bubbles. Scale bar, 1 mm. Higher magnification (5) of the granulation tissue is shown on the right. Martino, M. M., Tortelli, F., Mochizuki, M.,
Traub, S., Ben-David, D., Kuhn, G., Muller, R., Livne, E., Eming, S. and Hubbell, J. A. (2011). Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing.
Science Translational Medicine 3, 100RA89.
581
Fig. 5 (A) Schematic showing the preparation of the MMP-responsive nanofibrous mesh and the proposed mechanism for delivering siRNA into
diabetic ulcers with the modified nanofibers. (B) Wound recovery of diabetic ulcers in C57BL/6 mice where wound recovery rate was calculated by
comparing the closed wound area. (C) In vivo expression levels of MMP-2, keratin 5, keratin 14 and GAPDH in re-epithelialized tissues at day 3 and
day 7. (B) Normalized expression levels of MMP-2, keratin 5 and keratin 14 with respect to GAPDH expression based on the electrophoresis results
in a. * Indicates a statistical significance (P > .05). Kim, H. S. and Yoo, H. S. (2013). Matrix metalloproteinase-inspired suicidal treatments of diabetic
ulcers with siRNA-decorated nanofibrous meshes. Gene Therapy 20, 378–385.
biologically inspired cellular interactions with the delivery system have the potential to enhance therapeutic potency and in turn
reduce therapeutic dosage requirements. Pursuit of better, holistic delivery strategies may overcome the clinical barriers that
have largely inhibited the commercial use of growth factors and prevented the approval of gene-based therapies.
Further Reading
Andreadis, S. T., & Geer, D. J. (2006). Biomimetic approaches to protein and gene delivery for tissue regeneration. Trends in Biotechnology, 24, 331–337.
Briquez, P. S., Hubbell, J. A., & Martino, M. M. (2015). Extracellular matrix-inspired growth factor delivery systems for skin wound healing. Advances in Wound Care, 4, 479–489.
Cao, S. G., Hu, B. H., & Liu, H. Q. (2009). Synthesis of pH-responsive crosslinked poly styrene-co-(maleic sodium anhydride) and cellulose composite hydrogel nanofibers by
electrospinning. Polymer International, 58, 545–551.
Cui, W. G., Qi, M. B., Li, X. H., Huang, S. Z., Zhou, S. B., & Weng, J. (2008). Electrospun fibers of acid-labile biodegradable polymers with acetal groups as potential drug carriers.
International Journal of Pharmaceutics, 361, 47–55.
Davis, H. E., & Leach, J. K. (2011). Designing bioactive delivery systems for tissue regeneration. Annals of Biomedical Engineering, 39, 1–13.
Foster, A. A., Greco, C. T., Green, M. D., Epps, T. H., & Sullivan, M. O. (2015). Light-mediated activation of siRNA release in diblock copolymer assemblies for controlled gene
silencing. Advanced Healthcare Materials, 4, 760–770.
Fu, G. D., Xu, L. Q., Yao, F., Li, G. L., & Kang, E. T. (2009). Smart nanofibers with a photoresponsive surface for controlled release. ACS Applied Materials & Interfaces, 1,
2424–2427.
Ganta, S., Devalapally, H., Shahiwala, A., & Amiji, M. (2008). A review of stimuli-responsive nanocarriers for drug and gene delivery. Journal of Controlled Release, 126, 187–204.
Hastings, C. L., Roche, E. T., Ruiz-Hernandez, E., Schenke-Layland, K., Walsh, C. J., & Duffy, G. P. (2015). Drug and cell delivery for cardiac regeneration. Advanced Drug Delivery
Reviews, 84, 85–106.
Hoare, T. R., & Kohane, D. S. (2008). Hydrogels in drug delivery: Progress and challenges. Polymer, 49, 1993–2007.
Jeon, O., Song, S. J., Kang, S. W., Putnam, A. J., & Kim, B. S. (2007). Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-
conjugated poly(L-lactic-co-glycolic acid) scaffold. Biomaterials, 28, 2763–2771.
Jin, G. R., Prabhakaran, M. P., & Ramakrishna, S. (2014). Photosensitive and biomimetic core–shell nanofibrous scaffolds as wound dressing. Photochemistry and Photobiology,
90, 673–681.
Kim, H. S., & Yoo, H. S. (2013). Matrix metalloproteinase-inspired suicidal treatments of diabetic ulcers with siRNA-decorated nanofibrous meshes. Gene Therapy, 20, 378–385.
Lai, H. J., Kuan, C. H., Wu, H. C., Tsai, J. C., Chen, T. M., Hsieh, D. J., & Wang, T. W. (2014). Tailored design of electrospun composite nanofibers with staged release of multiple
angiogenic growth factors for chronic wound healing. Acta Biomaterialia, 10, 4156–4166.
Lee, K., Bae, K. H., Lee, Y., Lee, S. H., Ahn, C. H., & Park, T. G. (2010). Pluronic/polyethylenimine shell cross linked nanocapsules with embedded magnetite nanocrystals for
magnetically triggered delivery of siRNA. Macromolecular Bioscience, 10, 239–245.
Lu, H. D., Dai, Y. H., Lv, L. L., & Zhao, H. Q. (2014). Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis. PLoS One,
9, e84703.
Martino, M. M., Tortelli, F., Mochizuki, M., Traub, S., Ben-David, D., Kuhn, G., Muller, R., Livne, E., Eming, S., & Hubbell, J. A. (2011). Engineering the growth factor micro-
environment with fibronectin domains to promote wound and bone tissue healing. Science Translational Medicine, 3, 100RA89.
Munsell, E. V., Ross, N. L., & Sullivan, M. O. (2016). Journey to the center of the cell: Current nanocarrier design strategies targeting biopharmaceuticals to the cytoplasm and
nucleus. Current Pharmaceutical Design, 22, 1227–1244.
Nie, H. M., & Wang, C. H. (2007). Fabrication and characterization of PLGA/HAp scaffolds for delivery of BMP-2 plasmid composite DNA. Journal of Controlled Release, 120,
111–121.
Nie, H., Soh, B. W., Fu, Y. C., & Wang, C. H. (2008). Three-dimensional fibrous PLGA/HAp composite scaffold for BMP-2 delivery. Biotechnology and Bioengineering, 99,
223–234.
Ordeig, O., Chin, S., Kim, S., Chitnis, P. V., & Sia, S. K. (2016). An implantable compound-releasing capsule triggered on demand by ultrasound. Scientific Reports, 6, 22803.
Saravanakumar, G., Kim, J., & Kim, W. J. (2017). Reactive-oxygen-species-responsive drug delivery systems: Promises and challenges. Advanced Science, 4, 1600124.
Schmaljohann, D. (2006). Thermo- and pH-responsive polymers in drug delivery. Advanced Drug Delivery Reviews, 58, 1655–1670.
Turner, C. T., McInnes, S. J. P., Melville, E., Cowin, A. J., & Voelcker, N. H. (2017). Delivery of flightless I neutralizing antibody from porous silicon nanoparticles improves wound
healing in diabetic mice. Advanced Healthcare Materials, 6.
Urello, M. A., Kiick, K. L., & Sullivan, K. L. (2014). A CMP-based method for tunable, cell-mediated gene delivery from collagen scaffolds. Journal of Materials Chemistry B, 2,
8174–8185.
Ethics of Issues and Stem Cell Research: the Unresolved Issues
Z Master, Albany Medical College, Albany, NY, USA; and University of Alberta, Edmonton, AB, Canada
Introduction 584
In the Beginning: The Discovery of hESCs 584
Hype Surrounding Stem Cell Research 585
Personhood and the Moral Status of the Human Embryo 585
Harms to Women 587
Alternative Technologies to the Derivation of Pluripotent Stem Cells 588
Nondestructive Techniques 588
Altered Nuclear Transfer 588
Cytoplasmic Hybrids 589
Parthenotes 589
hESC-Derived Ova 589
Nonegg and Nonembryo Techniques to Derive Pluripotent Stem Cells 589
The Reality of SCR: IPSCs Are Not Replacing hESCs 590
Stem Cell Translation and Commercialization Challenges 590
Stem Cell Markets 591
Stem Cell Tourism 591
Patient and Public Perceptions 592
Strategies to Mitigate Stem Cell Tourism 592
Summary 592
Acknowledgments 593
References 593
Introduction
There has been considerable hype surrounding stem cell research (SCR). This hype, presented as exaggerated positive and negative
portrayals of SCR, can skew public perception and sway ethics discourse toward a particular point of view (Master and Resnik,
2013). Upon the isolation of human embryonic stem cells (hESCs) in 1998, ethics discussions focused primarily on the moral
status of human embryos and to a lesser degree, the physical and social harms to women egg providers. This moral discourse
had received so much attention and publicity that it influenced, at least in part, the conduct of science to identify new ways of
creating stem cells. In 2007, the discovery of induced pluripotent stem cells (iPSCs) was heralded in the popular press as absolving
the ethical issues surrounding the moral status of human embryos and harms to women (Caulfield and Rachul, 2011) and thus,
moral conversations began shifting toward the translation and commercialization of SCR, and importantly, the premature trans-
lation of stem cell interventions delivered to the public – a phenomenon called ‘stem cell tourism.’ But to date, no satisfactory
or ethically sustainable solution to the initial moral issues has been resolved. As the science of SCR continues to advance and scien-
tists discover new ways of creating embryos, the unresolved ethical issues surrounding moral status and harms to women resurface.
In this article, I will discuss how the hype surrounding stem cells has influenced ethics discourse and how both scientific discov-
eries and ethics debates influence one another. I will cover topics such as the moral status of human embryos and the harms to
women debates, the challenges of having a translation and commercialization ethos, and the ethics of stem cell tourism.
In the Beginning: The Discovery of hESCs
Stem cells can be derived from a host of sources including embryonic, fetal, and adult tissue. These cells are different than most
differentiated adult cells because they are capable of dividing into other cell types through a process called differentiation and
can self-renew creating more undifferentiated stem cells. Different cell types vary in their ability to differentiate (denoted as poten-
tiality). Adult stem cells have limited potentiality and can differentiate into a few cell types (multipotent) or a single cell type (uni-
potent). The hematopoietic stem cell is an example of a multipotent stem cell capable of differentiating into all of the cells of
hematopoietic origin including T and B lymphocytes, erythrocytes (red blood cells), megakaryocytes (platelets), eosinophils,
neutrophils, monocytes, and mast cells. hESCs are pluripotent and can divide into each and every cell type of the human body.

Regenerative Engineering j Ethics of Issues and Stem Cell Research: the Unresolved Issues 585
Totipotency is when a cell(s) (e.g., a single cell embryo) can divide to become a new organism. Both adult and hESCs are desirable
candidates for regenerative medicine to repair or replace diseased, destroyed, or degenerated cells and tissues.
The derivation of hESCs was first accomplished by isolating cells from the inner cell mass (ICM) of day 3–5 embryos called blas-
tocysts, which leads to its destruction (Shamblott et al., 1998; Thomson et al., 1998). Because of their potentiality and higher divi-
sion rate, hESCs were sought after more than adult stem cells and hence ethical debates honed in on the destruction of human
embryos. Yet another layer of scientific and ethical complexity was added to the mix – how would transplanted stem cells bypass
immune rejection?
The transplantation of allogeneic hESCs would not likely survive the host immune response. Scientists explained that cloning
hESCs to genetically match the host through a technique known as somatic cell nuclear transfer (SCNT) may bypass immune rejec-
tion. SCNT is a technique where oocytes are enucleated of their genetic material and the nucleus of a diploid cell (e.g., from
a patient’s skin cell) can be transferred into the enucleated egg. After nuclear transfer, the egg can be parthenogenically activated
either through chemical or electrical means permitting it to undergo development. At the blastocyst stage, hESCs can be derived
from the ICM and this time, the cells are a nuclear genetic match to the patient and when they are transplanted into the patient,
they should be recognized as self and will not be immune rejected. This process was commonly referred to as therapeutic or research
cloning. Instead of deriving hESCs, if the cloned embryo was transferred into a woman, then the resulting child would be a living
clone of the patient – a process referred to as reproductive cloning, the technique used to clone Dolly (Campbell et al., 1996). Yet
both reproductive cloned animals and cloned animal ESCs by SCNT exhibit a series of problems (reviewed in Master et al., 2007a).
Performing SCNT to derive hESCs has recently been accomplished and the technique has been made more efficient so fewer ova are
required (Tachibana et al., 2013).
Hype Surrounding Stem Cell Research
With the advantage of hindsight, it is safe to say that many players engaged in the debate (e.g., scientists, reporters, governmental
spokespersons, and politicians) made outlandish exaggerations of stem cell and cloning research merely as a means to swing moral
discourse and scientific practice in a particular direction, or simply halt the science (Nisbet, 2004; Nisbet and Lewenstein, 2002).
Hype can be positive or negative exaggerated representations predicting the future of SCR (Master and Resnik, 2013). There are
several ethical issues with hyping SCR: (1) as science is communicated to the public primarily through the media (Nelkin,
1995), hyping SCR can distort the public’s understanding of science; (2) positive portrayals of cures can promote premature trans-
lation of research (Knowles, 2009; Ryan et al., 2010); and (3) unmet promises can lead to a loss of public trust in SCR (Ogbogu,
2006; Doerflinger, 2008; Downey and Geransar, 2008; Bubela et al., 2009; Wilson, 2009).
Several popular media sources have demonstrated positive and negative hype as it relates to SCR (Kitzinger and Williams, 2005;
Zarzeczny et al., 2010; Murdoch et al., 2011). The positive hype surrounding SCR has focused on (1) promising cures for many
diseases; (2) creating gametes that may be used in assisted reproduction (Master, 2006); (3) developing new diagnostic and ther-
apeutic means to treat cancer based on cancer SCR; and (4) being a major economic engine (Caulfield, 2010). The negative hype
surrounding SCR and cloning has focused on cloning human armies, creating grotesque animal–human chimeras, the commodi-
fication of life including human ova, and killing unborn life (tantamount to killing babies). Despite the hyped promises and perils
of SCR, several cogent moral arguments can be made to permit or prohibit the use of embryos for SCR. (Ethical issues related to
reproductive cloning, the banking of stem cells, and research ethics are beyond the scope of this article.)
Personhood and the Moral Status of the Human Embryo
From 1998 till 2007, the moral status of the human embryo and defining personhood dominated moral discussions on SCR. Not
surprisingly, questions about when life begins and what is the moral status of human embryos stemmed from earlier arguments on
abortion, which created the appropriate conceptual basis for a similar set of arguments delivered by the same set of actors (Jasanoff,
2005). Persons are ascribed inalienable rights such as the right to life, liberty, and security; interference of privacy; freedom of
thought, belief, opinion, and expression; and freedom from persecution, among others (UN General Assembly, 1948). If persons
are given the right to life, it was believed that if embryos were labeled as persons, they deserve protection from being destroyed. The
attention given to defining personhood and ascribing moral characteristics of human embryos took foothold and became instru-
mental as it was believed to be the key to winning the debate. One logical flaw in this line of argument is that being a person does
not automatically guarantee moral protection and not being a person does not automatically mean one can treat it as a means for
research. One’s view of the moral status of human embryos, whether it is religious or secular, would at minimum, influence, if not
fully guide, their views on the ethical permissibility of SCR. This is likely to include whether embryos can be purposely created for
SCR, whether only excess embryos can be used, or whether embryos cannot be used for SCR.
Human embryos can be created for different purposes. They may be created specifically for reproductive purposes (assisted
reproduction) or they may be created specifically for research. Because assisted reproductive procedures require the creation of
several embryos of which one or more may be transferred into a woman in order to achieve pregnancy, excess or surplus embryos
may remain. Surplus embryos originally created for reproductive purposes that are no longer needed can be donated for research.
Some observers of the SCR debate maintain that it is ethical to use only surplus embryos, but not create embryos specifically for SCR
586 Regenerative Engineering j Ethics of Issues and Stem Cell Research: the Unresolved Issues
because these excess embryos would otherwise be destroyed or remain in a state of cryopreservation. Others, however, claim that
excess embryos should only be used for reproductive purposes and cannot be used for research. These positions, in part, depend on
an individual’s views of moral status.
Perhaps a central and powerful voice in US debates on SCR stems from the notion that a human embryo has full moral status
equivalent to persons. In general, upholders of this view are unlikely to accept the destruction of human embryos for any purpose
irrespective of how laudable the research goals are or for what purpose the embryos were created, although some believe that even if
embryos had full person status they can be used for hESC research (Rizzieri, 2012). Many such critics of SCR have strong moral
convictions that destroying human embryos is an illicit act because it suppresses innocent human lives that ought to be protected
(Paul, 2003; Vatican, 2008). In other words, the destruction of human embryos treats them only as a means for research and not as
ends.
According to this view, human embryos at the moment of conception have full moral status. One logical problem with this view-
point is that it conflates species identity with membership in a moral community permitting anything genetically human to be
considered moral (Marquis, 2002). Analogously, a plate of human epithelial cells would deserve the same moral status as embryos
because they are merely human. A second problem with this line of argument is when, developmentally speaking, does an embryo
become a person? Many supporters of this view would argue personhood is attained at the moment of conception, which may be
biologically defined as the moment a sperm penetrates the outer membrane of the egg and deposits its nuclear genetic material. But
at this stage, the two nuclear genomes from the male and female gametes exist separately as pronuclei and have not yet combined,
which occurs a bit later in development during syngamy. (Syngamy is the point in development where the nuclear membranes of
the two pronuclei – one from the sperm and another from the egg – breakdown and the nuclear genetic material combine and form
a single nucleus.) So perhaps personhood is attained during syngamy. But even after syngamy, an embryo can split naturally to
create twins until embryonic day 14 during gastrulation and the formation of the primitive streak when natural twinning cannot
occur afterward. Identical twins in society are considered separate persons so perhaps embryonic day 14 is the pinnacle moment
when personhood is attained. Yet perhaps when neurogenesis or other identifiable events occur throughout development are
morally important time points. Claiming personhood at discrete developmental events in a continuous developmental process
is to some degree arbitrary, and perhaps based on one’s own moral value and importance placed on particular biological points
(Green, 2002).
Instead of claiming full moral status at discrete developmental time points, another view generally used to justify full prohibi-
tion of using embryos for SCR is the potentiality view. The potentiality view states that if an embryo is allowed to develop fully, it
will inherit the moral characteristics to the status of persons because if allowed to develop and be born, it will come to have a future
like ours (FLO) and thus, should be protected from unjust killing (Marquis, 2002). As moral persons, we have values, interests, and
cherish our lives and hence it is wrong to kill potential persons as it is to kill actual persons because it deprives potential persons
with an FLO. Devolder claims that individuals who assign a moral difference permitting the use of surplus embryos, but prohibit the
creation of embryos for SCR have professed beliefs on the potentiality theory of moral status, which she claims cannot justify this
distinction (Devolder, 2005). Yet to many others, an embryo is not a person because it does not have any intrinsic value or interests,
and irrespective of whether it will eventually be a person, at its current stage of development it simply does not have full moral
status. It logically flows that if the embryo does not have moral status it does not deserve moral protection.
An argument generally made by proponents of SCR is that persons must have cognitive capacities. Because persons are conscious
and have the capacity to feel joy, pain, and other emotions is why persons make plans, have values, and take an interest in their
future lives (Feinberg, 1992). Mary Anne Warren outlines five cognitive criteria to warrant membership in the moral community:
“(1) consciousness (of objects and events external and/or internal to the being), and in particular the capacity to feel pain; (2)
reasoning (the developed capacity to solve new and relatively complex problems); (3) self-motivated activity (activity which is rela-
tively independent of genetic or direct external control); (4) the capacity to communicate, by whatever means, messages of an indef-
inite variety of types, that is, not just with an indefinite number of possible contents, but on indefinitely many possible topics; (5)
the presence of self-concepts, and self-awareness, either individual or racial, or both” (Warren, 1973). Others believe that sentience
is a requirement for moral community membership as sentient beings are rational, have an interest in avoiding pain, and are self-
aware (Bortolotti and Harris, 2005). Although none of these criteria are independently necessary, an entity lacking all of them
cannot be a moral person. Yet the problem in this line of argument is that individuals with severe developmental delay, unconscious
individuals, newborn infants, and perhaps the elderly senile may be excluded from the moral community, but society grants these
individuals the rights of persons. Moreover, sentience can also be ascribed to nonhuman animals making it morally unpermissive to
use certain animals in research or for other uses.
One issue with all of these moral theories that ascribe personhood characteristics is that they equate personhood with moral
protection and nonpersonhood with using embryos for SCR. Albeit this argument has an intuitive appeal, it is not so straightfor-
ward as we afford many nonperson entities moral protections. Second, many in the public will not clearly articulate their professed
beliefs of a certain moral theory and neatly align it to their choice on the conduct of human embryo research. Human embryos can
be respected whether they are persons or nonpersons (Steinbock, 1992, 2001). Embryos are nonsentient beings incapable of feeling
pain, pleasure, or having interests in their own welfare, but does this mean we can do whatever we want with them? We respect art,
the environment, monuments, flags, burial grounds, and other nonmoral entities. Neither a van Gogh painting nor the deceased
have moral status, but we still refrain from defacing art, or tramping around in cemeteries or digging up the dead. Similarly, we
respect human embryos because they are developing human life and have moral value even if they do not have full moral status
(Steinbock, 1992). Perhaps respect should be given because a human embryo is a salient symbol of human life and how we treat
human embryos as a society demonstrates how we value human life more generally (Robertson, 1995). In this sense, we respect
human embryos more than other human cells and tissues and perhaps this is why we are reluctant to use them in instrumental
ways. The issue with the respect for embryo argument is that it is unclear how much respect should be paid to human embryos
and whether this means they can or cannot be used for SCR. Some would grant respect to mean that human embryos can be sacri-
ficed for socially valuable ends including SCR or for improving assisted reproduction procedures, but they are not to be wasted; they
certainly cannot be used for trivial purposes such as making jewelry or for cosmetic testing (Steinbock, 2001). Others might grant
permission to use surplus embryos for SCR because of their symbolic representation of life, but not to create embryos specifically for
research (Ryan, 2001).
Similar to respecting human embryos for their moral value and/or symbolic nature, several theorists contend that human
embryos are sacred and as such should not be violated. The belief in the sacredness of human embryos can be based on secular
beliefs or religious doctrines of life and the living. The Oxford Dictionary of English first defines sacred in a religious context as
something “connected with God or a god or dedicated to a religious purpose and so deserving veneration”; it also defines sacred
in secular ways as something “regarded with great respect and reverence by a particular religion, group or individual” and “regarded
as too valuable to be interfered with” (The Oxford Dictionary of English, 2005). Religious-based sacredness is generally well under-
stood, but a secular meaning of the term requires further explanation. Dworkin claims that human life is intrinsically valuable
despite whether someone places value in it (Dworkin, 1993). For example, most believe that death of someone young or even
an abortion is bad because it prematurely ends life even if it is not bad for a particular individual. This would mean that embryos
are intrinsically valuable irrespective of whether some people have an interest in the life of embryos. Yet, many also believe that
premature death is only bad if it fails to satisfy someone else’s desires or interests and as such, life is extrinsically sacred; these indi-
viduals believe that for something to have intrinsic value, it must have inherent value meaning value must derive from its internal
features (Uniacke, 2004). Thus, being biologically alive does not give the entity intrinsic value and for life to have intrinsic value it
must be conscious and have value in itself. In other words, many would believe that ending embryonic life is bad, not because an
embryo has interests in its own existence, but because it leads to a negative experience to others. Most people who believe embryos
are sacred would not want to have them deliberately destroyed for SCR, while a few may believe that despite embryos being ‘sacred,’
sacred life can be sacrificed for certain purposes like SCR.
Although many commentators engaged in the embryo and SCR debate use terms like sacredness, dignity, and respect (including
terms like ‘commodification’ or ‘instrumentalization’), many have argued that their amorphous and undefined nature makes these
terms less action guiding and more about a visceral or emotional response using it as a way to stop or prohibit an action like using
embryos for SCR (Macklin, 2003; Harmon, 2009; Caulfield and Ogbogu, 2011). Yet despite the limitation of these concepts being
difficult to articulate and understand what they mean in terms of policy choices on SCR, many in the general public, politicians, and
scholars readily use these terms toward establishing policies on SCR (Master and Crozier, 2011). These terms mean different things
to different people and are highly personal. They have been used in ethics and policy debates not only in SCR, but in genetics and
biotechnology time and time again. Albeit not cleanly defined and well-theorized, they should not be so easily dismissed as they
seem to play a significant role in public ethics discourse and in shaping policy.
Harms to Women
A major concern in the embryo–SCR debate is the possible physical and social harms to women who provide ova for research.
Whether we want to create an international bank of hESCs or develop individual clonal lines for each patient’s needs, the
number of eggs needed for such efforts would be unimaginable and possibly create a social dependency on women for their
eggs (Dickenson, 2004; Testa and Harris, 2005; Beeson and Lippman, 2006; George, 2007; Baylis, 2008). Yet despite this significant
moral concern, moral reflection and dialog on the harms to women have received far less play in comparison to defining moral
status (Dickenson, 2006).
Although human ova can be obtained from a variety of sources including oophorectomies, ovariectomies, cadavers, and fetal
ovaries, most oocytes used for research are obtained from women egg providers. Obtaining eggs from women requires providing
ovulatory drugs to stimulate follicular growth and development in order to retrieve several mature ova. Generally, oocytes are
retrieved surgically through transvaginal follicular aspiration using ultrasound guidance under sedation. In cases where ovaries
are inaccessible from the vagina, ova can be retrieved laproscopically under light anesthesia. The physical risks associated with
ovarian stimulation is ovarian hyperstimulation syndrome (OHSS), which occurs through the loss of controlled ovarian stimula-
tion. OHSS symptoms ranges in severity from minor abdominal distension and possible diarrhea to increased abdominal pain, low
urine production, blood clots, respiratory distress, and thromboembolic episodes (George, 2007). Although uncertain, there
remains a possible risk of ovarian and uterine cancer from long-term ovarian stimulation (Rossing et al., 1994; Wakeley and
Grendys, 2000; Ness et al., 2002; Althuis et al., 2005). Surgical risks include bleeding, vaginal hemorrhage, pelvic infections and
injuries, and risk from low-dose anesthetics.
Beyond the physical risks, there are social risks in the possible solicitation and exploitation of women for their eggs. There are
several reports of advertisements targeted to women egg providers offering substantial payments for eggs from young fecund
women (Padawer, 2002; Steinbock, 2004). Although much of these substantial payments are to recruit egg donors for reproductive
purposes, payments can also be offered to women to provide eggs strictly for research purposes (Dickenson, 2004). Giving eggs
strictly for research alters the risk-benefit ratio as women assume all the risks and receive no direct benefit, whereas benefits are
afforded to science and society and other individuals such as researchers (Dickenson, 2006; George, 2007). The ethical concern is
that substantial financial incentives for obtaining eggs for research may influence a woman, particularly one in financial need, to
assume the physical risks associated with ova stimulation and retrieval that they would normally otherwise not consider (Steinbock,
2004). As such, substantive financial inducements may compromise the woman’s ability to make an autonomous decision and
exercise informed consent about donating ova for research. Countries with populations facing higher poverty rates with limited
research ethics oversight may be more easily exploited (Waldby and Cooper, 2008). A demand for ova for research may also estab-
lish some form of reproductive tourism for eggs (Crozier and Martin, 2012). Yet is it prima facie unethical to pay women for their
eggs? If a woman understands her choice and agrees on an arrangement where she will receive financial benefit it would not neces-
sarily be considered exploitive or unfair since both parties obtain their desires (Gruen, 2007; Nuffield Council on Bioethics, 2011).
It seems ethically acceptable for models to be paid for their looks and talents, athletes for their physical endurance and abilities, so
why is it unethical for a woman to sell her ova? Perhaps this can be explained by drawing analogy that selling ‘sacred’ things like
organs, blood, and gametes for profane things like cash seems morally offensive where it is permissible to exchange like things, i.e.,
cash for a television (Hirschman, 1991). In this sense, some believe that egg donation should be done on a strictly altruistic basis.
Alternative Technologies to the Derivation of Pluripotent Stem Cells
Partly as a reaction to the moral status and other ethical issues, scientists began developing alternative technologies to derive plurip-
otent stem cells in order to circumvent ethical issues.
Nondestructive Techniques
The first is a set of two nondestructive techniques – namely, blastomere biopsy and blastocyst transfer method (BTM) – aimed to
derive pluripotent stem cells from part of the embryo and without destroying it, transfer the rest of the embryo into a woman to
create a child. Blastomere biopsy is a relatively standardized procedure used in preimplantation genetic diagnosis (PGD) where one
or two blastomeres from six- to eight-cell embryos are extracted and used for genetic analysis. After genetic analysis, the desired
embryo is transferred into a woman. Several children have been born from PGD. In our case, blastomere biopsy would be used
to derive pluripotent stem cells from blastomeres as demonstrated in mice (Chung et al., 2006) and humans (Klimanskaya
et al., 2006) while the rest of the embryo is transferred to create a child. The BTM is a theoretical procedure that isolates a substantive
portion of the ICM of a blastocyst to derive pluripotent stem cells while the remainder of the embryo is transferred into a woman to
create a child (Liao, 2005). Isolation of ICM cells would be done using a needle similar to the one used in intracytoplasmic sperm
injection (ICSI) to create embryos for male infertility.
There are several issues with these nondestructive techniques. First, both the procedures attempt to absolve the moral status issue
by treating the embryo not only as a means for research, but also as an end by permitting it to be born. Blastomeres in mice have
been shown to be totipotent and can give rise to a new organism suggesting the same is probably true for human blastomeres (Kelly,
1977). Similarly, it has been shown that ICM and hESCs when introduced into human embryos serve to create an entirely new
organism (Nagy et al., 1990, 1993; Ueda et al., 1995). If blastomeres, and potentially ICM cells, are totipotent and can be coaxed
to create an organism anew, then these two techniques are not absolving the moral status issue, but merely preventing the creation
of a second individual – a genetic twin (Devolder and Ward, 2007; Master et al., 2007a). A second problem with these nondestruc-
tive techniques is that they involve micromanipulation, more extensively in the case of BTM, which purposely subjects the embryo
to potential physical harm that could affect is survivability and developmental potential in vitro and in the worst situation, present
higher risk for congenital malformations in children (Master, 2005, 2006; Pearson, 2006). Two meta-analyses on preimplantation
genetic screening (PGS) have shown that PGS has a lower pregnancy and birth rate than in vitro fertilization (IVF) or IVF with ICSI
alone (Checa et al., 2009; Mastenbroek et al., 2011). The third issue is that the proposal, although both ethically and scientifically
interesting, is completely impractical. No individuals undergoing assisted reproduction would opt to place their embryos at risk and
potentially lower their chances for a successful pregnancy all in the name of science, and perhaps receiving a vial of stem cells genet-
ically matched to their offspring (Master, 2005). Fourth, the nondestructive techniques still require ova from women and thus the
potential harms to women remains an issue.
Altered Nuclear Transfer

William Hurlbut proposed a technique called altered nuclear transfer (ANT), which uses SCNT to derive genetically matched hESCs,
but where the somatic cell has a mutation that prematurely arrests the embryo from further development, generally preventing
implantation, but the embryo can develop to a blastocyst stage where hESCs can be derived (Hurlbut, 2005). Proof of concept
of ANT has been shown in mice (Meissner and Jaenisch, 2006). Hurlbut claims that because these embryos cannot implant,
they are ‘nonembryos’ and as such they can be used to derive hESCs; this technique is a morally superior alternative to SCNT or
IVF embryos because these latter methods create and destroy human embryos that undergo full development (Hurlbut et al.,
2006). However, many scholars have questioned the classification of ANT-created embryos as ‘nonembryos’ (Melton et al.,
2004; Guenin, 2005; Devolder, 2006). Hurlbut’s proposal focuses on the capacity of an embryo to undergo normal development,
but it remains ambiguous at which point in development we would classify an entity that develops abnormally or ceases
development prematurely a ‘nonembryo’ (Gruen and Grabel, 2006). Certainly, embryos gone awry and developing into a teratoma
would not be classified as an embryo, but it is unclear whether an embryo that ceases development before implantation would be
classified differently. In addition, just because an embryo does not develop normally and classifying it as a ‘nonembryo’ does not
preclude it from moral protection (Guenin, 2005). Why cannot this entity be classified as an embryo with limited potential? We
must also not ignore the fact that we are purposely manipulating cells and creating an embryo-like entity in order to harvest hESCs,
and as such what does this say about how we regard early human life (Cheshire and Jones, 2005; Jones and Cheshire, 2005). It is
thus unlikely that opponents of embryo research will permit the creation of entities by ANT as a source of hESCs. In addition, ANT,
like the nondestructive techniques, still requires ova from women.
Cytoplasmic Hybrids
An alternative to using human eggs that has received some attention is to create pluripotent stem cells from hybrid embryos
commonly known as cytoplasmic hybrids or interspecific cytoplasmic hybrids. In some cases, this technique has been advertised
as a way to sidestep the shortage of human eggs for SCR, not as a means to circumvent the harms to women (Tecirlioglu et al.,
2006; Minger, 2007; Bahadur et al., 2008; Camporesi and Boniolo, 2008; Hammond and Holm, 2008). The technique uses ova
obtained from nonhuman animals such as rabbits (Chen et al., 2003) or cows (Chang et al., 2003) for nuclear transfer experiments
using the nucleus of a human cell. Similar to SCNT, the hybrid human–animal ovum would be parthenogenically activated and
pluripotent stem cells could be derived. The use of animal eggs for nuclear transfer experiments may be advantageous for many
basic scientific research endeavors such as to study diseases (i.e., mitochondrial disorders), nuclear reprogramming, how cells differ-
entiate, and how embryos develop (Crawford et al., 2008; Swerdlow, 2007), but it is unlikely to be used for human transplantation
and regenerative medicine.
Scientifically, there may be reasons to doubt the efficacy of cytoplasmic hybrid research to resolve social justice concerns arising
from the demand for human ova because (1) the cells derived may display differential gene expression patterns than their human
embryo counterparts (Chung et al., 2009; Ledford, 2009); (2) the resulting pluripotent stem cells may display differences in energy
levels than normal hESCs due to dissimilar mitochondria combinations from the animal ovum (St John and Lovell-Badge, 2007);
(3) the generation of pluripotent stem cells from cytoplasmic hybrid embryos may be at risk of carrying cross-species contamination
(HFEA, 2007); and (4) cytoplasmic hybrids may cause moral confusion by crossing species boundaries (Baylis, 2008).
Parthenotes
Diploid oocytes can be activated by parthenogenesis (denoted as parthenotes) and can undergo embryonic development until
implantation. Because parthenotes lack imprinted paternal genes, they are unable to implant and arrest development prematurely
(Lyle, 1997), but they can be used to derive ESCs (Cibelli et al., 2002; Vrana et al., 2003; Kim et al., 2007). As parthenotes cannot
develop similar to embryos, several commentators have argued that they can be used to derive hESCs and sidestep ethical objections
related to moral status (Fangerau, 2005; Kiessling, 2005; Marchant, 2006; Rodriguez et al., 2011). However, many people may still
find it morally offensive to create these embryo-like entities and it is unclear how useable hESCs derived from parthenotes would be
as a source for cellular replacement therapies. Lastly, the ethical issue surrounding the harms to women remains with the use of
parthenotes as a source of hESCs.
hESC-Derived Ova
A second alternative to lessen the harms to women would be to create ova from the differentiation of hESCs instead of obtaining
them from women (Newson and Smajdor, 2005; Testa and Harris, 2005; Master, 2006; Hammond and Holm, 2008). Both mouse
and human oocytes have been shown to be created from ESC differentiation (Hubner et al., 2003; Clark et al., 2004; Lacham-Kaplan
et al., 2006). There may be several scientific and safety issues of using hESC-derived ova for nuclear transfer experiments: (1) hESC-
derived gametes may not be effectively reprogrammed to initiate de novo embryonic development to the blastocyst stage in order to
derive pluripotent stem cells (Testa and Harris, 2005); (2) embryos created from hESC-derived ova may display altered gene expres-
sion and the resulting pluripotent stem cells may not function properly; and (3) hESC-derived ova will still be used to create
embryos that would be considered by some to have significant moral weight and would not resolve the moral status debate.
Although the use of this technology is beneficial for basic research, much more research is required before they can be used for clin-
ical ends (Marques-Mari et al., 2009).
Nonegg and Nonembryo Techniques to Derive Pluripotent Stem Cells
The debates surrounding the moral status are intractable and unlikely to be resolved upon further moral reflection or from a greater
understanding of human development. The alternative technologies to derive pluripotent stem cells fail to resolve one or more ethical
challenges and in some cases create new ones. One way to absolve many of these ethical issues is to derive pluripotent stem cells from
nonovum and nonembryo sources (Master and Crozier, 2012). Here, I review two strategies that aim to derive pluripotent stem cells
from nonegg and nonembryonic sources by inducing pluripotency of adult differentiated cells in some manner.
The first technique involves inducing pluripotency of somatic cells through fusion with stem cells (Do et al., 2006). In particular,
Cowan and colleagues demonstrated that the fusion of hESCs with human fibroblasts resulted in a tetraploid hybrid cell that had
similar morphology, growth rates, DNA methylation patterns, antigen expression profiles, and differentiation patterns to hESCs
(Cowan et al., 2005). However, several moral concerns remain: (1) there is continued dependence on using established hESCs
for fusion experiments since they were the products of human embryos that were intentionally destroyed in creating them; (2) it
remains unclear whether the somatic genomes of hybrid cells have been sufficiently reprogrammed such that the fused cells can
differentiate into a diverse population of cell types required for cell restorative therapies (Yamanaka, 2007); (3) most of the reagents
used to fuse cells are cytotoxic, immunogenic, and nonspecific allowing many cell types to fuse in vitro (Sullivan and Eggan, 2006);
(4) using tetraploid cells could pose a tumorigenic risk (Sullivan and Eggan, 2006) and may also be immune rejected (Ambrosi and
Rasmussen, 2005); and (5) the resulting cells may have different properties and potentially posttransplantation rendering them
clinically unusable (Phimister, 2005). Despite the scientific unknowns and feasibility in utilizing cell fusion to create pluripotent
stem cells, the technique does seem to liberate the need for ova from women and reduce the moral risks surrounding the destruction
of human embryos, but has received relatively little interest as a way to move the SCR field forward, both ethically and scientifically.
A second nonegg and nonembryo technique that has truly revolutionized the field of SCR is iPSC technology. The discovery of
iPSCs was a remarkable accomplishment and has swept how SCR is practiced today and for this discovery, Professors Shinya Yama-
naka and John Gurdon were jointly awarded the Nobel prize for reprogramming differentiated cells into a pluripotent state – Yama-
naka for iPSCs and Gurdon for SCNT (Abbott, 2012). Pluripotency can be induced in adult differentiated cells through the
overexpression of key genes that reprogram the cell into a pluripotent state (Takahashi and Yamanaka, 2006). The original tech-
nique used viruses to infect cells in order to overexpress genes of interest, but since then, nonviral vectors, exogenous factors,
and modified culture medium have been used to generate iPSCs (Huangfu et al., 2008; Yu et al., 2009; Zhao et al., 2010).
The discovery of iPSCs has been a major accomplishment in understanding cellular reprogramming, but much work still needs
to be done if iPSCs are to proceed to the clinic. Several reports comparing iPSCs with hESCs have shown lower efficiencies of iPSCs
to differentiate into certain cell types (Hu et al., 2010); differences in gene expression profiles (Chin et al., 2009; Ghosh et al., 2010);
iPSCs carrying epigenetic memory (Kim et al., 2010; Polo et al., 2010; Lister et al., 2011); iPSCs carrying more genetic variations and
mutations or chromosomal aberrations (Mayshar et al., 2010; Gore et al., 2011; Laurent et al., 2011; Hussein et al., 2011); an
increase in tumorigenic markers found in iPSCs (Malchenko et al., 2010); and potential immune recognition of iPSCs (Zhao
et al., 2011). These studies demonstrate the need for further research to understand the molecular and cellular mechanisms of differ-
entiation and development.
The Reality of SCR: IPSCs Are Not Replacing hESCs
The hype surrounding iPSC research in the media has been astronomical and the greatest benefit portrayed is that iPSCs are free of
moral concern (Caulfield and Rachul, 2011). Even George W. Bush in his Eight State of the Union address echoed that the iPSC
breakthrough can extend the frontiers of medicine without destroying human life (Bush, 2001; Gottweis and Minger, 2008).
Despite iPSCs obviating many ethical concerns (Meyer, 2008), they are certainly not replacing hESC research and instead, both
are used as research tools (Scott et al., 2011). Moreover, there are ethical challenges to iPSC research, a significant one surrounding
moral complicity where supporters of iPSC research are morally complicit in embryo destruction because iPSC research continues to
need and depend on hESC research (Brown, 2009, 2013; Hyun, 2011). There are also a host of other ethical issues regarding
banking of iPSC or other cell types, i.e., informed consent, genetic privacy, and withdrawal (Sugarman, 2008; Zarzeczny et al.,
2009). Yet given that iPSC research requires and depends on hESC research and thus the continued use of human embryos and
need for ova from women donors, can there be a moral and political compromise? Although many believe a loss of personal integ-
rity in a moral compromise (Devolder, 2005; Devolder and Harris, 2005; Tannsjo, 2007), there is value in compromising ethically
and politically in SCR and a policy framework could be designed to permit SCR using embryos and eggs for a certain period and
then prohibiting their use when nonegg and nonembryo techniques are sufficiently well developed and can replace hESC research
(Master and Crozier, 2012). Yet despite whether a compromise can or cannot be settled, it seems that bioethical exchange
surrounding moral status and harms to women has been significantly stunted after the discovery of iPSCs and instead, new ethical
and policy issues focus toward the translation and commercialization of SCR and stem cell tourism (Zarzeczny et al., 2009;
Caulfield et al., 2012a; Kato et al., 2012). Although understanding the ethical and scientific challenges to stem cell translation
and commercialization is an understandable and required next step, as basic science continues to explore the molecular mecha-
nisms of differentiation, reprogramming, and development, new types of embryos are created and the same moral issues
surrounding personhood, cloning, and harms to women are revisited (Annas, 2011; Caplan, 2011; Callaway, 2011; Darnovsky
et al., 2011; Kaiser, 2011, 2013; Young, 2011). Moral reflection and discourse by bioethicists on these ‘older’ debates should still
continue strongly.
Stem Cell Translation and Commercialization Challenges
Beginning in the early 1980s, there has been a change in culture in academic research toward a movement to translate and commer-
cialize research. In the United States, several factors may account for the cultural orientation from basic academic research toward
translation and commercialization including the passing of the Bayh-Dole Act (1980), public perception of the importance of
biomedical research, intellectual property decisions to commercialize research (i.e., Diamond vs Chakrabarty decision), and John
Moore vs Regents of University of California court decision that property rights are awarded to scientists for their intellectual labor
as opposed to the biovalue of human tissue (Annas, 1988; Mowery et al., 2001; Lemmens, 2004). This shift is seen through increases
in patents and licenses (Mowery et al., 2001; Shane, 2004), funding policy (Woolf, 2008; Wadman, 2010; Nature Cell Biology,
2012; Rohn, 2012; Reed et al., 2012; Trounson and Dewitt, 2012), and the potential pressure to translate and commercialize results
(Caulfield et al., 2008; Murdoch and Caulfield, 2009; Bubela and Caulfield, 2010; Caulfield et al., 2012). Academic success has
always been measured by the quality and quantity of publications and funding for research, but the new metrics of success for
academic researchers include patents, licenses, and the creation of start-up companies (Lemmens, 2004).
The commercialization crusade has been deemed to raise a specter of ethical and integrity issues (Maienschein et al., 2008)
including (1) minimizing data sharing and increase in secrecy (Louis et al., 2001; Caulfield et al., 2008; Hong and Walsh, 2009;
Joly et al., 2010); (2) increase in bias of research during data collection, interpretation, and reporting (Sismondo, 2008); (3) ghost
authorship (Sismondo, 2007; Wislar et al., 2011); and (4) premature translation and harms to research participants and the public
(Knowles, 2009; Wilson, 2009; Ryan et al., 2010). Yet despite the push to translate research into products and services that directly
profit society, only modest clinical success in SCR has been seen to date (Geffner et al., 2008; Baker, 2009; Davis, 2012; Gupta et al.,
2012; Hare et al., 2012; Holding, 2012; Makkar et al., 2012; Schwartz et al., 2012; Underwood, 2012). An analysis of clinical trials
shows that current clinical research focuses on hematopoietic and mesenchymal stem cells compared to what is reported in the
news, which emphasizes hESCs, and neurological and cardiovascular conditions (Bubela et al., 2012).
There are several challenges to the clinical translation of SCR. The first challenge would be to identify and utilize the appropriate
animal model for human translation (Lindvall, 2012) as not all animal data may be good predictors in determining effectiveness in
humans (Knight, 2008; Regenberg et al., 2009b). As the push to translate and commercialize may promote bias, scientists may over-
estimate effectiveness and underestimate contrary results (Weaver, 2010). Second, stem cells offer novel challenges as a therapeutic
when compared to drug-based interventions. There is a risk of tumor formation, undesired motility of stem cells into other areas,
genomic instability, and potential risks from viral and other vectors that may be used to modify cells in order to control their
behavior posttransplantation (Glass et al., 1999; Master et al., 2007b; Fink, 2009; Goldring et al., 2011). Third, stem cell translation
into humans requires microbiological, viral, and other contamination testing, assessing purity of populations, and scaling up to
manufacture sufficient quantities of cells in a controlled manner (Halme and Kessler, 2006; ISSCR, 2008; Rayment and Williams,
2010; Goldring et al., 2011). Lastly, stem cells and stem cell products are captured within national regulatory frameworks; their
classification and evaluation is different between different countries making it difficult for sponsors to seek regulatory approval
(von Tigerstrom, 2008; Goldring et al., 2011; von Tigerstrom, 2011). First-in-human trials are also likely to be difficult because
of the unknown outcomes and probabilities in assessing risks, which in turn can undermine a systematic analysis of risk during
ethics review (Fung and Kerridge, 2013; Kimmelman, 2012).
Stem Cell Markets
The positive hype surrounding SCR has spawned several markets that play on the theme that stem cells have rejuvenating and regen-
erative properties. The new fountain of youth in the beauty industry centers on products containing the revitalizing power of stem
cells. These stem cell–based products are sold directly to consumers and include antiaging hand and facial creams, soaps, shampoos,
moisturizers, mascara, eye liner, and other products. A second stem cell–based industry that also hypes the regenerative properties of
stem cells is stem cell–based vitamins. Stem cell vitamins aim to increase the endogenous production of adult stem cells and ‘may
aid in the repair of damaged tissue’ and ‘in the regeneration of new tissue’ (Vita-Stim Concentrate, 2012). Although the use of stem
cell–based lotions and vitamins are relatively benign, the same is unfortunately not true for stem cell tourism (Master and Resnik,
2011a).
Stem Cell Tourism

The term stem cell tourism is commonly used, perhaps less than ideally, to describe the phenomena of patients seeking stem cell
treatments/interventions that are understudied and untested (Zarzeczny et al., 2012). Stem cell tourism originally received its name
as a form of medical tourism where patients traveled to destinations to receive stem cell interventions: patients from countries like
the United States, Canada, Australia, and the United Kingdom traveled to China, India, and Mexico where possible lax regulations
and enforcement permitted the existence of such clinics (Ryan et al., 2010; Zarzeczny et al., 2010; Sipp, 2011). Yet more clinics are
also popping up in highly regulated countries. For example, a few clinics in the United States are offering autologous adult stem cell
transplantation therapies for back pain and minor injuries (Cyranoski, 2010a; Lysaght and Campbell, 2011; von Tigerstrom, 2011).
Stem cell tourism is a direct-to-consumer, Internet-based market. It is unclear how many people have actually sought stem cell
interventions, but it can be estimated to be at least in the tens of thousands and likely a lot more (Master and Ogbogu, 2012). We
know a fair bit about stem cell tourism from patient and public web blogs and interviews, media reports, and the analysis of
provider Web sites: (1) providers offer to treat a range of diseases varying in severity from backaches and sport injuries, hair
loss, erectile dysfunction, to very severe debilitating diseases and injuries such as blindness, cerebral palsy, Parkinson’s disease,
and spinal cord injury; (2) providers use all sorts of cell types including adult, fetal, and ESCs, which are administered surgically,
topically, intravenously, and orally; (3) providers charge hefty sums and require repeated treatments (Lau et al., 2008; Regenberg
et al., 2009a; Ryan et al., 2010); (4) clinics and the media offer mainly positive portrayals of the interventions and underemphasize
risks (Lau et al., 2008; Zarzeczny et al., 2010); (5) clinics rely on patient testimonials as evidence of efficacy; and (6) there are several
risks including tumors, lesions, meningitis, tremors, and death (Dobkin et al., 2006; Amariglio et al., 2009; Barclay, 2009; Miller,
2009; Cyranoski, 2010b; Mendick and Palmer, 2010; Thirabanjasak et al., 2010; Mendick and Hall, 2011; Vogel, 2011).
Patient and Public Perceptions

Patients and the public have a diverse array of perspectives on SCR, therapies, and tourism. Many patients who seek stem cell inter-
ventions have dealt with conventional medical treatments and feel they have exhausted all their options and that a stem cell treat-
ment is their only viable opportunity (Rachul, 2011). Patients believe they are informed and understand the experimental nature of
the therapy, that stem cells can cure them, they are contributing to the advancement of science, and patients seem to distrust the
health care and research systems in their home countries (Ryan et al., 2010; Chen and Gottweis, 2013; Rachul, 2011; Einsiedel
and Adamson, 2012; Master et al., 2013). There are also some patients who received an intervention with no improvement or relief
of symptoms who have become skeptical of the exaggerated claims of stem cell treatments. Interestingly, most healthy volunteers
see positive aspects about a decision to go and pursue stem cell treatments (Einsiedel and Adamson, 2012).
Strategies to Mitigate Stem Cell Tourism

There are three types of proposals that aim to reduce or ameliorate stem cell tourism. The first involves developing and further
strengthening both domestic and international regulations and enforcement to curtail the market. Such strategies have been per-
formed with varying success in China (Cyranoksi, 2009; Nature, 2010), Germany (Stafford, 2009; Vogel, 2011), and several other
nations. Regulatory authorities and medical licensure boards are pursing and disciplining providers who have gone off the reserva-
tion (Ward, 2010; FBI, 2011a, 2011b). Legal action is a powerful means to deter illegal stem cell activity, yet the nature of the inter-
national market makes it challenging to enforce and regulate stem cell tourism. Moreover, international laws, regulations, or
policies may take time to develop, and those who wish to evade the rules can move to a more permissive regulatory environment
or simply break the rules and accept the penalties, especially when they are not severe (Master and Resnik, 2011b).
A second means to quell stem cell tourism is to educate the public and patients. There are several amazing sources of patient
information, but it seems that patients who are severely ill and desperate for a possible treatment are not likely to heed the warnings
of untested stem cell treatments (Master and Resnik, 2011b; Einsiedel and Adamson, 2012). If patients are distrusting of their
national health research and regulatory governance systems as some perception data indicate, it is also unlikely that educating
them about the safety and efficacy of stem cell treatments will help in promoting trust or dissuade future stem cell tourists from
seeking treatment; however, it is still a considerably worthwhile effort to provide robust information to potential tourists even if
it does not deter them from undertaking the intervention (Master et al., 2013).
A third response to stem cell tourism is for frontline physicians to inform their patients about the dangers of stem cell tourism
(Caulfield et al., 2012b; Zarzeczny and Caulfield, 2010). A similar frontline approach involves scientists sharing stem cell reagents
and materials for specific ‘research’ purposes and not to provide such materials to potential providers who wish to start up their stem
cell tourism business; this would require scientists to evaluate the curriculum vitae and protocols from the requesters of materials
and to stipulate specifically how the materials are to be used in material transfer agreements (Master and Resnik, 2011b).
Although much is known about stem cell tourism, further qualitative research understanding the perceptions of a range of stake-
holders is needed. For example, many celebrities and public figures have recently sought stem cell interventions, i.e., athletes for
sports-related injuries. How is this shaping the public’s view on stem cell treatments? If physicians are to stop stem cell tourism,
how much do they know about SCR, how many patients talk with their physicians, and what advice do physicians provide? Simi-
larly, if education is going to stop stem cell tourism, how much education is out there, is it being targeted to the right audiences, and
how effective will it be as a deterrent to future stem cell tourists? These and many other research questions still need to be addressed
(Master and Ogbogu, 2012).
Summary
The ethics and hype in science and technology have been especially rampant in SCR to the point where it has influenced the conduct
of science (to make ethical stem cells) and where the hype may have contributed to prematurely market regenerative products and
interventions – including the stem cell tourism market. The hype and promises surrounding SCR have also likely contributed to
developing a translational and commercialization ethos within the field. While many of the problematic issues surrounding moral
status and harms to women who provide eggs for research are difficult to resolve, deriving stem cells from nonegg and nonem-
bryonic sources (e.g., through iPSC research) does seem to be a step in the right direction as they skirt around these ethical issues.
As iPSC and SCR show tremendous promise as a possible curative for many diseases and injuries, much more scientific research is
still needed. Accurate portrayals of SCR need to be emphasized and hype needs to be tempered so that the public has realistic expec-
tations of where the science is, where it is heading, and when the fruits of labor will be delivered to them.
Acknowledgments
Part of this work was supported by the Cancer Stem Cell Consortium (CSCC) with funding from Genome Canada and the Ontario Genomics
Institute (OGI-047); the Canadian Stem Cell Network; and the Interdisciplinary Chronic Disease Collaboration funded by Alberta Innovates. I would
like to thank Professor Timothy Caulfield and Dr Lisa Campo-Engelstein for providing helpful feedback to the article and project support by Mr
Daniel G. Frederick, Ms Robyn Hyde-Lay, and Ms Faria Grant.
References
Abbott, A. (2012). Cell rewind wins medicine Nobel. Nature, 490, 151–152.
Althuis, M. D., Moghissi, K. S., Westhoff, C. L., Scoccia, B., Lamb, E. J., Lubin, J. H., & Brinton, L. A. (2005). Uterine cancer after use of clomiphene citrate to induce ovulation. Am.
J. Epidemiol., 161, 607–615.
Amariglio, N., Hirshberg, A., Scheithauer, B. W., Cohen, Y., Loewenthal, R., Trakhtenbrot, L., Paz, N., Koren-Michowitz, M., Waldman, D., Leider-Trejo, L., Toren, A., Constantini, S.,
& Rechavi, G. (2009). Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med., 6, e10000029. https://doi.org/
10.1371/journal. pmed.1000029.
Ambrosi, D. J., & Rasmussen, T. P. (2005). Reprogramming mediated by stem cell fusion. J. Cell. Mol. Med., 9, 320–330.
Annas, G. J. (1988). Whose waste is it anyway? The case of John Moore. Hastings Cent. Rep., 18, 37–39.
Annas, G. J. (2011). Sudden death for a challenge to federal funding of stem-cell research. N. Engl. J. Med., 364, e47.
Bahadur, G., Iqbal, M., Malik, S., Sanyal, A., Wafa, R., & Nobel, R. (2008). Admixed human embryos and stem cells: legislative, ethical and scientific advances. Reprod. Biomed.
Online, 17, 25–32.
Baker, M. (2009). Bone marrow stem cells, surgery assessed for spinal cord injury. Nat. Rep. Stem Cells. https://doi.org/10.1038/stemcells.2009.57. http://www.nature.com/
stemcells/index.html.
Barclay, E. (2009). Stem-cell experts raise concerns about medical tourism. Lancet, 373, 883–884.
Baylis, F. (2008). Animal eggs for stem cell research: a path not worth taking. Am. J. Bioeth., 8, 18–32.
Beeson, D., & Lippman, A. (2006). Egg harvesting for stem cell research: medical risks and ethical problems. Reprod. Biomed. Online, 13, 573–579.
Bortolotti, L., & Harris, J. (2005). Stem cell research, personhood and sentience. Reprod. Biomed. Online, 10, 68–75.
Brown, M. (2013). No ethical bypass of moral status in stem cell research. Bioethics, 27, 12–19.
Brown, M. T. (2009). Moral complicity in induced pluripotent stem cell research. Kennedy Inst. Ethics J., 19, 1–22.
Bubela, T., Nisbet, M. C., Borchelt, R., Brunger, F., Critchley, C., Einsiedel, E., Geller, G., Gupta, A., Hampel, J., Hyde-Lay, R., Jandciu, E. W., Jones, S. A., Kolopack, P., Lane, S.,
Lougheed, T., Nerlich, B., Ogbogu, U., O’Riordan, K., Ouellette, C., Spear, M., Strauss, S., Thavaratnam, T., Willemse, L., & Caulfield, T. (2009). Science communication
reconsidered. Nat. Biotechnol., 27, 514–518.
Bubela, T., Li, M. D., Hafez, M., Bieber, M., & Atkins, H. (2012). Is belief larger than fact: expectations, optimism and reality for translational stem cell research. BMC Med., 10, 133.
https://doi.org/10.1186/1741-7015-10-133.
Bubela, T. M., & Caulfield, T. (2010). Role and reality: technology transfer at Canadian universities. Trends Biotechnol., 28, 447–451.
Bush, G. (9 August 2001). President Discusses Stem Cell Research. http://www.whitehouse.gov/.
Callaway, E. (2011). Give Your Eggs to Science, Get Paid, Suggests New Report. http://blogs.nature.com/news/.
Campbell, K. H., McWhir, J., Ritchie, W. A., & Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature, 380, 64–66.
Camporesi, S., & Boniolo, G. (2008). Fearing a non-existing minotaur? The ethical challenges of research on cytoplasmic hybrid embryos. J. Med. Ethics, 34, 821–825.
Caplan, A. (2011). Don’t Fear the Cloned Stem Cells: They’re Not People. http://www.nbcnews.com.
Caulfield, T. (2010). Stem cell research and economic promises. J. Law Med. Ethics, 38, 303–313.
Caulfield, T., Harmon, S. H., & Joly, Y. (2012). Open science versus commercialization: a modern research conflict? Genome Med., 4, 17.
Caulfield, T., & Ogbogu, U. (2011). Stem cell research, scientific freedom and the commodification concern. EMBO Rep., 13, 12–16.
Caulfield, T., Ogbogu, U., Murdoch, C., & Einsiedel, E. (2008). Patents, commercialization and the Canadian stem cell research community. Regen. Med., 3, 483–496.
Caulfield, T., & Rachul, C. (2011). Science spin: iPS cell research in the news. Clin. Pharmacol. Ther., 89, 644–646.
Caulfield, T., Rachul, C., & Zarzeczny, A. (2012a). The evolution of policy issues in stem cell research: an international survey. Stem Cell Rev., 8, 1037–1042.
Caulfield, T., Zarzeczny, A., & Toronto Stem Cell Working Group. (2012b). Stem cell tourism and Canadian family physicians. Can. Fam. Physician, 58, 365–368.
Chang, K. H., Lim, J. M., Kang, S. K., Lee, B. C., Moon, S. Y., & Hwang, W. S. (2003). Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from
nuclear transfer of human cord fibroblasts into enucleated bovine oocytes. Fertil. Steril., 80, 1380–1387.
Checa, M. A., Alonso-Coello, P., Solà, I., Robles, A., Carreras, R., & Balasch, J. (2009). IVF/ICSI with or without preimplantation genetic screening for aneuploidy in couples without
genetic disorders: a systematic review and meta-analysis. J. Assist. Reprod. Genet., 26, 273–283.
Chen, H., & Gottweis, H. (2013). Stem cell treatments in China: rethinking the patient role in the global bio-economy. Bioethics, 27, 194–207. https://doi.org/10.1111/j.1467–
8519.2011.01929.x.
Chen, Y., He, Z. X., Liu, A., Wang, K., Mao, W. W., Chu, J. X., Lu, Y., Fang, Z. F., Shi, Y. T., Yang, Q. Z., Chen da, Y., Wang, M. K., Li, J. S., Huang, S. L., Kong, X. Y., Shi, Y. Z.,
Wang, Z. Q., Xia, J. H., Long, Z. G., Xue, Z. G., Ding, W. X., & Sheng, H. Z. (2003). Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit
oocytes. Cell Res., 13, 251–263.
Cheshire, W. P., & Jones, N. L. (2005). Can artificial techniques supply morally neutral human embryos for research? Part II. The meaning of artificial life. Ethics Med., 21, 73–88.
Chin, M. H., Mason, M. J., Xie, W., Volinia, S., Singer, S., Peterson, C., Ambartsumyan, G., Aimiuwu, O., Richter, L., Zhang, J., Khvorostov, I., Ott, V., Grunstein, M., Lavon, N.,
Benvenisty, N., Croce, C. M., Clark, A. T., Baxter, T., Pyle, A. D., Teitell, M. A., Pelegrini, M., Plath, K., & Lowry, W. E. (2009). Induced pluripotent stem cells and embryonic stem
cells are distinguished by gene expression signatures. Cell Stem Cell, 5, 111–123.
Chung, Y., Bishop, C. E., Treff, N. R., Walker, S. J., Sandler, V. M., Becker, S., Klimanskaya, I., Wun, W. S., Dunn, R., Hall, R. M., Su, J., Lu, S. J., Maserati, M., Choi, Y. H.,
Scott, R., Atala, A., Dittman, R., & Lanza, R. (2009). Reprogramming of human somatic cells using human and animal oocytes. Cloning Stem Cells, 11, 213–223.
Chung, Y., Klimanskaya, I., Becker, S., Marh, J., Lu, S. J., Johnson, J., Meisner, L., & Lanza, R. (2006). Embryonic and extraembryonic stem cell lines derived from single mouse
blastomeres. Nature, 439, 216–219.
Cibelli, J. B., Grant, K. A., Chapman, K. B., Cunniff, K., Worst, T., Green, H. L., Walker, S. J., Gutin, P. H., Vilner, L., Tabar, V., Dominko, T., Kane, J., Wettstein, P. J., Lanza, R. P.,
Studer, L., Vrana, K. E., & West, M. D. (2002). Parthenogenetic stem cells in nonhuman primates. Science, 295, 819.
Clark, A. T., Bodnar, M. S., Fox, M., Rodriquez, R. T., Abeyta, M. J., Firpo, M. T., & Pera, R. A. R. (2004). Spontaneous differentiation of germ cells from human embryonic stem
cells in vitro. Hum. Mol. Gen., 13, 727–739.
Cowan, C. A., Atienza, J., Melton, D. A., & Eggan, K. (2005). Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science, 309, 1369–1373.
Crawford, L., Laforce, D., & Master, Z. (2008). A problematic principle. Am. J. Bioeth., 8, 40–42.
Crozier, G. K., & Martin, D. (2012). How to address the ethics of reproductive travel to developing countries: a comparison of national self-sufficiency and regulated market
approaches. Dev. World Bioeth., 12, 45–54.
Cyranoksi, D. (2009). Stem cell therapy faces more scrutiny in China. Nature, 459, 146–147.
Cyranoski, D. (2010a). FDA challenges stem-cell clinic. Nature, 466, 909.
Cyranoski, D. (2010b). Korean deaths spark inquiry. Nature, 468, 485.
Darnovsky, M., Fogel, S. B., & Norsigian, J. (2011). Cloning technology control the bonanza for research eggs. Nature, 480, 39.
Davis, N. (2012). Donor stem cells as safe and effective as patient’s own in heart attack trial. BioNews. http://bionews.org.uk.
Devolder, K. (2005). Human embryonic stem cell research why the discarded-created-distinction cannot be based on the potentiality argument. Bioethics, 19, 167–186.
Devolder, K. (2006). What’s in a name? Embryos, entities, and ANTities in the stem cell debate. J. Med. Ethics, 32, 43–48.
Devolder, K., & Harris, J. (2005). Compromise and moral complicity in the embryonic stem cell debate. In N. Athanassoulis (Ed.), Philosophical Reflections on Medical Ethics (pp.
88–108). Hampshire, England: Palgrave Macmillan.
Devolder, K., & Ward, C. M. (2007). Rescuing human embryonic stem cell research: the possibility of embryo reconstitution after stem cell derivation. In L. Gruen, L. Grabel, &
P. Singer (Eds.), Stem Cell Research: The Ethical Issues (pp. 105–123). Oxford, UK: Blackwell.
Dickenson, D. (2004). The threatened trade in human ova. Nat. Rev. Genet., 5, 167.
Dickenson, D. (2006). The lady vanishes: what’s missing from the stem cell debate. J. Bioeth. Inq., 3, 43–54.
Do, J. T., Han, D. W., & Schöler, H. R. (2006). Reprogramming somatic gene activity by fusion with pluripotent cells. Stem Cell Rev., 2, 257–264.
Dobkin, B., Curt, A., & Guest, J. (2006). Cellular transplants in China observational study from the largest human experiment in chronic spinal cord injury. Neurorehabil. Neural
Repair, 20, 5–13.
Doerflinger, R. M. (2008). The problem of deception in embryonic stem cell research. Cell Prolif., 41(Suppl. 1), 65–70.
Downey, R., & Geransar, R. (2008). Stem cell research, publics’ and stakeholder views. Health L. Rev., 16, 69–85.
Dworkin, R. (1993). Life’s Dominion: An Argument about Abortion, Euthanasia, and Individual Freedom. New York: Vintage Books.
Einsiedel, E., & Adamson, H. (2012). Stem cell tourism and future stem cell tourists policy and ethical implications. Dev. World Bioeth., 12, 35–44.
Fangerau, H. (2005). Can artificial parthenogenesis sidestep ethical pitfalls in human therapeutic cloning? An historical perspective. J. Med. Ethics, 31, 733–735.
Federal Bureau of Investigation (FBI). (18 August 2011a). Owner of Arizona Company Charged and Convicted of Introducing an Unapproved New DrugdStem Cellsdinto Interstate
Commerce. San Antonio Division, U.S. Attorney’s Office(a). Press Release www.fbi.gov.
Federal Bureau of Investigation (FBI). (December 28, 2011b). Federal Indictments Lead to Arrests in Stem Cell Case. San Antonio Division, U.S. Attorney’s Office(b). Press Release
www.fbi.gov.
Feinberg, J. (1992). Freedom & Fulfillment. New Jersey: Princeton University Press.
Fertilisation, Human, and Embryology Authority. (2007). Hybrids and Chimeras: A Report on the Findings of the Consultation. London: HFEA.
Fink, D. W., Jr. (2009). FDA regulation of stem cell-based products. Science (New York, NY), 324, 1662–1663.
Fung, R. K., & Kerridge, I. H. (2013). Uncertain translation, uncertain benefit and uncertain risk: ethical challenges facing first-in-human trials of induced pluripotent stem (IPS) cells.
Bioethics, 27, 89–96.
Geffner, L. F., Santacruz, P., Izurieta, M., Flor, L., Maldonado, B., Auad, A. H., Montenegro, X., Gonzalez, R., & Silva, F. (2008). Administration of autologous bone marrow stem cells
into spinal cord injury patients via multiple routes is safe and improves their quality of life comprehensive case studies. Cell Transplant., 17, 1277–1293.
George, K. (2007). What about the women? Ethical and policy aspects of egg supply for cloning research. Reprod. Biomed. Online, 15, 127–133.
Ghosh, Z., Wilson, K. D., Wu, Y., Hu, S., Quertermous, T., & Wu, J. C. (2010). Persistent donor cell gene expression among human induced pluripotent stem cells contributes to
differences with human embryonic stem cells. PLoS One, 5, e8975.
Glass, K. C., Weijer, C., Cournoyer, D., Lemmens, T., Palmour, R. M., Shapiro, S. H., & Freedman, B. (1999). Structuring the review of human genetics protocols, part III: gene
therapy studies. IRB, 21, 1–9.
Goldring, C. E., Duffy, P. A., Benvenisty, N., Andrews, P. W., Ben-David, U., Eakins, R., French, N., Hanley, N. A., Kelly, L., Kitteringham, N. R., Kurth, J., Ladenheim, D., Laverty, H.,
McBlane, J., Narayanan, G., Patel, S., Reinhardt, J., Rossi, A., Sharpe, M., & Park, B. K. (2011). Assessing the safety of stem cell therapeutics. Cell Stem Cell, 8, 618–628.
Gore, A., Li, Z., Fung, H. L., Young, J. E., Agarwal, S., Antosiewicz-Bourget, J., Canto, I., Giorgetti, A., Israel, M. A., Kiskinis, E., Lee, J. H., Loh, Y. H., Manos, P. D., Montserrat, N.,
Panopoulos, A. D., Ruiz, S., Wilbert, M. L., Yu, J., Kirkness, E. F., Belmonte, J. C. I., Rossi, D. J., Thomson, J. A., Eggan, K., Daley, G. Q., Goldstein, L. S. B., & Zhang, K.
(2011). Somatic coding mutations in human induced pluripotent stem cells. Nature, 471, 63–67.
Gottweis, H., & Minger, S. (2008). iPS cells and the politics of promise. Nat. Biotechnol., 26, 271–272.
Green, R. M. (2002). Stem cell research: a target article collection: part IIIddetermining moral status. Am. J. Bioeth., 2, 20–30.
Gruen, L. (2007). Oocytes for sale? Metaphilosophy, 38, 285–308.
Gruen, L., & Grabel, L. (2006). Concise review: scientific and ethical roadblocks to human embryonic stem cell therapy. Stem Cells, 24, 2162–2169.
Guenin, L. M. (2005). Wishful thinking will not obviate embryo use. Stem Cell Rev., 1, 309–315.
Gupta, N., Henry, R. G., Strober, J., Kang, S. M., Lim, D. A., Bucci, M., Caverzasi, E., Gaetano, L., Mandelli, M. L., Ryan, T., Perry, R., Farrell, J., Jeremy, R. J., Ulman, M.,
Huhn, S. L., Barkovich, A. J., & Rowitch, D. H. (2012). Neural stem cell engraftment and myelination in the human brain. Sci. Transl. Med., 4, 155ra137.
Halme, D. G., & Kessler, D. A. (2006). FDA regulation of stem-cell-based therapies. N. Engl. J. Med., 355, 1730–1735.
Hammond, N., & Holm, S. (2008). Resolving the ‘egg supply problem’ in human embryonic stem cell derivation through technical means: a legal and ethical analysis. Med. Law, 27,
167–178.
Hare, J. M., Fishman, J. E., Gerstenblith, G., DiFede Velazquez, D. L., Zambrano, J. P., Suncion, V. Y., Tracy, M., Ghersin, E., Johnston, P. V., Brinker, J. A., Breton, E., Davis-
Sproul, J., Schulman, I. H., Byrnes, J., Mendizabal, A. M., Lowery, M. H., Rouy, D., Altman, P., Wong, P. F. C., Ruiz, P., Amador, A., Da Silva, J., McNiece, I. K., &
Heldman, A. W. (2012). Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic
cardiomyopathy: the POSEIDON randomized trial. JAMA, 308, 2369–2379.
Harmon, S. H. E. (2009). Of plants and people: why do we care about dignity? EMBO Rep., 10, 946–948.
Hirschman, E. C. (1991). Babies for sale: market ethics and the new reproductive technologies. J. Consum. Aff., 25, 358–390.
Holding, C. (2012). Heart attack scars healed in stem cell safety trial. BioNews. http://bionews.org.uk.
Hong, W., & Walsh, J. P. (2009). For money or glory? Commercialization, competition, and secrecy in the entrepreneurial university. Sociol. Q., 50, 145–171.
Hu, B. Y., Weick, J. P., Yu, J., Ma, L. X., Zhang, X. Q., Thomson, J. A., & Zhang, S. C. (2010). Neural differentiation of human induced pluripotent stem cells follows developmental
principles but with variable potency. Proc. Natl Acad. Sci. U. S. A., 107, 4335–4340.
Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E., & Melton, D. A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by
small-molecule compounds. Nat. Biotechnol., 26, 795–797.
Hubner, K., Fuhrmann, G., Christenson, L. K., Kehler, J., Reinbold, R., De La, F. R., Wood, J., Strauss, J. F., III, Boiani, M., & Scholer, H. R. (2003). Derivation of oocytes from mouse
embryonic stem cells. Science, 300, 1251–1256.
Hurlbut, W. B. (2005). Altered nuclear transfer as a morally acceptable means for the procurement of human embryonic stem cells. Perspect. Biol. Med., 48, 211–228.
Hurlbut, W. B., George, R. P., & Grompe, M. (2006). Seeking consensus: a clarification and defense of altered nuclear transfer. Hastings Cent. Rep., 36, 42–50.
Hussein, S. M., Batada, N. N., Vuoristo, S., Ching, R. W., Autio, R., Narva, E., Ng, S., Sourour, M., Hamalainen, R., Olsson, C., Lundin, K., Mikkola, M., Trokovic, R., Peitz, M.,
Brustle, O., Bazett-Jones, D. P., Alitalo, K., Lahesmaa, R., Nagy, A., & Otonkoski, T. (2011). Copy number variation and selection during reprogramming to pluripotency. Nature,
471, 58–62.
Hyun, I. (2011). The bioethics of iPS cell-based drug discovery. Clin. Pharmacol. Ther., 89, 646–647.
ISSCR International Society for Stem Cell Research. (3 December 2008). Guidelines for the Clinical Translation of Stem Cells. http://www.isscr.org/.
Jasanoff, S. (2005). Designs on Nature. New Jersey: Princeton University Press.
Joly, Y., Caulfield, T., Knoppers, B. M., Harmsen, E., & Pastinen, T. (2010). The commercialization of genomic research in Canada. Healthc. Policy, 6, 24–32.
Jones, N. L., & Cheshire, W. P. (2005). Can artificial techniques supply morally neutral human embryos for research? Part I. Creating novel categories of human embryos. Ethics
Med., 21, 29–40.
Kaiser, J. (2011). NIH wins suit challenging legality of research. Science, 333, 683.
Kaiser, J. (2013). Stem Cell Lawsuit Finally Over. Science Insider. http://news.sciencemag.org/scienceinsider/.
Kato, K., Kimmelman, J., Robert, J., Sipp, D., & Sugarman, J. (2012). Ethical and policy issues in the clinical translation of stem cells: report of a focus session at the ISSCR tenth
annual meeting. Cell Stem Cell, 11, 765–767.
Kelly, S. J. (1977). Studies on the developmental potential of 4- and 8-cell stage mouse blastomeres. J. Exp. Zool., 200, 365–376.
Kiessling, A. A. (2005). Eggs alone. Nature, 434, 145.
Kim, K., Lerou, P., Yabuuchi, A., Lengerke, C., Ng, K., West, J., Kirby, A., Daly, M. J., & Daley, G. Q. (2007). Histocompatible embryonic stem cells by parthenogenesis. Science,
315, 482–486.
Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., Kim, J., Aryee, M. J., Ji, H., Ehrlich, L. I. R., Yabuuchi, A., Takeuchi, A., Cunniff, K. C., Hongguang, H., Mckinney-
Freeman, S., Naveiras, O., Yoon, T. J., Irizarry, R. A., Jung, N., Seita, J., Hanna, J., Murakami, P., Jaenisch, R., Weissleder, R., Orkin, S. H., Weissman, I. L.,
Feinberg, A. P., & Daley, G. Q. (2010). Epigenetic memory in induced pluripotent stem cells. Nature, 467, 285–290.
Kimmelman, J. (2012). Ethics, ambiguity aversion, and the review of complex translational clinical trials. Bioethics, 26, 242–250.
Kitzinger, J., & Williams, C. (2005). Forecasting science futures: legitimising hope and calming fears in the embryo stem cell debate. Soc. Sci. Med., 61, 731–740.
Klimanskaya, I., Chung, Y., Becker, S., Lu, S. J., & Lanza, R. (2006). Human embryonic stem cell lines derived from single blastomeres. Nature, 444, 481–485.
Knight, A. (2008). Systematic reviews of animal experiments demonstrate poor contributions toward human healthcare. Rev. Recent Clin. Trials, 3, 89–96.
Knowles, L. P. (2009). Stem Cell Hype and the Dangers of Stem Cell ‘Tourism’. Ethics White Paper for the Stem Cell Network. http://www.stemcellnetwork.ca.
Lacham-Kaplan, O., Chy, O., & Trounson, A. (2006). Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.
Stem Cells, 24, 266–273.
Lau, D., Ogbogu, U., Taylor, B., Stanfinski, T., Menon, D., & Caulfield, T. (2008). Stem cell clinics online: the direct-to-consumer portrayal of stem cell medicine. Cell Stem Cell, 3,
591–594.
Laurent, L. C., Ulitsky, I., Slavin, I., Tran, H., Schork, A., Morey, R., Lynch, C., Harness, J. V., Lee, S., Barrero, M. J., Ku, S., Martynova, M., Semechkin, R., Galat, V., Gottesfeld, J.,
Belmonte, J. C. I., Murry, C., Keirstead, H. S., Park, H. S., Schmidt, U., Laslett, A. L., Muller, F. J., Nievergelt, C. M., Shamir, R., & Loring, J. F. (2011). Dynamic changes in the
copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture. Cell Stem Cell, 8, 106–118.
Ledford, H. (2009). Hybrid embryos fail to live up to stem-cell hopes. Nature, 457, 642.
Lemmens, T. (2004). Leopards in the temple: restoring scientific integrity to the commercialized scene. J. Law Med. Ethics, 32, 641–657.
Liao, S. M. (2005). Rescuing human embryonic stem cell research: the Blastocyst Transfer Method. Am. J. Bioeth., 5, 8–16.
Lindvall, O. (2012). Why is it taking so long to develop clinically competitive stem cell therapies for CNS disorders? Cell Stem Cell, 10, 660–662.
Lister, R., Pelizzola, M., Kida, Y. S., Hawkins, R. D., Nery, J. R., Hon, G., Antosiewicz-Bourget, J., O’Malley, R., Castanon, R., Klugman, S., Downes, M., Yu, R., Stewart, R., Ren, B.,
Thomson, J. A., Evans, R. M., & Ecker, J. R. (2011). Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature, 471, 68–73.
Louis, K. S., Jones, L. M., Anderson, M. S., Blumenthal, D., & Campbell, E. G. (2001). Entrepreneurship, secrecy, and productivity: a comparison of clinical and non-clinical life
sciences faculty. J. Technol. Transf., 26, 233–245.
Lyle, R. (1997). Gametic imprinting in development and disease. J. Endocrinol., 155, 1–12.
Lysaght, T., & Campbell, A. (2011). Regulating autologous adult stem cells: the FDA steps up. Cell Stem Cell, 9, 393–396.
Macklin, R. (2003). Dignity is a useless concept. BMJ, 327, 1419–1420.
Maienschein, J., Sunderland, M., Ankeny, R. A., & Robert, J. S. (2008). The ethos and ethics of translational research. Am. J. Bioeth., 8, 43–51.
Makkar, R. R., Smith, R. R., Cheng, K., Malliaras, K., Thomson, L. E. J., Berman, D., Czer, L. S. C., Marban, L., Mendizabal, A., Johnston, P. V., Russell, S. D., Schuleri, K. H.,
Lardo, A. C., Gerstenblith, G., & Marban, E. (2012). Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective,
randomised phase 1 trial. Lancet, 379, 895–904.
Malchenko, S., Galat, V., Seftor, E. A., Vanin, E. F., Costa, F. F., Seftor, R. E. B., Soares, M. B., & Hendriz, M. J. C. (2010). Cancer hallmarks in induced pluripotent cells: new
insights. J. Cell. Physiol., 225, 390–393.
Marchant, J. (2006). Human eggs supply ’ethical’ stem cells. Nature, 441, 1038.
Marques-Mari, A. I., Lacham-Kaplan, O., Medrano, J. V., Pellicer, A., & Simón, C. (2009). Differentiation of germ cells and gametes from stem cells. Hum. Reprod. Update, 15,
379–390.
Marquis, D. (2002). An argument that abortion is wrong. In J. Arthur (Ed.), Morality and Moral Controversies. Readings in Moral, Social and Political Philosophy (pp. 218–227).
New Jersey: Prentice Hall.
Mastenbroek, S., Twisk, M., Van der Veen, F., & Repping, S. (2011). Preimplantation genetic screening: a systematic review and meta-analysis of RCTs. Hum. Reprod. Update, 17,
454–466.
Master, Z. (2005). Can we really bypass the moral debate for embryo research? Am. J. Bioeth., 5, 27–28.
Master, Z. (2006). ES cell gametes: the new frontier in human reproduction. Hum. Reprod., 21, 857–863.
Master, Z., & Crozier, G. K. (2011). Symbolism and sacredness of human parthenotes. Am. J. Bioeth., 11, 37–39.
Master, Z., & Resnik, D. B. (2011a). Reforming stem cell tourism. Scientist. http://the-scientist.com.
Master, Z., & Resnik, D. B. (2011b). Stem cell tourism and scientific responsibility. EMBO Rep., 12, 992–995.
Master, Z., & Crozier, G. K. (2012). The ethics of moral compromise for stem cell research policy. Health Care Anal., 20, 50–65.
Master, Z., & Ogbogu, U. (2012). Stem cell tourism in the era of personalized medicine: what we know, and what we need to know. Curr. Pharmacogenomics Person. Med., 10,
106–110.
Master, Z., & Resnik, D. B. (2013). Hype and public trust in science. Sci. Eng. Ethics, 19, 321–335. https://doi.org/10.1007/s11948-011-9327-6.
Master, Z., Laforce, D., McLeod, M., & Williams-Jones, B. (2007a). Moral and scientific considerations in embryonic stem cell research. Stem Cell Res. J., 1, 189–239.
Master, Z., McLeod, M., & Mendez, I. (2007b). Benefits, risks and ethical considerations in translation of stem cell research into clinical applications in Parkinson’s disease. J. Med.
Ethics, 33, 169–173.
Master, Z., Zarzeczny, A., Rachul, C., & Caulfield, T. (2013inpress). What’s missing? Discussing stem cell translational research in educational information on stem cell “tourism”.
J. Law Med. Ethics, 41, 254–268.
Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J. C., Yakir, B., Clark, A. T., Plath, K., Lowry, W. E., & Benvenisty, N. (2010). Identification and classification of chromosomal
aberrations in human induced pluripotent stem cells. Cell Stem Cell, 7, 521–531.
Meissner, A., & Jaenisch, R. (2006). Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature, 439, 212–215.
Melton, D. A., Daley, G. Q., & Jennings, C. G. (2004). Altered nuclear transfer in stem-cell researchda flawed proposal. N. Engl. J. Med., 351, 2791–2792.
Mendick, R., & Palmer, A. (23 October 2010). Baby death scandal at stem cell clinic which treats hundreds of British patients a year. Telegraph. http://www.telegraph.co.uk/news/.
Mendick, R., & Hall, A. (8 May 2011). Europe’s largest stem cell clinic shut down after death of baby. Telegraph. http://www.telegraph.co.uk/news.
Meyer, J. R. (2008). The significance of induced pluripotent stem cells for basic research and clinical therapy. J. Med. Ethics, 34, 849–851.
Miller, N. (22 November 2009). Stem cell therapy sees man “infected” with cancer. WAToday. http://www.watoday.com.au.
Minger, S. (2007). Interspecies SCNT-derived human embryosda new way forward for regenerative medicine. Regen. Med., 2, 103–106.
Mowery, D., Nelson, R. R., Sampat, B. N., & Ziedonis, A. A. (2001). The growth of patenting and licensing by U.S. universities: an assessment of the effects of the Bayh-Dole act of
1980. Res. Policy, 30, 99–119.
Murdoch, B., Rachul, C., & Caulfield, T. (2011). Biotechnology and science in video games: a destructive portrayal? Health Law Rev., 20, 13–17.
Murdoch, C., & Caulfield, T. (2009). Commercialization, patenting and genomics: researcher perspectives. Genome Med., 1, 22.
Nagy, A., Gócza, E., Diaz, E. M., Prideaux, V. R., Iványi, E., Markkula, M., & Rossant, J. (1990). Embryonic stem cells alone are able to support fetal development in the mouse.
Development, 110, 815–821.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., & Roder, J. C. (1993). Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl
Acad. Sci., 90, 8424–8428.
Nature. (2010). Stem-cell laws in China fall short. Nature, 467, 633.
Nature Cell Biology. (2012). Stem cell research: regulating translational application. Nat. Cell Biol., 14, 557.
Nelkin, D. (1995). Selling Science (revised ed.). New York, NY: WH Freeman.
Ness, R. B., Cramer, D. W., Goodman, M. T., Kjaer, S. K., Mallin, K., Mosgaard, B. J., Purdie, D. M., Risch, H. A., Vergona, R., & Wu, A. H. (2002). Infertility, fertility drugs, and
ovarian cancer: a pooled analysis of case-control studies. Am. J. Epidemiol., 155, 217–224.
Newson, A. J., & Smajdor, A. C. (2005). Artificial gametes: new paths to parenthood? J. Med. Ethics, 31, 184–186.
Nisbet, M. C. (2004). Explaining majority support for stem cell research. Skept. Inq. http://www.csicop.org.
Nisbet, M. C., & Lewenstein, B. V. (2002). Biotechnology and the American Media: the policy process and the elite press, 1970 to 1999. Sci. Commun., 23, 359–391.
Nuffield Council on Bioethics. (October 2011). Human Bodies: Donation for Medicine and Research. http://www.nuffieldbioethics.org/donation.
Ogbogu, U. (2006). A review of pressing ethical issues relevant to stem cell translational research. Health Law Rev., 14, 39–43.
Padawer, R. (2002). Soaring Egg Donation Prices Causing Ethical Concerns. http://www.mindfully.org/GE/GE4/Egg-Donation-Ethical-Concerns7oct02.htm.
Paul, P. J., II (2003). The unspeakable crime of abortion. In B. Steinbock, J. D. Arras, & A. London (Eds.), Ethical Issues in Modern Medicine (sixth ed., pp. 461–463). New York:
McGraw-Hill.
Pearson, H. (2006). Early embryos can yield stem cells. and survive. Nature, 442, 858.
Phimister, E. G. (2005). A tetraploid twist on the embryonic stem cell. N. Engl. J. Med., 353, 1646–1647.
Polo, J. M., Liu, S., Figueroa, M. E., Kulalert, W., Eminli, S., Tan, K. Y., Apostolou, E., Stadtfeld, M., Li, Y., Shioda, T., Natesan, S., Wagers, A. J., Melnick, A., Evans, T., &
Hochedlinger, K. (2010). Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat. Biotechnol., 28, 848–855.
Rachul, C. (2011). What have I got to lose? An analysis of stem cell therapy patients’ blogs. Health Law Rev., 20, 5–12.
Rayment, E. A., & Williams, D. J. (2010). Concise review: mind the gap: challenges in characterizing and quantifying cell- and tissue-based therapies for clinical translation. Stem
Cells, 28, 996–1004.
Reed, J. C., White, E. L., Aubé, J., Lindsley, C., Li, M., Sklar, L., & Schreiber, S. (2012). The NIH’s role in accelerating translational sciences. Nat. Biotechnol., 30, 16–19.
Regenberg, A., Hutchinson, L., Schanker, B., & Mathews, D. (2009a). Medicine on the fringe: stem cell-based interventions in advance of evidence. Stem Cells, 27, 2312–2319.
Regenberg, A., Mathews, D. J., Blass, D. M., Bok, H., Coyle, J. T., Duggan, P., Faden, R., Finkel, J., Gearhart, J. D., Hillis, A., Hoke, A., Johnson, R., Johnston, M., Kahn, J.,
Kerr, D., King, P., Kurtzberg, J., Liao, S. M., McDonald, J. W., McKhann, G., Nelson, K. B., Rao, M., Siegel, A. W., Smith, K., Solter, D., Song, H., Sugarman, J., Vescovi, A.,
Young, W., Greely, H. T., & Traystman, R. J. (2009b). The role of animal models in evaluating reasonable safety and efficacy for human trials of cell-based interventions for
neurologic conditions. J. Cereb. Blood Flow Metab., 29, 1–9.
Rizzieri, A. (2012). Stem cell research on embryonic persons is just. J. Bioeth. Inq., 9, 195–203.
Robertson, J. A. (1995). Symbolic issues in embryo research. Hastings Cent. Rep., 25, 37–38.
Rodriguez, S., Campo-Engelstein, L., Tingen, C., & Woodruff, T. (2011). An obscure rider obstructing science: the conflation of parthenotes with embryos in the Dickey-Wicker
amendment. Am. J. Bioeth., 11, 20–28.
Rohn, J. (2012). California’s CIRM courts industry. Nat. Biotechnol., 30, 572.
Rossing, M. A., Daling, J. R., Weiss, N. S., Moore, D. E., & Self, S. G. (1994). Ovarian tumors in a cohort of infertile women. N. Engl. J. Med., 331, 771–776.
Ryan, K. A., Sanders, A. N., Wang, D. D., & Levine, A. D. (2010). Tracking the rise of stem cell tourism. Regen. Med., 5, 27–33.
Ryan, M. A. (2001). Creating embryos for research: on weighing symbolic costs. In P. Lauritzen (Ed.), Cloning and the Future of Human Embryo Research (pp. 50–66). New York:
Oxford University Press.
Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., & Lanza, R. (2012). Embryonic stem cell
trials for macular degeneration: a preliminary report. Lancet, 379, 713–720.
Scott, C. T., McCormick, J. B., DeRouen, M. C., & Owen-Smith, J. (2011). Democracy derived? New trajectories in pluripotent stem cell research. Cell, 145, 820–826.
Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M., Littlefield, J. W., Donovan, P. J., Blumenthal, P. D., Huggins, G. R., & Gearhart, J. D. (1998). Derivation of pluripotent stem
cells from cultured human primordial germ cells. Proc. Natl Acad. Sci. U.S.A., 95, 13726–13731.
Shane, S. (2004). Encouraging university entrepreneurship? The effect of the Bayh-Dole Act on university patenting in the United States. J. Bus. Venturing, 19, 127–151.
Sipp, D. (5 September 2011). Stem Cell Pseudomedicine: Calling a Spade a Spade. Blog: Stem Cell Treatment Monitor. http://sctmonitor.blogspot.com.
Sismondo, S. (2007). Ghost management: how much of the medical literature is shaped behind the scenes by the pharmaceutical industry? PLoS Med., 4, e286.
Sismondo, S. (2008). How pharmaceutical industry funding affects trial outcomes: causal structures and responses. Soc. Sci. Med., 66, 1909–1914.
St John, J., & Lovell-Badge, R. (2007). Human-animal cytoplasmic hybrid embryos, mitochondria, and an energetic debate. Nat. Cell Biol., 9, 988–992.
Stafford, N. (2009). Germany tightens law on stem cell treatments. BML, 339, b2967.
Steinbock, B. (1992). Life before Birth the Moral and Legal Status of Embryos and Fetuses. New York: Oxford University Press.
Steinbock, B. (2001). Respect for human embryos. In P. Lauritzen (Ed.), Cloning and the Future of Human Embryo Research (pp. 21–33). New York: Oxford University Press.
Steinbock, B. (2004). Payment for egg donation and surrogacy. Mt Sinai J. Med., 71, 255–265.
Sugarman, J. (2008). Human stem cell ethics: beyond the embryo. Cell Stem Cell, 2, 529–533.
Sullivan, S., & Eggan, K. (2006). The potential of cell fusion for human therapy. Stem Cell Rev., 2, 341–349.
Swerdlow, R. H. (2007). Mitochondria in cybrids containing mtDNA from persons with mitochondriopathies. J. Neurosci. Res., 85, 3416–3428.
Tachibana, M., Amato, P., Sparman, M., Gutierrez, N. M., Tippner-Hedges, R., Ma, H., Kang, E., Fulati, A., Lee, H. S., Sritanaudomchai, H., Masterson, K., Larson, J., Eaton, D.,
Sadler-Fredd, K., Battaglia, D., Lee, D., Wu, D., Jensen, J., Patton, P., Gokhale, S., Stouffer, R. L., Wolf, D., & Mitalipov, S. (2013). Human embryonic stem cells derived by
somatic cell nuclear transfer. Cell, 153, 1228–1238.
Tannsjo, T. (2007). Why no compromise is possible. In L. Gruen, L. Grabel, & P. Singer (Eds.), Stem Cell Research: The Ethical Issues (pp. 188–201). Oxford, UK: Blackwell.
Tecirlioglu, R. T., Guo, J., & Trounson, A. O. (2006). Interspecies somatic cell nuclear transfer and preliminary data for horse-cow/mouse iSCNT. Stem Cell Rev., 2, 277–287.
Testa, G., & Harris, J. (2005). Ethics and synthetic gametes. Bioethics, 19, 146–166.
The Oxford Dictionary of English. (2005). Sacred adjective. In C. Soanes, & A. Stevenson (Eds.), Oxford Reference Online (revised ed.). Oxford: Oxford University Press.
Thirabanjasak, D., Tantiwongse, K., & Thorner, P. (2010). Angiomyeloproliferative lesions following autologous stem cell therapy. J. Am. Soc. Nephrol., 21, 1218–1222.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., & Jones, J. M. (1998). Embryonic stem cell lines derived from human blastocysts.
Science, 282, 1145–1147.
Trounson, A., & Dewitt, N. D. (2012). Stem cell biology: towards the reality of cell therapeutics. Nat. Cell Biol., 14, 331.
Ueda, O., Jishage, K., Kamada, N., Uchida, S., & Suzuki, H. (1995). Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells
with cytochalasin B induced tetraploid embryos. Exp. Anim., 44, 205–210.
Underwood, E. (2012). Stem cells safe for rare brain disorder. ScienceNOW. http://news.sciencemag.org/sciencenow/.
Uniacke, S. (2004). Is life sacred? In B. Rogers (Ed.), Is Nothing Sacred? (pp. 59–80). New York: Routledge.
United Nations, 1948. Universal declaration of human rights. General Assembly Resolution 217A (III). (Unenacted Bill/Resolution).
Vatican. (2008). Instruction Dignitas Personae on Certain Bioethical Questions. http://www.vatican.va/.
Vita-Stim Concentrate, 2012. http://www.stemcellvitaminstore.com/.
Vogel, G. (10 May 2011). Authorities shut controversial German stem cell clinic. Science Insider. http://news.sciencemag.org/scienceinsider.
von Tigerstrom, B. J. (2008). The challenges of regulating stem cell-based products. Trends Biotechnol., 26, 653–658.
von Tigerstrom, B. J. (2011). The Food and Drug Administration, regenerative sciences, and the regulation of autologous stem cell therapies. Food Drug Law J., 66, 479–506.
Vrana, K. E., Hipp, J. D., Goss, A. M., McCool, B. A., Riddle, D. R., Walker, S. J., Wettstein, P. J., Studer, L. P., Tabar, V., Cunniff, K., Chapman, K., Vilner, L., West, M. D.,
Grant, K. A., & Cibelli, J. B. (2003). Nonhuman primate parthenogenetic stem cells. Proc. Natl Acad. Sci. U.S.A., 100(Suppl. 1), 11911–11916.
Wadman, M. (2010). The bridge between lab and clinic. Nature, 468, 877.
Wakeley, K. E., & Grendys, E. C. (2000). Reproductive technologies and risk of ovarian cancer. Curr. Opin. Obstet. Gynecol., 12, 43–47.
Waldby, C., & Cooper, M. (2008). The biopolitics of reproduction. Aust. Feminist Stud., 23, 57–73.
Ward, V. (30 September 2010). Stem cell doctor Robert Trossel struck off medical register. Telegraph. http://www.telegraph.co.uk.
Warren, M. A. (1973). On the moral and legal status of abortion. Monist, 57, 43–61.
Weaver, J. (2010). Animal studies paint misleading picture. Sci. Am. http://www.scientificamerican.com/.
Wilson, J. (2009). A history lesson for stem cells. Science, 324, 727–728.
Wislar, J. S., Flanagin, A., Fontanarosa, P. B., & Deangelis, C. D. (2011). Honorary and ghost authorship in high impact biomedical journals: a cross sectional survey. BMJ, 343,
d6128.
Woolf, S. H. (2008). The meaning of translational research and why it matters. JAMA, 299, 211–213.
Yamanaka, S. (2007). Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell, 1, 39–49.
Young, S. (2011). Mississippi to vote on ‘personhood’. Nature, 479, 13–14.
Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, I. I., & Thomson, J. A. (2009). Human induced pluripotent stem cells free of vector and transgene sequences. Science,
324, 797–801.
Zarzeczny, A., & Caulfield, T. (2010). Stem cell tourism and doctors’ duties to minors. Am. J. Bioeth., 10, 3–15.
Zarzeczny, A., Scott, C., Hyun, I., Bennett, J., Chandler, J., Chargé, S., Heine, H., Isasi, R., Kato, K., Lovell-Badge, R., McNagny, K., Pei, D., Rossant, J., Surani, A., Taylor, P. L.,
Ogbogu, U., & Caulfield, T. (2009). iPS cells: mapping the policy issues. Cell, 139, 1032–1037.
Zarzeczny, A., Rachul, C., Nisbet, M., & Caulfield, T. (2010). Stem cell clinics in the news. Nat. Biotechnol., 28, 1243–1246.
Zarzeczny, A., Rachul, C., & Caulfield, T. (2012). The phenomenon of stem cell tourism. In A. Atala (Ed.), Progenitor and Stem Cell Technologies and Therapies: Principles and
Issues (vol. 1, pp. 206–233). Woodhead Publishing.
Zhao, T., Zhang, Z. N., Rong, Z., & Xu, Y. (2011). Immunogenicity of induced pluripotent stem cells. Nature, 474, 212–215.
Zhao, X. Y., Li, W., Lv, Z., Liu, L., Tong, M., Hai, T., & Hao, J. (2010). Efficient and rapid generation of induced pluripotent stem cells using an alternative culture medium. Cell Res.,
20, 383–386.
Eye Diseases and Stem Cells
H Ouyang, DH Nguyen, and K Zhang, Shiley Eye Center and Institute for Genomic Medicine, University of California at San Diego, La
Jolla, CA, USA
Introduction 599
Anatomy of the Eye 599
Schematic Overview of the Retina 600
Diseases of the Retina 600
Age-Related Macular Degeneration 600
Pathophysiology of AMD 600
Diagnosis of AMD 601
Treatment of AMD 601
Glaucoma 602
Pathophysiology of POAG 602
Diagnosis of POAG 602
Treatment of POAG 602
Retinitis Pigmentosa 603
Diabetic Retinopathy 603
Sources of Stem Cells 603
Embryonic Stem Cells 603
Mesenchymal Stem Cells 603
Retinal Progenitor Cells 604
Müller Glial Cells 604
Stem Cell Therapy in Retinal Diseases 604
RPE Transplantation 604
Photoreceptors Cells Transplantation 605
Ganglion Cells Transplantation 605
Cornea Injury and Stem Cell Therapy 605
Potentials and Limitations of Stem Cell-Based Therapy 606
References 606
Glossary
AMD Age-related macular degeneration
CLET Cultivated corneal limbal epithelial transplantation
CNS Central nervous system
DP Diabetic retinopathy
ESCs Embryonic stem cells
IOP Intraocular pressure
iPSCs Induced pluripotent stem cells
LESC Limbal epithelial stem cells
MGCs Müller glial cells
MSCs Mesenchymal stem cells
POAG Primary open-angle glaucoma
RD Retina degeneration
RGCs Retinal ganglion cells
RP Retinitis pigmentosa
RPE Retinal pigment epithelium
RPCs Retinal progenitor cells
SC Stem cell
TACs Transit amplifying cells
VEGF Vascular endothelial growth factors

Regenerative Engineering j Eye Diseases and Stem Cells 599
Introduction
Visual impairment or blindness can dramatically reduce the quality of life for an individual. It can be devastating to suddenly or
gradually lose the ability to read, drive, or recognize one’s family and friends. Vision loss can result from common eye diseases
which lead to damage of the retina and optic nerve, such as age-related macular degeneration (AMD), diabetic retinopathy
(DR), retinitis pigmentosa (RP), and glaucoma. Other injuries to the cornea can also cause scarring and subsequent blindness.
Vision loss is a considerable public health burden. According to recent statistics reported by the Prevent Blindness America organi-
zation, the annual cost of adult vision problems in the U.S. is around $51.4 billion, and the cost will continue to rise with 50 000 or
more people becoming blind each year. Transplantation of stem cells in animal models of eye diseases or clinical trials demon-
strated that stem cell-based therapy is a promising treatment to restore vision in patients with retinal diseases or ocular surface
injury.
Anatomy of the Eye
The eyeball contains three main layers: the outer fibrous layer, the middle uveal layer, and the inner neural layer (Figure 1). The
surface of the eye is protected by the conjunctiva, which is composed of stratified columnar epithelium. Underneath the conjunctiva
is the sclera, commonly known as the white part of the eye. The sclera is made of a tough tissue that connects with the cornea. The
border between the sclera and the cornea is called the limbus. The pupil is located in the center of the iris and can change size to
accommodate the amount of incoming light.
The middle layer of the eyeball houses the aqueous humor, lens, blood vessels, lymphatic vessels, ciliary body and its extension,
the iris. The lens can change shape and in doing so change how much light rays are ‘bent’ before they go through a viscous and
jellylike substance called the vitreous, before finally arriving at the retina. The retina is a crucial light sensing structure by which
visual information is translated into electrical impulses and transmitted to the visual cortex of the brain by the optic nerve.
The pathway light travels consist of first traveling through the cornea and pupil. An amount of refraction happens at the cornea,
after which light is further focused by the lens. It continues through the vitreous and onto the retina. The retina transfers light signals
to the brain which processes the information into images that we see.
Figure 1 Schematic diagram of the anatomy of the eye and layers of the retina.
600 Regenerative Engineering j Eye Diseases and Stem Cells
Schematic Overview of the Retina

There are nine layers of the retina (Figure 1). Starting most internally and immediately behind the vitreous, the layers are the
internal limiting membrane, the stratum opticum, the ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the outer
plexiform layer, the outer nuclear layer, the external limiting membrane, and the layer of photoreceptors.
There are millions of photoreceptors called cones and rods, which sense color or light intensity, respectively. Rods are distributed
more heavily around the peripheral retina while cones are mainly concentrated in the macula that is at the back of the retina. The
central part of the macula is called the fovea, containing the highest amounts of cones and thus providing the sharpest sense of sight.
Rods are totally absent at the fovea.
Rods and cones transmit visual signals to neurons called bipolar cells, which in turn pass the signals to retinal ganglion cells
(RGCs) whose nerve fibers merge to form the optic nerve. Support cells, called horizontal cells, facilitate communications between
photoreceptors. Amacrine cells are support cells that control communications between bipolar cells and RGCs. Müller glia and
astrocytes are another two support cells among the neural retina.
Diseases of the Retina

Age-Related Macular Degeneration
AMD is a progressive disease of the macula, which is an important component in the retina that is responsible for visual acuity and
color vision. AMD is among the top three causes of irreversible blindness among elderly people worldwide, while in the United
States it is the leading cause of visual impairment (Pascolini et al., 2004). It is estimated that about 1.75 million people in the
United States are affected by AMD, and this figure is expected to increase to 3 million by 2020 due to the rapid aging of the American
population. Of note, about 7 million Americans currently have evidence of early changes in the retina that are at risk for progression
to AMD (Friedman et al., 2004). Visual impairment is a feared disability, and is related to a significant reduction in the quality of life
and overall health status.
Pathophysiology of AMD
AMD is a multifactorial disease with a complex mechanism of development that is not completely understood. It has been well
documented that AMD results from genetic and environmental factors. Risk factors for developing AMD include age, cigarette
smoking, obesity, cardiovascular disease, sunlight exposure, and certain genetic variants in the physiologic pathways that regulate
inflammation, angiogenesis, and innate immunity (Lim et al., 2012).
Evidence of early AMD development is drusen, which are accumulations of extracellular debris under the retinal pigment epithe-
lium (RPE), a crucial component of the retina. Drusen are categorized by location, size, and texture. They are observable on clinical
exams and photographs of the retina, and their presence without evidence of vision loss defines early AMD. Many studies have
found that substantial progression in size and number, as well as extensive involvement of drusen in the area of the macula, is asso-
ciated with the risk of developing the two advanced forms of AMD with vision loss, including geographic atrophy (GA) and
choroidal neovascularization.
Compared to the normal eye (Figure 2), GA, or ‘dry’ AMD, is defined as the presence of a well-demarcated area of at least 175 mm
in diameter in the retina where there is marked atrophy of the RPE, which manifests as focal depigmentation without evidence of
abnormal blood vessel growth (Bird et al., 1995) (Figure 3). Choroidal neovascularization, or ‘wet’ AMD, is diagnosed when there is
Figure 2 Normal eye. Fundus photograph.

Figure 3 ‘Dry’ age-related macular degeneration, or geographic atrophy. Fundus photograph.
Figure 4 ‘Wet’ age-related macular degeneration, or choroidal neovascularization. Optical coherence tomography image.
presence of growth of new blood vessels under the retina that can cause bleeding, fibrosis, and detachment of the retina (Figure 4).
The two advanced forms of AMD can lead to significant deterioration of vision.
Diagnosis of AMD
Patients with AMD typically present with a progressive decrease in central vision, which manifests as difficulty seeing straight lines,
dark patches in the visual field, and decrease of color vision. In some cases of ‘wet’ AMD in which bleeding from the abnormal blood
vessels cause a more acute insult to the retina, sudden and more severe decreases in vision may occur due to detachment of the
retina. Diagnosis of AMD requires a complete ophthalmologic examination, which typically includes visual acuity measurements,
intraocular pressure (IOP) reading, slit-lamp examination, indirect ophthalmoscopy, and ocular imaging studies. Fundus photog-
raphy, optical coherence tomography, and fundus fluorescein angiography are several imaging modalities that are very helpful in
the diagnosis of AMD and categorization of the stage and type of the disease. They are instrumental in detecting the presence of fluid
under the retina and disruptions of the layers of the retina that reflect advanced AMD, as well as provide objective means to evaluate
effectiveness of treatment in the ‘wet’ type of AMD.
Treatment of AMD
Currently, there is no treatment for ‘dry’ AMD. In the case of ‘wet’ AMD, intraocular injection of antiangiogenic medications is avail-
able to halt and/or minimize the growth of abnormal blood vessels. These medications target vascular endothelial growth factors
(VEGF), which are key components in the generation of new blood vessels. Current anti-VEGF therapy includes ranibizumab
(‘Lucentis’), bevacizumab (‘Avastin’), and aflibercept (‘Eylea’). Several short-term studies have indicated that intravitreal bevacizu-
mab results in improvement in visual acuity that is similar to the improvement seen with ranibizuma (Spaide et al., 2006; Algvere
et al., 2008). In 2011, an NIH sponsored randomized clinical trial provided further evidence that ranibizumab and bevacizumab
provided similar effects in neovascular AMD when administered on the same schedule (Martin et al., 2011). Other treatment
modalities include laser photocoagulation, in which direct laser beams target choroidal vessels to stop their growth.
Glaucoma
Glaucoma is a chronic and progressive disease of the optic nerve, which causes the loss of peripheral vision that may progress to
total loss of vision if untreated. Glaucoma is categorized based on the angle of the iridocorneal angle, or the angle between the
iris and the cornea. Primary open-angle glaucoma (POAG) is the most common type of glaucoma in the Western world, and is
characterized by a normal and open iridocorneal angle, in contrast to angle-closure glaucoma in which the iridocorneal angle creates
obstruction of the outflow of the aqueous humor, the circulation that nourishes the cornea and lens, and causes an acute increase in
IOP. POAG can be, but is not always, associated with elevated IOP. The molecular pathogenesis of this disease is largely unknown,
and lowering IOP is the only available treatment. However, POAG patients eventually progress to blindness despite aggressive IOP
treatment. Risk factors for POAG include age, elevated IOP, family history of POAG, myopia, and being of African descent. Some
research studies have also found that having a low diastolic pressure is another risk factor (Kwon et al., 2009).
Glaucoma poses a heavy public health burden, and is currently the second most common cause of blindness worldwide. In the
United States, adult-onset POAG is defined as occurring after 40 years of age and is estimated to affect two out of every 10 adults
aged 40 and older. In people with African ancestry, glaucoma is the leading cause of irreversible blindness, and about one-third of
the blindness is due to POAG (Leske, 2007).
Pathophysiology of POAG
POAG is characterized by slow and progressive optic nerve degeneration due to the death of RGCs, which are neurons with critical
functions in the visual pathway. Loss of RGCs and their axons lead to characteristic appearances of the optic nerve that are observ-
able on clinical examination and imaging studies. Typically, compared to the normal eye (Figure 2), the glaucomatous eye has
evidence of thinning of the neuroretinal rim which enlarges the optic nerve cup, and increases the optic cup to optic disk ratio
(Figure 5). At the same time, there is also loss of surrounding support cells and vasculature. The loss of RGCs and their axons
are the cause of visual impairment in POAG (Quigley, 2011).
Diagnosis of POAG
As POAG is a chronic and progressive disease that causes painless loss of vision, many patients with POAG may not seek medical
attention until the damage is advanced with a substantial loss of visual field. Besides a complete ophthalmologic examination
which includes measurement of the IOP, imaging studies to assess the severity of damage to the optic nerve are crucial. Automated
visual field testing provides an objective assessment of the degree of loss of visual field, while direct photography of the optic nerve
provides a direct evaluation of optic nerve cupping.
Treatment of POAG
Controlling IOP is important in the management of POAG. Surgical and medical alternatives exist for lowering IOP. Typically,
a patient with POAG is started on eye drops that lower IOP and maintained on the medication or combination of medications
as long as the pressure is kept within a normal range. When medical treatment fails, more invasive options are needed such as laser
therapy to the drainage network of the eye to enhance outflow of the aqueous humor, or surgical procedures where drainage devices
are placed inside the eye to facilitate the drainage of intraocular fluid and therefore lower the IOP.
Figure 5 Primary open-angle glaucoma. Image of the optic nerve.

Retinitis Pigmentosa
A degenerative disease of photoreceptor cells in the retina, RP (Kempen et al., 2004) is a group of diffuse retinal dystrophies that is
genetically and clinically diverse. RP is an inheritable disease, with many cases due to mutations of the rhodopsin gene, which is
responsible for the production of the pigment of photoreceptors. In some cases, RP is a sporadic mutation without any previous
family history. RP can present as a solitary ocular defect or in combination with other systemic pathologies such as hearing loss
(Usher syndrome) and kidney disease (Bardet–Biedl syndrome). Patients typically present with loss of peripheral and night vision,
usually in the second decade of life but may manifest earlier depending on the pattern of inheritance. RP is characterized by pigmen-
tary changes in the retina which give the disease its name, as well as changes in the retinal blood vessels and degeneration of the
photoreceptors. Optical imaging studies to detect pigmentary changes and disruption of retinal layers, as well as eletroretinography,
or the study of the function of various retinal cells including photoreceptors, are extremely helpful in the diagnosis of RP. Currently
there is no cure for RP.
Diabetic Retinopathy
Diabetes is one of the most common diseases worldwide. It is estimated that the number of patients with diabetes in the world will
reach 429 million by 2030. Approximately 4.1 million people in the United States have DR, of which almost 1 million has sight-
threatening retinopathy (Kempen et al., 2004). It is most prevalent in Latino Americans. DR (Jiang et al., 2002) is a disease of the
retinal blood vessel networks that can lead to irreversible blindness. It is a severe and the most common complication of diabetes.
The two most important risk factors for DR are the duration of diabetes and the adequacy of glucose control. DR can be classified
into two forms, nonproliferative and proliferative, depending respectively on the absence or presence of abnormal retinal blood
vessels and bleeding. The major mechanisms by which proliferative DR results in vision loss are (1) retinal vascular leakage and
exudation resulting in macular edema (Thompson et al., 1996), and (2) retinal capillary closure leading to retinal ischemia and
secondary neovascular proliferation with its attendant complications of vitreous hemorrhage and tractional retinal detachment.
On ocular imaging studies, disruption of the retinal layers due to accumulation of fluid and blood from abnormal vessels leakage
can be observed. Treatment of proliferative DR includes laser therapy to target abnormal fluid accumulation in the eye, injection of
antiangiogenic medications to limit the growth of aberrant blood vessels, and in severe cases surgeries to prevent further injuries to
the retina. DR is a chronic disease, and timely diagnosis and close ophthalmologic monitoring are crucial to prevent the onset or
progression of vision loss.
Sources of Stem Cells

Embryonic Stem Cells
Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst-stage embryos. They possess unlimited self-
renewal capabilities and the ability to differentiate into any adult cell types within human tissue (Evans and Kaufman, 1981).
ESCs hold great therapeutic promises in the regeneration of functional cell types including neurons (Reubinoff et al., 2001; Zhang
et al., 2001), hepatocytes (Shirahashi et al., 2004), and cardiomyocytes (Laflamme et al., 2007), among others.
Successful differentiation to eye field cells from human ESCs has been done since 2009. Data have shown that ESCs can differ-
entiate into photoreceptor progenitors and RPE in a sequence and time course that mimic in vivo human retinal development
(Meyer et al., 2009).
The Sasai group has demonstrated that an optic cup can form by self-organization both in human and mouse ESC culture
(Nakano et al., 2012; Eiraku et al., 2011). This ESC-derived retina structure grows into multilayered tissue containing two types
of photoreceptors, rods and cones. Whether this optic cup can be induced to form an entire eye remains to be seen, but ESCs
do hold potential applications to the treatment of retinal degenerative disorders.

Induced pluripotent stem cells (iPSCs) are adult cells that have been made to resemble ESCs. iPSCs are induced in vitro to express
certain genes and factors such as Oct3/4, Sox2, Klf4, and c-Myc that are used in ESCs. iPSCs have been shown to have regenerative
properties and have also been shown to be similar in characteristics and abilities to ESCs (Takahashi and Yamanaka, 2006).
It has been proven that human iPSCs have a similar potential as ESCs to form RPE and photoreceptors (Meyer et al., 2009).
iPSCs transplantation does not require immunosuppression and has no ethical issues. However, iPSCs and ESCs differ in clinical
outcomes. Moreover, potential risks exist during integration because of the oncogene expression for iPSCs generation (Puzio-Kuter
and Levine, 2009). Transplantation of iPSCs was observed to form tumors in the three germ layers in mice.
Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are cells that are collected from bone marrow biopsies and umbilical cord blood, which are used
for autologous cell transplantation (Bianco and Robey, 2001). They can also be harvested from adipose tissue, placenta (In ’t Anker
et al., 2004), cord blood, and liver (Campagnoli et al., 2001). Haematopoetic stem cells are mobilized from bone marrow to the
bloodstream which makes it less invasive to obtain these cells, though methods of mobilizing mesenchymal cells have proven to be
challenging. MSCs differentiate into mesodermal cells (bone, cartilage, and muscle), but have also been shown to have the ability to
dedifferentiate into nonmesoderm cells in vitro (Jiang et al., 2002). An in vivo animal model has shown that when MSCs was injected
into the subretinal space, the stem cells were integrated and differentiated into photoreceptors, and also slowed the degradation of
retinal cells in Royal College of Surgeons rats (Inoue et al., 2007).
Retinal Progenitor Cells

Retinal progenitor cells (RPCs) are found from fetal or neonatal retinas. These cells are used to create all of the retinal cells that are
formed during embryonic development. In the ectoderm, cells are pushed by transcription factors into a multistep cascade to
develop retinal tissue. RPCs can be used to grow neuronal and glial cells (Müller cells), retinal support cells, and can enter all retinal
layers to adopt characteristics of several retinal cells (Reh, 2006). This ability suggests that RPCs can be useful in treating retinal
degenerative diseases.
Müller Glial Cells

It is more accepted that the central nervous system could be regenerated from radial glial cells in recent years. Müller glia is a kind of
radial glia located in the retina. Studies have demonstrated that species like zebra fish could regenerate retina from Müller glia by
reentering the cell cycle and producing neuronal progenitor cells (Fimbel et al., 2007). A population of Müller glia with stem cell
characteristics has also been found in the human adult retina. In vitro markers of neural progenitors are expressed and some of these
cells even express markers of mature retinal neurons under different culture conditions (Lawrence et al., 2007). Experiments on the
transplantation of Müller glial cells (MGCs) to rodent ganglion cell injury model showed that predifferentiated cells can integrate
into the host RGC layer and restore partial scotopic threshold response in electroretinography studies (Singhal et al., 2010).
Stem Cell Therapy in Retinal Diseases
The eye is thought to be an ideal organ for cell transplantation due to its special structural characteristics. The optical media of the
eye, composed of cornea, lens, and vitreous, are extremely clear when healthy, which facilitate intravitreal and subretinal injection of
cells. After surgery, transplanted cells are assessed by imaging techniques such as scanning laser ophthalmoscope and optical coher-
ence tomography. Furthermore, cells could be ablated by laser treatment if overproliferation happened.
RPE Transplantation
The RPE is a monolayer of polarized pigmented cells that lies between the photoreceptor outer segments and the choriocapillaris. It
plays a crucial role in maintaining the outer blood retinal barrier. The apical surface of the RPE interdigitates with the adjacent
photoreceptor outer segments and participates in phagocytosis, transportation, and production of essential components. RPE
dysfunction usually results in photoreceptor degeneration and leads to several retinal disorders, such as AMD and RP (Kempen
et al., 2004).
Despite attempts to transplant autologous RPE to the macular region in patients with AMD, there was no postoperative improve-
ment in visual acuity at 1 year (Del Priore et al., 2001). Other disadvantages for autologous RPE grafts still remain, including the
difficulty and impracticality of obtaining sufficient quantities of autologous RPE cells, and the disadvantage that autologous cells
always carry the same genetic defect that led to the host disease. Fresh fetal and adult RPE cells have been employed to replace lost
RPE in patients with GA (Weisz et al., 1999), but considerable variability in the quality of the obtained RPE is one of its limitations.
These issues highlight the urgent need of exploring stem cell-based therapy to create functional RPE for transplantation.
Recently, the US Food and Drug Administration has approved the use of human ESC (hESC)-RPE in two human clinical trials in
patients with Stargardt’s dystrophy and AMD. A preliminary report in 2012 showed the description of hESC-derived cells trans-
planted into human patients with Stargardt’s macular dystrophy and dry AMD. In this report, hESCs are differentiated into greater
than 99% pure RPE under defined condition. Submacular injection of 5 104 hESC-RPE cells was delivered into a preselected
macular site with native albeit compromised RPE and photoreceptors. After 4 months, there were no identified signs of hyperpro-
liferation, tumorigenicity, or immune-mediated transplant rejection in either patient. And it is encouraging that during the obser-
vation period neither patient lost vision. Best corrected visual acuity improved from hand motions, and vision also seemed to
improve in the patient with dry AMD (Schwartz et al., 2012).
Successful RPE transplantation is not only determined by the quality and integration of cells, but also how these cells could
survive the rejection environment in the subretinal space. Moreover, the use of hESCs is still ethically controversial. In this case,
RPE-like cells generated from patients’ own iPSCs may be considered a better source. These iPSC-RPE cells are similar to RPE cells
according to the morphology, function of photoreceptor outer segment phagocytosis and shared markers of developing and mature
RPE cells.
Photoreceptors Cells Transplantation

Rods and cones are two types of photoreceptors in the retina. Rods are responsible for sensitivity to light and dark changes, move-
ment and shape of objects, while cones provide color vision (green, red and blue) and sharp images. When photoreceptors are
completely lost in retinal diseases, such as RP, vision restoration is impossible even with therapy of RPE transplantation. Therefore,
a replacement of photoreceptors is necessary in such cases.
Preliminary data showed that transplantation of fetal retinal cells into the midbrain was capable of making pretectal connections
and enabled the response to light (Klassen and Lund, 1990). Fetal retinal sheets as a cell mixture has also been used in patients with
AMD and RP and showed variable effects (Seiler and Aramant, 2012). Some improvement in visual function is more likely contrib-
uted by the positive factors secretion from transplanted tissue.
The feasibility of photoreceptor transplantation as a therapeutic strategy was shown by transplanted photoreceptor precursors
into adult Gnat1/ mice, which lack rod function and thus provide a model of congenital stationary night blindness. These
precursors could form synaptic connections with other retinal cells in the recipient retina and are light-responsive with similar func-
tion to normal photoreceptors in adults (Pearson et al., 2012).
RPCs transplantation in animal studies also demonstrated the ability to migrate and differentiate into photoreceptors (Bartsch
et al., 2008). Unlike ESCs and iPSCs, RPCs do not have unlimited self-renewal ability and have to be reobtained from fetus. Alter-
native strategies including generating photoreceptors from ESCs and iPSCs could be used as inexhaustible sources. Human
ESC-derived RPCs have been transplanted into Crx-deficient mice, an animal model for Lebers Congenital Amaurosis, with the
improvement in light response (Lamba et al., 2009).
An optimized protocol for differentiating photoreceptors from ESCs has been established recently that offers a direct transplan-
tation of photoreceptors to patients (Meyer et al., 2009). However, besides the ethical restrictions and immune rejection following
implantation, other disadvantages of poor cell quality, difficulty in purification and viability of photoreceptors remain to be solved.
Ganglion Cells Transplantation

RGCs receive visual signal from photoreceptors. Their axons bundle together to form the optic nerve and extend into the brain. The
death of RGCs is the cause of injury to the optic nerve in the common neurodegenerative disease of glaucoma (Quigley, 2011).
ESCs (Aoki et al., 2008) and MSCs (Johnson et al., 2010) could serve as a potential source of replacement for damaged RGCs.
Transplanted cells have been shown to migrate and integrate into the retina. However, to date, there has been no evidence of func-
tional improvement. There are protection effects in transplantation to rat model of glaucoma, but the reason is thought to be due to
the secretion of neurotrophic factors (Cho et al., 2005).
MGCs have been reported to retain the ability to divide indefinitely in vitro (Limb et al., 2002). Recently, it has been reported that
after an intravitreal injection of MGCs into RGC-depleted retina, vision is partly restored and the injected cells can be differentiated
into RGC precursors with the integration into the retina (Singhal et al., 2010). Cell replacement by MGCs transplantation may there-
fore offer a more promising therapy after irreversible damage or RGC loss in glaucoma.
Of note, a successful transplants/replacement means the transplanted cells must not only migrate and integrate into the ganglion
cell layer, but also have the ability to differentiate into functional RGCs and generate axons which could extend to the optic nerve
and beyond, which is a big additional challenge when compared to RPE and photoreceptor cell transplantation.
Cornea Injury and Stem Cell Therapy
The cornea is composed of five layers, including the outermost stratified corneal epithelium, Bowman’s layer, corneal stroma, Desce-
met’s membrane, and innermost corneal endothelium. Highly resistant tight junctions formed between neighboring corneal epithe-
lium cells protect the eye from injury and foreign body (Klyce, 1972). The transparency of the cornea depends on the epithelial
integrity and stromal avascularity. The cornea also provides the majority (two thirds) of the refractive power of the eye (Meek
et al., 2003). So the clarity of the cornea is essential for visual acuity.
There is a population called limbal epithelial stem cells (LESCs), which are located at the junction of cornea and sclera in an area
known as the limbus. These LESCs are presumed to be primitive. They can symmetrically divide to self renew and asymmetrically
produce daughter transit amplifying cells (TACs) which migrate centripetally to populate the basal layer of the corneal epithelium
(Tseng, 1989). The TACs divide and migrate superficially, becoming more differentiated and finally turning into postmitotic termi-
nally differentiated cells. LESCs deficiency can be the result of primary or acquired disease which affects corneal wound healing and
surface integrity, such as chemical (alkali/acid) or thermal burns, aniridia and Stevens-Johnson syndrome. Conjunctivalization,
neovascularization, chronic inflammation, recurrent erosions, ulceration, and scarring are possible sequelae, among pain, opacity,
and vision loss (Holland and Schwartz, 1996).
Allogeneic corneal transplantations, a surgical procedure of transplanting the cornea from a donor, have been successful in
restoring patients’ vision to some extent. Still there are several issues to be faced. First, the number of available donors is not suffi-
cient to meet demand and second, the increasingly popular laser eye surgery for refractive error often makes the cornea unsuitable
for transplantation.
Recently, cultivated corneal limbal epithelial transplantation has been described as a promising treatment for limbal stem cell
deficiency patients. Pellegrini et al. reported that cultured human limbal stem cells from biopsies of limbal tissue could be a viable
source of cells for transplantation to treat burned human corneas. They obtained limbal stem cells from the healthy eyes of 112
patients with ocular burns, and transplanted the cultured cells onto the patients’ damaged eyes. After a 10-year follow-up, perma-
nent restoration of a transparent and self-renewing corneal epithelium was found in three-quarters of the study patients (Rama
et al., 2010).
Since then, various substrates have been used for limbal stem cell expansion, such as human amniotic membrane (Mariappan
et al., 2010), fibrin gels, and temperature-responsive culture inserts. Other sources of epithelial stem cells have also been tried as an
alternative option. Transplantation of autologous oral mucosal epithelial stem cells showed a well-reconstructed cornea for patients
who have limbal stem cell deficiency, which presented benefits for the treatment of bilateral severe disorders of ocular surface
without immunosuppression requirement.
Potentials and Limitations of Stem Cell-Based Therapy
With the characteristics of unlimited proliferation and differentiation ability for multiple cell types, stem cells represent a promising
approach for vision restoration. A successful replacement of dysfunctional cells by transplantation holds great hope for patients.
However, although ESCs have been considered to be an ideal candidate for stem cell-based therapy, ethical objections exist
because of its source of human embryo donation. For iPSCs, the use of viruses in generating the cells raises a serious safety issue
that transgene integration may lead to mutation within the host genome. In addition, along with their high pluripotency, ESCs
and iPSCs also showed the ability of teratoma formation. Tumor growth has been found in mice after the transplantation of
a neutrally selected ESC (Arnhold et al., 2004). Indeed, controlling the direction of stem cell differentiation remains a major chal-
lenge. To date, the protocol used for differentiation of specific cells requires the use of complex factors and takes many steps. In
addition, immune rejection of grafted cells is also a considerable cause of transplantation failure.
Despite many challenges that will need to be solved before stem cells become effective clinical therapy, the unique characteristics
of stem cells and several successful clinical trials make stem cell-based therapy applicable and promising for vision restoration. Keep
in mind that if an optic cup could be generated from a single stem cell, miracle is just about time.
References
Algvere, P. V., Steen, B., Seregard, S., & Kvanta, A. (2008). A prospective study on intravitreal bevacizumab (avastin) for neovascular age-related macular degeneration of different
durations. Acta Ophthalmol., 86, 482–489.
Aoki, H., Hara, A., Niwa, M., Motohashi, T., Suzuki, T., & Kunisada, T. (2008). Transplantation of cells from eye-like structures differentiated from embryonic stem cells in vitro and
in vivo regeneration of retinal ganglion-like cells. Graefes Arch. Clin. Exp. Ophthalmol., 246, 255–265.
Arnhold, S., Klein, H., Semkova, I., Addicks, K., & Schraermeyer, U. (2004). Neurally selected embryonic stem cells induce tumor formation after long-term survival following
engraftment into the subretinal space. Invest. Ophthalmol. Vis. Sci., 45, 4251–4255.
Bartsch, U., Oriyakhel, W., Kenna, P. F., Linke, S., Richard, G., Petrowitz, B., Humphries, P., Farrar, G. J., & Ader, M. (2008). Retinal cells integrate into the outer nuclear layer and
differentiate into mature photoreceptors after subretinal transplantation into adult mice. Exp. Eye Res., 86, 691–700.
Bianco, P., & Robey, P. G. (2001). Stem cells in tissue engineering. Nature, 414, 118–121.
Bird, A. C., Bressler, N. M., Bressler, S. B., Chisholm, I. H., Coscas, G., Davis, M. D., De Jong, P. T., Klaver, C. C., Klein, B. E., Klein, R., Mitchell, P., Sarks, J. P., Sarks, S. H.,
Soubrane, G., Taylor, H. R., Vingerling, J. R., & The International ARM Epidemiological study group. (1995). An international classification and grading system for age-related
maculopathy and age-related macular degeneration. Surv. Ophthalmol., 39, 367–374.
Campagnoli, C., Roberts, I. A., Kumar, S., Bennett, P. R., Bellantuono, I., & Fisk, N. M. (2001). Identification of mesenchymal stem/progenitor cells in human first-trimester fetal
blood, liver, and bone marrow. Blood, 98, 2396–2402.
Cho, K. J., Trzaska, K. A., Greco, S. J., Mcardle, J., Wang, F. S., Ye, J. H., & Rameshwar, P. (2005). Neurons derived from human mesenchymal stem cells show synaptic
transmission and can be induced to produce the neurotransmitter substance P by interleukin-1 alpha. Stem Cells, 23, 383–391.
Del Priore, L. V., Kaplan, H. J., Tezel, T. H., Hayashi, N., Berger, A. S., & Green, W. R. (2001). Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-
related macular degeneration: clinicopathologic correlation. Am. J. Ophthalmol., 131, 472–480.
Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, K., Adachi, T., & Sasai, Y. (2011). Self-organizing optic-cup morphogenesis in three-
dimensional culture. Nature, 472, 51–56.
Evans, M. J., & Kaufman, M. H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature, 292, 154–156.
Fimbel, S. M., Montgomery, J. E., Burket, C. T., & Hyde, D. R. (2007). Regeneration of inner retinal neurons after intravitreal injection of ouabain in zebrafish. J. Neurosci., 27,
1712–1724.
Friedman, D. S., O’colmain, B. J., Munoz, B., Tomany, S. C., Mccarty, C., De Jong, P. T., Nemesure, B., Mitchell, P., & Kempen, J. (2004). Prevalence of age-related macular
degeneration in the United States. Arch. Ophthalmol., 122, 564–572.
Holland, E. J., & Schwartz, G. S. (1996). The evolution of epithelial transplantation for severe ocular surface disease and a proposed classification system. Cornea, 15, 549–556.
In ’t Anker, P. S., Scherjon, S. A., Kleijburg-Van Der Keur, C., De Groot-Swings, G. M., Claas, F. H., Fibbe, W. E., & Kanhai, H. H. (2004). Isolation of mesenchymal stem cells of fetal
or maternal origin from human placenta. Stem Cells, 22, 1338–1345.
Inoue, Y., Iriyama, A., Ueno, S., Takahashi, H., Kondo, M., Tamaki, Y., Araie, M., & Yanagi, Y. (2007). Subretinal transplantation of bone marrow mesenchymal stem cells delays
retinal degeneration in the RCS rat model of retinal degeneration. Exp. Eye Res., 85, 234–241.
Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A.,
Low, W. C., Largaespada, D. A., & Verfaillie, C. M. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418, 41–49.
Johnson, T. V., Bull, N. D., Hunt, D. P., Marina, N., Tomarev, S. I., & Martin, K. R. (2010). Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in
experimental glaucoma. Invest. Ophthalmol. Vis. Sci., 51, 2051–2059.
Kempen, J. H., O’colmam, B. J., Leske, C., Haffner, S. M., Klein, R., Moss, S. E., Taylor, H. R., Hamman, R. F., West, S. K., Wang, J. J., Congdon, N. G., Friedman, D. S., &
GRP, E. D. P. R. (2004). The prevalence of diabetic retinopathy among adults in the United States. Arch. Ophthalmol., 122, 552–563.
Klassen, H., & Lund, R. D. (1990). Retinal graft-mediated pupillary responses in rats: restoration of a reflex function in the mature mammalian brain. J. Neurosci., 10, 578–587.
Klyce, S. D. (1972). Electrical profiles in the corneal epithelium. J. Physiol., 226, 407–429.
Kwon, Y. H., Fingert, J. H., Kuehn, M. H., & Alward, W. L. (2009). Primary open-angle glaucoma. N. Engl. J. Med., 360, 1113–1124.
Laflamme, M. A., Chen, K. Y., Naumova, A. V., Muskheli, V., Fugate, J. A., Dupras, S. K., Reinecke, H., Xu, C., Hassanipour, M., Police, S., O’sullivan, C., Collins, L., Chen, Y.,
Minami, E., Gill, E. A., Ueno, S., Yuan, C., Gold, J., & Murry, C. E. (2007). Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of
infarcted rat hearts. Nat. Biotechnol., 25, 1015–1024.
Lamba, D. A., Gust, J., & Reh, T. A. (2009). Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem
Cell, 4, 73–79.
Lawrence, J. M., Singhal, S., Bhatia, B., Keegan, D. J., Reh, T. A., Luthert, P. J., Khaw, P. T., & Limb, G. A. (2007). MIO-M1 cells and similar Muller glial cell lines derived from
adult human retina exhibit neural stem cell characteristics. Stem Cells, 25, 2033–2043.
Leske, M. C. (2007). Open-angle glaucoma – an epidemiologic overview. Ophthalmic Epidemiol., 14, 166–172.
Lim, L. S., Mitchell, P., Seddon, J. M., Holz, F. G., & Wong, T. Y. (2012). Age-related macular degeneration. Lancet, 379, 1728–1738.
Limb, G. A., Salt, T. E., Munro, P. M., Moss, S. E., & Khaw, P. T. (2002). In vitro characterization of a spontaneously immortalized human Muller cell line (MIO-M1). Invest.
Ophthalmol. Vis. Sci., 43, 864–869.
Mariappan, I., Maddileti, S., Savy, S., Tiwari, S., Gaddipati, S., Fatima, A., Sangwan, V. S., Balasubramanian, D., & Vemuganti, G. K. (2010). In vitro culture and expansion of human
limbal epithelial cells. Nat. Protoc., 5, 1470–1479.
Martin, D. F., Maguire, M. G., Ying, G. S., Grunwald, J. E., Fine, S. L., & Jaffe, G. J. (2011). Ranibizumab and bevacizumab for neovascular age-related macular degeneration.
N. Engl. J. Med., 364, 1897–1908.
Meek, K. M., Dennis, S., & Khan, S. (2003). Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells. Biophys. J., 85, 2205–2212.
Meyer, J. S., Shearer, R. L., Capowski, E. E., Wright, L. S., Wallace, K. A., Mcmillan, E. L., Zhang, S. C., & Gamm, D. M. (2009). Modeling early retinal development with human
embryonic and induced pluripotent stem cells. Proc. Natl. Acad. Sci. U.S.A., 106, 16698–16703.
Nakano, T., Ando, S., Takata, N., Kawada, M., Muguruma, K., Sekiguchi, K., Saito, K., Yonemura, S., Eiraku, M., & Sasai, Y. (2012). Self-formation of optic cups and storable
stratified neural retina from human ESCs. Cell Stem Cell, 10, 771–785.
Pascolini, D., Mariotti, S. P., Pokharel, G. P., Pararajasegaram, R., Etya’ale, D., Negrel, A. D., & Resnikoff, S. (2004). 2002 global update of available data on visual impairment:
a compilation of population-based prevalence studies. Ophthalmic Epidemiol., 11, 67–115.
Pearson, R. A., Barber, A. C., Rizzi, M., Hippert, C., Xue, T., West, E. L., Duran, Y., Smith, A. J., Chuang, J. Z., Azam, S. A., Luhmann, U. F., Benucci, A., Sung, C. H.,
Bainbridge, J. W., Carandini, M., Yau, K. W., Sowden, J. C., & Ali, R. R. (2012). Restoration of vision after transplantation of photoreceptors. Nature, 485, 99–103.
Puzio-Kuter, A. M., & Levine, A. J. (2009). Stem cell biology meets p53. Nat. Biotechnol., 27, 914–915.
Quigley, H. A. (2011). Glaucoma. Lancet, 377, 1367–1377.
Rama, P., Matuska, S., Paganoni, G., Spinelli, A., De Luca, M., & Pellegrini, G. (2010). Limbal stem-cell therapy and long-term corneal regeneration. N. Engl. J. Med., 363,
147–155.
Reh, T. A. (2006). Neurobiology: right timing for retina repair. Nature, 444, 156–157.
Reubinoff, B. E., Itsykson, P., Turetsky, T., Pera, M. F., Reinhartz, E., Itzik, A., & Ben-Hur, T. (2001). Neural progenitors from human embryonic stem cells. Nat. Biotechnol., 19,
1134–1140.
Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas, V., Pan, C. K., Ostrick, R. M., Mickunas, E., Gay, R., Klimanskaya, I., & Lanza, R. (2012). Embryonic stem cell
trials for macular degeneration: a preliminary report. Lancet, 379, 713–720.
Seiler, M. J., & Aramant, R. B. (2012). Cell replacement and visual restoration by retinal sheet transplants. Prog. Retin. Eye Res., 31, 661–687.
Shirahashi, H., Wu, J., Yamamoto, N., Catana, A., Wege, H., Wager, B., Okita, K., & Zern, M. A. (2004). Differentiation of human and mouse embryonic stem cells along
a hepatocyte lineage. Cell Transplant, 13, 197–211.
Singhal, S., Lawrence, J. M., Salt, T. E., Khaw, P. T., & Limb, G. A. (2010). Triamcinolone attenuates macrophage/microglia accumulation associated with NMDA-induced RGC
death and facilitates survival of Muller stem cell grafts. Exp. Eye Res., 90, 308–315.
Spaide, R. F., Laud, K., Fine, H. F., Klancnik, J. M., Jr., Meyerle, C. B., Yannuzzi, L. A., Sorenson, J., Slakter, J., Fisher, Y. L., & Cooney, M. J. (2006). Intravitreal bevacizumab
treatment of choroidal neovascularization secondary to age-related macular degeneration. Retina, 26, 383–390.
Thompson, M. J., Toomre, J., Anderson, E. R., Antia, H. M., Berthomieu, G., Burtonclay, D., Chitre, S. M., Christensen-Dalsgaard, J., Corbard, T., Derosa, M., Genovese, C. R.,
Gough, D. O., Haber, D. A., Harvey, J. W., Hill, F., Howe, R., Korzennik, S. G., Kosovichev, A. G., Leibacher, J. W., Pijpers, F. P., Provost, J., Rhodes, E. J., Jr., Schou, J.,
Sekii, T., Stark, P. B., & Wilson, P. R. (1996). Differential rotation and dynamics of the solar interior. Science, 272, 1300–1305.
Tseng, S. C. (1989). Concept and application of limbal stem cells. Eye (London), 3(Pt 2), 141–157.
Weisz, J. M., Humayun, M. S., De Juan, E., Jr., Del Cerro, M., Sunness, J. S., Dagnelie, G., Soylu, M., Rizzo, L., & Nussenblatt, R. B. (1999). Allogenic fetal retinal pigment epithelial
cell transplant in a patient with geographic atrophy. Retina, 19, 540–545.
Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., & Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat.
Biotechnol., 19, 1129–1133.
Human Parthenogenetic Pluripotent Stem Cells
N Turovets, University of California, Irvine, CA, USA
M Csete, Huntington Medical Research Institutes, Pasadena, CA, USA
Parthenogenesis and Parthenogenetic Stem Cells. General Terms 608

Parthenogenesis 608
Names and Acronyms 609
hpSC Line Derivation 609
Human Oocytes 609
Parthenogenetic Activation of Unfertilized Oocytes 609
hpSC Isolation 612
HLA Homozygous and HLA Heterozygous hpSC Lines 613
History of hpSC Line Creation 615
Properties of Undifferentiated hpSC 615
Morphology and Gene Expression Patterns 615
Karyotype 615
Homozygosity of Pericentric Region 615
Pluripotency 616
Differentiation Capacity of hpSC 616
Are hpSC a More Risky Cell Source for Transplantation than Other Pluripotent Cell Types? 616
Acknowledgments 617
References 617
Glossary
Aneuploidy Abnormal number of chromosomes (too many or too few). Aneuploidy can cause tumor development or birth
defects.
Blastocyst Stage of the early embryo that contains two cell types: (i) inner cell mass (ICM) which (if implanted) forms the
embryo and (ii) trophoblast, the cellular source of placenta. The trophoblast surrounds the ICM in a fluid-filled blastocyst
cavity.
Diploid chromosome set Normal set of 46 chromosomes (human) made of two 23-chromosome sets.
Haploid chromosome set Set of 23 chromosomes (human), half the normal chromosome set.
HLA Human leukocyte antigens are expressed on almost every cell and immunologically distinguish one person from others.
IVF (in vitro fertilization) Medical procedure performed for infertility, in which oocytes are fertilized in a lab (in vitro), grown
into early embryos, and then transferred into the uterus.
Karyotype The number and arrangement of chromosomes. Normal human karyotype is 46 chromosomes including sex
chromosomes XY (male) or XX (female). ‘46,XX’ means ‘cell(s) contain 46 chromosomes including two X chromosomes’.
‘47,XX,þ6’ means ‘cell(s) contain 47 chromosomes including two X chromosomes and one extra (pathologic) number 6
chromosome.’ ‘45,X0’ means ‘cell(s) contains 45 chromosomes where one X chromosome is missing.’
Mosaic karyotype Tissue/organ with cells of different karyotypes.
Pericentric chromosome region Chromosome region surrounding the centromere.
Parthenogenesis and Parthenogenetic Stem Cells. General Terms

Parthenogenesis
Parthenogenesis is a form of asexual reproduction in which an egg (oocyte) develops without fertilization (by spermatozoa) and
therefore without male contribution into the embryo genome. Parthenogenetically activated oocytes pass through similar stages of
embryonic development as do fertilized eggs: They form one or several pronuclei, undergo cleavage, form morulae, and generate
blastocyst-like structures.
Human parthenogenetic stem cells (hpSC) are pluripotent stem cells derived from a parthenogenetically activated oocyte.

Regenerative Engineering j Human Parthenogenetic Pluripotent Stem Cells 609
Names and Acronyms

Because the field of hpSC is relatively new, a consensus name for hpSC has not emerged. In the literature the cells may be called
parthenogenetic stem cells, parthenogenetic pluripotent stem cells, parthenogenetic embryonic stem cells, parthenote stem cells,
p-cells, or parthenotes, with ‘human parthenogenetic stem cells’ the most common. To emphasize the equivalent pluripotentiality
of hpSC with human embryonic stem cell (hESC) and induced pluripotent stem cells (iPSC), hpSC are also commonly
called ‘human pluripotent parthenogenetic stem cells.’ Here we use ‘hpSC.’
hpSC Line Derivation

Human Oocytes
All reported hpSC lines were derived from the cells of p-blastocysts (from parthenogenetically activated oocytes). Unlike hESC, no
blastomere- or morula-derived hpSC lines are known.
Generation of hpSC lines takes place in three stages: (1) the process of obtaining of human oocytes, (2) parthenogenetic acti-
vation of the oocyte and in vitro cultivation to the p-blastocyst stage, and (3) derivation/isolation of hpSC lines from the blastocyst
ICM.
Obtaining human eggs, similar to donation for research on any human tissue or cells, requires review and approval of legal and
ethical protocols and should be done only after approval of a local Institutional Review Board and, in some jurisdictions, an Embry-
onic Stem Cell Research Oversight (ESCRO) committee, respecting international standards for human subjects protection. The
oocyte donor should be healthy and able to tolerate hormone stimulation and anesthesia. Generally, only volunteer oocyte dona-
tion without any financial compensation is approved by IRBs in the United States.
Human oocytes are collected from preovulatory follicles by ultrasound-guided follicular puncture after controlled ovarian
hyperstimulation or a natural menstrual cycle without any or minimal hormone stimulation. The natural cycle approach is not
complicated by the massive stimulation of ovaries, but only one oocyte per cycle can be obtained. Ovarian hyperstimulation
with hormones is more common for patients undergoing in vitro fertilization (IVF) and yields multiple oocytes per cycle. The daily
high-dose hormone injections over long periods of time are often associated with severe side effects and so should only be done in
the context of fertility treatments.
In spite of a number of attempts to use fertilization-failed oocytes (oocytes that underwent in vitro addition of sperm but did not
show signs of sperm penetration), or frozen oocytes to derive hpSC, no hpSC lines have emerged from these alternative egg sources.
With the exception of the chHES-32 line (Table 1) from an oocyte that underwent in vitro maturation, all hpSC lines reported orig-
inated from fresh (not frozen) metaphase II-arrested (MII) oocytes.
Parthenogenetic Activation of Unfertilized Oocytes

Oocyte activation can be induced by a variety of stimuli including electrical, chemical, and mechanical stimuli, and even sponta-
neous activation can happen. With the exception of two hpSC lines (P-TJ and chHES-32; Table 1) from spontaneously activated
oocytes, all other reported hpSC lines originated from oocytes stimulated by chemical agents. (Other lines may have been derived
in industry settings or not reported.) The chemicals used are chosen because they initiate transients in calcium concentration in the
egg cytoplasm similar to sperm-induced repetitive Ca2þ oscillations (also called calcium waves) that persist for several hours after
spermatozoon penetration. These calcium waves are critical for moving the egg out of meiotic arrest and into further development
(Figures 1(a) and 2(a)).
The most commonly used method of activation is serial treatment of oocytes with ionomycin (a calcium ionophore) and 6-
dimethylaminopurine (6-DMAP, a protein kinase inhibitor). Short (5 min) calcium ionophore incubation causes the first large
calcium wave, and 3–5 h of 6-DMAP treatment stimulates slightly prolonged calcium oscillations (Figure 1(b)). 6-DMAP also
blocks extrusion of the second polar body so that the activated oocytes retain all their genetic material, which ultimately contributes
to a diploid chromosome set (Figure 2(b)).
In 2008 three hpSC lines (hpSC-Hhom-2, hpSC-Hhom-3, and hpSC-Hhom-4; Table 1) were derived using ionomycin and puro-
mycin. Similar to 6-DMAP, puromycin supported oocyte activation, but unlike 6-DMAP, puromycin has no effect on spindle integ-
rity and the second polar body was extruded. The puromycin-activated oocyte therefore contained only half of a set of metaphase II
chromosomes but they resulted in diploid stem cells with homozygous genotypes. These cells’ genome contained a duplicated set of
half of the genes derived from the oocyte donor, including HLA genes (Figure 2(c)). The exact mechanism and timing of duplication
of haploid genetic material following oocyte activation are unclear. More than likely, DNA replication occurs in the absence of cell
cleavage or division. Prior studies suggest that 80% of parthenogenetically activated mouse oocytes preserve their haploid state until
the morula stage, although stem cell lines derived from these embryos become diploid.
Five other HLA homozygous hpSC lines were derived using yet other approaches. The chHES-32 line originates from a sponta-
neously activated oocyte that extruded its second polar body. hpSC-Hhom-1, pSC, and SCNT-hES-1 lines originate from oocytes
activated by ionomycin and 6-DMAP, and FY-phES-018 is from an oocyte activated by ionomycin, 6-DMAP, and trichostatin A
(TSA) (a histone deacetylase inhibitor) (Table 1).
610
Regenerative Engineering j Human Parthenogenetic Pluripotent Stem Cells
Table 1 Human pluripotent stem cell lines published by July 2013
Oocyte Genome HLA In vivo In vitro

hpSC line name stage Origin Method of activation status status Karyotype tumorigenicity pluripotency hpSC derivation method References
SCNT-hES-1 MII p-blastocyst Ionomycin6-DMAP Hetero Homo 45,X0a Teratoma Yes Immuno surgery Hwang et al. (2004)
Kim et al. (2007),b
phESC-1 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero 46,XXc Teratoma Yes Immuno surgery, NSF feeder Revazova et al. (2007)
phESC-3 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero 46,XXd Teratoma Yes Whole p-blastocyst plating Revazova et al. (2007)
on NSF
phESC-4 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero 46,XXe Teratoma Yes Whole p-blastocyst plating Revazova et al. (2007)
on NSF
phESC-5 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero 46,XXf Teratoma Yes Whole p-blastocyst plating Revazova et al. (2007)
on NSF
phESC-6 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero 46,XXg Teratoma Yes Whole p-blastocyst plating Revazova et al. (2007)
on NSF
phESC-7 MII p-blastocyst Ionomycin6-DMAP Hetero Hetero Mosaich: Teratoma Yes Whole p-blastocyst plating Revazova et al. (2007)
47,XXX; on NSF
48,XXX,þ6
hPES-1 MII p-blastocyst ElectricalIonomycin6- Hetero ND 46,XXi Teratoma Yes Immune surgery, MEF feeder Mai et al. (2007)
DMAP
hPES-2 MII p-blastocyst ElectricalIonomycin6- Hetero ND 46,XX, Failed to form Yes Immune surgery, MEF feeder Mai et al. (2007)
DMAP translocationsj any teratoma
chHES-32 ND P-blastocyst Spontaneous Homok Homol 46,XXm Teratoma Yes Whole p-blastocyst plating Lin et al. (2007)
on HEF
hpSC-Hhom-1 MII p-blastocyst Ionomycin6-DMAP Hetero Homo 46,XX Teratoma Yes Whole p-blastocyst plating Revazova et al. (2008)
on NSF
hpSC-Hhom-2 MII p-blastocyst IonomycinPuromycin Homo Homo Mosaicn: Teratoma Yes Whole p-blastocyst plating Revazova et al. (2008)
46,XX on NSF
47,XX,þ8
hpSC-Hhom-3 MII p-blastocyst IonomycinPuromycin Homo Homo Mosaico: Teratoma Yes Whole p-blastocyst plating Revazova et al. (2008)
46,XX on NSF
47,XX,þ1
hpSC-Hhom-4 MII p-blastocyst IonomycinPuromycin Homo Homo 46,XX Teratoma Yes Whole p-blastocyst plating Revazova et al.
on NSF
HP1 MII p-blastocyst Ionomycin6-DMAP ND ND NDp Myofibrosarcoma Yes Microsurgical ICM removal Brevini et al. (2009)
HP3 MII p-blastocyst Ionomycin6-DMAP ND ND NDq Myofibrosarcoma Yes Microsurgical ICM removal Brevini et al. (2009)
P-TJ MIIr p-blastocyst Spontaneous ND ND 46,XX ND Yes Immunesurgery, hFF feeder Lu et al., 2010
pSC MII p-blastocyst Ionomycin6-DMAP ND Homo 46,XX Teratoma Yes Whole p-blastocyst plating Vassena et al., 2012
on HFF
FY-phES-018 MII p-blastocyst Ionomycin6- Homos Homo 46,XXt ND Yes Immunesurgery, MEF feeder Liu et al., 2011
DMAPTrichostatin A
a
The original (when line was reported as somatic nuclear transfer originated instead of parthenogenetically originated) karyotype was reported as 46,XX (Hwang, W.S., et al., 2004. Evidence of a pluripotent human embryonic stem cell line derived from
a cloned blastocyst. Science 303, 1669–1674.), but independent investigation (Kim, K., Lerou, P., Yabuuchi, A., Lengerke, C., Ng, K., West, J., Kirby, A., Daly, M.J., Daley, G.Q., 26 January 2007. Histocompatible embryonic stem cells by
parthenogenesis. Science 315 (5811), 482–486.) demonstrated 45,X0 karyotype for this line.
b
Line was reported to be of somatic cell nuclear transfer origin (Hwang, W.S., et al., 2004. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 303, 1669–1674.), but later after detection homozygosity in
pericentric region by single nucleotide polymorphism (SNP) analysis was assumed as parthenogenetically originated (Kim, K., Lerou, P., Yabuuchi, A., Lengerke, C., Ng, K., West, J., Kirby, A., Daly, M.J., Daley, G.Q., 26 January 2007. Histocompatible
embryonic stem cells by parthenogenesis. Science 315 (5811), 482–486.).
Regenerative Engineering j Human Parthenogenetic Pluripotent Stem Cells

c
12% of cells show X chromosome heteromorphism.
d
e
f
g
h
Aneuploid mosaic karyotype: 91% of cells have a 47,XXX karyotype and 9% of the cells have a 48,XXX,þ6 karyotype. 70% of cells show X chromosome heteromorphism.
i
Over 100 passages; karyotyping was performed every 20 passages from passage 20 to 120.
j
Chromosome translocations beyond passage 50.
k
The homozygous sites comprised >99% out of SNP sites tested (500 447).
l
The status of HLA-A, -B, and -DRB loci was determined.
m
As determined at passage 6 and 49.
n
Aneuploid mosaic karyotype: 85% of cells have a 46,XX karyotype and 15% of the cells have a 47,XX,þ8 karyotype.
o
Aneuploid mosaic karyotype: 95.8% of cells have a 46,XX karyotype and 4.2% of the cells have a 47,XX,þ1 karyotype.
p
At the later work (Brevini et al., 2012) line was described as aneuploid contained (from hypo-haploid to hypo-diploid configurations).
q
At the later work (Brevini et al., 2012) line was described as aneuploid (from hypo-haploid to hypo-diploid configurations).
r
In vitro maturation from MI oocyte.
s
Determined by short tandem repeat analysis.
t
FY-phES-018 line demonstrates unstable karyotype. Before passage 20: 46, XX. At passage 35, almost all the cells displayed a 45,XO karyotype. At passage 45: the mosaic ratio of 46,XX to 45,XO was 67:33. After passage 60, most cells displayed the
46,XX karyotype with a mosaic ratio of 97:3.
611
612 Regenerative Engineering j Human Parthenogenetic Pluripotent Stem Cells
Figure 1 Parthenogenetic activation simulates spermatozoon-caused calcium waves in oocyte. (a) The penetration of human spermatozoon causes
calcium oscillations that are part of signal releasing oocyte from meiotic arrest and permitting embryonic development (oocyte activation). The graph
represents time-dependent fluctuations of intracellular free calcium. Spermatozoon-activated human oocyte follows development program through
release of second polar body and formation of zygote with male and female pronuclei, subsequent passing the cleavage from two to eight blasto-
meres, formation of morula and blastocyst. (b) Similar to fertilization, parthenogenetic activation causes calcium oscillations in oocyte (graph repre-
sents tendency and not actual oscillations): short-time ionomycin treatment simulates first large calcium oscillations and long-term 6-DMAP
treatment supports prolonged calcium waves. Parthenogenetically activated human oocyte follows development program similar to fertilized oocyte.
Human parthenogenetic stem cells can be isolated from p-blastocyst. P-zygote, p-morula, and p-blastocyst are the structures that are similar to
human zygote, morula, and blastocyst (developed from fertilized oocyte) but originated from parthenogenetically activated human oocyte.
hpSC Isolation
Under proper in vitro culture conditions (usually commercially available culture systems developed and used for IVF) parthenoge-
netically activated oocytes are able to follow the same developmental program as fertilized oocytes, through cleavage and morula
stages, followed by development into p-blastocysts (Figures 3(a)–3(c)). The derivation of the stem cell line from the p-blastocyst is
similar to derivation of hESC lines from the ICM. Two approaches to isolating pluripotent stem cells from blastocysts have been
used. In the first, ICM is isolated by removing the outer layer of the blastocyst or through immuno- or micro- surgery to open
the blastocyst for plating ICM cells on a feeder cell layer. Because the quality of p-blastocyts is often very poor and the ICM is
not always easy to identify microscopically, most parthenogenetic lines are derived through a second approach in which the whole
p-blastocyst is plated on feeder cells. The ICM cells grow out from the plated blastocyst and can be further mechanically isolated and
transferred to fresh feeder cells.
Figure 2 Formation of diploid karyotype in human parthenogenetic stem cells. (a) Normal fertilization leads to the diploid nuclear genome. Human
spermatozoon brings male genes (23 chromosomes) to metaphase II-arrested oocyte (MII) that preserves a diploid chromosome set via the spindle.
After spermatozoon penetration, the oocyte releases 23 chromosomes with the second polar body. The remaining 23 chromosomes (oocyte) form
the female pronucleus, while 23 chromosomes contributed by the spermatozoon form the male pronucleus. The resultant oocyte diploid genome is
retained throughout development (and preserved in derived pluripotent cell lines). (b) Formation of diploid heterozygous hpSC. In addition to sup-
porting prolonged calcium oscillations in the parthenogenetically activated oocyte, 6-DMAP also furthers spindle degradation, preventing extrusion of
the second polar body. The entire MII oocyte genome (46 chromosomes) resulting from parthenogenetic activation remains in the activated oocyte
and through development of the p-blastocyst (as well as in derived hpSC). (c) Formation of diploid homozygous hpSC. The example here results
from ionomycin and puromycin activation but presumably the mechanism leading to diploid stem cells from spontaneously activated oocytes is
similar. Puromycin treatment does not affect spindle integrity; therefore parthenogenetically activated oocytes release 23 chromosomes with the
second polar body and retain a haploid (23) chromosome set. The timing of duplication of these 23 chromosomes is not known. In theory, duplica-
tion can occur at any stage (activated oocyte, zygote, during cleavage or during morula or blastocyst formation, or even during stem cell line isola-
tion) after spontaneous activation of oocytes. (Cleavage and morula stages of parthenote development are not shown.)
HLA Homozygous and HLA Heterozygous hpSC Lines

Transplantation of allogeneic (from another human) organs or cells initiates an immune response in the host, in which the host
immune system recognizes HLA antigens of the donor. The risk of transplant rejection is generally proportional to the degree of
cell surface antigen disparity between the donor and recipient cells. Matching donor and recipient tissue for HLA antigens reduces
the chance of a cytotoxic T-cell response in the recipient and thus increases the likelihood of transplant survival. Since a normal HLA
repertoire has some maternal and paternal antigens, successful immune matching of donor to host requires matching both maternal
and paternal antigens, it is easy to see that the complexity of immune matching is greatly reduced if the donor cells are HLA
homozygous.
Using various activation approaches, it is possible to create hpSC that are HLA heterozygous or HLA homozygous. With HLA
homozygous hpSC the immune matching to a potential recipient is easier because there is simply less diversity in the HLA
Figure 3 Morphology of an MII oocyte, embryos derived from parthenogenetically activated oocytes, and hpSC. (a) Metaphase II-arrested (MII)
human oocyte with clearly visible first polar body. To date all hpSC lines have been derived from MII oocytes. (Figure source: Thomas Elliott, http://
www.ivf.net/ivf/stripped-oocyte-o682.html, reproduced with permission.) (b) Cleavage embryo (eight blastomeres) from a parthenogenetically acti-
vated human oocyte (source of P-TJ hpSC). (Figure source: Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., Zhang, Y.A., Zhang, Y., Zhou, Q., June
2010. Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. J. Assist. Reprod. Genet. 27
(6), 285–291, reproduced with permission.) (c) Blastocyst from a parthenogenetically activated human oocyte. The expanded blastocyst (left) and
hatching blastocyst with a clearly visible ICM extruding out of the zona pellucida (right). (Left figure source: Mai, Q., Yu, Y., Li, T., et al., 2007. Deri-
vation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res. 17, 1008–1019, right figure source: Lu, Z., Zhu, W., Yu, Y.,
Jin, D., Guan, Y., Yao, R., Zhang, Y.A., Zhang, Y., Zhou, Q., June 2010. Derivation and long-term culture of human parthenogenetic embryonic stem
cells using human foreskin feeders. J. Assist. Reprod. Genet. 27 (6), 285–291, reproduced with permission.) (d) hpSC morphology: Undifferentiated
chHES-32 colonies growing on an inactivated human feeder layer. (Inset is higher magnification of one colony). (Figure source: Lin, G., OuYang, Q.,
Zhou, X., et al., 2007. A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following
in vitro fertilization procedure. Cell Res. 17, 999–1007, reproduced with permission.) (e) hpSC express normal human pluripotent stem cell markers.
OCT4 expression in the hpSC-Hhom-1 line. (Figure source: Revazova, E.S., Turovets, N.A., Kochetkova, O.D., Agapova, L.S., Sebastian, J.L., Pryzh-
kova, M.V., Smolnikova, V.I., Kuzmichev, L.N., Janus, J.D., March 2008. HLA homozygous stem cell lines derived from human parthenogenetic blas-
tocysts. Cloning Stem Cells 10 (1), 11–24, reproduced with permission. SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression in the P-TJ line.
Figures source: Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., Zhang, Y.A., Zhang, Y., Zhou, Q., June 2010. Derivation and long-term culture of
human parthenogenetic embryonic stem cells using human foreskin feeders. J. Assist. Reprod. Genet. 27 (6), 285–291, reproduced with permis-
sion.) (f) Normal 46,XX karyotype of hpSC-Hhom-4. (Figure source: Revazova, E.S., Turovets, N.A., Kochetkova, O.D., Agapova, L.S., Sebastian, J.L.,
Pryzhkova, M.V., Smolnikova, V.I., Kuzmichev, L.N., Janus, J.D., March 2008. HLA homozygous stem cell lines derived from human parthenogenetic
blastocysts. Cloning Stem Cells 10 (1), 11–24, reproduced with permission.) (g) Abnormal aneuploid karyotype of hpSC-Hhom-3. This hpSC line has
a mosaic karyotype: 95.8% of cells are 46,XX (left) and 4.2% are 47,XX, þ1 (right) with an extra chromosome 1. Figure source: Revazova, E.S., Tur-
ovets, N.A., Kochetkova, O.D., Agapova, L.S., Sebastian, J.L., Pryzhkova, M.V., Smolnikova, V.I., Kuzmichev, L.N., Janus, J.D., March 2008. HLA
homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 10 (1), 11–24, reproduced with permission.
genotype of HLA homozygous hpSC. In theory, this means that HLA homozygous hpSC could be generated to match large
segments of the population with significantly fewer lines required for allogeneic matching than would be possible using HLA-
characterized hES cells or iPS cells. (Of course, iPS cells have the potential advantage of autologous use without inducing an
immune response.)
History of hpSC Line Creation

The first intentional creation of hpSC lines was described by Revazova et al. in 2007: Six pluripotent hpSC lines were derived from
chemically activated human oocytes. Additional lines were derived by this group and by others with 19 lines published to date.
Properties of Undifferentiated hpSC

Morphology and Gene Expression Patterns
Undifferentiated hpSC are morphologically similar to other types of human pluripotent stem cells such as hESC and hiPSC. Specif-
ically in culture, undifferentiated hpSC form colonies of tightly packed cells. Individual cells of hpSC and other undifferentiated
pluripotent cells have characteristic features including prominent nucleoli and a small cytoplasm to nucleus ratio (Figure 3(d)).
hpSC demonstrate high levels of alkaline phosphatase activity, a marker of pluripotent stem cells, and express other ‘traditional’
pluripotent stem cell markers including OCT4 (also known as POU5F1), SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81
(Figure 3(e)). In addition, hpSC demonstrate high levels of telomerase activity associated with indefinite proliferation potential,
unlike adult stem cells that invariably undergo proliferative senescence in culture. Similar to hESC and hiPSC, hpSC can be main-
tained in undifferentiated state for a long period of time using growth factors and/or feeder cell layers.
hpSC demonstrate similar global gene expression patterns to hESC and hiPSC and DNA methylation patterns, with exception of
imprinted genes. Genomic imprinting is the mechanism by which monoallelic gene expression of certain genes (imprinted genes) is
achieved in a parent-of-origin-specific manner based on DNA methylation (an epigenetic change, not a change in sequence).
Because hpSC originate from oocytes without contribution of male genetic material, hpSC express only maternal imprinted genes.
It is important to note that methylation patterns are dynamic and can be altered by in vitro culture conditions.
Karyotype
All hpSC lines are diploid and XX (Figure 3(f); Table 1) except one line that had loss of one X chromosome. Some hpSC lines
demonstrate aneuploidy including aneuploid mosaic variants (Figure 3(g)). Different degrees of X chromosome heteromorphism
are reported for some hpSC lines. Chromosome instability during in vitro cultivation has also been reported. The absence of paternal
centriole in the parthenogenetic zygote (normally inherited through the sperm) leads to a centrosome amplification process and
may contribute to chromosome instability and aneuploidy.
Homozygosity of Pericentric Region

The distinguishing property of hpSC is homozygosity of chromosome pericentric regions adjacent to centromeres. For hpSC lines
derived from oocytes that did not release the second polar body after activation, homozygosity of chromosome pericentric regions
reflects the failure of independent segregation of the sister chromatids during meiosis and is consistent with meiotic recombination
events (Figure 4). Pericentric homozygosity is uniquely found in hpSC lines and is not observed in either hESC or iPSC. The analysis
of single nucleotide polymorphisms in genome regions adjacent to the centromere can reliably distinguish hpSC from other cell types.
Figure 4 Formation of the homozygous pericentric region in parthenogenetically activated oocytes. Germinal vesicle (GV) oocyte contains dupli-
cated diploid chromosome set (4n ¼ 2 46); each homologous chromosome contains two sister chromatids joined at the centromere (homozygous
sister chromatid). During MI, recombination occurs between both sister chromatid pairs of homologous chromosomes, yielding heterozygosity of the
distal ends of sister chromatids, but recombination does not occur at the centromere region which remains homozygous. During formation of MII
oocytes (first meiotic division) one of the recombined chromosomes (from a pair of homologous chromosomes) is segregated into the first polar
body, resulting in a diploid oocyte (second recombined chromosome from the other homologous chromosome pair) represented as 23 pairs of chro-
matids still joined at the centromere. During a normal MII in a fertilized egg, the second polar body would be extruded. However, after parthenoge-
netic activation with ionomycin þ 6-DMAP, the second polar body is not extruded, leaving an oocyte diploid chromosome. After chromatid
disjunction each becomes a separate chromosome.
Pluripotency
hpSC lines demonstrate ability to give rise to derivatives of three germ layers (endoderm, ectoderm, and mesoderm) in embryoid
body formation assays. Most hpSC are capable of forming teratomas approximately 2–3 months after injection into immunocom-
promised rodents. Teratomas are benign tumors that contain cell types from all three germ layers. Teratoma formation is the current
gold standard confirmatory test of pluripotency. Some hpSC lines, however, fail to form teratomas and other aberrant differentia-
tion patterns have also been observed.
Differentiation Capacity of hpSC
Differentiation of hpSC into definitive endoderm (DE) and early hepatocytes has been accomplished in four hpSC lines (Table 2).
hpSC are able to respond to well-characterized developmental signals which direct differentiation of pluripotent cells into DE, the
precursor for number of cell types including hepatocytes. hpSC (like hESC) exposed to the proper developmental signals demon-
strate a similar temporal sequence of gene expression to that which occurs in the course of in vivo DE differentiation during verte-
brate gastrulation. However, in the few lines studied, the yield of DE and hepatocytes from hpSC lines is lower than from hESC.
Treatment of undifferentiated hpSC by TSA, a potent histone deacetylase inhibitor, before this directed differentiation significantly
increases the efficiency of DE differentiation from hpSC, but significant numbers of hpSC do not respond to the differentiation cues
and remain undifferentiated. In anticipating clinical manufacture of cells for therapeutic applications, it is undesirable to have
undifferentiated cells in the cell graft because of their potential to form teratomas.
Differentiation into neural progenitor cells was reported for two hpSC lines: phESC-3 and phESC-5 (Table 2). Again yield was
lower in comparison with hESC lines, and differentiation was less efficient.
hpSC differentiation into retinal pigment epithelium (RPE) is quite similar to hESC differentiation of RPE (Table 2). Differen-
tiation toward hematopoietic fate was reported by just one group for two hpSC lines (Table 2) with generation of CD34/CD45-
positive cells that were able to form colonies in methylcellulose after 3 weeks. Lymphoid, erythroid, and myeloid subpopulations
were generated.
Under appropriate conditions, hpSC lines are also able to generate mesenchymal stem cells (MSC) (Table 2). In addition, MSC
derived from hpSC show the usual differentiation potential of MSC, that is, in vitro differentiation of chondrocyte (cartilage), oste-
ocyte (bone), and adipocyte (fat) lineages.
Are hpSC a More Risky Cell Source for Transplantation than Other Pluripotent Cell Types?
The answer to this question is not known because the definitive experiments have not yet been performed. Nonetheless, the absence
of sperm centriole in parthenotes is a potential risk factor for safe clinical application of hpSC-derived cell therapies. Abnormal
number of centrioles as well as aberrant levels of molecules related to mitotic spindle formation and spindle assembly checkpoint
have been reported in some hpSC lines and may also contribute to the high incidence of aneuploidy observed in hpSC lines.
Table 2 Differentiation capacity of hpSC
hpSC line name Specific cell type/direction of differentiation References
phESC-1 Definitive endoderm Turovets et al. (2011a)

Fetal-like hepatocytes Turovets et al. (2012)
Retinal pigment epithelium Harness et al. (2011)
Neural progenitor cells Harness et al. (2011)
Neural progenitor cells Harness et al. (2011)
chHES-32 Mesenchymal stem cells Chen et al. (2012)
hpSC-Hhom-4 Definitive endoderm Turovets et al. (2011a)
HP1 Hematopoietic fate Brevini et al. (2009)
HP3 Hematopoietic fate Brevini et al. (2009)
P-TJ Retinal pigment epithelium Li et al. (2012)
pSC Mesenchymal stem cells Vassena et al. (2012)
Concerns that these abnormalities will lead to malignant transformation are a major hurdle for hpSC-based therapies. For some
applications, though, hpSC may find a place in regenerative medicine if these concerns can be addressed in rigorous preclinical
studies.
Acknowledgments
We thank Irina Turovets for the translation of our ideas and thoughts into graphics and for the creation illustrations and schemes for this article.
References
Brevini, T. A., Pennarossa, G., Maffei, S., Tettamanti, G., Vanelli, A., Isaac, S., Eden, A., Ledda, S., de Eguileor, M., & Gandolfi, F. (2012). Centrosome amplification and
chromosomal instability in human and animal parthenogenetic cell lines. Stem Cell Rev., 8(4), 1076–1087.
Brevini, T. A., Pennarossa, G., Antonini, S., Paffoni, A., Tettamanti, G., Montemurro, T., Radaelli, E., Lazzari, L., Rebulla, P., Scanziani, E., de Eguileor, M., Benvenisty, N., Ragni, G.,
& Gandolfi, F. (December 2009). Cell lines derived from human parthenogenetic embryos can display aberrant centriole distribution and altered expression levels of mitotic
spindle check-point transcripts. Stem Cell Rev., 5(4), 340–352.
Chen, Y., Ai, A., Tang, Z. Y., Zhou, G. D., Liu, W., Cao, Y., & Zhang, W. J. (January 2012). Mesenchymal-like stem cells derived from human parthenogenetic embryonic stem cells.
Stem Cells Dev., 21(1), 143–151.
Fried, E. P., Ross, P., Zang, G., Divita, A., Cunniff, K., Denaday, F., Salamone, D., Kiessling, A., & Cibelli, J. (April 2008). Human parthenogenetic blastocysts derived from
noninseminated cryopreserved human oocytes. Fertil. Steril., 89(4), 943–947.
Harness, J. V., Turovets, N. A., Seiler, M. J., Nistor, G., Altun, G., Agapova, L. S., Ferguson, D., Laurent, L. C., Loring, J. F., & Keirstead, H. S. (7 January 2011). Equivalence of
conventionally-derived and parthenote-derived human embryonic stem cells. PLoS One, 6(1), e14499.
Hwang, W. S., et al. (2004). Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science, 303, 1669–1674.
Kim, K., Lerou, P., Yabuuchi, A., Lengerke, C., Ng, K., West, J., Kirby, A., Daly, M. J., & Daley, G. Q. (26 January 2007). Histocompatible embryonic stem cells by parthenogenesis.
Science, 315(5811), 482–486.
Li, W. B., Zhang, Y. S., Lu, Z. Y., Dong, L. J., Wang, F. E., Dong, R., & Li, X. R. (August 2012). Development of retinal pigment epithelium from human parthenogenetic embryonic
stem cells and microRNA signature. Invest Ophthalmol Vis. Sci., 53(9), 5334–5343.
Lin, G., OuYang, Q., Zhou, X., et al. (2007). A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro
fertilization procedure. Cell Res., 17, 999–1007.
Lu, Z., Zhu, W., Yu, Y., Jin, D., Guan, Y., Yao, R., Zhang, Y. A., Zhang, Y., & Zhou, Q. (June 2010). Derivation and long-term culture of human parthenogenetic embryonic stem
cells using human foreskin feeders. J. Assist. Reprod. Genet., 27(6), 285–291.
Mai, Q., Yu, Y., Li, T., et al. (2007). Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res., 17, 1008–1019.
Nazor, K. L., Altun, G., Lynch, C., Tran, H., Harness, J. V., Slavin, I., Garitaonandia, I., Müller, F. J., Wang, Y. C., Boscolo, F. S., Fakunle, E., Dumevska, B., Lee, S., Park, H. S.,
Olee, T., D’Lima, D. D., Semechkin, R., Parast, M. M., Galat, V., Laslett, A. L., Schmidt, U., Keirstead, H. S., Loring, J. F., & Laurent, L. C. (May 4, 2012). Recurrent variations in
DNA methylation in human pluripotent stem cells and their differentiated derivatives. Cell Stem Cell, 10(5), 620–634.
Revazova, E. S., Turovets, N. A., Kochetkova, O. D., Agapova, L. S., Sebastian, J. L., Pryzhkova, M. V., Smolnikova, V. I., Kuzmichev, L. N., & Janus, J. D. (March 2008). HLA
homozygous stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells, 10(1), 11–24.
Revazova, E. S., Turovets, N. A., Kochetkova, O. D., Kindarova, L. B., Kuzmichev, L. N., Janus, J. D., & Pryzhkova, M. V. (2007). Patient-specific stem cell lines derived from human
parthenogenetic blastocysts. Cloning Stem Cells, 9(3), 432–449. Fall.
Turovets, N., D’Amour, K. A., Agapov, V., Turovets, I., Kochetkova, O., Janus, J., Semechkin, A., Moorman, M. A., & Agapova, L. (June 2011a). Human parthenogenetic stem cells
produce enriched populations of definitive endoderm cells after trichostatin A pretreatment. Differentiation, 81(5), 292–298.
Turovets, N., Semechkin, A., Kuzmichev, L., Janus, J., Agapova, L., & Revazova, E. (2011b). Derivation of human parthenogenetic stem cell lines. Methods Mol. Biol., 767, 37–54.
Turovets, N., Fair, J., West, R., Ostrowska, A., Semechkin, R., Janus, J., Cui, L., Agapov, V., Turovets, I., Semechkin, A., Csete, M., & Agapova, L. (2012). Derivation of high-purity
definitive endoderm from human parthenogenetic stem cells using an in vitro analog of the primitive streak. Cell Transplant., 21(1), 217–234.
Vassena, R., Montserrat, N., Carrasco Canal, B., Aran, B., de Oñate, L., Veiga, A., & Izpisua Belmonte, J. C. (1 August 2012August). Accumulation of instability in serial
differentiation and reprogramming of parthenogenetic human cells. Hum. Mol. Genet., 21(15), 3366–3373.
Human Pluripotent Stem Cells
P Rajan, The Scripps Research Institute, La Jolla, CA, USA
Introduction 618
Definition of hESCs 619
Unique Properties of hESCs 619
Methods of Generation of hESCs 620
‘Traditional’ Derivations 620
Somatic Cell Nuclear Transfer 621
Cell Fusion 621
Current Applications of hESCs 621
Legal and Social Aspects of hESC Research 622
References 623
Glossary
Adherent culture Cell cultures which are adherent to the surface of the culture dish.
ADME Absorption, distribution, metabolism, excretion. These are characteristics of therapeutic compounds, which need to be
defined before clinical application.
Allele One of a pair of a specific gene located on sister chromatids.
Companion diagnostics Clinical tests which are performed in conjunction with the administration of a therapeutic.
Diploid A cell which has the normal cohort of two sister chromatids per chromosome.
Germline transmission Transmission of genes to the next generation by presence in sperm and egg.
In vitro Experiment performed in a representative experimental platform, which is outside the body of the animal model under
study.
In vivo Experiment performed within the body of the experimental animal model.
Passaging Technique of expanding a culture by removal from original culture dish and replating in new culture dishes.
Pharmacogenomics Science that combines information about characteristics of genome of a particular individual with the
effect of a pharmaceutical drug.
Suspension culture Cell cultures that proliferate while floating in the culture medium.
Telomere Ends of chromosomes, which have specific sequence repeats.
Teratology The study of mutations and defects in developing embryos.
Tetraploid A cell having four sister chromatids per chromosome – twice the normal number.
Untransformed Cells that have not undergone a transformation event, which causes uncontrolled proliferation and abnormal
maintenance of differentiation phenotype.
Introduction
Stem cells are ‘normal,’ untransformed cells which have dual intrinsic properties of proliferation and differentiation. While empir-
ical evidence for the existence of these cells has existed for several decades especially with relation to the study of embryology of
invertebrates and lower vertebrates, they have recaptured the imagination of biologists recently due to their enormous potential
in human disease therapies, and the establishment of sophisticated techniques by which they may be studied. Broadly, stem cells
may be classified as somatic stem cells (SSCs) and embryonic stem cells (ESCs). SSCs are derived from developing and mature
tissues in which they are resident and multipotent as they are restricted in their differentiation potential, and can only give rise
to some or all of the cells present in their tissue of origin. ESCs are pluripotent as they are capable of differentiation into any
cell type of the body, and may be derived from the inner cell mass (ICM) of the blastocyst of a fertilized embryo.
The potential of stem cells may be realized for human therapeutics in two ways: firstly, stem cells may be used as a therapeutic
themselves and used for regenerative medicine endpoints where derivatives of stem cells functionally substitute for deficits caused
by the disease. Secondly, stem cells may be useful in promoting endogenous regenerative processes. Thirdly, stem cells may be used

Regenerative Engineering j Human Pluripotent Stem Cells 619
to create laboratory platforms, which are used to model diseases and functional tissues. These models serve as experimental plat-
forms to study disease mechanisms, and identify molecules, which reverse disease phenotypes. This article will describe the prop-
erties of human embryonic stem cells (hESCs), methods for their derivation and their properties, and the manner in which their
enormous potential may be realized for human benefit.
Definition of hESCs
Human embryonic stem cells are identified and characterized based on combinations of cellular markers, and the functional criteria
of self-renewal and pluripotency. They are created in vitro by manipulations in culture, and most closely resemble epiblast cells in
early mouse development which give rise to the three germ layers of ectoderm, mesoderm, and definitive endoderm, rather than the
more primitive mouse ESCs which differentiate into the extraembryonic endoderm in addition to the three germ layers mentioned
above (Tesar et al., 2007). The cellular markers which are used to define hESCs are Oct4, Nanog, Sox2, SSEA4, Tra1-60, Tra1-81,
alkaline phosphatase, and high telomerase activity (Figure 1). Functionally, hESCs are defined by their property of pluripotency,
which may be demonstrated in vitro by directed differentiation of the cultures to defined fates representative of the three germ layer
lineages, and in vivo by the formation of teratomas. For instance, in vitro differentiation of the culture into neuronal (ectodermal),
blood and muscle (mesodermal), and liver and pancreas (endodermal) would have to be demonstrated by the same hESC line.
Teratomas are tumors which comprise cells of ectodermal, mesodermal, and endodermal lineages, and serve as a surrogate assay
of in vivo differentiation potential, as germline transmission and formation of entire embryos cannot be demonstrated in humans
as it has been shown in mice and other animal models due to ethical reasons.
Unique Properties of hESCs
There is an enormous effort being made to study the mechanisms by which stem hESCs maintain their dual properties of self-
renewal (proliferation) and pluripotency (differentiation). One hypothesis considers that the pluripotency of a cell arises due to
the fact that it is capable of responding to diverse stimuli hence allowing it to differentiate along several lineages. The enhanced
responsiveness of an hESC could be due to the expression of a greater cohort of proteins, which gets culled down to a defined
set of proteins as commitment to a particular lineage occurs, thus restricting responsiveness. In fact, ESCs have the potential to acti-
vate the majority of the gene expression programs present in embryonic and adult cell lineages. The regulation of the cohort of genes
expressed within the hESC is brought about by a combination of genetic and epigenetic characteristics, and is realized by inter- and
Figure 1 Colony of hESC (a). Phase contrast micrograph of an hESC colony. Undifferentiated colony stained with SSEA4 (b). Nuclei are labeled in
blue (DAPI), and the cell surface SSEA antigen in red. Undifferentiated colony stained with Nanog (c). Nuclei are labeled in blue (DAPI), and the cyto-
plasmic antigen Nanog in red. MAP2 staining of neuronal rosettes in hESC colony differentiated into neuronal precursors (d). SSEA, stage-specific
embryonic antigen; DAPI, diamidino-2-phenylindole, MAP2, microtubule-Associated Protein 2.
620 Regenerative Engineering j Human Pluripotent Stem Cells
intracellular signaling mechanisms. Interestingly, with the advent of the technology of generating induced pluripotent stem cells
(iPSCs), it appears that the limited cohort of proteins expressed in mature cells may be expanded to give rise to cells, which
have properties of pluripotency similar to hESCs.
Pluripotency in hESCs is maintained by cellular programs activated by the growth factors such as fibroblast growth factor 2
(FGF2) and Activin/Nodal, along with some involvement of the Wnt (wingless-related integration) proteins. The creation of
methods to regulate and control pluripotency in hESCs would be advantageous for the maintenance of these lines in culture in
an undifferentiated pluripotent state until they are required to differentiate upon stimulation. A pharmacological inhibitor of
GSK3b (glycogen synthase kinase), a kinase central to the Wnt signaling pathway, 6-bromoindirubin-30 -oxime is an effector of
human ESC self-renewal, and induces the expression of pluripotency markers Oct3/4, Rex1, and Nanog. Epigenetic modifications,
defined as heritable changes of the genome which are not coded in the sequence of the DNA, also contribute to the maintenance of
pluripotency. At a gross level, differences have been found in the regulation of acetylation and methylation of DNA, histones and
transcription factors in ESCs when compared to differentiated cells. DNA methylation of promoter sequences was detected in
hESCs, which may account for regulating about 30% of genes in these cells.
A related area of study is the mechanisms, and the creation of improved methods, by which stem cells differentiate into cells of
interest. This is particularly relevant to regenerative medicine and cell-replacement therapies where hESC derivatives may function-
ally replace lost cells or tissues. For instance, dopaminergic neurons are lost in the basal ganglia structures of the brains of Parkin-
son’s disease patients, and insulin-secreting pancreatic b-cells are lost in diabetes type 1 patients. The replacement of these particular
cell types derived from hESCs in these patients may serve as an effective therapy. Similar differentiation techniques are also useful
for modeling of several diseases in the laboratory, identification and validation of novel targets of disease, and screening for new
entities, which ameliorate disease phenotypes.
Methods of Generation of hESCs
ESCs of various species including frogs, rodents, livestock, and monkeys have been created and are in use for over 40 years, for
cloning and research purposes. However the first human ESC lines were created by culturing human blastocysts in 1998, almost
simultaneously with the report of the first cultures of embryonic germ cells (hEGC; Thomson et al., 1998; Shamblott et al.,
1998). hEGCs are derived from the gonadal ridge and mesenchyma of 5- to 9-week fetal tissue, and differentiate into the three
germ layers in vitro, but unlike hESCs do not form teratomas in vivo.
hESCs or hESC-like cells may be developed using four strategies. While the first method described below involves selective
culture of hESC cells from a cultured blastocyst, the latter three methods involve the process of nuclear reprogramming. During
the process of nuclear reprogramming, a nucleus from a mature cell is reprogrammed to resemble a more stemlike state, presumably
by factors in the cytoplasm of the cell/egg into which the nucleus is transplanted, or by the transient overexpression of reprogram-
ming factors using specialized gene expression methods and vectors (Figure 2).
‘Traditional’ Derivations
Human blastocysts are obtained from discarded fertilized eggs from in vitro fertilization clinics. Fertilized eggs are cultured to a blas-
tocyst over 5 days. The ICM is released from the blastocyst either by mechanical dissection with a microscalpel or by hatching the
blastocyst using acid Tyrode solution (pH 2.2) and immunodissection using alkaline phosphatase and complement-mediated inter-
actions. The ICM is cultured with or without feeder cultures, and colonies of hESCs form at low frequency, which develop into
Figure 2 Schematic of hESC derivation methods. Traditional derivations involve developing a blastocyst from fertilized eggs, and culturing an hESC
line from the inner cell mass (ICM). SCNT also involves the establishment of a blastocyst, but it occurs from introducing a nucleus from a donor cell
into the egg, and then stimulating it to develop a blastocyst. iPSC allows for development of a cell line without involvement of a blastocyst, but
directly from reprogrammed cells (fibroblasts in this case).
a stable line over several weeks. The hESC lines have been traditionally derived on mouse embryonic fibroblasts (MEFs), which
secrete requisite supporting factors and function as a feeder layer. However, several lines have now been derived on human fibro-
blast feeder layers, or in feeder-free conditions. The latter is preferred for cell lines that have been created with the intention of use in
clinical applications, as mouse glycoproteins have been detected on hESCs grown on MEFs. hESC proliferate in colonies, and are
usually passaged mechanically or using mild enzymatic treatment with Accutase. hESC lines that have been passaged using harsher
enzymes like trypsin appear to progressively acquire mutations. hESC colonies typically are comprised of small cells with high
nuclear: cytoplasm ratios, and are positive for the markers mentioned above. They also need to have a stable karyotype in order
to give rise to a successful and usable line (Rajan et al., 2007).
The hESC lines have also been obtained from human eggs, which have been activated parthenogenetically to give rise to blas-
tocysts. Although these lines are diploid (2n), since they have not arisen from the fusion of a sperm with an egg, they have some
homozygosity at HLA loci, which may be advantageous for immunologic matching in the context of regenerative therapies.
Somatic Cell Nuclear Transfer

This procedure was used successfully to clone the first sheep Dolly. In this procedure a diploid nucleus from a parent cell is reprog-
rammed to a stemlike state by introduction into an enucleated egg. After appropriate stimulation, which causes the egg to divide,
formation of a blastocyst (with cells containing the donor nucleus) can be cultured as described for traditional hESC derivations.
Somatic cell nuclear transfer (SCNT) requires the donation of eggs from healthy human volunteers, and is the platform necessary for
“therapeutic cloning”. Therapeutic cloning involves the creation of cells or tissues (but not entire organisms) with known nuclear
chromosome input, for research purposes.
Cell Fusion
This procedure results in cells, which are tetraploid (4n), and hence derivatives may be used only as research tools. In this procedure
a mature cell such as a fibroblast of the desired genotype is chemically fused with an established hESC cell. Reprogramming factors
that are presumably in the cytoplasm of the hESC cause the progeny to have hESC-like characteristics. Although 4n, these cells
exhibit the pluripotent properties of hESCs and resemble hESCs in gene expression and methylation profiles.

This remarkable procedure which garnered the Nobel Prize for Physiology and Medicine in 2012 (awarded to Dr Shinya Yamanaka)
involves the overexpression of four to six proteins which include at least four transcription factors that are usually expressed in
hESCs, and leads to reprogramming of cells in cultures derived from mature differentiated tissues to culture with characteristics
of hESCs. Oct4, Klf4, Sox2, and Myc are the genes used for induction of pluripotency, in the absence or presence of other genes
including SV40 Large-T and Nanog. The exact nature of the ‘mature’ cell is not clear, and iPSC reprogramming appears to be
more efficient with stemlike cells are used as the source of iPSC. While the original iPSC experiments were performed with the
use of retroviral and lentiviral transduction to overexpress proteins, several refinements have since been reported involving the
use of small molecules, microRNAs, protein transductions, plasmid transfections, transposons, adenoviral vectors, and more. These
refinements make the procedure more accessible to laboratories around the world. The genetic/epigenetic regulation of reprogram-
ming events is complex and takes place in a series of steps that are still being elucidated. Thus, although the use of these cells in
regenerative medicine therapies requires more research, the power of this technology is already being realized for in vitro disease
modeling and drug discovery applications. iPSC technology has already been used successfully to model muscular dystrophies,
neurological diseases including amyotrophic lateral sclerosis, Huntington’s disease, and Alzheimer’s disease, psychiatric diseases
including autism, and metabolic diseases including juvenile diabetes mellitus, Lesch–Nyhan syndrome, and Gaucher’s disease.
Current Applications of hESCs
Several hESC lines exist around the globe, and while the majority of them have been created using good laboratory practices proce-
dures, a few have been created under current good manufacturing practices (cGMP). While the former lines are useful in research
and development projects, it is only the latter that may be developed for use in regenerative applications. The list of approved cell
lines which may be used with US Federal funding is present on the NIH website (see website list), and other similar lists of lines
approved by other countries including the UK is also included in the website list. Cell lines specific to other countries are also in the
process of being created, notably in India, Brazil, China, and Japan. It is essential that genotypes of most countries are eventually
represented in the global library of hESC and iPSC lines, as this will be extremely relevant to present drug discovery efforts, and
future regenerative medicine technologies.
Currently hESCs (including those derived using iPSC technology) have found excellent use in drug discovery applications, where
their in vitro differentiation potential can be appropriately exploited. hESCs are developed into relevant in vitro platforms for vali-
dating targets, creating screening platforms, and developing toxicology platforms where they may be differentiated into liver and
cardiac tissues. These toxicology platforms are invaluable in creating physiologically relevant laboratory paradigms of these tissues,
622 Regenerative Engineering j Human Pluripotent Stem Cells
and may eventually be developed into companion diagnostics using pharmacogenomic technologies. This would enable the
screening of compounds, which may be particularly toxic or effective in selected populations or groups of patients, based on the
genotypes of the hESC platforms created.
While the inherent property of pluripotency of hESCs makes them an ideal candidate for regenerative medicine and cell-
replacement therapies, the promise of this potential remains to be realized. Due to the possibility of cancer arising from the trans-
planted hESCs, only partially differentiated hESCs are used as a therapeutic. Geron, USA, spearheaded the effort of using hESC
therapy for spinal cord injury with their FDA approved Phase I clinical trial. This program has since been discontinued by the
company. Currently, Advanced Cell Technology, Inc. is determining the safety and efficacy of hESCs in macular degeneration in
an FDA approved trial, while Viacyte is at the stage of preclinical testing in the development of an hESC-derived therapeutic for
diabetes.
Legal and Social Aspects of hESC Research
Since the creation of hESC lines involves the use of a fertilized human egg, and hESCs possess the enormous potential to create
tissues, organs, or even clone a human, there are considerable social and religious overtones to this field of research. Legislation
in the USA had restricted the use of federal funds for the creation of new hESC lines, although this has recently been relaxed. Other
countries are more permissive to this research, but regulate the field tightly. The patent landscape was also tightly controlled by the
published inventors of the technology, Wisconsin Alumni Research Foundation, and control of access of cell lines by creators of
other cell lines have had a somewhat restrictive influence on progress in the field. These situations are also partially due to the
fact that appreciable expense in time and effort is expended to create these lines.
Future Perspectives
In order for the enormous potential of hESCs to be realized in both regenerative medicine and drug discovery, several aspects of
hESC need to be clarified. These include determination of the factors controlling stability of genotype and phenotype, predictability
of differentiation phenotype and efficiency of differentiation upon providing defined stimuli, control of length of telomeres and
‘aging’ of the cell line, control of mutation (deletions and duplications) of hot spots in the genome with continuous passaging,
control of tumor formation after administration, and need for better assays of pluripotency and efficacy.
The use of patient-specific cells for regenerative medicine provides a therapeutic which will not induce classic cytotoxic rejec-
tion. However, this is a possibility only when the hESCs do not carry a genetic mutation, which is instrumental in manifestation
of the disease. The creation of patient-specific hESCs would likely use iPSC technology, and newer methods need to be developed
where no retroviral transduction or oncogene expression is involved. Also, additional methods need to be developed for large-
scale culture of cGMP grade cells so that ample numbers are generated for differentiation and transplantation into patients. This
could involve suspension or adherent culture formats depending on the projected use. Finally, as instances of trials and treat-
ments evolve and increase, a streamlined regulatory process needs to be developed for cellular replacement and regenerative
medicine therapies.
The power of hESC technology has already been proven for in vitro applications. It has been successfully used for disease
modeling as mentioned above, and in selected instances proved to be an effective platform for reversal of disease phenotype
(Raya et al., 2009). iPSC technology can be used very effectively for creating models of disease using tissue from patient
samples with relevant disease backgrounds. Deficits due to these particular mutations can be studied in dynamic and physi-
ologically relevant laboratory platforms where processes such as differentiation, maturation, survival/apoptosis, proliferation,
etc. may be dissected. hESC and iPSC technology will prove extremely useful for identification of novel therapeutic targets of
disease, the validation of known and novel targets, and the prosecution of validated targets. Sophisticated disease-specific
screening platforms and assays may be developed using high content screening and similar technologies. Targeted transgenic
and knockout lines may also be created as required and this will be valuable in target validation. hESC lines will also be used
for toxicology and selected in vitro ADME assays. Toxicology may be studied at several levels such as differentiation of
functional liver, cardiac and neural platforms for studying mechanisms of toxicity of therapeutic molecules, and teratological
screening of molecules on undifferentiated hESC lines. The power of hESC/iPSC technology will be realized when these
physiological studies are performed on panels of cultures of known genotypes, so that companion diagnostics may be devel-
oped using pharmacogenomic methods. This will allow predictive toxicology of medications on individuals of known
genotypes.
The question of “are all hESC cultures equal” remains. As mentioned above, hESCs may be created by culture of ICM, SCNT, and
iPSC techniques, among others. The similarities, differences, and limitations of each of the stem cell types needs to be further
defined. Are the final hESC lines which are formed distinct based on the method of derivation, the inducing factor/s, and the char-
acteristics of the cells, which have undergone selection to create the hESC line? As well, the reasons for different differentiation
thresholds and potentials for each individual heSC line requires further definition.
References
Rajan, P., Ross, R., Mackler, A., Smotrich, D., & Loring, J. (2007). Derivation of human embryonic stem cells. In J. Loring (Ed.), Human Stem Cells: A Laboratory Guide (pp. 291–
308). Elsevier Publications.
Raya, A., Rodríguez-Pizà, I., Guenechea, G., et al. (2009). Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature, 460, 53–59.
Shamblott, M. J., Axelman, J., Wang, S., et al. (1998). Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl. Acad. Sci. U.S.A., 95, 13726–
13731.
Tesar, P. J., Chenoweth, J., Brook, F. A., et al. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature, 448, 196–199.
Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.
Relevant Websites
http://clinicaltrials.gov/ct2/show/NCT01469832?term¼advancedþCellþTherapy&cond¼%22MacularþDegeneration%22&rank¼3 – ACT hESC clinical trial.

http://www.roslin.ed.ac.uk/public-interest/dolly-the-sheep/ – Dolly the cloned sheep.
http://grants.nih.gov/stem_cells/registry/current.htm – List of NIH approved hESC lines.
http://stemcells.nih.gov/Pages/Default.aspx – NIH General Stem Cell information.
http://stemcells.nih.gov/info/scireport/pages/chapter3.aspx – NIH hESC information site.
http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/yamanaka-facts.html – Shinya Yamanaka.
http://www.ukstemcellbank.org.uk/ – UK Stem Cell Bank.
http://viacyte.com/products/vc-01-diabetes-therapy/ – Viacyte diabetes treatment.
Introduction to Regenerative Engineering
Manisha Jassal and Radoslaw Junka, Stevens Institute of Technology, Hoboken, NJ, United States
Cato T Laurencin, University of Connecticut Health Center, Farmington, CT, United States
Xiaojun Yu, Stevens Institute of Technology, Hoboken, NJ, United States
Introduction 624
Regenerative Engineering 624
Clinical Need for Regenerative Engineering 624
Different Strategies for Regenerative Engineering 625
Top-down engineering approach 625
Bottom-up engineering approach 625
Key Elements of Regenerative Engineering 625
Stem Cells: The Fundamental Building Block of New Tissues 626
Morphogenetic Signals: Importance of Transition From Individual Cells to Structured Tissues 626
Biomaterials 627
Regenerative Engineering Application Areas 628
Current Challenges and Future Directions 628
Acknowledgment 628
References 629
Further Reading 630
Glossary
Blastema Mass of undifferentiated cells that has the capability to develop into an organ or appendage.
Inducerons Small-molecule inducers of cell differentiation.
Introduction
One of the most fascinating clinical issues being faced by scientists of the 20th century has been the regeneration of complex tissues
and organ systems such as a knee or whole limb. Compared to urodele amphibians that exhibit a remarkable ability to completely
regrow severed limbs during any time point in their life, the humans do not possess such regenerative ability for amputated limbs.
Rehabilitation of with patients amputated limbs has been achieved using prosthetic devices. However, these devices still cannot
perform complex functions such as normal gait and movement feedback. Limitations of current biological and engineering
approaches toward limb regeneration have led to emergence of a new field called “Regenerative Engineering.”
Regenerative Engineering, an interdisciplinary field, is defined as the convergence of advanced Materials Science, Stem Cell
Sciences, Physics, Developmental Biology, and Clinical Translation for the regeneration of complex tissues and organ systems. Built
constructs are produced using a combination of cells, growth factors, and synthetic scaffolds with an aim to achieve functionality
either equivalent to or greater than that of damaged or lost native tissue (El-Amin et al., 2013). The focus of regenerative engineering
has advanced from replace/restore function to regenerate the living tissue, making it the future of tissue engineering. To achieve this
goal, advances made in gaining knowledge of phenomenon taking place in nanoregime combined with advanced materials science,
a mature stem cell science field that gives the opportunity of using stem cells as an everyday tool, and deeper understanding of devel-
opmental biology mechanisms has given scientists the tools to work toward regeneration of whole tissue (Laurencin and Khan,
2012). Success would be achieved for regenerative engineering field when elements of science, engineering, and medicine are
combined together to create constructs that would ultimately improve body’s ability to regenerate its diseased/lost organs.
Clinical Need for Regenerative Engineering

Blood and bone represent two systems inside of a human body that can regenerate. Bone regeneration is limited, because it cannot
regenerate beyond a critical sized defect (Nair and Laurencin, 2015). Similarly, liver has a regenerative capacity that is seen in all
vertebrate organisms (Michalopoulos, 2007). Most of the other tissues present in human body undergo a repair mechanism instead
of regeneration that leads to formation of a scar tissue. This scar tissue lacks necessary biological and mechanical properties that are
present in the native tissue, hence leading to functional impairment (Nair and Laurencin, 2015). In order to address the issue of

Regenerative Engineering j Introduction to Regenerative Engineering 625
functional impairment, efforts were focused on different types of biological grafts, namely autograft, allograft, and xenografts. Auto-
grafts are considered the gold standard, because they do not present complications associated with other grafts such as adverse
immune response. The limited availability of autografts and complications associated with other grafts led to engineering tissues
as substitutes for clinical use to replace diseased organs or heal damaged tissues (Nair and Laurencin, 2015). However, the clinical
success of tissue-engineered products is still limited due to various limitations such as insufficient vascularization that leads to lack
of nutrients and oxygen and insufficient removal of waste products including scaffold degradation products (Liu et al., 2013; Selik-
tar et al., 2014). Tissue engineering has given successful outcomes in terms of regenerating small segments of bone and hallow
organs, but regeneration of complex structures has still not been realized clinically. This has led the scientific community to focus
on developing novel strategies to regenerate complex tissue and organ systems such as a functional knee or whole limb, hence giving
rise to a much advanced research field of regenerative engineering (Reichert et al., 2011).
Different Strategies for Regenerative Engineering

The success of regenerative engineering will depend on recapitulating the complex architecture of organs and the native arrangement
of cells and extracellular matrix (ECM) at the functional level. A comprehensive approach is required for the design of regenerative
constructs based on understanding of cellular and morphogenic processes, biological factors, scaffolds, physical forces, and the
interrelation between these essential components (Nair and Laurencin, 2015). Regenerative engineering could make complex tissue
regeneration possible using the top-down and bottom-up approaches that are described below.
Top-down engineering approach

The top-down approach for limb regeneration considers the limb structure as an aggregate of individual specific tissues integrated
one by one into a unified structure. The top-down approaches are built on the integration of advanced material science and engi-
neering, cells with high regenerating ability, physical forces, and immune system modulation (Nair and Laurencin, 2015). Cells are
seeded on prefabricated porous polymeric scaffolds that serve as temporary template for new tissue growth. Bioreactors can be
utilized to simulate physiological conditions so that cells can differentiate and secrete their own ECM (Urciuolo et al., 2013).
The main advantage associated with top-down approaches is that these processes are cost-effective, scalable and able to generate
clinically relevant nanostructured matrices. Limitations of this approach includes difficulty in recreating the complex microarchitec-
ture of tissues, slow vascularization, and diffusion barriers which restrict the potential of this strategy to be used effectively for regen-
eration of complex tissues (Urciuolo et al., 2013; Lu et al., 2013).
Bottom-up engineering approach

The bottom-up approach is based on the principle of molecular self-assembly. This phenomenon is defined as a spontaneous orga-
nization of molecules under near thermodynamic equilibrium conditions to form stable structures. A larger tissue construct can be
generated by assembling smaller building blocks via multiple assembling methods, such as surface tension assembly, acoustic
assembly or magnetic assembly. The advantage of the bottom-up approach is spacial control over features and composition of indi-
vidual blocks (Lu et al., 2013).
The successful regeneration of complex tissues and organ systems will depend on the ability to integrate both approaches, while
optimizing advantages offered by each. Many amphibians have a remarkable ability to regenerate complex tissues and organ
systems by the self-assembly of proliferating and differentiating cells in blastema. Humans lack such capability, except repair of
a small injury in tissues such as bone or skin (Nair and Laurencin, 2015). The bottom-up approach will help to understand the
highly orchestrated process of tissue development as it is unfolding through a tightly controlled spatial and temporal expression
of various morphogenetic cues (Nair and Laurencin, 2015). The top-down approach will aids in designing strategies to deliver bio-
logically active effector molecules and inducerons in a tissue-engineered construct that would replicate the complex architecture of
the organ system (Nair and Laurencin, 2015).
Key Elements of Regenerative Engineering
The epimorphic regeneration exhibited by the urodele amphibians occurs through the formation of a blastema that consists of
progenitor cell population with intrinsic morphogenetic cues. The blastema presents a highly complex gene profile, and the unique
ECM of the blastema provides directional cues to modulate cellular functions. Some of the pathways that control embryonic limb
formation such as fibroblast growth factor (FGF) and Wnt-ß catenin signaling, along with retinoic acid and Shh signaling, are essen-
tial in patterning and morphogenesis during limb regeneration. A combination of these signaling pathways combined with the role
played by the immune system and innervation gives rise to a favorable regenerative environment in the blastema. The under-
standing achieved from the regenerative process in urodele amphibians, combined with interdisciplinary scientific approaches,
can lead to exciting new solutions that address current challenges to human organ regeneration.
A deeper understanding of adult and embryonic stem cells over the past few decades led researchers to appreciate similarities
these cells have with blastema cells. Similarly, induced pluripotent stem cells (iPSCs) derived from adult differentiated cells undergo
a process of cellular dedifferentiation also present in blastema. Considering blastema cells molecularly similar to a cell in a more
undifferentiated state, the possibility of controlling the extent of cellular dedifferentiation may have significant impact on
626 Regenerative Engineering j Introduction to Regenerative Engineering
developing a translational protocol for limb regeneration in humans. Another key component of regenerative engineering that has
advanced significantly in the past two decades is the biomaterials science. The current research focuses on developing advanced
biomaterials, wherein the physical, mechanical, and biological properties of the scaffold can be fine-tuned to enhance the natural
regenerative process in the body. In the following sections, essential components for regenerative engineering are described in detail
including stem cells, morphogenetic signals, and biomaterials. In addition to these, physical forces such as electrical forces influence
morphogenesis and patterning, and modulate cellular functions leading to a more permissive microenvironment for tissue
regeneration.
Stem Cells: The Fundamental Building Block of New Tissues

Regeneration of a damaged tissue largely depends on cells’ ability to repopulate the defect or the substituting scaffolding material
and reestablish the native architecture of the tissue. To this end, of a particular interest to engineers are populations of regeneration-
competent cells, called stem cells. Stem cells are phenotypically characterized by their ability to divide continuously through asym-
metric mitosis to yield two different populations of cells, daughter stem cells of the same phenotype and progenitor cells that are
more differentiated than the original cell (Zhong, 2008). This self-renewal capability allows for stem cells to undergo almost infinite
number of replications, generating in the process any mass of tissue needed. Therefore, they can produce populations of cells
capable of maintaining this special phenotypic state and simultaneously differentiate into other cell types. These progenitor cells
are plastic; meaning they have the ability to alter their genetic profile expression and adopt functional phenotype of cells present
in developed tissues through a process of transdifferentiation. Transdifferentiation can result directly through interaction of stem
cells with another tissue of a different phenotype, or it can be achieved indirectly via artificially manipulation of the cell environ-
ment. The indirect transdifferentiation transforms an adult differentiated cell into a more primitive state, which is then differenti-
ated into another cell type (Lodi et al., 2011). However, stem cells’ plasticity depends on the origin of a tissue type, embryo or the
adult tissue.
Stem cells are categorized into three populations depending on the tissue of origin, such as embryonic stem cells (ESCs), induced
pluripotent stem cells (iPSCs), and postnatal or adult stem cells. ESCs have the greatest potential for self-renewal and expansion and
are able to differentiate into any cell type (Lenoir, 2000). While these characteristics make ESCs a promising candidate for the use in
tissue regeneration, their implementation is often entangled with serious ethical considerations. iPSCs are derived from adult differ-
entiated cells, for example, skin fibroblasts, by induction of a more primitive state or dedifferentiation (Takahashi and Yamanaka,
2006). They do not have the same self-renewal and differentiation potential; however this is an area of intense ongoing investiga-
tion. Although both of these stem cell populations present a tremendous therapeutic potential, their application encompasses
serious risks and limitations such as formation of cancerous tissues, immune response to ESCs, and difficulty in eliciting specific
genetic profiles in iPSCs (Ben-David and Benvenisty, 2011). Postnatal adult stem cells, on the other hand, do not pose these
difficulties in culture. Two of the most widely studied adult stem cell populations are mesenchymal stem cells (MSCs) and
adipose-derived stem cells (ASCs). Likewise, these cell populations are limited in their ability to undergo self-renewal and transdif-
ferentiation process, hence they are termed as multipotent and not pluripotent as ESCs (Mizuno et al., 2012).
MSCs can be isolated from postnatal organs and connective tissues, such as bone (Song et al., 2005; Choi et al., 2008), synovial
membrane (De Bari et al., 2001), skeletal muscle (Dodson et al., 2010), peripheral blood (Shi et al., 2009), periodontal ligament
(Seo et al., 2004), and umbilical cord (Baksh et al., 2007; Musina et al., 2005) among other tissues. However, these sources usually
yield low number of cells that can be harvested. Thus, to become a viable option for the clinical use, strategies implementing the use
of ESCs, iPSCs, or MSCs require labor intensive and expensive ex vivo culture methods. In contrast, ASCs overcome this limitation
and hold greater promise for future therapeutic use as adipose tissue can be easily harvested in large quantities with little donor site
morbidity (Zuk et al., 2001). This advantage is manifested in a large number of preclinical and clinical studies of injury and disease
already underway (Gimble et al., 2007; Tobita et al., 2011; Rosado-de-Castro et al., 2013).
Nevertheless, there remain several challenges to translation of stem cell therapies to a wide clinical use. The limited under-
standing of motility, survival, proliferation, and differentiation in in vivo setting is a major obstacle. Human and animal studies
show poor engraftment of stem cells as reflected in only a few percent of cells remaining several weeks after injection (Terrovitis
et al., 2010). Likewise, long-term safety and efficacy is a concern as previous studies demonstrate these cells’ ability to form tumors
(Hentze et al., 2009). Once implanted, stem cells, progenitor cells derived from them, and the surrounding endogenous cells
encounter and exchange a multitude of signals in a highly dynamic niche that affects their phenotype. Thus, greater efforts are neces-
sary to elucidate and control molecular processes that guide expansion and transdifferentiation of these cells in vivo. Recent devel-
opments in the science of stem cells and their associated morphogenic cues offer exciting possibilities for the future use of stem cells
to regenerate tissues and, eventually, to replace entire organs (Platt, 2004).
Morphogenetic Signals: Importance of Transition From Individual Cells to Structured Tissues

Complex interrelationship between mechanical forces and a number of protein families orchestrate the process of tissue morpho-
genesis during development. The mechanical stimuli are known to affect cells’ size, shape, and phenotype. These changes in gene
expression in turn induce cells to secrete a host of protein products that further influence tissue formation (Heisenberg and Bel-
laïche, 2013). These molecules regulate self-renewal, migration, and differentiation uncommitted stem or progenitor cells. Their
secretion is tightly controlled in space and time (Murry and Keller, 2008). Interestingly, many of the same morphogenetic processes
observed during development also take place in some adult human tissues as they regenerate (Stocum, 1998; Lutolf and Hubbell,
2005). Thus, scientists and engineers aim to augment the self-healing capacity in certain tissues by artificially recapitulating this
intricate balance between mechanical forces and cell signaling molecules. Application of one or more of these morphogenic
cues guides cells toward a desired tissue formation by enhancing several of the processes important for healing. A category of these
signaling molecules, called growth factors, are often added to cultures, delivered via polymeric vehicles, or bound to scaffolds.
During development, these factors are actively secreted by stem cells and nearby niche cells. Tissue growth and differentiation occurs
as a result of the interpretation of specific concentrations or a gradient of these morphogenetic signals (Discher et al., 2009). Concur-
rently, local cell–cell interactions between stimulated and unstimulated cells establish boundaries of different cell phenotypes.
Functional tissue recovery is thought to be possible only when undifferentiated or genetically modified cells are recruited into
a regeneration or controlled growth pathway by the presence of these morphogenetic signals. Taking into consideration each tissue’s
own ability to heal, functional recovery might also depend on suppression of inhibitory signals originating from rapid scar forma-
tion that curtail the regenerative process. Therefore, to achieve successful tissue regeneration, it is imperative to create and control
over time the artificial environment that enables cells to proliferate and differentiate by carefully manipulating timing and concen-
trations of morphogenic cues. The dependence of successful tissue regeneration on growth factors is perhaps best exemplified in
their direct induction of angiogenesis, sprouting of immature capillaries, which supply oxygen and nutrients to cells. This process
is necessary to maintain biological functions of cells in immature regenerating tissue of a clinically relevant size (Tabata, 2003).
Although biological effects of growth factors are greatly enumerated, successful long-term delivery of these morphogenetic
factors evades scientists due to their poor in vivo stability. Direct injections are deemed impractical, because their systemic effects
are likely to be short-lived or cause undesired cancerous growth. Therefore, scientists turned toward various drug-delivery systems
to achieve sustained long-term targeted delivery of these factors. Despite fervent efforts, application of growth factors in clinical
treatment remains limited due to inefficient loading of these large molecules into their carriers and subsequent burst release
upon administration. In addition, future studies should bring vast improvement in production of recombinant growth factors to
drive down the cost of production. Emphasis on robust preclinical models and detailed pharmacokinetic studies should ensure
safety and efficacy of these recombinant factors. Remaining important caveats to be accounted for are the likely required use of
a combination of these factors, their corresponding therapeutic concentrations, and the native time sequence at which they are
secreted during regeneration (Koria, 2012). To achieve more efficient delivery systems and realize the therapeutic potential of these
morphogenic cues requires novel biomaterials with tunable physical and chemical properties.
Biomaterials
Biomaterials act as extracellular matrix scaffolding as they provide physical structure to retain cell population after implantation,
guidance, and template for cells to lay new extracellular matrix. However, over time, biomaterials have evolved from just func-
tioning as a template where cells can attach and lay their own extracellular matrix to multifunctional systems that actively regulate
various aspects of tissue regeneration. Apart from being biocompatible and biodegradable, they can now incorporate biological and
structural cues to induce favorable cell response such as enhanced cell attachment and directed cell differentiation to achieve a partic-
ular outcome. One of the examples includes biomaterials utilized for bone regeneration that could be made osteoconductive (e.g.,
by selecting appropriate chemical composition) and osteoinducive (e.g., by varying the microstructure of the material) (Yu et al.,
2015).
Biomaterials can be classified as natural and synthetic, based on their origin with both offering certain advantages and disadvan-
tages. Natural biomaterials can be subclassified based on their chemical structure into proteins, polysaccharides, and polyesters
(Mano et al., 2007). These offer advantages such as being similar to biological macromolecules that might aid in reducing the
inflammation and toxicity when implanted in body. Some examples of natural protein biomaterials include collagen, fibrin, fibro-
nectin, and silk. Chitin, chitosan, alginate, and hyaluronic acid are some examples of polysaccharide-based natural biomaterials.
Polyhydroxyalkanoates, derived from microorganisms, are examples of natural thermoplastic polyesters that are biocompatible
and biodegradable. Decellularized tissue-derived biomaterials can also be considered another category of natural biomaterials
that are obtained by the elimination of cellular/nuclear components and retaining just the extracellular matrix components. Exam-
ples include decellularized dermis, heart valves, blood vessels, small-intestinal submucosa, and liver (Chen and Liu, 2016). The
main limitations of natural biomaterials include insufficient mechanical strength, batch-to batch variation due to complex purifi-
cation process, possibility of adverse immunogenic reaction, and inability to control in-vivo degradation rate.
Synthetic biomaterials, on the other hand, offer advantages such as tunable mechanical and degradation properties by varying
the chemical composition of the polymers, and the ability to be fabricated into various shapes with controllable micro and macro-
structure. The most extensively studied synthetic biomaterials include polycaprolactone, polyglycolide, polylactide, polyhydroxy-
butyrate, and their copolymers. Their disadvantages include reduced bioactivity and harmful degradation by-products produced
in-vivo.
Studies have shown that the bioactivity of the advanced biomaterials can be significantly enhanced using biological proteins/
peptides as well as biologically active effector molecules and inducerons. The three-dimensional structure of the biomaterial scaffold
also plays an important role in modulating cellular behavior that could positively impact the regenerative capability of tissues and
organs. The developments in the micro- and nanotechnologies have led to creation of novel 3D biomimetic scaffolds and the last
decade exhibited significant growth in the fabrication and characterization of nanofibrous 3D structures as biomimetic scaffolds.
The latest innovation in this direction is focused on additive manufacturing or 3D printing that can create patient-specific complex
3D structures. The success of regenerative engineering would depend on our ability to use these biomaterial scaffolds, natural,
synthetic, or a blend of the two, as a delivery system for growth factors, adhesion peptides, and cytokines as well as a mechanical
support structure with favorable micro- and macrostructure to induce regeneration of specific tissue/organ (O’brien, 2011).
Regenerative Engineering Application Areas
Recent progress made in understanding stem cells science and developmental biology, combined with advances made in material
science and nanotechnology have brought the scientific community closer to realizing their goal of regenerating complex tissues,
organs, or organ systems (Nair and Laurencin, 2015). Regenerative engineering principles have been applied for musculoskeletal
tissue, cardiovascular tissue, and neural tissue regeneration. Apart from these, other organs of interest include the liver, lung,
kidneys, and pancreas.
Regenerative engineering clinical applications based on biodegradable scaffolds include the artificial skin, nerve conduits, bone,
articular cartilage, blood vessels, and bladder. Another technique utilized for cell-dense-tissues is cell sheet technology where autol-
ogous cells are grown and collected as a contiguous sheet of cells while preserving their natural extracellular matrix (Elloumi-
Hannachi et al., 2010). This technology has been utilized for corneal surface reconstruction, periodontal tissue, and myocardial
tissue reconstruction.
Various clinical trials have been undertaken to establish the efficacy of different scaffolds for their regeneration potential in-vivo,
but only a few have been approved till date. For bone regeneration, PCL scaffolds in various forms and shapes such as Osteoplug,
Osteoplug-C, Osteomesh, and Osteomesh-Osteostrip have been approved for craniofacial applications (Liu et al., 2013). Many clin-
ical trials for bone regeneration are currently in progress that involves a combination of stem cells, growth factors, and/or scaffold-
based technologies.
For nerve regeneration applications, many natural resorbable and synthetic resorbable devices have been approved by FDA that
include Type I collagen based devices (NeuraGenÒ, NeuroflexÔ, NeuromatrixÔ, NeuraWrapÔ, and NeuroMendÔ), polyglycolic
acid-based device (NeurotubeÒ), and poly D,L lactide-co-3-carprolactone-based device (NeurolacÒ) (Kehoe et al., 2012). Type I
collagen-based devices fall under the category of natural resorbable materials, whereas polyglycolic acid and poly D,L lactide-co-3-car-
prolactone-based devices fall under synthetic resorbable materials utilized for nerve regeneration applications. Similarly, various
trials are underway in the lung, liver, and pancreas regeneration field that utilize stem-cell-based therapies. Examples include infu-
sion of mesenchymal stem cells (MSCs) for lung repair and regeneration (Kotton, 2012) and transplantation of bone-marrow-
derived stem cells that initiated endogenous pancreatic regeneration (Hess et al., 2003).
Current Challenges and Future Directions
Although enormous advances have been made in the application of regenerative engineering principles to regenerate various
human tissues, the regenerative field still far from achieving its ultimate goal of regeneration of a whole organ system. One of
the major challenges is to recreate the complex architecture of tissues and organs as it directly influences the function of that partic-
ular organ/tissue. Various strategies to capture native organ structure and material composition include decellularization of organs
followed by recellularization before transplantation or use of biomaterials seeded with cells. The limitation associated with these
techniques is inadequate control of cell placement within the scaffolds. 3D bioprinting can address some of these limitations, but
they suffer from trade-offs such as feature resolution, cell viability, and print resolution (Mao and Mooney, 2015).
Another challenge lies in properly integrating the implanted grafts with the body in terms of vascularization and innervation.
Vascularization can either be achieved by exploiting body’s own angiogenic response via presentation of angiogenic growth factors
through controlled delivery or prevascularization of the graft. Similarly, innervation of engineered tissues/biomaterials can also be
induced by use of growth factors (Mao and Mooney, 2015).
Modulating the immune system in order to avoid rejection of the implanted biomaterial/decellularized tissue presents compli-
cation that can be addressed by either engineering the responses of immune cells or changing the properties of the implanted mate-
rial such as increasing hydrophilicity or modifying the surface with adhesion ligands that promote cell attachment and proliferation
(Mao and Mooney, 2015).
The future success of regenerative engineering holds promise for patients whose tissue/organ systems cannot recover from
disease or trauma by currently available drugs or therapeutic methods. The advances made in design of biomaterials that mimic
the natural architecture of tissues along with the ability to deliver biomolecules in a controlled manner, better understanding of
developmental biology along with advances made in stem cell research would pave the way to regenerate complex tissues and
organs to heal patients.
Acknowledgment
This work was partly supported by the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under
Award No. W81XWH-16-1-0132.
References
Baksh, D., Yao, R., & Tuan, R. S. (2007). Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and
bone marrow. Stem Cells, 25(6), 1384–1392.
Ben-David, U., & Benvenisty, N. (2011). The tumorigenicity of human embryonic and induced pluripotent stem cells. Nature Review Cancer, 11(4), 268–277.
Chen, F.-M., & Liu, X. (2016). Advancing biomaterials of human origin for tissue engineering. Progress in Polymer Science, 53, 86–168.
Choi, Y. S., Noh, S. E., & Lim, S. M. (2008). Multipotency and growth characteristic of periosteum-derived progenitor cells for chondrogenic, osteogenic, and adipogenic
differentiation. Biotechnol Letters, 30(4), 593–601.
De Bari, C., Dell’Accio, F., Tylzanowski, P., & Luyten, F. P. (2001). Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis & Rheumatology, 44(4),
1928–1942.
Discher, D. E., Mooney, D. J., & Zandstra, P. W. (2009). Growth factors, matrices, and forces combine and control stem cells. Science, 324(5935), 1673–1677.
Dodson, M. V., Hausman, G. J., et al. (2010). Skeletal muscle stem cells from animals I. Basic cell biology. International Journal of Biological Sciences, 6(5), 465–474.
El-Amin, S. F., III, Thomas, J. W. L., Ihekweazu, U. N., Woods, M. D., & Gupta, A. (2013). Regenerative engineering: The future of medicine. In C. T. Laurencin, & Y. S. Khan (Eds.),
Regenerative engineering (1st edn., pp. 1–20). Florida: CRC Press.
Elloumi-Hannachi, I., Yamato, M., & Okano, T. (2010). Cell sheet engineering: A unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in
regenerative medicine. Journal of Internal Medicine, 267(1), 54–70.
Gimble, J. M., Katz, A. J., & Bunnell, B. A. (2007). Adipose-derived stem cells for regenerative medicine. Circulation Research, 100(9), 1249–1260.
Heisenberg, C. P., & Bellaïche, Y. (2013). Forces in tissue morphogenesis and patterning. Cell, 153(5), 948–962.
Hentze, H., Soong, P. L., Wang, S. T., Phillips, B. W., Putti, T. C., & Dunn, N. R. (2009). Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for
future safety studies. Stem Cell Research, 2(3), 198–210.
Hess, D., Li, L., Martin, M., Sakano, S., Hill, D., Strutt, B., … Bhatia, M. (2003). Bone marrow–derived stem cells initiate pancreatic regeneration. Nature Biotechnology, 21(7),
763–770.
Kehoe, S., Zhang, X. F., & Boyd, D. (2012). FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy. Injury, 43(5),
553–572.
Koria, P. (2012). Delivery of growth factors for tissue regeneration and wound healing. BioDrugs, 26(3), 163–175.
Kotton, D. N. (2012). Next-generation regeneration: The hope and hype of lung stem cell research. American Journal of Respiratory and Critical Care Medicine, 185(12),
1255–1260.
Laurencin, C. T., & Khan, Y. (2012). Regenerative engineering. Science Translational Medicine, 4, 160ed9.
Lenoir, N. (2000). Europe confronts the embryonic stem cell research challenge. Science, 287(5457), 1425–1427.
Liu, Y., Lim, J., & Teoh, S. H. (2013). Review: Development of clinically relevant scaffolds for vascularised bone tissue engineering. Biotechnology Advances, 31(5),
688–705.
Lodi, D., Iannitti, T., & Palmieri, B. (2011). Stem cells in clinical practice: applications and warnings. Journal of Experimental & Clinical Cancer Research, 30(1), 9.
Lu, T., Li, Y., & Chen, T. (2013). Techniques for fabrication and construction of three-dimensional scaffolds for tissue engineering. International Journal of Nanomedicine, 8(2),
337–350.
Lutolf, M. P., & Hubbell, J. A. (2005). Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnology, 23(1),
47–55.
Mano, J. F., Silva, G. A., Azevedo, H. S., Malafaya, P. B., Sousa, R. A., Silva, S. S., Boesel, L. F., Oliveira, J. M., Santos, T. C., Marques, A. P., Neves, N. M., & Reis, R. L. (2007).
Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. Journal of the Royal Society Interface, 4(17),
999–1030.
Mao, A. S., & Mooney, D. J. (2015). Regenerative medicine: Current therapies and future directions. Proceedings of the National Academy of Sciences, 112(47), 14452–
14459.
Michalopoulos, G. K. (2007). Liver regeneration. Journal of Cellular Physiology, 213(2), 286–300.
Mizuno, H., Tobita, M., & Uysal, C. A. (2012). Concise review: Adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells, 30(5), 804–810.
Murry, C. E., & Keller, G. (2008). Differentiation of embryonic stem cells to clinically relevant populations: Lessons from embryonic development. Cell, 132(4), 661–680.
Musina, R. A., Bekchanova, E. S., & Sukhikh, G. T. (2005). Comparison of mesenchymal stem cells obtained from different human tissues. Bulletin of Experimental Biology and
Medicine, 139(4), 504–509.
Nair, L. S., & Laurencin, C. T. (2015). Regenerative engineering: Approaches to limb regeneration and other grand challenges. Regenerative Engineering and Translational Medicine,
1, 1–3.
O’brien, F. J. (2011). Biomaterials & scaffolds for tissue engineering. Materials Today, 14(3), 88–95.
Platt, L. J. (2004). Preface: Future approaches to replacement of organs. American Journal of Transplantation, 4(S6), 5–6.
Reichert, W. M., Ratner, B. D., Anderson, J., Coury, A., Hoffman, A. S., Laurencin, C. T., & Tirrell, D. (2011). 2010 panel on the biomaterials grand challenges. Journal of
Biomedical Materials Research. Part A, 96(2), 275–287.
Rosado-de-Castro, P. H., Pimentel-Coelho, P. M., da Fonseca, L. M., de Freitas, G. R., & Mendez-Otero, R. (2013). The rise of cell therapy trials for stroke: Review of published and
registered studies. Stem Cells Development, 22(15), 2095–2111.
Seliktar, D., Berdicheviski, A., Meroni-Harpaz, I., & Shapira-Schweitzer, K. (2014). Heart regeneration: The bioengineering approach. In G. Orlando, J. P. Lerut, S. Shoker, &
R. J. Strata (Eds.), Regenerative medicine applications in organ transplantation (1st ed., pp. 445–456). Cambridge, MA: Academic Press.
Seo, B. M., Miura, M., Gronthos, S., et al. (2004). Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet, 364(9429), 149–155.
Shi, M., Ishikawa, M., Kamei, N., et al. (2009). Acceleration of skeletal muscle regeneration in a rat skeletal muscle injury model by local injection of human peripheral blood-derived
cd133-positive cells. Stem Cells, 27(4), 949–960.
Song, L., Young, N. J., Webb, N. E., & Tuan, R. S. (2005). Origin and characterization of multipotential mesenchymal stem cells derived from adult human trabecular bone. Stem
Cells Development, 14(6), 712–721.
Stocum, D. L. (1998). Regenerative biology and engineering: Strategies for tissue restoration. Wound Repair Regeneration, 6(4), 276–290.
Tabata, Y. (2003). Tissue regeneration based on growth factor release. Tissue Engineering, 9(S1), 1–15.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–676.
Terrovitis, J. V., Ruckdeschel Smith, R., & Marbán, E. (2010). Assessment and optimization of cell engraftment after transplantation into the heart. Circulation Research, 106(3),
479–494.
Tobita, M., Orbay, H., & Mizuno, H. (2011). Adipose-derived stem cells: Current findings and future perspectives. Discovery Medicine, 11(57), 160–170.
Urciuolo, F., Imparato, G., Totaro, A., & Netti, P. A. (2013). Building a tissue in vitro from the bottom up: Implications in regenerative medicine. Methodist DeBakey Cardiovascular
Journal, 9(4), 213–217.
Yu, X., Tang, X., Gohil, S. V., & Laurencin, C. T. (2015). Biomaterials for bone regenerative engineering. Advanced Healthcare Materials, 4(9), 1268–1285.
Zhong, W. (2008). Timing cell-fate determination during asymmetric cell divisions. Current Opinion in Neurobiology, 18(5), 472–478.
Zuk, P. A., Zhu, M., Mizuno, H., et al. (2001). Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Engineering, 7(2), 211–228.
Further Reading
Laurencin, C. T., & Khan, Y. S. (Eds.). (2013). Regenerative engineering. Florida: CRC Press.
Laurencin, C. T., & Nair, L. S. (Eds.). (2015). Nanotechnology and regenerative engineering: The Scaffold. Florida, USA: CRC Press.
Liu, S. Q. (2007). Bioregenerative engineering: Principles and applications. New Jersey: Wiley.
Berthiaume, F., Maguire, T. J., & Yarmush, M. L. (2011). Tissue engineering and regenerative medicine: History, progress, and challenges. Annual Review of Chemical and
Biomolecular Engineering, 2, 403–430.
Orlando, G., Wood, K. J., Stratta, R. J., Yoo, J. J., Atala, A., & Soker, S. (2011). Regenerative medicine and organ transplantation: Past, present, and future. Transplantation,
91(12), 1310–1317.
Dawson, J. I., & Oreffo, R. O. (2008). Bridging the regeneration gap: Stem cells, biomaterials and clinical translation in bone tissue engineering. Archives of Biochemistry and
Biophysics, 473(2), 124–131.
Huey, D. J., Hu, J. C., & Athanasiou, K. A. (2012). Unlike bone, cartilage regeneration remains elusive. Science, 338(6109), 917–921.
Nanoelectronics for Neuroscience
Sahil Kumar Rastogi and Tzahi Cohen-Karni, Carnegie Mellon University, Pittsburgh, PA, United States
Introduction 632
Bioelectrical Signals Recording 633
Extracellular Recordings 634
MEAs 634
Planar passive electrodes 635
Flexible passive electrodes 635
FETs 638
2D FETs 639
3D FETs 639
Injectable Electronics 641
Intracellular Recordings 642
MEA 642
Functionalized gold-spine electrode (FGSE) 642
Vertical Si nanowire (SiNW) electrode array (VNEA) 642
Pt nanopillars 643
FETs 644
3D-kinked NW probes 644
Branched intracellular nanotube-FET (BIT-FET) 644
Active Si nanotube transistor (ANTT) 644
Conclusion 646
References 647
Further Reading 649
Glossary
Action potential Localized change in the electric potential across the cell membrane of an electrically active cell, from about
70 mV to þ 30 mV.
Electrocorticography (ECoG) Technique of recording brain electrical field potentials with metal electrodes placed directly on
the cortical surface.
Electroencephalogram (EEG) Technique of recording brain electrical field potentials with metal electrodes placed on the
scalp.
Electrophysiology Study of the electrical properties of electrically active cells.
Electroporation A technique in which an electrical field is applied to cells to increase the permeability of the cell membrane.
Excitatory postsynaptic potentials (EPSP) Depolarization of the membrane potential of a postsynaptic neuron caused by the
binding of an excitatory neurotransmitter from a presynaptic cell to a postsynaptic receptor.
Extracellular recording Monitoring the change in the potential across cell membrane of cells from outside the cell.
Field-effect transistor (FET) A type of electrical device where the current across source and drain terminals is controlled by the
potential applied to a third terminal named gate.
fMRI Functional neuroimaging procedure using magnetic resonance imaging (MRI) technology that measures brain activity by
detecting changes associated with blood flow.
Glia Nonneuronal cells that maintain homeostasis, form myelin, and provide support and protection for neurons in the
central and peripheral nervous systems.
Graphene One atom thick layer of sp2-hybridized carbon atoms arranged in hexagonal lattice.
Hyperpolarization Change in the membrane potential of the cell making it more negative.
Inhibitory postsynaptic potentials (IPSP) Hyperpolarization of the membrane potential of a postsynaptic neuron caused by
the binding of an inhibitory neurotransmitter from a presynaptic cell to a postsynaptic receptor.
Intracellular recordings Monitoring electrical activity of cells from within the cell cytosol.
Ion channels Proteins that act as pores in a cell membrane and permit the selective passage of ions such as potassium, sodium,
and calcium ions.
Microelectrode array (MEA) An array of passive electrodes, ranging from 10 mm to 500 mm in dimensions, that enable
simultaneous multisite recording of electrical signals from the electrically active cells.

632 Regenerative Engineering j Nanoelectronics for Neuroscience
Neuron An electrically excitable cell in the nervous system that processes and transmits information through electrical and
chemical signals.
Neurotransmitter A chemical released from an electrically excited presynaptic neuron that leads to either excitation or
inhibition of the postsynaptic neuron.
Optogenetics A technique in which genes for light-sensitive proteins are introduced into neurons to monitor and control their
activity precisely using light signals.
Patch clamp Type of intracellular recording that involves attaching a glass pipette to the outer membrane of a single cell and
then recording the activity of ion channels in the cell membrane.
Phospholipid Amphipathic compounds with hydrophilic head and hydrophobic lipophilic tail that serve as a major structural
component of cell membranes.
Positron emission tomography Imaging technique that measures emissions from radioactively labeled metabolically active
chemicals that have been injected into the bloodstream.
Resting membrane potential (RM) Voltage difference across the cell membrane when the cell is at a resting or quiescent state.
Seal resistance Resistance generated in the cleft between the cell and the sensing element.
Subthreshold potential A stimulus that leads to change in membrane potential of a neuron with a magnitude smaller than the
threshold that is required to initiate an action potential.
Synapse Junction that permits a neuron to pass an electrical or chemical signal to another neuron.
Introduction
Nervous system, the most complex network in the body, consists of two classes of specialized cells called glia and neurons. Glia are
cells that mechanically support the neural growth, form the immune system, supply the neurons with nutrients, and electrically
isolate the neurons from each other (Kandel et al., 2000). Neurons, on the other hand, are electrically excitable cells that receive
signals, process them, and transmit information throughout the body (Kandel et al., 2000). Typical morphology of a neuron
consists of a cell body called soma, thin branched outgrowths called dendrites, and a filament that extends away from the cell
body called axon, as represented in Fig. 1A. Dendrites are the main apparatus for receiving incoming signals from other nerve cells,
whereas axon is the main conducting unit for carrying signals to other neurons. An axon can convey electrical signals, called action
potentials (APs), along the distances ranging from 0.1 mm to 3 m (Kandel et al., 2000). Neurons communicate with one another at
junctions called synapses. The communication between neurons, also called neurotransmission, is accomplished by the movement
of chemicals across the chemical synapse or electrical signals across the electrical synapse. At electrical synapses, two neurons are
physically connected to one another through gap junctions (Nicholls et al., 2001). Gap junctions permit changes in the electrical
properties of one neuron to effect the other, and vice versa. In chemical neurotransmission, the presynaptic neuron and the post-
synaptic neuron are separated by a small gap called the synaptic cleft. The AP in the transmitting neuron releases a chemical
messenger (a neurotransmitter) across the synaptic cleft. The neurotransmitter binds to receptor proteins on the receiving neuron,
and the resulting reaction transduces the potential chemical energy of the transmitter into electrical energy (Nicholls et al., 2001).
The cellular membrane is composed of a phospholipid bilayer that separates cytoplasm of the cell from the extracellular envi-
ronment. In general, the major ionic constituents of the external tissue fluid are Naþ and Cl ions, whereas there is high Kþ ion
concentration inside the cell. Since the ions are charged, they cannot pass directly through the hydrophobic lipid regions of the
membrane. Therefore, in order to cross the membrane, they have to use specialized ion-specific channel proteins such as (i)
voltage-gated ion channels that are activated by changes in the membrane electrical potential, (ii) leak ion channels that are always
permeable to the ions, and (iii) ATP-dependant Naþ–Kþ pumps that pump Naþ and Kþ ions against their concentration gradients
(Fig. 1B). The movement of the ions across the cell membrane is influenced by two energetic factors: the concentration gradient and
the electrical potential difference (caused due to the charge on ions). When the electrical potential difference across the cell
membrane exactly balances the concentration gradient for an ion, it is known as the equilibrium potential of that ion. When
the cell is at rest, the potential across the neuronal membrane is referred to as resting membrane potential (VM). Resting VM is deter-
mined by the uneven distribution of ions between the inside and the outside of the cell, and by the different permeability of the
membrane to different types of ions.
At the resting state, the voltage-gated ion channels are closed, and the mode the ions can cross the membrane is through leak ion
channels. Since the resting membrane comprises of high number of leak channels specific to Kþ ions, therefore the resting
membrane potential is closer to the Kþ equilibrium potential than it is to the Naþ equilibrium potential, and lies around 85
to 60 mV. However, if an external stimulus or summation of multiple stimuli at the origin of the axon called the axon hillock
increases VM above a threshold value of ca. 55 mV, it initiates an AP which gets conducted down the axon without failure or distor-
tion at rates of 1–100 ms 1. Initiation of an AP involves opening of voltage-gated Naþ channels (Fig. 1C). When the channels open,
Naþ ions flow inside the cell and depolarize the cell, thus generating a positive intracellular and a negative extracellular potential
change (Fig. 1D). The reversed VM results in the closing of Naþ channels and activation of the voltage-gated Kþ channels. The
outflow of Kþ ions repolarizes the cell which eventually crosses the resting VM leading to hyperpolarization. With the help of
Regenerative Engineering j Nanoelectronics for Neuroscience 633
(A) (D)
Inhibitory
input
0
Terminal
d d arborization
d Axon hillock Axon –20
Vintra (mV)
d
Excitatory –40
input
d S
d Myelin
d Postsynaptic –60
element
(B) (C) 0.4

Cell interior 60 Action potential 70
40 60
K+ Na+ conductance 0.2
C
Vextra (mV)
G (mS/cm2)
20 50
V (mV)
0 40 0.0
K+ conductance
–20 30
–0.2
–40 20
–60 10 –0.4
+ 0 2 4 6 8 10
Na 0 5 10 15 20
Cell exterior Time (ms) Time (ms)
Fig. 1 Morphology and electrical activity of neurons. (A) Schematic illustration of a mammalian neuron (Reprinted with permission from Koeppen,
B. M. and Stanton, B. A. (2009). Berne & Levy physiology. Elsevier Health Sciences. Copyright 2009 Mosby, Elsevier). (B) Neuronal membrane con-
taining voltage-gated Kþ ion (blue) and Naþ ion (orange) channels, as well as Kþ–Naþ pumps (green). (C) Coarse of the membrane potential and the
channel conductance of Naþ and Kþ ion channels during an action potential. Red arrow indicates the threshold value and blue arrow the after hyper-
polarization (Reprinted with permission from Bezanilla, F. (2006). The action potential: From voltage-gated conductances to molecular structures. Bio-
logical Research 39 (3), 425–435. Copyright 2006 BioMed Central). (D) Intracellular (top) and extracellular (bottom) action potentials measured
simultaneously using a patch clamp (intra), and a Si-FET (extra) interfaced to the axon of a leech neuron (Reprinted with permission from Fromherz,
P. (2002). Electrical interfacing of nerve cells and semiconductor chips. ChemPhysChem 3 (3), 276–284 Copyright 2002 John Wiley & Sons).
the Naþ–Kþ pumps that exchange three intracellular Naþ for two extracellular Kþ, the cell gets back to its resting VM. Once a single
AP spike is initiated on a small section of the membrane, it triggers an AP in the subsequent section, ensuring the signal propagation
along the membrane. At the same time a transient negative shift due to the hyperpolarization prevents the back-propagation of the
signal. The amplitude of an AP remains constant while traveling down the axon because the AP is an all-or-none impulse that is
regenerated at regular intervals along the axon (Nicholls et al., 2001). APs lead to multitude of functions from signal propagation
to information encoded in their firing frequency and pattern (Nicholls et al., 2001). A stimulus that is insufficient to initiate an AP is
known as a subthreshold stimulus. It can either lead to depolarization of the cells called excitatory postsynaptic potentials (EPSPs)
or hyperpolarization of the cells called inhibitory postsynaptic potentials (IPSPs).
Bioelectrical Signals Recording

In the 18th century, Luigi Galvani discovered that the function of the nervous system is intrinsically linked to its electrical activity
(Galvani, 1791). By the late 1930s, researchers started to understand the conduction of electrical signals within neurons. Kenneth
Cole and Howard Curtis demonstrated that the AP is associated with a large increase in membrane conductance (Cole and Curtis,
1939). During the same time, Alan Hodgkin and Andrew Huxley demonstrated the first intracellular recording of an AP in a giant
squid axon model (Hodgkin and Huxley, 1939; Hodgkin and Katz, 1949; Hodgkin and Huxley, 1952a; Hodgkin and Huxley, 1945;
Hodgkin and Huxley, 1952a; Hodgkin and Huxley, 1952b; Hodgkin and Huxley, 1952c; Hodgkin et al., 1952). Their mathematical
model explained the initiation and propagation of AP and the flow of currents across the cell membrane (Hodgkin and Huxley,
1952d). Hodgkin and Huxley’s work allowed researchers a step-by-step view of the processes involved in an AP. The effect of their
work was tremendous, leading to an increase in the interest in electrophysiology.
Since then, generations of investigators have invested immense effort into building and developing tools capable of recording
and controlling the electrical activity of neurons to enable better understanding of the brain in health and disease, enable early diag-
nosis and treatment of neurodegenerative disorders, and help restore lost neuronal function associated with neurological diseases
and injuries (Marconi et al., 2012; Kanagasabapathi et al., 2012; Ruaro et al., 2005; Liu et al., 2007; Sun, 2002; DeLorenzo et al.,
2000; Yuan et al., 2016; Marg and Adams, 1967; Evarts, 1966; Wolpaw et al., 2002; Wolpaw and McFarland, 2004; Lebedev and
Nicolelis, 2006).
The recording platforms that have been developed until now can be categorized into three groups. First, neuroimaging
approaches, such as functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) (Kornblum et al.,
2000; Poldrack and Farah, 2015). Their advantages include noninvasive nature and successful long-term monitoring of brain
activity. However, the detected signal represents a low bandwidth superimposition of the activity of large neuron populations
leading to low spatiotemporal resolutions which limit the investigations and study of circuit dynamics/neural circuitry at the
cellular level (Poldrack and Farah, 2015). Second, optical imaging techniques, such as voltage and Ca2 þ sensitive dyes (Hamel
et al., 2015) and optogenetics (Hochbaum et al., 2014). These techniques use reporter chemicals or proteins that convert changes
in VM to an optical signal. Their advantages include outstanding spatial resolution, allowing signals in even the smallest neuronal
structures to be resolved, and the possibility for simultaneous measurement from a wide range of spatial locations that have enabled
mapping at a tissue as well as cellular level. However, these optical imaging techniques are limited by the penetration depth of light
in the tissue, the toxicity of the fluorescent dyes, genetic incorporation of the proteins, and slow time-resolution in macroscopic
three-dimensional (3D) imaging (Hamel et al., 2015). Third category involves devices that can interface directly with the cells or
tissues and enable electrical recording and stimulation. The primary strength of this technique is its combination of high temporal
resolution and sensitivity with higher signal-to-noise ratio (SNR) (Scanziani and Häusser, 2009). It has been the preferred means of
analyzing brain activity due to the ability to capture a wide range of neural phenomena, from the spiking activity of individual
neurons to the slower network oscillations of small populations (Llinás, 1988; Contreras, 2004; Assad et al., 2014).
In this article, we will discuss the advancements made in the development of the nanomaterials-based platforms that lie in the
third category. Over the years, several groups came up with different kinds of materials, platforms, and designs to improve the capa-
bilities of these recording platforms.
In principle, a device could be considered as an ideal recording system if it has the following capabilities (Scanziani and
Häusser, 2009; Spira and Hai, 2013; Fattahi et al., 2014): (i) it is biocompatible and mimics properties of biological tissue
to minimize any mechanical mismatch, immune response, scar tissue formation, and tissue disintegration, (ii) it supports inte-
gration with neurons to avoid shear motion at the cell-electrode interface, and remain functional for long period of time, (iii) it
has low impedance to enable high SNR, (iv) it enables readout that covers the entire spectrum of membrane potential events,
that is, APs, subthreshold potentials (EPSPs and IPSPs) and subthreshold membrane oscillations, (v) it provides high spatial and
temporal resolution to allow recordings at single cell level with submillisecond resolution, and (vi) it simultaneously records/
stimulates multiple neurons to enable the study of network dynamics and signal propagation in a neuronal network.
In addition to recording signals, seamless integration of these biomaterials with engineered or native 3D tissues is essential. This
would not only enable monitoring but also simultaneously controlling the functional or electrical activity of the cells. This two-way
interaction would provide a platform for both understanding and promoting treatment of disorders associated with neural circuitry
and injuries in the cells. The development of such a platform would be an important advancement for the fusion of nanoelectronics
with the field of regenerative engineering.
Currently two common recording paradigms exist: passive devices, that is, microelectrode arrays (MEAs) and active devices, that
is, field-effect transistors (FETs). Depending on the design of the device, the electrical activity can either be recorded extracellularly or
intracellularly.
Extracellular Recordings
The change in potential across the neuronal cell membrane gives rise to transmembrane current in the extracellular medium. The
current contributions of neurons superimpose in the extracellular medium and generate a potential with respect to a reference
potential. This potential gradient across the brain tissue results in an electrical field, which can be monitored by extracellularly
placed electrodes. The characteristics of the detected electrical signal, such as amplitude and frequency, depend on the propor-
tional contribution of the multiple sources, the properties of the brain area, and the distance of the recording electrode from the
source. Depending on the electrode position, the recorded potentials can be categorized into four groups: First, electroencephalo-
gram (EEG) signals, which are recorded from the scalp, are slow rhythms (5–300 mV, < 100 Hz); second, electrocorticogram
(ECoG) signals, which are recorded from the cortical surface, are medium rhythms (0.01–5 mV, < 200 Hz); third, local field
potentials (LFP), which are generated through the superimposition of all ionic processes, including action potentials, synaptic
activity, and Ca2 þ spikes, are fast rhythms (few hundred mV–1 mV, < 200 Hz); and fourth, AP, which is measured at a single
cell level, can be up to ca. 500 mV at 0.1–7 kHz when measured extracellularly (Fig. 2A,B) (Fattahi et al., 2014; Buzsáki et al.,
2012).
Extracellular recording techniques are less invasive and therefore, are compatible for long-term measurements, enabling simul-
taneous recording and stimulation of large populations of excitable cells for days and months without inflicting mechanical damage
to the neuronal cell membrane. Reducing the dimension of electrical transducers to the micron and nanometer scale significantly
improves the spatial resolution and enables high scalability and multifunctional platforms.
MEAs
The flow of ions across the neuronal cell membrane gives rise to an extracellular current that spreads across the gap between the cell
and the surface of the electrode called cleft with resistance Rcleft. This results in an extracellular potential difference, Vextra with respect
to the bath solution. Vextra can be detected by MEAs since the extracellular potential change leads to a charge transfer at the electrode
surface, resulting in a small capacitive/faradaic current through the microelectrode owing to the electrochemical impedance (Spira
and Hai, 2013).
(A) (B)
EEG 5–300 µV NI signals and sensors

< 100 Hz Signal
Sensor
ECoG location
Signal name Signal type (Band) information
state/signs
invasive
ERP P300
Field potentials
Non
0.01–5 mV
Scalp EEG Slow
< 200 Hz
rhythms BP
Medium µ (Alpha)
Epicortical ECoG
rhythms
Beta
Invasive
< 1 mV
< 200 Hz
Fast
LFP Gamma
LFP Intra rhythms
~500 µV
spikes 0.1–7 kHz
paren Single neuron
Action potentials
chymal MUA
(spikes) populations Signals
Fig. 2 Detection of neuronal field potentials and single spike events. (A) Schematic of sensor position with corresponding neural signals. (B)
Different categories of neural signals and their properties (Reprinted with permission from Fattahi, P., et al. (2014). A review of organic and inorganic
biomaterials for neural interfaces. Advanced Materials 26 (12), 1846–1885. Copyright 2014 John Wiley & Sons).
MEA was first introduced in 1972 as a new platform for studying cultured cardiac myocytes (Thomas et al., 1972). Within few
years, more platforms were developed to record from cultured neurons (Pine, 1980; Novak and Wheeler, 1986). Since then it has
become a popular experimental platform for electrophysiological studies of neural networks. MEAs allow for the simultaneous
recording of signals from multiple neurons at multiple locations, thereby improving the understanding of signal propagation in
a neural network. Each electrode in an MEA can be individually addressed for signal amplification and processing. Also, the elec-
trodes can be used to apply a voltage transient to depolarize the cell membrane and thus, stimulate electrical activity of neurons.
These capabilities make MEAs a powerful tool to investigate the spatiotemporal neuronal network dynamics as well as allow active
influence and control of neuronal activity both in vitro and in vivo (Wise et al., 2008).
Planar passive electrodes

Typically, planar MEAs are based on recording sites made of metallic conductors, such as Au (Fig. 3A) (Oka et al., 1999) and Pt
(Thiebaud et al., 1997). Sputtering of materials on metal electrodes, such as IrOx (Fig. 3B) (Gawad et al., 2009) and TiN (Janders
et al., 1996), has also been demonstrated to enhance the capacitive properties of the electrodes. These platforms enable study of
neurons at a network scale by enabling multisite recording and stimulation of dissociated cultured neurons or brain slices. However,
being on a flat, stiff two-dimensional (2D) surface these are restricted to in vitro experiments involving cultured cells.
To enable multisite recording and stimulation of neurons in in vivo, numerous attempts have been made to obtain recordings
from the brain tissue using arrays of microwires (Schwartz, 2004; Nicolelis et al., 2003; Yuen and Agnew, 1995; Kralik et al., 2001).
These electrodes consist of fine wires 20–50 mm in diameter that are mainly composed of metallic conductors, such as Pt, Au, Ir,
stainless steel, and W. Spacing between wires is usually around 100–300 mm and is maintained either with polyethylene glycol
or methyl methacrylate. These probes can enable recording from up to 2 mm below the brain tissue surface. However, post-
insertion, a key issue associated with these probes in the change in the relative position of the electrode tip from the recording
site, thus moving it away from the required site in the brain (Schwartz, 2004). To overcome the technical challenges associated
with microwire technology, penetrating electrodes, such as Utah MEAs and Michigan arrays, have been developed (Kipke et al.,
2008; Wise and Najafi, 1991; Rousche and Normann, 1998; Maynard et al., 1999; Hatsopoulos et al., 1998; Hochberg et al.,
2006). Utah electrode arrays (Fig. 3C) (Hochberg et al., 2006) are composed of 100 Si needle electrodes with exposed tips of diam-
eter 10–30 mm, and Michigan electrodes (Fig. 3D) (Kipke et al., 2008) comprise of multiple recording sites with the surface area of
100–400 mm2, arranged along a flattened shank of dimensions 15 mm thick, 3 mm long, and 90 mm wide at the base. While these
electrodes allow localized recordings from specific brain areas, such as motor or visual cortex at high spatial resolution, their appli-
cation in neuroscience is limited due to the highly invasive nature of the recordings. These probes are fabricated from rigid materials
such as Si with different mechanical properties from the brain tissue, leading to mechanical mismatch (Schwartz, 2004; Polikov
et al., 2005; Lagoa et al., 2006; Leach et al., 2010; Biran et al., 2005). Furthermore, the poor biocompatibility of these electrodes’
materials leads to chronic immune responses (Maynard et al., 1999; Biran et al., 2005; Szarowski et al., 2003; Turner et al., 1999;
Edell et al., 1992; Biran et al., 2007). The mechanical mismatch and poor biocompatibility result in shear motion, glial scar forma-
tion, and neuron depletion at electrode-tissue interfaces leading to degradation of recording and stimulation capabilities over time
(Kralik et al., 2001; Rousche and Normann, 1998; Maynard et al., 1999; Hatsopoulos et al., 1998; Polikov et al., 2005; Leach et al.,
2010; Edell et al., 1992; Ludwig et al., 2006; Kim et al., 2009).
Flexible passive electrodes

In implantable MEA technology, one of the main focuses lies in the development of stable electrode/tissue interfaces with reduced
chronic inflammatory response. Some strategies for bio-integrated electronics have been incorporated to overcome challenges
Fig. 3 Extracellular recordings using passive planar electrodes. (A) Au planar microelectrode array (MEA) with 64 microelectrodes of
50 mm 50 mm area each arranged in an 8 8 array with an interpolar distance of 150 mm. Scale bar: 150 mm (Reprinted with permission from
Oka, H., et al. (1999). A new planar multielectrode array for extracellular recording: application to hippocampal acute slice. Journal of Neuroscience
Methods 93 (1), 61–67. Copyright 1999 Elsevier). (B) IrOx planar MEA. (I) Top view of the full MEA assembled on a PCB. (II) Close-up of the
substrate electrode array showing 16 electrode sites and the insulated leads. Each microelectrode is 30 mm in diameter, with a 100 mm spacing.
Scale bars: (I) 6 mm, (II) 100 mm (Reprinted with permission from Gawad, S., et al. (2009). Substrate arrays of iridium oxide microelectrodes for
in vitro neuronal interfacing. Frontiers in Neuroengineering 2, 1. Copyright 2009 Frontiers Media). (C) Utah array with 100 Si-based electrode sensors
for neural recording. Individual electrodes are 1 mm long and spaced 400 mm apart, in a 10 10 grid. Scale bar: 1 mm (Reprinted with permission
from Hochberg, L. R. et al. (2006). Neuronal ensemble control of prosthetic devices by a human with tetraplegia. Nature 442 (7099), 164–171.
Copyright 2006 Macmillan Publishers). (D) Michigan probes. Photograph of (I) a four-shank Si neural probe having four electrode sites arranged
near the tip, each terminated in a bond pad at the tab, (II) four different types of sites layouts for specialized interfaces, and (III) modular 128-site,
three-dimensional array made from several multishank planar arrays (Reprinted with permission from Kipke, D. R. et al. (2008). Advanced
neurotechnologies for chronic neural interfaces: New horizons and clinical opportunities. The Journal of Neuroscience 28 (46), 11830–11838.
Copyright 2008 Society for Neuroscience).
associated with the mechanical mismatch between the hard, planar surfaces of the recording platform and the soft, curvilinear
nature of biological systems.
Silk-based MEAs
One such strategy to overcome the challenges associated with rigid substrates relies on the fabrication of ultrathin Au electrode array
supported by silk fibroin substrate which is highly flexible, biocompatible, biodegradable, transparent, and mechanically robust
(Fig. 4A-I) (Kim et al., 2010a). Mounting such devices on tissue and then allowing the silk to dissolve and resorb initiates a spon-
taneous, conformal wrapping process driven by capillary forces at the biotic/abiotic interface (Fig. 4A-II). No evidence of immune
response or inflammation was observed even after 4 weeks enabling long-term stable integration with the brain tissue. The high
degree of conformal contact with the neural tissue enables recording of field potentials with good signal amplitude and high
SNR (Fig. 4A-III). However, the spatial resolution is compromised due to the large electrode size of 500 mm 500 mm. And, scaling
down the dimensions of the microelectrode while maintaining a high enough SNR has been a major challenge.
Graphene-based MEAs
To better decode the functions of individual circuit elements, it is important to achieve recording from a network level to a single cell
level at high temporal resolution. Performing electrophysiology and optical imaging simultaneously could leverage the temporal
and spatial resolution advantages of both techniques. However, metal-based MEAs that are commonly used for recording neural
activity are opaque in nature because of which they generate optical shadows and are prone to producing light-induced artifacts
in the recordings (Kuzum et al., 2014). Using a transparent material as electrodes can enable simultaneous optical and electrophys-
iology studies. Indium tin oxide (ITO), with transmittance of 80%, can be used for transparent neural electrode array. However,
ITO does not have a flat transmittance across the visible spectrum, and the brittleness of ITO may limit the conformability of the
device to the brain surface (Kuzum et al., 2014).
Graphene (Novoselov et al., 2004; Geim, 2009), a one-atom-thick 2D honeycombed arrangement of sp2 hybridized carbon
lattice, has been explored in as a building block in bioelectronics owing to its transparency, electrical and thermal conductivity, flex-
ibility, and biocompatibility (Kuzum et al., 2014; Geim, 2009; Park et al., 2016; Park et al., 2014; Rastogi et al., 2017).
Fig. 4 Extracellular recordings using passive flexible electrodes. (A) MEA on biodegradable silk. Image of electrode arrays (I) wrapped onto a glass
hemisphere and (II) on a feline brain. Scale bars: 2 mm (Reprinted with permission from Kim, D.-H., et al. (2010). Dissolvable films of silk fibroin for
ultrathin conformal bio-integrated electronics. Nature Materials 9 (6), 511–517. Copyright 2010 Macmillan Publishers). (B) Graphene-based MEA. (I)
Schematic illustration of a flexible graphene neural electrode array. (II) Photograph of a 50 50 mm2 graphene electrode and 500 500 mm2 Au
electrode placed on the cortical surface of the left and right hemispheres, respectively. The inset shows the flexibility of the electrodes. (III) Interictal-
like spiking activity recorded by doped-graphene (black) and Au electrodes (red) (Reprinted with permission from Kuzum, D., et al. (2014). Trans-
parent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging. Nature Communications, 5. Copyright 2014
Macmillan Publishers). (C) Stretchable MEA (SMEA). (I) Top view of the prototype SMEA array with 11 recording electrodes and 1 reference elec-
trode. (II) Hippocampal slice placed on the SMEA; the parallel horizontal lines are part of a mesh that holds down the tissue. (III) Spontaneous and
(IV) evoked field potentials recorded from the hippocampal slice. Scale bars: (I) 8 mm, (II) 0.5 mm (Reprinted with permission from Graudejus, O.,
et al. (2009). Characterization of an elastically stretchable microelectrode array and its application to neural field potential recordings. Journal of the
Electrochemical Society 156 (6), P85–P94. Copyright 2009 The Electrochemical Society).
Graphene-based flexible and transparent electrodes enable simultaneous optical and electrophysiology studies as demonstrated in
the reference (Kuzum et al., 2014). To fabricate such electrodes, the chemical vapor deposition-synthesized graphene was trans-
ferred on to flexible polyimide (PI) films and patterned to obtain 50 mm 50 mm active electrode area (Fig. 4B-I). The graphene
electrode was then compared with Au electrode by recording from the cortical surface of the left and right hemispheres simulta-
neously (Fig. 4B-II). When compared to Au electrodes, graphene electrodes showed five- to sixfold improvement in SNR and
100-fold reduction in electrical interference noise (Fig. 4B-III). The in vivo neural recording experiments performed using graphene
electrodes in conjunction with optical recordings demonstrated that graphene electrodes can detect interictal and ictal activity and
fast population spikes with durations < 5 ms, and the Ca2 þ transients within the electrode area captured by the confocal microscope
showed an increase in Ca2 þ signal coinciding with the interictal-like event recorded by the graphene electrode. In summary, the
temporal resolution of the graphene electrode recordings enabled detection of high-frequency population discharges, which could
not be resolved by the Ca2 þ fluorescence responses. In contrast, Ca2 þ imaging responses were able to capture complex network
contributions of individual neurons, which were not evident in the electrical recordings using graphene electrodes. Therefore,
combination of both techniques revealed temporal and spatial characteristics of high-frequency bursting activity and synaptic
potentials in hippocampal slices with high precision. In addition to low noise and transparency, graphene-based electrodes were
shown to be highly robust, stable for long term, and resistive to corrosion, thus enabling long-term stable recordings from brain
tissues at high temporal resolution. However, the major drawback that still remains is the inability to perform long-term recordings
at high spatial resolution from the deep regions in the brain due to the limitations of optical dyes.
Stretchable MEAs
Fabricating MEAs on PI-based substrate (Kuzum et al., 2014) reduce the problems associated with mechanical mismatch of the MEA
materials with the underlying soft neural tissue as the elastic modulus of PI is ca. 2.8 GPa which is much lower than the Si-based
rigid electrodes with elastic modulus of > 100 GPa. However, it is still several orders of magnitude higher than the neural tissue
which has an elastic modulus of < 1 kPa. To further improve on the mechanical mismatch between the electrodes and the under-
lying tissue, stretchable MEAs (SMEAs) have been fabricated on soft, elastically stretchable, silicone membranes with an elastic
modulus of 1 MPa (Fig. 4C-I) (Graudejus et al., 2009). The purpose behind fabrication of stretchable array was to enable the
study of traumatic brain injury (TBI) which requires stable integration of MEAs with the tissue and allow direct comparison of elec-
trical activity from the same location on the tissue before and after traumatic injury (or a similar mechanical stimulation). To
develop SMEA, < 100 mm wide Au electrodes were fabricated on PDMS that could be stretched repeatedly and reproducibly by
> 30% while remaining electrically conducting. The recording sites of SMEA were electroplated with Pt to reduce the impedance
of the electrode/electrolyte interface. This leads to reduction in the noise during recording and the stimulus artifacts during stim-
ulation. The SMEAs were then used to record extracellular spontaneous and evoked electrophysiological activity of neurons in
an organotypic hippocampal slice culture while the electrodes were under biaxial strain (Fig. 4C-II). The SMEAs enable recordings
of both spontaneous and evoked responses (Fig. 4C-III, IV). Also, the electrical attributes of the electrodes did not change even by
severe stretching (up to 30% strain) of the substrate, thus enabling stable and functional integration without any motion at the
electrode-tissue interface.
Even though MEAs have been modified to achieve stable integration with neural tissues at high temporal resolutions, nonethe-
less, achieving high spatial resolution still remains a challenge. Ideally, for high spatial resolution, the site of a microelectrode
should have a small geometric area to enable communication with individual neurons. And for high sensitivity, the device should
have a low impedance and high injection charge density during recording and stimulation (Kovacs et al., 1994; Paik and Park, 2003;
Cogan, 2008). However, in the case of planar MEAs, there is a trade-off between spatial resolution and sensitivity since decreasing
the geometric area of a recording site causes an increase in the impedance and a decrease in the capacity of the injection charge
density of neural microelectrodes (Scanziani and Häusser, 2009; Kovacs et al., 1994; Drake et al., 1988; Robinson, 1968; Frey
et al., 2009). Reducing the impedance by surface modification of the electrodes has led to an improvement in the performance
of the MEAs. Pt black has been used to coat the metal electrodes since its porous structure is effective for impedance reduction
(Novak and Wheeler, 1986; Chang et al., 2000; Mathieson et al., 2004; Maher et al., 1999). Various Au nanostructures have also
been reported that involve deposition of nanoflake (Kim et al., 2010b), nanograin (Kim et al., 2013), and fuzzy Au (Cui and Martin,
2003) structures. The rough surface formed due to the nanostructures lead to increase in the net surface area, thus reducing the
impedance of the microelectrodes. To increase the effective surface area, the microelectrodes have also been modified with carbon
nanotubes (CNTs) by direct synthesis on the electrode surface (Gabay et al., 2007), electroplating (Suzuki et al., 2013; Keefer et al.,
2008), and microcontact printing (Fuchsberger et al., 2011) methods. Application of conductive polymers to MEA such as poly(3,4-
ethylenedioxythiophene) (PEDOT) and polypyrrole has also been demonstrated to reduce the impedance of the electrodes (Cui
et al., 2001; Ludwig et al., 2011; Venkatraman et al., 2011). In addition, the use of PEDOT/CNT composites (Zhou et al., 2013)
and polypyrrole/graphene oxide composites (Deng et al., 2011) has shown to increase stability and charge injection capacity of
the microelectrodes (> 200 mCcm 2) by two orders of magnitude when compared with planar Pt electrodes (1.4 mCcm 2).
Even though the surface modifications improve the sensitivity of the MEAs by reducing the surface impedance and increasing the
SNR, the electrodes geometry of tens of microns limits the capability for recording signals from subcellular structures, such as single
axon and dendrites (Scanziani and Häusser, 2009). In addition, due to the large-size geometry of MEAs, the recorded signals are
often comprised of average signals from multiple neurons and thus, complicating the interpretation of recorded signals (Spira
and Hai, 2013).
FETs
FET is a semiconductor device that exhibits a conductance change in response to variations in the charge or potential at the surface of
the channel region. A standard planar FET consists of three terminals: (i) source, through which the carriers enter the channel; (ii)
drain, through which the carriers leave the channel; and (iii) gate, the terminal that modulates the channel conductivity. The gate
electrode is capacitively coupled to the semiconductor channel by an insulating oxide layer. When there is no gate voltage, the FET is
in its off state as there is no flow of current. When gate voltage exceeds a threshold voltage, charge carriers (e.g., holes for p-Si and
electrons for n-Si) are induced at the semiconductor–oxide interface, and the potential barrier of the channel drops, resulting in the
current flow. Therefore, the conductance of the semiconductor channel between the source and drain regions can be switched from
off to on and modulated in the on-state by the potential at the gate electrode.
When a FET is integrated with a neuron, the potential change across the neuronal membrane acts as a gate and modulates the
conductivity of the transistor channel by changing the charge carrier concentration. This conductance change can be easily detected
by recording the net current through the FET channel at a constant bias voltage. The detected current change depends on the
transconductance of the device, and thus, is independent of gate impedance unlike MEAs. Development of a FET device for neuro-
physiological measurements was first demonstrated in 1970s (Bergveld, 1970; Bergveld et al., 1976). Since then, many studies have
demonstrated the use of planar FETs to enable recordings from electrogenic cells (Weis et al., 1996; Patolsky et al., 2006; Lambacher
et al., 2004; Fromherz, 2002; Fromherz and Offenhausser, 1991; Voelker and Fromherz, 2005; Merz and Fromherz, 2005; Besl and
Fromherz, 2002; Vassanelli and Fromherz, 1999; Vassanelli and Fromherz, 1998; Offenhäusser and Knoll, 2001; Offenhäusser et al.,
1997; Ingebrandt et al., 2005). Some of the recent advances in the development of FETs for cellular recordings are discussed later.
2D FETs
Since FETs are independent of gate impedance, it makes it possible to go down to nanoscale, thus making them one of the most
promising nanoscale bioelectronics devices for recording with subcellular resolution (Patolsky et al., 2006; Duan et al., 2012;
Cohen-Karni et al., 2010; Tian et al., 2012; Zhang and Lieber, 2016; Duan et al., 2013; Gao et al., 2012; Lieber, 2011; Tian and
Lieber, 2013; Dai et al., 2016; Tian et al., 2010).
Graphene and nanowire (NW) FET

To achieve single cell resolution with high SNR, graphene and SiNW FETs have been interfaced with cultured cardiomyocytes
(Fig. 5A-I,II) (Cohen-Karni et al., 2010). The advantage of using NWs in FETs lies in the 1D nanoscale morphology of NWs that
enhances the net surface area of the device active region. High surface area leads to enhanced interactions of NW FETs with the
cell membrane and thus, high sensitivity when compared with their planar counterparts. On the other hand, graphene FETs
have an advantage due to their ambipolar behavior that enables both n- and p-type recording with the same device. This charac-
teristic was demonstrated by signal shape flip of recorded extracellular potentials across the Dirac point. In addition, when
compared with other planar structures, one atom thick graphene FETs demonstrate better performance by yielding well-defined
extracellular signals with SNR > 4, exceeding typical values for other planar devices (Cohen-Karni et al., 2010). However, the
measured signal is dependent on the size of the graphene flake. A large graphene FET with active channel of 20.8 mm 9.8 mm
recorded signals with peak-to-peak width of 1.31 0.04 ms (Fig. 5A-III). Whereas signals recorded from a much smaller graphene
FET with active channel dimensions of 2.4 mm 3.4 mm yielded peak-to-peak width of 0.73 0.04 ms, which is almost a factor of
2 smaller than that obtained from the larger device (Fig. 5A-IV). Signals recorded from a 0.07 mm2 active area SiNW-FET had a peak-
to-peak width of 0.76 0.04 ms, which is similar to the value for the smaller graphene device (Fig. 5A-IV). These results indicate
that the signals recorded with the larger graphene device represent an average of the extracellular potential from sufficiently distinct
sources of the beating cell. Although NW FET device yielded similar peak-to-peak widths as the 100 bigger area graphene FET,
NW devices still have an advantage for spatially resolved multiplexed measurements in high-density device arrays.
3D FETs
When compared with cells cultured in 2D, 3D cellular networks mimic more to an in vivo like environment: spherical cell
morphology, high cell-to-cell interaction, and neurite outgrowth in all directions (Irons et al., 2008). Coupling recording platforms
with 3D networks would represent a powerful in vitro model capable of better emulating in vivo physiology.
Coupling of electronics with the 3D tissues using flexible (Kim et al., 2010a; Kuzum et al., 2014) and stretchable planar devices
(Graudejus et al., 2009) that conform to natural tissue surfaces was recently reported. However, these planar devices have been used
to probe electrical activities near surfaces of the tissue and lack seamless 3D integration of the electronics with the tissue.
NW nanoelectronic scaffolds (NanoES)

To enable recordings from cells in 3D, nanoelectronics and synthetic tissues have been integrated with macroporous nanoelectronic
scaffolds (nanoES) (Tian et al., 2012). The aims behind building such recording platform involved: developing macroporous elec-
tronic structures to enable 3D interpenetration with biomaterials; nanometer to micrometer scale features of electronic network
comparable to biomaterial scaffolds; and 3D interconnected electronic network with mechanical properties similar to biomaterials.
The electronic scaffold was based on SiNW FETs to achieve subcellular resolutions. The fabrication of the nanoES involved the
following steps: first, chemically synthesized SiNWs were deposited, and then NW FET devices were lithographically patterned to
create two types of nanoES designsdReticular and Mesh. Reticular nanoES were made to mimic the size scale and morphology
of submicron ECM features, such as the fibrous meshwork of brain ECM. Open mesh nanoES were made with a regular structure,
similar to the ECM of the ventricular myocardium. These scaffolds were fabricated on sacrificial layers, which were subsequently
removed, yielding freestanding macroporous scaffolds (Fig. 5B-I). The nanoES were designed to be 3D and mimic ECM structures,
having nanometer to micrometer features with high (> 99%) porosity, and to be highly flexible and biocompatible. NanoES were
then combined with synthetic or natural macroporous ECMs providing ECMs with electrical sensory function and nanoES with
biochemical environments suitable for tissue culture. Finally, cells were cultured within the nanoES to yield 3D hybrid nanoelec-
tronics tissue constructs (Fig. 5B-II). The emphasis on the nanoscale and biomimetic bottom-up pathway allows minimally invasive
integration of electronic devices with cells and ECM components at the subcellular level in 3D. Simultaneous recordings from four
NW FETs with separations up to 6.8 mm in a nanoES-cardiac construct demonstrated multiplexed sensing of a coherently beating
cardiac patch (Fig. 5B-III). The data revealed submillisecond time resolution with regularly spaced spikes with a frequency of
1 Hz, calibrated potential change of 2–3 mV and 2 ms width, and SNR of 3. This platform enabled good integration
with engineered tissue allowing access to the cellular response with single-shot submillisecond time resolution. However, the
size and the design of the device precluded 3D tissue mapping.
Fig. 5 Extracellular recordings using field-effect transistors (FETs). (A) Si nanowire (NW) and graphene (Gra)-FETs. (I) Schematic illustrating the
cardiomyocyte cell interfaced with Gra-FET and SiNW-FET devices. (II) Optical microscope image of PDMS/cells interfaced with devices. White-
dashed line, red and blue arrows represent graphene flake, Gra-FET and SiNW-FET devices, respectively. (III) Electrical signal recorded from the Gra-
FET (active channel of 20.8 mm 9.8 mm). (IV) Electrical signals recorded from the Gra-FET (red, active channel of 2.4 mm 3.4 mm) and SiNW-FET
(blue, active channel area of 0.07 mm2). Scale bar: (II) 13.6 mm (Reprinted with permission from Cohen-Karni, T., et al. (2010). Graphene and nano-
wire transistors for cellular interfaces and electrical recording. Nano letters 10 (3), 1098–1102. Copyright 2010 American Chemical Society). (B) NW
nanoelectronic scaffolds (NanoES). (I) Device fabrication schematics of (i) reticular NW FET devices and (ii) mesh NW FET devices. Light blue, dark
blue, green, and yellow represent SiO2 substrates, nickel sacrificial layers, nanoES, and individual NW FETs, respectively. (II) 3D reconstructed
confocal images (x: 127 mm; y: 127 mm; z: 68 mm) of rat hippocampal neurons after a 2-week culture in Matrigel on reticular nanoES. Red (Alexa
Fluor 546) and yellow (rhodamine 6G) represent neuronal b-tubulin and epoxy ribbons, respectively. The white arrow highlights a neurite passing
through a ring-like structure supporting a NW FET. (III) Multiplex electrical recording from four NW FETs in a mesh nanoES (Reprinted with permis-
sion from Tian, B., et al. (2012). Macroporous nanowire nanoelectronic scaffolds for synthetic tissues. Nature Materials 11 (11), 986–994. Copyright
2012 Macmillan Publishers). (C) 3D-folded scaffold integrated NW FET. Schematics of (I) freestanding macroporous nanoelectronic scaffold with NW
FET arrays (red dots); Inset: one NW FET, (II) folded 3D freestanding scaffolds with four layers of individually addressable FET sensors, and (III)
nanoelectronic scaffold/cardiac tissue resulting from the culturing of cardiac cells within the 3D folded scaffold; Inset: the nanoelectronic sensors
(blue circles) innervate the 3D cell network. (IV) Simultaneous traces recorded from 16 sensors in the top layer (L1) of the nanoelectronics–cardiac
tissue. The (x,y) coordinates correspond to each FET sensor in the 4 4 array (Reprinted with permission from Dai, X., et al. (2016). Three-
dimensional mapping and regulation of action potential propagation in nanoelectronics-innervated tissues. Nature Nanotechnology. Copyright 2016
Macmillan Publishers).
3D folded scaffold integrated NW FET

Real-time 3D spatiotemporal mapping of APs in engineered cardiac tissues with submillisecond temporal resolution was achieved
using nanoelectronics that mimic tissue scaffolds (Dai et al., 2016). In this work, 3D macroporous nanoelectronic networks were
fabricated using high-density SiNW FETs, with miniaturized features designed to achieve ca. 2 mm dimensions and ca. 0.29–
2.8 10 16 N m 2 bending stiffness values that are comparable to conventional electro spun fiber tissue scaffolds (Fig. 5C-I).
The freestanding macroporous nanoelectronic networks were then folded into 3D scaffolds (Fig. 5C-II) and neonatal rat cardiac
cells were cultured within the scaffolds to yield nanoelectronics cardiac tissues (Fig. 5C-III). Extracellular cardiac AP signals
recorded from 4 4 FET sensors in a single layer across a 5 5 mm2 domain show a synchronized beating rate of 1.8 Hz, an
amplitude of 1–2 mV, and a peak width of 1 ms from all 16 channels. Higher-resolution examination of these peaks reveals
a submillisecond time latency between any given set of AP peaks recorded by the 16 FET sensors, where the intrinsic temporal
resolution of the device is 0.01–0.05 ms (Fig. 5C-IV). This platform enables 3D electrophysiology mapping at high temporal
resolution.
Integrating the FET sensors in 3D scaffolds did allow recording from cells that mimic morphology as in vivo models, however
these platforms are incapable of achieving measurements from intact brain tissue which involves much more complicated neuronal
circuitry when compared with the 3D tissue engineered for the earlier-mentioned devices.
Injectable Electronics
To address the challenge of integrating electronics with the 3D tissue in vivo, the syringe injection of macroporous mesh electronics
in the brain tissue was recently demonstrated (Liu et al., 2015). The seamless and minimally invasive delivery of macroporous NW
nanoelectronic probes into tissue enables long-term and intimate electronics-tissue interface due to their flexibility, nonplanar struc-
ture, and tissue-scaffold mimicking properties. The syringe injection concept involves (i) loading the macroporous mesh electronics
into a syringe needle, (ii) inserting the needle into the biological tissue such as the brain, and (iii) injecting the mesh nanoelec-
tronics and delivering the input/output region of the mesh outside the material for subsequent bonding and measurements
(Fig. 6A). To enable injection of the electronic mesh into the tissue, the structure of mesh network was designed with transverse
bending stiffness values sufficiently small and with flexibility closer to that of tissue, to allow the mesh electronics to roll up
and smoothly go through the needle during injection. To demonstrate the capabilities of the mesh electronics, it was injected stereo-
taxically into the lateral ventricle and hippocampus of live rodents for in vivo brain activity recordings (Fig. 6B). The mesh was able
to record signals with modulation amplitude of 200–400 mV and dominant modulation frequency of 1–4 Hz which are character-
istic of d- wave LFPs (Fig. 6C). The recorded spike had an average duration of 2 ms and peak-to-peak amplitude of 70 mV which
is a characteristic of single-unit APs (Fig. 6D). These results suggested substantial promise of using injectable electronics to mobilize
and monitor neural networks in in vivo models.
Those examples illustrate how extracellular recording platforms have been modified and improved over the years in order to
enable long-term measurements as well as simultaneous recording and stimulating large populations of excitable cells for days
and months at high spatiotemporal resolution. However, the major limitation of these technologies is the extraction of less detailed
information from the excitable cells and their network since these electrodes record spikes with 2–3 orders of magnitude lower than
the true AP across the cell membrane. In addition, the extracellular recordings are blind to the subthreshold potentials such as
EPSPs, IPSPs, and membrane oscillations. These potentials play a significant role in the functioning of neuronal circuit, and a great
deal of neuroplasticity is associated with changes in the amplitude of synaptic potentials. Hence, it is crucial to enable recordings of
the entire dynamic range of transmembrane voltage changes to better understand the functioning and connectivity of neuronal
circuit (Spira and Hai, 2013).
Fig. 6 Injectable electronics. (A) Schematics showing the (I) insertion and (II) retraction of the needle and (III) placement of the mesh electronics in
the cavity. (B) Optical image of the stereotaxic injection of mesh electronics into the brain of an anesthetized mouse. Scale bar: 5 mm. (C) (I) A 16-
channel recording with the mesh electronics following injection into the brain of a live mouse. (II) Superimposed single-unit neural recordings from
one channel (Reprinted with permission from Liu, J., et al. (2015). Syringe-injectable electronics. Nature Nanotechnology 10 (7), 629–636. Copyright
2015 Macmillan Publishers).
Intracellular Recordings
Unlike extracellular recording, intracellular recording can enable accurate readout of the entire dynamic range of transmembrane
voltage changes with little signal distortion and therefore, can monitor subthreshold events and very slow changes of transmem-
brane potentials due to the synaptic interactions (Spira and Hai, 2013; Purves, 1981). As a result, intracellular recording can
provide faithful amplitude and shape information of APs in individual cells, as well as help in the investigations of neuronal
circuits.
The glass micropipette patch-clamp technique developed in the late 1970s has been the most widely used approach to record
intracellularly (Molleman, 2003). This technique monitors the voltage across the cell membrane by either creating a tight seal
between the cell membrane and micropipette, referred to as cell-attached patch clamp, or accessing the cell interior by puncturing
the cell membrane using a glass micropipette, referred to as whole-cell patch clamp (Molleman, 2003). However, there are some
limitations associated with these patch clamp techniques (Spira and Hai, 2013; Edwards et al., 1989). The patch-clamp measure-
ment involves a bulky setup, including 3D manipulators for precise and delicate interfacing between a micropipette and individual
cells. This impedes the development of multiplexed intracellular recording (Spira and Hai, 2013). In addition, the whole-cell patch
technique leads to mixing of cell cytosol and the exogenous solution filling the pipette, which results in an irreversible change to the
cells, making long-term measurements difficult (Edwards et al., 1989). To address these limitations, substantial effort has been
placed on the development of micro- and nanoscale 3D electrode arrays to enable long-term, multiplexed intracellular recording
and stimulation from cells in cellular networks.
MEA
When neurons are cultured on a recording device, a gap or cleft exists between the electrode and cell membrane where this cleft
determines the seal resistance. Enhancing the adhesion between electrode and cell surface leads to an increase in electrode-cell
seal resistance. A number of approaches such as surface modification, substrate modulation, and electrode shape control have
been focused on to increase the neuron-microelectrode electrical coupling coefficient and decrease the cleft size, thus increasing
the seal resistance as a means to enhance signals and achieve intracellular-like recordings (Spira and Hai, 2013; Hai et al., 2010;
Hai and Spira, 2012; Hai et al., 2009; Xie et al., 2012; Robinson et al., 2012).
Functionalized gold-spine electrode (FGSE)

An enhancement in tight coupling of the electrodes with the cell membrane was achieved by fabrication of functionalized
microscale 3D mushroom-shaped Au protrusions, also referred to as functionalized gold-spine electrode (FGSE) (Fig. 7A-I)
(Spira and Hai, 2013; Hai et al., 2010; Hai et al., 2009). These Au electrodes were fabricated with a stem height of approxi-
mately 1 mm, a stem diameter of approximately 800 nm, and a mushroom-shaped cap of 1.8–3 mm in diameter (Fig. 7A-II).
The shape and the dimension of the electrodes were selected to mimic the geometry and dimensions of dendritic spines.
The electrodes were chemically functionalized by multiple Arg-Gly-Asp (RGD) peptides and engulfment-promoting peptide
(EPP) to facilitate the engulfment by the cultured neurons and other types of cells, as observed from the transmission electron
microscopic analysis of the interfaces formed between cells and mushroom-like microelectrodes (Fig. 7A-III). The use of an
FGSE as the sensing electrode led to the activation of endocytotic-like mechanisms in the neurons that increased the
neuron-microelectrode electrical coupling coefficient to 50% as compared to approximately 0.1% as recorded by a planar extra-
cellular MEA. The generation of high Rseal ( 100 MU) between the neuronal membrane and the engulfed FGSE, and the
increased junctional membrane conductance led to intracellular-like recording enabling successful monitoring of APs in the
range of tens of mV (as compared to 100 mV recorded by planar Au electrodes) as well as subthreshold synaptic potentials
(Fig. 7A-IV).
Vertical Si nanowire (SiNW) electrode array (VNEA)

A scalable multiplexed intracellular nanoelectrode platform based on arrays of vertical nanopillars (VNEA) was recently demon-
strated (Robinson et al., 2012). In this work, 3 3 arrays of 9 nanopillars, 150 nm in diameter, 3 mm in height at 2 mm pitch
were grown on 16 sensing pads (Fig. 7B-I). The geometry of each NW array (a 4 mm square) was chosen to be smaller than the
size of a typical neuronal cell body so as to increase the probability of single-neuron coupling. The entire device is fabricated
from a Si-on-insulator (SOI) substrate so that each pad could be independently addressed electrically. Each NW in the array is
composed of a doped Si core, a Ti/Au tip, and an insulating SiO2 shell. The Si core and metal tip provide electrical access to the
interior of the cell, and the glass shell plays the dual role of preventing current leakage through the NW sidewalls and serving as
a material with which to make tight seals to the cell membrane. Because of its planar integrated geometry, the VNEA is well
suited to studying in vitro dissociated neuronal circuits and ex vivo preparations, such as brain slices. When embryonic rat
cortical neurons were cultured on the VNEAs, 50% of the VNEAs spontaneously penetrated through the plasma membrane
of the cells and created a high seal resistance of 100–500 MU (Fig. 7B-II). In cases where spontaneous penetration of the
membrane were not evident, an electroporating pulse (approximately 3 V, 100 ms) was applied to permeabilize the cell
membrane and promote NW penetration. Consistent with the intracellular positioning of the VNEA, all recorded APs were posi-
tive monophasic and intracellular-like with amplitude of ca. 5 mV (Fig. 7B-III). The nanoscale structure of the electrodes
enables recording and stimulation of individual neurons. Even though a single sensing pad carries multiple nanopillars,
Fig. 7 Intracellular-like recordings using passive electrodes. (A) Functionalized gold-spine electrode (FGSE). (I) Confocal microscopy image of three
Aplysia neurons cultured on an FGSE array. Green lines represent the conducting lines. (II) Scanning electron microscopic image of FGSE fabricated
on a glass surface. (III) Transmission electron microscopic image of electrode/cell interface. (IV) Recording of Aplysia LUQ neuron action potentials
using FGSE (blue) and patch pipette (red). Scale bars: (I) 50 mm, (II) 500 nm, (III) 2 mm (Reprinted with permission from Hai, A., J. Shappir, and
M.E. Spira (2010). In-cell recordings by extracellular microelectrodes. Nature Methods 7 (3), 200–202 and Hai, A., et al. (2009). Spine-shaped gold
protrusions improve the adherence and electrical coupling of neurons with the surface of microelectronic devices. Journal of the Royal Society Inter-
face rsif20090087. Copyright 2009, 2010 Macmillan Publishers). (B) Vertical Si nanowire (SiNW) electrode array (VNEA). SEM image of (I) nine
SiNW array with metal-coated tips (gray) and insulating silicon oxide (blue), and (II) rat cortical cell (3 DIV) on top of a VNEA pad. Inset represents
the cell/electrode interface. (III) Action potentials stimulated and recorded using a patch pipette (blue) and recorded by the NW array (magenta). Scale
bars: (I) 10 mm, (II) 2.5 mm (Reprinted with permission from Robinson, J. T., et al. (2012). Vertical nanowire electrode arrays as a scalable platform
for intracellular interfacing to neuronal circuits. Nature Nanotechnology 7 (3), 180–184. Copyright 2012 Macmillan Publishers). (C) Pt nanopillars. (I)
SEM image of an array of five vertical nanopillar electrodes with dimensions of 1.5 mm in height and 150 nm in width; Inset: schematic of a nano-
pillar electrode. (II) SEM image of HL-1 cell/nanopillar electrode interface. Electrical recording by Pt nanopillars (III) before and (IV) after electro-
poration. Scale bars: (I) 2 mm, (II) 200 nm (Reprinted with permission from Xie, C., et al. (2012). Intracellular recording of action potentials by
nanopillar electroporation. Nature Nanotechnology 7 (3), 185–190. Copyright 2012 Macmillan Publishers).
and a number of them penetrate the plasma membrane, the electrodes impedance is too high to enable recording of
subthreshold potentials.
Pt nanopillars
In parallel, the use of vertical Pt nanopillar electrodes (150 nm in diameter and 1–2 mm in height) (Fig. 7C-I) for intracellular-like
recording from cultured HL-1 cardiomyocytes (Fig. 7C-II) was also demonstrated (Xie et al., 2012). The aim was to increase the Rseal
by forming tight junctions between cell membranes and vertically aligned nanopillar electrodes. The study showed that transient
electroporation using the nanopillars leads to a change from biphasic extracellular signature (Fig. 7C-III) to two orders of magni-
tude higher monophasic intracellular-like signal signature (Fig. 7C-IV). The drastically improved signal is attributed to the reduction
in impedance between the electrode and the cell interior due to the formation of nanopores in the cell membrane by electropora-
tion. However, after electroporation, the amplitude of the recorded AP signals continuously decays and reduces to 30% of its orig-
inal amplitude in 120 s due to the subsequent self-sealing of electroporation-generated pores in the cell membrane. This transient
nature of the electrical coupling and the attenuation of the APs indicate that electroporation could not be used to obtain long-term
stable recordings from the cells.
Modifying the shape of the passive electrodes, as discussed earlier, does enable recording of spikes with 1–2 orders of magnitude
higher than their planar counterparts, thus providing more information about the electrical activity. However, the high impedance
associated with the small size of these electrodes prevents the recording of the full-amplitude AP which is an order of magnitude
higher than what these electrodes can record.
FETs
NW FETs have a potential to be ideal for intracellular probes because (i) their nanoscale dimensions allow for minimally invasive
insertion and (ii) the FET device can enable true intracellular recording with high SNR since the size of the device is not dependent
on interfacial impedance in contrast to passive electrode techniques.
3D-kinked NW probes
The overall size of nanoFETs is larger than the active FET component due to the source and drain electrical contacts. The necessity of
having two contacts makes minimally invasive insertion of a nanoFET into cells challenging and less feasible. One of the methods to
overcome the geometry-size constraint was achieved by developing nanoFETs based on kinked NWs (Tian et al., 2010). The method
involved synthesis of SiNWs with two 120o kinks. Leveraging on the bottom-up synthesis approach, contacts (source/drain) to the
nanoprobe were defined by in situ degenerately n-doping the SiNW arms. In addition, a nanoprobe was synthetically encoded close
to the kinks site by changing the dopant to lightly n-doped. The SiNWs were transferred on SU8 supported on a sacrificial layer and
mechanical supports (SU8) and metallization lines (Cr/Pd) were defined using electron beam lithography. The interfacial stress
between materials was used to bend the probe upward off the surface leading to a freestanding nanoFET (Fig. 8A-I). The acute-
angle kinked NW geometry and extended source/drain arms spatially separate the functional nanoscale FET from the bulky metallic
interconnects, thus allowing better interface of the active region of the device with the cell with minimal interference from the
source/drain contacts. An interesting feature of these nanoFET probes was modification with phospholipid bilayers to promote
spontaneous cellular internalization without external forces. Contact of cultured cardiomyocyte cells to a 3D-kinked SiNW bio-
probe showed three distinguishable recording stages during internalization: extracellular (Fig. 8A-II), extracellular to intracellular
transition (Fig. 8A-III), and stead-state intracellular recordings (Fig. 8A-IV). The initial extracellular signals had an amplitude of
3–5 mV and a submillisecond width. Without applying external force, the initial extracellular signal gradually disappeared with
a concomitant emergence of intracellular peaks featured with an opposite polarization, a much larger amplitude of 80 mV,
and a longer duration of 200 ms. This study demonstrated for the first time intracellular electrical recording platform based
on FET nanoprobes that are capable of recording true AP with high spatial and temporal resolution and high SNR. However,
the kink configuration and device design places limits on the probe size and the potential for multiplexing.
Branched intracellular nanotube-FET (BIT-FET)

Overcoming the challenge of multiplexing of the FET probes to achieve intracellular recordings was demonstrated by using
branched intracellular nanotube FET (BIT-FET) (Duan et al., 2012). BIT-FET is a combination of a SiNW FET and an electrically
insulating SiO2 nanotube. An NW FET is fabricated on the chip as the recording transducer and a vertical nonconductive nanotube
is created directly on active channel region of the nanowire FET to interface the FET to the cell interior (cytosol) for intracellular
recording (Fig. 8B-I). The fabrication of the BIT-FET probe involved combination of bottom-up synthesis/assembly and top-
down lithographic patterning. The steps involved growth of Germanium NW (GeNW) branches on top of SiNWs using the Au-
nanocluster-catalyzed vapor–liquid–solid mechanism. The GeNWs were then coated with conformal thin SiO2 layer. Following
selective removal of the topmost part of the SiO2 shell and etching off the Ge nanowire core, a vertical hollow SiO2 nanotube
was created on the SiNW FET. To facilitate internalization of BIT-FET probes in cells for intracellular recording, the probes were
modified with phospholipids. The capability of BIT-FET probes was demonstrated by recording intracellular signals from sponta-
neously beating cardiomyocyte cells cultured on the PDMS sheets. As shown in Fig. 8B-II, two BIT-FET devices can be interfaced with
a single cardiomyocyte cell to achieve simultaneous two-channel recording from the same cell (Fig. 8B-III). The nanoscale dimen-
sions of the probe along with the modification with the phospholipid led to easy access to the cell interior, leading to intracellular
measurements with full amplitude of 75–100 mV. This suggests the potential to implement large arrays of BIT-FET probes for multi-
plexed intracellular recording in cellular networks.
Active Si nanotube transistor (ANTT)

Complementing the BIT-FET probe, needle-shaped intracellular nanoprobes based on an active Si nanotube transistor (ANTT) were
demonstrated (Gao et al., 2012). The ANTT probe consists of a single semiconductor nanotube. The source/drain contacts of the
nanotube transistor are defined on one end of the Si nanotube and insulated from external solution so that the active channel
regions of nanotube FET can sense the transmembrane potential changes through the solution filling the nanotube. Therefore, if
the free end of an ANTT probe is inserted into the interior of an electrogenic cell, the time-dependent changes associated with
an AP spike will give rise to time-varying conductance signal that maps the intracellular AP (Fig. 8C-I). The fabrication of ANTT
probes involved synthesis of GeNWs followed by p-type Si shell deposition, and contact printing of these Ge/Si NWs onto a pre-
baked SU-8 layer, which was initially deposited on a sacrificial Ni sacrificial layer. The next steps involved registration of positions of
Ge/Si NWs, definition of the bottom SU-8 layer, and definition of source/drain metal contacts followed by the top SU-8 passivation
layer (Fig. 8C-II). Finally, etching the Ni sacrificial layer and Ge core of the Ge/Si NW yielded the p-type Si ANTT probe (Fig. 8C-
III,IV). The capability of ANTT probes was demonstrated by recording intracellular signals from spontaneously beating cardiomyo-
cytes cultured on PDMS sheets. To enable better seal between the ANTT probes and the cells for intracellular recording, the probes
Fig. 8 Intracellular recordings using FETs. (A) 3D-kinked NW probes. (I) Schematics of cellular recording from the cardiomyocyte monolayer on
PDMS (left) and highlight of extracellular (middle) and intracellular (right) NW/cell interfaces. Inset: SEM image of the kinked NW device. Purple lines
represent the cell membrane and NW lipid coatings. Plots corresponding to (II) extracellular, (III) extracellular to intracellular transition, and (IV)
steady-state intracellular recording. Green and pink stars denote the peak positions of intracellular and extracellular signal components, respectively.
Scale bar: (I) 5 mm (Reprinted with permission from Tian, B., et al. (2010). Three-dimensional, flexible nanoscale field-effect transistors as localized
bioprobes. Science 329 (5993), 830–834. Copyright 2010 American Association for the Advancement of Science). (B) Branched intracellular
nanotube-FET (BIT-FET). (I) Schematic of a cell coupled to a BIT-FET. S and D indicate source and drain electrodes, respectively. (II) Differential
interference contrast (DIC) image of two BIT-FET devices (positions marked with dots) coupled to a single cardiomyocyte cell. Yellow-dashed line
represents the cell boundary. (III) Representative trace of stable intracellular action potentials recorded 120 s after internalization of the device.
Scale bar: (II) 10 mm (Reprinted with permission from Duan, X., et al., Intracellular recordings of action potentials by an extracellular nanoscale field-
effect transistor. Nature Nanotechnology 7 (3), 174–179. Copyright 2012 Macmillan Publishers). (C) Active Si nanotube transistor (ANTT). (I) Sche-
matic of an ANTT probe inserted into a cell. (II) Overview of the steps used for ANTT probe fabrication: (1) Transfer of NWs to a SU-8 layer on
a sacrificial layer (silver), (2) registration of positions of NWs and definition of the bottom SU-8 layer, and (3) definition of S/D metal contacts fol-
lowed by the top SU-8 passivation layer. (III) Schematic of the ANTT probe released from the substrate. (IV) SEM image of an ANTT probe; Inset:
zoom in of the probe tip from the dashed red box. Representative potential versus time data recorded (V) immediately following contact between the
ANTT probe and a single cardiomyocyte, (VI) ca. 100 s following contact, and (VII) ca. 5 min following trace VI. The tick marks in V-VII correspond
to 1 s. Scale bars: (IV) 10 mm, (inset) 100 nm (Reprinted with permission from Gao, R., et al. (2012). Outside looking in: Nanotube transistor intra-
cellular sensors. Nano Letters 12 (6), 3329–3333. Copyright 2012 American Chemical Society).
were modified with phospholipid. Bringing the cell in contact with the probe led to regularly spaced spikes with a frequency of
1.8 Hz that is correlated with cell beating (Fig. 8C-V). These peaks had widths of 0.7 ms and amplitudes up to 10 mV that are
consistent with extracellular cardiomyocyte APs. After ca. 100 s following contact between the ANTT probe and PDMS-
supported cell, the recorded periodic signals changed substantially with an increase in amplitude and duration to 40–50 mV
and ca. 200 ms, respectively (Fig. 8C-VI). Over a period of several minutes, the probe was able to achieve stable intracellular record-
ings with amplitude and duration of ca. 80 mV and 200 ms, respectively (Fig. 8C-VII). The nanoscale dimensions of the probe allow
stable interface with the cell. The straightforward fabrication of the devices enables multiple ANTTs at the end of single probes,
demonstrating the potential to achieve multiplexed recording of full-amplitude intracellular APs from single cells as well as cellular
networks.
Conclusion
In this article, we have discussed the current state of the recording platforms and the significant advances in approaches that took
place over the last several years. Various groups around the globe have been working on (i) modifying the design and optimizing the
shape, size, number, tip geometry, and flexibility of the electrodes to gain access to the cell surface or interior and maximize inter-
actions with large cell population; (ii) using materials such as metals, semiconductors, composites, and polymers to minimize the
impedance and making the devices more sensitive and achieve high signal-to-noise ratio; and (iii) modifying the surface of the
devices with bioactive coatings to mimic the biological environment and thus, enhancing electrode-tissue interface and minimize
immune response. As stated previously in the introduction, a device could be considered as an ideal recording system if it: (i) is
biocompatible and mimics properties of biological tissue to minimize any mechanical mismatch, immune response, scar tissue
formation, and tissue disintegration; (ii) supports seamless integration with neurons to avoid shear motion at the cell-electrode
interface, and remain functional for long period of time; (iii) enables readout that covers the entire spectrum of membrane potential
events, that is, APs, subthreshold potentials (EPSPs and IPSPs), and subthreshold membrane oscillations; (iv) provides high spatial
and temporal resolution to allow recordings at single cell level with submillisecond resolution; (v) simultaneously records/stimu-
lates multiple neurons to enable the study of network dynamics and signal propagation in a neuronal network (Scanziani and
Häusser, 2009; Spira and Hai, 2013; Fattahi et al., 2014).
Using flexible substrates, such as polyimide films, hydrogels and silk, have minimized mechanical mismatch of MEAs with the
brain tissue, thus improving the integration of devices with the cortical surface. However, the limitations that remain with these
systems include limited access to deeper brain regions, inability to record the entire spectrum of membrane potential events,
recording attenuated AP signals, and inability to achieve subcellular spatial resolution owing to the size of the electrodes.
Designing passive electrodes in 3D such as Au mushroom electrodes, vertical NWs and nanopillars lead to enhanced surface
area and high seal resistance between the probes and cell membrane, thus enabling multiplex stimulation and intracellular-
type recording with amplitude of one to two orders of magnitude higher compared with planar MEAs. However, the high
impedance associated with the small size of these electrodes prevents the recording of the full-amplitude AP which is an order
of magnitude higher than what these electrodes can record. Also, even though these platforms show promise for multisite record-
ings and stimulations of multiple cells, they have not been explored to achieve multiplex recordings from neuronal circuits in 3D
or in vivo model. Using extracellular nanoprobes, such as Si FETs, has enabled recording with submillisecond temporal resolution
and subcellular spatial resolution, and integrating the FET sensors in 3D scaffolds allowed recording from cells that mimic
morphology as in vivo models. However, these platforms are incapable of achieving measurements from intact brain tissue which
involves much more complex neuronal circuitry. Using injectable electronics have enabled access to the neuronal network deep in
the brain tissue. However, not being able to access the interior of the cells prevents these devices to record potentials of entire
spectrum. Modifying the nanoprobe design to access interior of the cells has enabled recording of full-amplitude intracellular
AP. However, there are some limitations associated with these platforms: there is a need to mechanically manipulate the cultured
cells and the substrate on which they grow into physical contact with the electrodes; these probes show promise of multiplex
recordings but have not been demonstrated to record intracellular signals from a large neuronal network; the design of these
probes might not enable recording from in vivo models, especially from deep regions of the brain; and these devices cannot
be used for stimulation purposes.
We believe that combining the best features of recently developed techniques will lead to new hybrid devices with multifunc-
tional capabilities that could enable long-term stable two-way interaction, that is, stimulation and mapping of in vivo brain activity
at high spatial and temporal resolution. This two-way interaction would not just allow understanding of neuronal circuitry but also
enable regenerative engineering where these platforms will aid in treatment of disorders and injuries in the electrically active tissues.
Another crucial aspect is to create smart nanomaterials by utilizing bio-inspired designs and biologically derived materials, such as
receptors and proteins. This would enable seamless integration of the devices into the complex cellular circuitry causing minimal
interference to the functioning of the brain. A promising route in this research field is the combination of the nanoelectronic devices
with other modalities, such as optogenetics to study the functional roles of different classes of neurons by manipulating the activity
of specific cellular subpopulations, and chemical sensing to study the biochemistry involved in the neuronal function. The advance-
ments in the development of smart hybrid electronic devices in combination with other modalities will enable greater insight into
the multitude of neural interactions, understanding complex biological systems and disease progression, and potential new ther-
apeutic directions.
References
Assad, J. A., et al. (2014). Brain function: Novel technologies driving novel understanding. In Bioinspired approaches for human-centric technologies (pp. 299–334). New York:
Springer.
Bergveld, P. (1970). Development of an ion-sensitive solid-state device for neurophysiological measurements. IEEE Transactions on Biomedical Engineering, 1, 70–71.
Bergveld, P., Wiersma, J., & Meertens, H. (1976). Extracellular potential recordings by means of a field effect transistor without gate metal, called OSFET. IEEE Transactions on
Besl, B., & Fromherz, P. (2002). Transistor array with an organotypic brain slice: field potential records and synaptic currents. European Journal of Neuroscience, 15(6), 999–1005.
Biran, R., Martin, D. C., & Tresco, P. A. (2005). Neuronal cell loss accompanies the brain tissue response to chronically implanted silicon microelectrode arrays. Experimental
Neurology, 195(1), 115–126.
Biran, R., Martin, D. C., & Tresco, P. A. (2007). The brain tissue response to implanted silicon microelectrode arrays is increased when the device is tethered to the skull. Journal of
Biomedical Materials Research Part A, 82(1), 169–178.
Buzsáki, G., Anastassiou, C. A., & Koch, C. (2012). The origin of extracellular fields and currentsdEEG, ECoG, LFP and spikes. Nature Reviews Neuroscience, 13(6), 407–420.
Chang, J. C., Brewer, G. J., & Wheeler, B. C. (2000). Microelectrode array recordings of patterned hippocampal neurons for four weeks. Biomedical Microdevices, 2(4), 245–253.
Cogan, S. F. (2008). Neural stimulation and recording electrodes. Annual Review of Biomedical Engineering, 10, 275–309.
Cohen-Karni, T., et al. (2010). Graphene and nanowire transistors for cellular interfaces and electrical recording. Nano Letters, 10(3), 1098–1102.
Cole, K. S., & Curtis, H. J. (1939). Electric impedance of the squid giant axon during activity. The Journal of General Physiology, 22(5), 649–670.
Contreras, D. (2004). Electrophysiological classes of neocortical neurons. Neural Networks, 17(5), 633–646.
Cui, X., & Martin, D. C. (2003). Fuzzy gold electrodes for lowering impedance and improving adhesion with electrodeposited conducting polymer films. Sensors and Actuators A:
Physical, 103(3), 384–394.
Cui, X., et al. (2001). Surface modification of neural recording electrodes with conducting polymer/biomolecule blends. Journal of Biomedical Materials Research, 56(2), 261–272.
Dai, X., et al. (2016). Three-dimensional mapping and regulation of action potential propagation in nanoelectronics-innervated tissues. Nature Nanotechnology, 11, 776–782.
DeLorenzo, R. J., Sombati, S., & Coulter, D. A. (2000). Effects of topiramate on sustained repetitive firing and spontaneous recurrent seizure discharges in cultured hippocampal
neurons. Epilepsia, 41(s1), 40–44.
Deng, M., et al. (2011). Electrochemical deposition of polypyrrole/graphene oxide composite on microelectrodes towards tuning the electrochemical properties of neural probes.
Sensors and Actuators B: Chemical, 158(1), 176–184.
Drake, K. L., et al. (1988). Performance of planar multisite microprobes in recording extracellular single-unit intracortical activity. IEEE Transactions on Biomedical Engineering,
35(9), 719–732.
Duan, X., et al. (2012). Intracellular recordings of action potentials by an extracellular nanoscale field-effect transistor. Nature Nanotechnology, 7(3), 174–179.
Duan, X., et al. (2013). Nanoelectronics-biology frontier: From nanoscopic probes for action potential recording in live cells to three-dimensional cyborg tissues. Nano Today, 8(4),
351–373.
Edell, D. J., et al. (1992). Factors influencing the biocompatibility of insertable silicon microshafts in cerebral cortex. IEEE Transactions on Biomedical Engineering, 39(6), 635–643.
Edwards, F. A., et al. (1989). A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflu¨gers Archiv European Journal of
Physiology, 414(5), 600–612.
Evarts, E. V. (1966). Pyramidal tract activity associated with a conditioned hand movement in the monkey. Journal of Neurophysiology, 29(6), 1011–1027.
Fattahi, P., et al. (2014). A review of organic and inorganic biomaterials for neural interfaces. Advanced Materials, 26(12), 1846–1885.
Frey, U., et al. (2009). Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices. Biosensors and Bioelectronics, 24(7), 2191–2198.
Fromherz, P. (2002). Electrical interfacing of nerve cells and semiconductor chips. Chemphyschem, 3(3), 276–284.
Fromherz, P., & Offenhausser, A. (1991). A neuron-silicon junction: A Retzius cell of the leech on an insulated-gate field-effect transistor. Science, 252(5010), 1290.
Fuchsberger, K., et al. (2011). Multiwalled carbon-nanotube-functionalized microelectrode arrays fabricated by microcontact printing: platform for studying chemical and electrical
neuronal signaling. Small, 7(4), 524–530.
Gabay, T., et al. (2007). Electro-chemical and biological properties of carbon nanotube based multi-electrode arrays. Nanotechnology, 18(3), 035201.
Galvani, L. (1791). De viribus electricitatis in motu musculari Commentarius. De bononiensi scientiarum et artium instituto atque academia Commentarii (Bononiae). cited in
Bernardi (2000).
Gao, R., et al. (2012). Outside looking in: Nanotube transistor intracellular sensors. Nano Letters, 12(6), 3329–3333.
Gawad, S., et al. (2009). Substrate arrays of iridium oxide microelectrodes for in vitro neuronal interfacing. Frontiers in Neuroengineering, 2, 1.
Geim, A. K. (2009). Graphene: Status and prospects. Science, 324(5934), 1530–1534.
Graudejus, O., et al. (2009). Characterization of an elastically stretchable microelectrode array and its application to neural field potential recordings. Journal of The Electrochemical
Society, 156(6), P85–P94.
Hai, A., Shappir, J., & Spira, M. E. (2010). In-cell recordings by extracellular microelectrodes. Nature Methods, 7(3), 200–202.
Hai, A., & Spira, M. E. (2012). On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes. Lab on
a Chip, 12(16), 2865–2873.
Hai, A., et al. (2009). Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices. Journal of The Royal
Society Interface, 6, 1153–1165.
Hamel, E. J., et al. (2015). Cellular level brain imaging in behaving mammals: An engineering approach. Neuron, 86(1), 140–159.
Hatsopoulos, N. G., et al. (1998). Information about movement direction obtained from synchronous activity of motor cortical neurons. Proceedings of the National Academy of
Sciences, 95(26), 15706–15711.
Hochbaum, D. R., et al. (2014). All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nature Methods, 11(8), 825–833.
Hochberg, L. R., et al. (2006). Neuronal ensemble control of prosthetic devices by a human with tetraplegia. Nature, 442(7099), 164–171.
Hodgkin, A., & Huxley, A. (1945). Resting and action potentials in single nerve fibres. The Journal of Physiology, 104(2), 176.
Hodgkin, A., & Huxley, A. (1952a). The components of membrane conductance in the giant axon of Loligo. The Journal of Physiology, 116(4), 473–496.
Hodgkin, A. L., & Huxley, A. F. (1939). Action potentials recorded from inside a nerve fibre. Nature, 144(3651), 710–711.
Hodgkin, A. L., & Huxley, A. F. (1952a). The dual effect of membrane potential on sodium conductance in the giant axon of Loligo. The Journal of Physiology, 116(4), 497.
Hodgkin, A. L., & Huxley, A. F. (1952b). Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. The Journal of Physiology, 116(4), 449.
Hodgkin, A. L., & Huxley, A. F. (1952c). Propagation of electrical signals along giant nerve fibres. Proceedings of the Royal Society of London. Series B, Biological Sciences, 140,
177–183.
Hodgkin, A. L., & Huxley, A. F. (1952d). A quantitative description of membrane current and its application to conduction and excitation in nerve. The Journal of Physiology,
117(4), 500.
Hodgkin, A. L., Huxley, A. F., & Katz, B. (1952). Measurement of current-voltage relations in the membrane of the giant axon of Loligo. The Journal of Physiology, 116(4), 424–448.
Hodgkin, A. L., & Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. The Journal of Physiology, 108(1), 37.
Ingebrandt, S., et al. (2005). Neuron–transistor coupling: Interpretation of individual extracellular recorded signals. European Biophysics Journal, 34(2), 144–154.
Irons, H. R., et al. (2008). Three-dimensional neural constructs: a novel platform for neurophysiological investigation. Journal of Neural Engineering, 5(3), 333.
Janders, M., et al. (1996). In Novel thin film titanium nitride micro-electrodes with excellent charge transfer capability for cell stimulation and sensing applicationsEngineering in
medicine and biology society. Bridging disciplines for biomedicine. Proceedings of the 18th annual international conference of the IEEE, IEEE.
Kanagasabapathi, T. T., et al. (2012). Functional connectivity and dynamics of cortical–thalamic networks co-cultured in a dual compartment device. Journal of Neural Engineering,
9(3), 036010.
Kandel, E. R., et al. (2000). Principles of neural science (Vol. 4). New York: McGraw-Hill.
Keefer, E. W., et al. (2008). Carbon nanotube coating improves neuronal recordings. Nature Nanotechnology, 3(7), 434–439.
Kim, D.-H., et al. (2010a). Dissolvable films of silk fibroin for ultrathin conformal bio-integrated electronics. Nature Materials, 9(6), 511–517.
Kim, J.-H., et al. (2010b). Surface-modified microelectrode array with flake nanostructure for neural recording and stimulation. Nanotechnology, 21(8), 085303.
Kim, R., Hong, N., & Nam, Y. (2013). Gold nanograin microelectrodes for neuroelectronic interfaces. Biotechnology Journal, 8(2), 206–214.
Kim, S., et al. (2009). Integrated wireless neural interface based on the Utah electrode array. Biomedical Microdevices, 11(2), 453–466.
Kipke, D. R., et al. (2008). Advanced neurotechnologies for chronic neural interfaces: New horizons and clinical opportunities. The Journal of Neuroscience, 28(46), 11830–11838.
Kornblum, H. I., et al. (2000). In vivo imaging of neuronal activation and plasticity in the rat brain by high resolution positron emission tomography (microPET). Nature Biotechnology,
18(6), 655–660.
Kovacs, G. T., Stenger, D., & McKenna, T. (1994). Enabling technologies for cultured neural networks (pp. 121–165). San Diego: Academic Press.
Kralik, J. D., et al. (2001). Techniques for chronic, multisite neuronal ensemble recordings in behaving animals. Methods, 25(2), 121–150.
Kuzum, D., et al. (2014). Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging. Nature Communications, 5.
Lagoa, C. E., et al. (2006). The role of initial trauma in the host’s response to injury and hemorrhage: Insights from a correlation of mathematical simulations and hepatic
transcriptomic analysis. Shock, 26(6), 592–600.
Lambacher, A., et al. (2004). Electrical imaging of neuronal activity by multi-transistor-array (MTA) recording at 7.8 mm resolution. Applied Physics A: Materials Science &
Processing, 79(7), 1607–1611.
Leach, J., Achyuta, A. K. H., & Murthy, S. K. (2010). Bridging the divide between neuroprosthetic design, tissue engineering and neurobiology. Frontiers in Neuroengineering, 2, 18.
Lebedev, M. A., & Nicolelis, M. A. (2006). Brain–machine interfaces: Past, present and future. Trends in Neurosciences, 29(9), 536–546.
Lieber, C. M. (2011). Semiconductor nanowires: a platform for nanoscience and nanotechnology. MRS Bulletin, 36(12), 1052–1063.
Liu, J., et al. (2015). Syringe-injectable electronics. Nature Nanotechnology, 10(7), 629–636.
Liu, Y., et al. (2007). NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. Journal of Neuroscience, 27(11), 2846–2857.
Llinás, R. R. (1988). Central nervous system function. Science, 242(4886), 1654–1664.
Ludwig, K. A., et al. (2006). Chronic neural recordings using silicon microelectrode arrays electrochemically deposited with a poly (3, 4-ethylenedioxythiophene) (PEDOT) film.
Journal of Neural Engineering, 3(1), 59.
Ludwig, K. A., et al. (2011). Poly (3, 4-ethylenedioxythiophene) (PEDOT) polymer coatings facilitate smaller neural recording electrodes. Journal of Neural Engineering, 8(1), 014001.
Maher, M., et al. (1999). The neurochip: a new multielectrode device for stimulating and recording from cultured neurons. Journal of Neuroscience Methods, 87(1), 45–56.
Marconi, E., et al. (2012). Emergent functional properties of neuronal networks with controlled topology. PLoS One, 7(4), e34648.
Marg, E., & Adams, J. E. (1967). Indwelling multiple micro-electrodes in the brain. Electroencephalography and Clinical Neurophysiology, 23(3), 277–280.
Mathieson, K., et al. (2004). Large-area microelectrode arrays for recording of neural signals. IEEE Transactions on Nuclear Science, 51(5), 2027–2031.
Maynard, E., et al. (1999). Neuronal interactions improve cortical population coding of movement direction. Journal of Neuroscience, 19(18), 8083–8093.
Merz, M., & Fromherz, P. (2005). Silicon chip interfaced with a geometrically defined net of snail neurons. Advanced Functional Materials, 15(5), 739–744.
Molleman, A. (2003). Patch clamping: an introductory guide to patch clamp electrophysiology. New York: Wiley.
Nicholls, J. G., et al. (2001). From neuron to brain (vol. 271). Sunderland, MA: Sinauer Associates.
Nicolelis, M. A., et al. (2003). Chronic, multisite, multielectrode recordings in macaque monkeys. Proceedings of the National Academy of Sciences, 100(19), 11041–11046.
Novak, J. L., & Wheeler, B. C. (1986). Recording from the Aplysia abdominal ganglion with a planar microelectrode array. IEEE Transactions on Biomedical Engineering, 2,
196–202.
Novoselov, K. S., et al. (2004). Electric field effect in atomically thin carbon films. Science, 306(5696), 666–669.
Offenhäusser, A., & Knoll, W. (2001). Cell-transistor hybrid systems and their potential applications. Trends in Biotechnology, 19(2), 62–66.
Offenhäusser, A., et al. (1997). Field-effect transistor array for monitoring electrical activity from mammalian neurons in culture. Biosensors and Bioelectronics, 12(8), 819–826.
Oka, H., et al. (1999). A new planar multielectrode array for extracellular recording: Application to hippocampal acute slice. Journal of Neuroscience Methods, 93(1), 61–67.
Paik, S.-J., & Park, Y. (2003). Roughened polysilicon for low impedance microelectrodes in neural probes. Journal of Micromechanics and Microengineering, 13(3), 373.
Park, D.-W., et al. (2014). Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications. Nature Communications, 5.
Park, D.-W., et al. (2016). Fabrication and utility of a transparent graphene neural electrode array for electrophysiology, in vivo imaging, and optogenetics. Nature Protocols, 11(11),
2201–2222.
Patolsky, F., et al. (2006). Detection, stimulation, and inhibition of neuronal signals with high-density nanowire transistor arrays. Science, 313(5790), 1100–1104.
Pine, J. (1980). Recording action potentials from cultured neurons with extracellular microcircuit electrodes. Journal of Neuroscience Methods, 2(1), 19–31.
Poldrack, R. A., & Farah, M. J. (2015). Progress and challenges in probing the human brain. Nature, 526(7573), 371–379.
Polikov, V. S., Tresco, P. A., & Reichert, W. M. (2005). Response of brain tissue to chronically implanted neural electrodes. Journal of Neuroscience Methods, 148(1), 1–18.
Purves, R. (1981). Microelectrode methods for intracellular recording and ionophoresis. New York: Academic Press.
Rastogi, S. K., et al. (2017). Effect of graphene on nonneuronal and neuronal cell viability and stress. Nano Letters, 17(5), 3297–3301.
Robinson, D. A. (1968). The electrical properties of metal microelectrodes. Proceedings of the IEEE, 56(6), 1065–1071.
Robinson, J. T., et al. (2012). Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits. Nature Nanotechnology, 7(3), 180–184.
Rousche, P. J., & Normann, R. A. (1998). Chronic recording capability of the Utah Intracortical Electrode Array in cat sensory cortex. Journal of Neuroscience Methods, 82(1), 1–15.
Ruaro, M. E., Bonifazi, P., & Torre, V. (2005). Toward the neurocomputer: image processing and pattern recognition with neuronal cultures. IEEE Transactions on Biomedical
Engineering, 52(3), 371–383.
Scanziani, M., & Häusser, M. (2009). Electrophysiology in the age of light. Nature, 461(7266), 930–939.
Schwartz, A. B. (2004). Cortical neural prosthetics. Annual Review of Neuroscience, 27, 487–507.
Spira, M. E., & Hai, A. (2013). Multi-electrode array technologies for neuroscience and cardiology. Nature Nanotechnology, 8(2), 83–94.
Sun, D. A. (2002). Glutamate injury-induced epileptogenesis in cultured hippocampal neurons: An in vitro model of stroke-induced epilepsy.
Suzuki, I., et al. (2013). Carbon nanotube multi-electrode array chips for noninvasive real-time measurement of dopamine, action potentials, and postsynaptic potentials. Biosensors
and Bioelectronics, 49, 270–275.
Szarowski, D., et al. (2003). Brain responses to micro-machined silicon devices. Brain Research, 983(1), 23–35.
Thiebaud, P., et al. (1997). Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures. IEEE Transactions on Biomedical Engineering,
44(11), 1159–1163.
Thomas, C., et al. (1972). A miniature microelectrode array to monitor the bioelectric activity of cultured cells. Experimental Cell Research, 74(1), 61–66.
Tian, B., & Lieber, C. M. (2013). Synthetic nanoelectronic probes for biological cells and tissues. Annual Review of Analytical Chemistry, 6, 31–51.
Tian, B., et al. (2010). Three-dimensional, flexible nanoscale field-effect transistors as localized bioprobes. Science, 329(5993), 830–834.
Tian, B., et al. (2012). Macroporous nanowire nanoelectronic scaffolds for synthetic tissues. Nature Materials, 11(11), 986–994.
Turner, J., et al. (1999). Cerebral astrocyte response to micromachined silicon implants. Experimental Neurology, 156(1), 33–49.
Vassanelli, S., & Fromherz, P. (1998). Transistor records of excitable neurons from rat brain. Applied Physics A: Materials Science & Processing, 66(4), 459–463.
Vassanelli, S., & Fromherz, P. (1999). Transistor probes local potassium conductances in the adhesion region of cultured rat hippocampal neurons. Journal of Neuroscience, 19(16),
6767–6773.
Venkatraman, S., et al. (2011). In vitro and in vivo evaluation of PEDOT microelectrodes for neural stimulation and recording. IEEE Transactions on Neural Systems and Rehabilitation
Engineering, 19(3), 307–316.
Voelker, M., & Fromherz, P. (2005). Signal transmission from individual mammalian nerve cell to field-effect transistor. Small, 1(2), 206–210.
Weis, R., Müller, B., & Fromherz, P. (1996). Neuron adhesion on a silicon chip probed by an array of field-effect transistors. Physical Review Letters, 76(2), 327.
Wise, K. D., & Najafi, K. (1991). Microfabrication techniques for integrated sensors and microsystems. Science, 254(5036), 1335.
Wise, K. D., et al. (2008). Microelectrodes, microelectronics, and implantable neural microsystems. Proceedings of the IEEE, 96(7), 1184–1202.
Wolpaw, J. R., & McFarland, D. J. (2004). Control of a two-dimensional movement signal by a noninvasive brain-computer interface in humans. Proceedings of the National
Academy of Sciences of the United States of America, 101(51), 17849–17854.
Wolpaw, J. R., et al. (2002). Brain–computer interfaces for communication and control. Clinical Neurophysiology, 113(6), 767–791.
Xie, C., et al. (2012). Intracellular recording of action potentials by nanopillar electroporation. Nature Nanotechnology, 7(3), 185–190.
Yuan, Q., et al. (2016). Regulation of brain-derived neurotrophic factor exocytosis and gamma-aminobutyric acidergic interneuron synapse by the schizophrenia susceptibility gene
dysbindin-1. Biological Psychiatry, 80(4), 312–322.
Yuen, T., & Agnew, W. (1995). Histological evaluation of polyesterimide-insulated gold wires in brain. Biomaterials, 16(12), 951–956.
Zhang, A., & Lieber, C. M. (2016). Nano-bioelectronics. Chemical Reviews, 116(1), 215.
Zhou, H., et al. (2013). Poly (3, 4-ethylenedioxythiophene)/multiwall carbon nanotube composite coatings for improving the stability of microelectrodes in neural prostheses
applications. Acta Biomaterialia, 9(5), 6439–6449.
Further Reading
Buzsáki, G., Anastassiou, C. A., & Koch, C. (2012). The origin of extracellular fields and currentsdEEG, ECoG, LFP and spikes. Nature Reviews Neuroscience, 13(6), 407–420.
Hodgkin, A. L., & Huxley, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. The Journal of Physiology,
117(4), 500.
Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A., & Hudspeth, A. J. (2000). Principles of neural science (Vol. 4). New York: McGraw-Hill.
Nicholls, J. G., Martin, A. R., Wallace, B. G., & Fuchs, P. A. (2001). From neuron to brain (vol. 271). Sunderland, MA: Sinauer Associates.
Offenhäusser, A., & Knoll, W. (2001). Cell-transistor hybrid systems and their potential applications. Trends in Biotechnology, 19(2), 62–66.
Spira, M. E., & Hai, A. (2013). Multi-electrode array technologies for neuroscience and cardiology. Nature Nanotechnology, 8(2), 83–94.
Zhang, A., & Lieber, C. M. (2016). Nano-bioelectronics. Chemical Reviews, 116(1), 215.
Bezanilla, F. (2006). The action potential: from voltage-gated conductances to molecular structures. Biological Research, 39(3), 425–435.
Koeppen, B. M., & Stanton, B. A. (2009). Berne and Levy physiology. St. Louis: Elsevier Health Sciences.
Neural Crest Stem Cells
T Hochgreb-Hägele and ME Bronner, California Institute of Technology, Pasadena, CA, USA
Origins of NC Cells 651

Epithelial to Mesenchymal Transition 651
Developmental Potential of NC Cells 651
Molecular Mechanisms of NC Formation 654
NC-Related Birth Defects and Cancers 656
NC Stem Cells and the Potential to Treat Disease 656
Acknowledgments 657
References 657
Further Reading 659
Glossary
CNS Central nervous system.
EMT Epithelial to mesenchymal transition.
GRN Gene regulatory network.
hESC Human embryonic stem cells.
NC Neural crest.
Stem cells are functionally defined as cells that are able to self-renew as well as capable of generating several distinct differentiated
cell types. Given their important therapeutic potential, there has been growing interest and steady progress in understanding the
biology of stem cells. Embryonic stem cells are totipotent cells, i.e., able to give rise to all cell types, and are generally derived
from the inner cell mass of the blastocyst. More restricted stem cell populations have been identified in association with differen-
tiated tissues or organs, such as skeletal muscle (Collins and Partridge, 2005; Zammit et al., 2006), heart (Laugwitz et al., 2005), skin
(Wong et al., 2006), and hematopoietic tissue (Bryder et al., 2006). These are more limited in their differentiation potential than
totipotent stem cells, but have greater significance for maintenance of tissue homeostasis. These tissue- or organ-specific populations
constitute important local sources of multipotent, proliferating cells, which can be activated and recruited in response to tissue
damage. In addition, these precursors play a constitutive role in replenishing differentiated tissue with newly generated cells
throughout life.
The embryonic neural crest (NC) is a unique population of highly migratory and multipotent cells that generate a variety of
derivatives, including peripheral neurons and associated glia, Schwann cells lining the peripheral nerves, and melanocytes respon-
sible for the pigmentation of our bodies. They also form endocrine cells of the adrenal medulla and thyroid calcitonin-producing
cells, as well as fibroblasts, myoblasts, adipocytes, and angioblasts (Le Douarin, 1982; Le Douarin and Kalcheim, 1999). In the head
region, cranial NC cells give rise to mesenchymal cells that differentiate into connective tissue and skeletal cells, such as chondro-
cytes and osteocytes, which contribute to cranial cartilage and bone. The latter comprises most of the skull and much of the facial
skeleton, most notably the jaws, as well as the odontoblasts of the tooth primordia. From an evolutionary perspective, NCs are
unique to vertebrates and one of their defining features. Evolutionarily NC is thought to have facilitated predatory ability by form-
ing a very efficient and powerful jaw. This in turn allowed vertebrate brains to get ever larger, making them the most efficient pred-
ators on the planet.
Cell lineage analysis of NC cell fate in vivo as well as clonal analysis in vitro have revealed that some individual NC cells indeed are
multipotent and able to contribute to several derivatives. Clonal studies in tissue culture show that some multipotent NC progen-
itors can differentiate into glia, neurons, melanocytes, and cartilage, or combinations of these cell types. In addition, NC cells have
a limited capacity for self-renewal, meaning that individual cells can divide and form progeny comprised of similar self-renewing
NC cells, plus sister cells that can differentiate into several types of derivatives (Trentin et al., 2004). In vivo, they retain the ability to
self-renew at least for a limited time. For example, crest-derived sympathoadrenal cells give rise to sympathetic neurons or
adrenomedullary cells, but remain mitotic even after expressing adrenergic markers (Morrison et al., 1999). Unlike true stem cells,
however, the self-renewal ability of NC cells is transient. Thus, it is more appropriate to call them ‘stemlike’ cells rather than true
stem cells.

Regenerative Engineering j Neural Crest Stem Cells 651
Origins of NC Cells
The early embryo contains three layers: the ectoderm and the endoderm lining the outer and inner surfaces of the embryo, respec-
tively, and the mesoderm localized in between. The ectoderm can be further subdivided in two regions: a middle portion which
becomes a thickened neural plate that will give rise to the central nervous system (CNS), while the adjacent tissue will form the
epidermis of the skin (Figure 1(a) and 1(a’)). Between the presumptive epidermis and neural ectoderm, ‘inductive’ signals define
the neural plate border, where induction of NC takes place (LaBonne and Bronner-Fraser, 1999). By definition, induction is an event
whereby two tissues are brought together and in response to an interaction between them, they form a new tissue. This is generally
mediated by the cross-talk of signaling molecules that travel between the two tissues.
The neural plate is initially a flat swathe of ectoderm that subsequently folds in upon itself during ‘neurulation.’ During this
event, the sheet of cells starts to fold in upon itself in a process called invagination. Concurrently, the neural folds containing
the NC precursors begin to elevate (Figure 1(b) and 1(b’)). These morphogenetic movements bring the neural folds together
(Figure 1(c) and 1(c’)) and their fusion in the dorsal aspect of the embryo results in closure of the neural tube, as a cylindrical
epithelium that becomes internalized and covered by the overlying epidermis (Figure 1(d)). The neural tube gives rise to the entire
CNS, forming the brain in the head region and spinal cord more posteriorly. Closure of the neural tube initiates in the head and
then proceeds progressively tailward in the embryo. Migration of NC cells is closely associated with neurulation, and in most verte-
brates, cells start migrating from the neuroepithelium shortly after neural tube closure. NC cells first emigrate in the cranial region,
then at vagal levels, and subsequently in the trunk, similar to the rostral to caudal direction of neural tube closure.
NC cells exhibit different patterns of migration and form distinct derivatives depending upon their site of origin in the neural
tube. At caudal forebrain and midbrain levels, cranial NC cells migrate as a broad and uniform sheet of cells that expands away from
the neural tube (Figure 1(d’)) and invades the cranial mesenchyme, where they populate the cranial ganglia and frontonasal
process. These cells contribute to neurons, glia, and cartilage of the face. At the hindbrain level, NC cell migration is segmental,
resulting in several distinct streams of migrating cells that invade the branchial arches. These cells then differentiate into portions
of the jaw and bones of the neck. The vagal NC undergoes the most extensive migrations of any embryonic cell type. These cells
migrate along the entire length of the gut, where they form the enteric ganglia that control gut motility. Trunk NC cells undergo
segmental migration through the somites. Some condense adjacent to the neural tube to form dorsal root ganglia, whereas others
migrate further ventrally to form sympathetic ganglia and the adrenal medulla. Melanocytes arise from NC cells at all axial levels.
Epithelial to Mesenchymal Transition
The neural tube is comprised of polarized epithelial cells with adherens and tight junctions establishing their intercellular connec-
tions. At the end of neurulation, the premigratory NC cells reside within the dorsal portion of the neuroepithelium, and thus
initially are part of the CNS. Subsequently, NC cells lose their polarity and undergo changes of adhesion. Their tight junctions
are disrupted, the cytoskeleton is reorganized, and they transition from a columnar epithelial arrangement to a migratory mesen-
chymal cell type. This process is known as epithelial to mesenchymal transition (EMT), and it involves great changes in cell
morphology, as well as in the repertoire of adhesion and recognition molecules that are expressed on the surface of the cells
(Figure 2).
The cellular changes of EMT imbue NC cells with the ability to delaminate from neural tube and migrate extensively through the
embryo along well-established pathways. They then differentiate into a variety of cell types in the head and trunk. Although it is an
essential step in the development of NC cells, EMT is not unique to this population but also occurs in several types of embryonic
cells. Importantly, it is characteristic of metastatic transformation of cancer cells. Interestingly, NC and other stem cells share many
biological properties with tumor cells: the ability to undergo EMT; their capacity to migrate extensively; and their ability to differ-
entiate into numerous cells.
Developmental Potential of NC Cells

NC cells have the ability to form derivatives as diverse and distinct as neurons, pigment cells, and cartilage. Initially, premigratory
NC cells appear to be multipotent. However, as these cells migrate along specific pathways, they encounter diverse environments
and are exposed to inductive signals that are differentially distributed within the embryo. These signals direct NC cells to assume
diverse identities and gradually restrict their developmental potential, ultimately resulting in acquisition of their differentiated state.
As a result, migrating NC cells are a heterogeneous population, including various types of intermediate precursors and highly multi-
potent cells, some of which retain the capacity for self-renewal. The latter can become both neuronal and nonneuronal derivatives,
including melanocytes, neurons and support cells of the peripheral nervous system, smooth muscle cells, and facial cartilage and
bones (Figure 3).
The kinds of derivatives that NC cells form depend upon their axial level from which they originate. For example, in vivo lineage
analysis has shown that the site of origin along the neural axis leads to generation of some distinct derivatives. Whereas melanocytes
are derived from NC cells from all axial levels, cranial NC cells have the unique potential to contribute to the bones and cartilage of
the face, in addition to neurons and glia of the cranial sensory ganglia. Even within the head region, NC cells contribute to different
derivatives. Those arising from the midbrain, rhombomeres (r) 1 and 2 contribute to the upper and lower jaws, and trigeminal
652 Regenerative Engineering j Neural Crest Stem Cells
Figure 1 The process of neural tube and neural crest (NC) formation. (Left) Schematic diagram illustrating the process of neural tube closure and
NC formation from the open neural plate stage to the closed neural tube. (Right) Whole mount in situ hybridization of bird embryos with gene
markers involved in NC development. (a) At the open neural plate stage, the neural plate border (green) is induced at the edges of the neural plate
(gray) and epidermal ectoderm (blue), as well as influenced by the underlying mesoderm (yellow) in some vertebrates. (a0 ) Expression of the neural
plate border gene, msx1 outlines the neural plate border, as seen from a dorsal view of an embryo at the open neural plate stage. (b) With time, the
neural folds (green) begin to elevate as the first step in invagination of the presumptive neural tube. (b0 ) Msx1 expression is retained on the elevating
neural folds as seen by whole mount in situ hybridization. (c) As the neural folds appose, bona fide NC markers like foxd3 (c0 ) initiate expression in
the newly closed neural tube. (d) The overlying epidermis (blue) closes over the neural tube. Premigratory NC cells are contained within the central
nervous system but some have already undergone an epithelial to mesenchymal transition to become migrating NC cells that migrate around and
through the mesodermal somites (yellow). (d0 ) Dorsal view of avian embryo expressing sox10 in migrating neural crest.
ganglion; those from r4 contribute to the proximal facial ganglion and the hyoid bone. The vagal NC forms the enteric nervous
system, the cardiac septum, and components of the aortic arch. In the trunk, NC cells form all of the peripheral ganglia as well
as the chromaffin cells in the adrenal medulla. However, unlike cranial crest, trunk NC never contribute to cartilage and bone,
even if transplanted to the head. These observations suggest that, although NC cells from all axial levels appear to have multiple
developmental potentials, the types of derivatives formed vary somewhat accordingly to axial level of origin.
Figure 2 The epithelial to mesenchymal transition (EMT) during neural crest (NC) emigration from the neural tube. After neural tube closure, premi-
gratory NC cells (green) reside within the dorsal or back portion of the neural tube. In this location, individual premigratory NC cells are polarized
epithelial cells with close connections mediated by tight junctions and adhesion molecules. As these cells undergo EMT, they lose their junctional
connections, depart from the neural tube, delaminate from the neural tube, and become migratory mesenchymal cells. This process of NC EMT is
similar to events occurring during cancer metastasis.
Figure 3 Neural crest (NC) cells are multipotent and form many distinct derivatives. The NC stem cell has the ability to give rise to progeny cells
that contribute to multiple and diverse lineages. For example, cranial NC cells can form bone and cartilage, some smooth muscle, as well as neurons
and glia of cranial ganglia. Vagal NC cells give rise to mesenchymal cells of the cardiac septum and to enteric ganglia that innervate the gut. Trunk
NC cells also give rise to neurons and glia of the dorsal root ganglia and sympathetic ganglia, and to chromaffin cells of the adrenal medulla. At all
axial levels, NC cells contribute to melanocytes.
The timing of migration from the neural tube also influences the type of derivative formed by NC cells at a particular axial level.
In the head, for example, the early migrating NC cells populate the branchial arches, where they contribute to bone, cartilage, and
connective tissue of the craniofacial skeleton. In contrast, the later wave of migrating NC stays close to the CNS and forms the glia
and some neurons of the cranial ganglia. In the trunk, earliest migrating NC cells contribute to ventrally located sympathetic ganglia,
while later migrating NC cells form more dorsal derivatives, such as dorsal root ganglia and melanocytes. However, if one ‘switches’
the positions of these early and late populations by performing transplants experiments in the embryos, they can behave appropri-
ately for their new location. Thus, it may be the environmental cues from the spots that are open to them rather than the inherent
knowledge that leads some cells to their final resting sites.
Molecular Mechanisms of NC Formation

Embryonic NC cells have a fascinating ability to maintain a stem-cell-like state prior to differentiating into very diverse derivatives.
This ability is retained as tissue-specific stem cells from the NC lineage are identified in niches of stem cells in adult tissues such as
the cornea and the dental pulp. Therefore, there has been great interest in uncovering the molecular mechanisms underlying NC
induction, how they maintain multipotency and acquire migratory ability, and finally what signals instruct them to choose their
final fates. Answers to these interesting topics are potentially useful in stem cell therapy and regenerative medicine.
The complex sequence of events, cooperating to transform naïve ectodermal cells into NC cells with broad differentiation poten-
tial, has been assembled into a putative NC gene regulatory network (GRN). This is essentially a circuit diagram describing the
molecular pathways that guide development of NC (Meulemans and Bronner-Fraser, 2004; Sauka-Spengler and Bronner-Fraser,
2008; Betancur et al., 2010). Each step is represented as distinct module that plays a role in (1) induction of the neural plate border,
(2) specification of neural plate border, (3) specification of NC progenitors, and (4) differentiation of NC derivatives. During this
complex set of interactions, factors operating at different hierarchical levels of the NC GRN work in concert to establish the NC
transcriptional state.
Formation of NC cells initiates during gastrulation, when signals mediated by diffusible growth factors emanating from the adja-
cent epidermal ectoderm and mesoderm are integrated to induce a presumptive NC territory at the neural plate border (Figure 4(a)
and 4(a’)). NC induction requires intermediate levels of BMPs (Bone Morphogenetic Proteins), secreted proteins that are members
of the TGF-b superfamily involved in many important developmental events such as dorsoventral patterning during early embry-
onic development (Knecht et al., 1995; Piccolo et al., 1996; Dale et al., 1992). In the ectoderm, BMPs play an early role in the induc-
tion of the neural plate and NC (LaBonne and Bronner-Fraser, 1998; Marchant et al., 1998; Wilson and Hemmati-Brivanlou, 1995;
Liem et al., 1995). Subsequently, expression of BMP inhibitors such as chordin, noggin, and follistatin establishes the intermediate
levels of BMP necessary for induction of NC cells (Lamb et al., 1993; Sasai et al., 1994; Piccolo et al., 1996; Hemmati-Brivanlou
et al., 1994). However, BMPs alone are not sufficient to induce NC progenitors, and other signaling systems also contribute to
NC formation (Streit et al., 1998; Marchant et al., 1998; LaBonne and Bronner-Fraser, 1998). For example, it is known that a combi-
nation of factors like FGF (Fibroblast Growth Factors), members of the Wnt signaling pathway and Notch-Delta is also necessary to
induce an NC-forming territory (Garcia-Castro et al., 2002; Mayor et al., 1995; Mayor et al., 1997; Monsoro-Burq et al., 2003; Endo
et al., 2002; Glavic et al., 2004; Lewis et al., 2004; LaBonne and Bronner-Fraser, 1998). FGFs are secreted from the paraxial meso-
derm, and Wnt signals emanate from the nonneural ectoderm and/or the paraxial mesoderm. Notch is expressed in the neural plate,
with higher levels in NC cells, while its ligand Delta is expressed in the epidermal ectoderm. Because of these signaling events, the
close interaction between the neuroepithelium, nonneural ectoderm, and paraxial mesoderm is critical for the establishment of
a presumptive NC territory. Integration of these various environmental signals is processed by the cells of the neural plate border
and manifested by expression of transcription factors (neural plate border specifier genes), whose overlapping expression at the
junction between neural and nonneural ectoderm specifies this region as the neural plate border.
The neural plate border specifiers include transcription factors such as Msx1/2, Dlx3/5, Pax3/7, Gbx2, as well as Zic (Figure 4(b))
(Monsoro-Burq et al., 2005; Tribulo et al., 2003; Sato et al., 2005; Monsoro-Burq et al., 2003; Khudyakov and Bronner-Fraser,
2009). Their regions of overlapping expression constitute a molecular signature of the neural plate border (Figure 4(b’)). Cells
within this territory are imbued with multipotency and, later, with the ability to respond to NC specifying signals. With time, posi-
tional information supplied by gradients of signaling molecules dictates the transcriptional state of NC precursors within the neural
plate border. Prospective NC cells integrate these signaling inputs to become premigratory NC cells. As the neural folds elevate and
position these cells within the dorsal portion of the developing neural tube, induction of bona fide NC cells is characterized by
expression of another group of transcription factors termed NC specifier genes (Figure 4(c) and 4(c’)). These include transcription
factors such as Snail2, Sox10, and FoxD3, as well as AP-2, Sox9, and c-Myc (Nikitina et al., 2008; Sauka-Spengler et al., 2007).
Expression of the NC specifier genes in premigratory and delaminating cell reflects the fact that these cells have been specified to
an NC cell fate. Functionally, these transcription factors control the expression of effector genes that confer unique migratory
and multipotent characteristics via changes in adhesion, shape, motility, and signaling repertoire of the NC precursors.
NC specifier genes are turned on at different phases during NC specification and directly activate gene batteries controlling
cellular processes like EMT, thus enabling cells to delaminate from the neural tube and migrate extensively throughout the embryo
to then differentiate into diverse derivatives (Figure 4(d) and 4(d’)) (Sauka-Spengler and Bronner-Fraser, 2008; Meulemans and
Bronner-Fraser, 2004). For example, expression of the transcriptional repressor Snail2 initiates during NC specification and plays
an important role in promoting EMT via repression of cell adhesion molecules called cadherins (Taneyhill et al., 2007; Batlle
et al., 2000; Cano et al., 2000). In addition, Snail influences the expression of proteins involved in cell motility and assembly of
junctions (Nieto, 2002; Ikenouchi et al., 2003). Whereas Snail2 is expressed transiently, Sox10 expression persists in migrating crest,
as well as in subsets of differentiating NC cells. Other early NC specifier genes like Id and c-Myc have been implicated in the main-
tenance of multipotency of NC cells by controlling expression of genes involved in cell cycle and cell fate decision (Bellmeyer et al.,
Figure 4 Gene regulatory network underlying progressive development of the neural crest (NC). A combination of signaling and transcriptional
events operates at progressive stages of NC development. For hypothetical purposes, these can be viewed as organized into distinct modules that
operate at defined stages of NC development. Collectively, this gene regulatory network describes the molecular pathways that guide development of
the NC from the open neural plate stage to the formation of differentiated derivatives. (a, a0 ) Signaling molecules like Wnts, BMPs, FGFs, and their
inhibitors transit inductive cues between the neural plate, nonneural ectoderm, and underlying mesoderm, resulting in formation of the neural plate
border at the junction between neural and nonneural ectoderm. (b, b0 ) These signals ultimately result in establishment of the neural plate border terri-
tory via upregulation of transcription factors, referred to as neural plate border specifier genes (e.g., Msx1/2, Pax3/7, Zic1, etc.), whose overlapping
expression defines the border region. (c, c0 ) The neural plate border specifiers cooperate with inductive signals to activate NC specifier genes (e.g.,
Snail, Ets1, Sox10, FoxD3, etc.) in the elevating neural folds and dorsal neural tube. (d, d0 ) After neural tube closure, the NC specifier genes, in turn,
influence expression of various effector genes that are involved in the process of epithelial to mesenchymal transition that allows emigration from the
neural tube and creates a population of migratory NC cells. (e, e0 ) NC specifier genes in combination with environmental factors also lead to activa-
tion of lineage-specific differentiation programs, facilitating the formation of various NC derivatives like neurons, glia, melanocytes, and craniofacial
cartilage.
2003; Kee and Bronner-Fraser, 2005; Light et al., 2005). FoxD3 also appears to maintain multipotency, by preventing early differ-
entiation of crest cells (Mundell and Labosky, 2011; Teng et al., 2008). Interestingly, FoxD3 is not only important in NC develop-
ment but also plays a role in maintaining pluripotency during early embryonic development and in stem cells (Hanna et al., 2002;
Tompers et al., 2005).
Finally, NC cells transition from their migratory and multipotent state and begin to differentiate into defined and diverse deriv-
atives like neurons and glia of peripheral ganglia, cartilage and bones of the face, or pigment cells (Figure 4(e)). After NC cells reach
their final destinations, expression of most early NC specifiers is downregulated. However, expression of certain genes persists in
subsets of derivatives, as in the case of FoxD3 for the neural and glial precursors in the dorsal root ganglia, Sox9 in the NC-
derived chondrocytes, as well as Sox10 in melanoblasts and elements of the peripheral nervous system. Effector genes involved
in differentiation of NC derivatives sometimes function to specify cell fate. For example, Mitf expression in melanoblasts, together
with Sox10, directly regulates expression of dopachrome tautomerase, an enzyme necessary for melanin synthesis (Ludwig et al.,
2004); in chondrocytes, Sox9 directly regulates expression of the chondrocyte matrix protein as Collagen type II (Lefebvre et al.,
1997).
NC-Related Birth Defects and Cancers
As a population, the NC and its derivatives are highly susceptible to errors at multiple steps, from specification to migration, prolif-
eration, and differentiation into distinct derivatives. In fact, pathologies that affect NC-derived tissues are among the most common
causes of birth defects in humans. For example, craniofacial defects represent 10% of human birth defects in term births. These
include cleft lip and palate, defects in dentition, and serious skull malformations such as missing or drastically reduced bones. Such
defects are critically important, as the frontal bones are necessary to protect the brain. TGF-b signaling has been shown to be impor-
tant during formation of the palate in both mouse and humans (Proetzel et al., 1995; Sanford et al., 1997; Kaartinen et al., 1995).
Some of these anomalies, including craniofacial and cardiac septation defects, are traditionally corrected surgically, it is clear that the
causes are complex and involve both gene–gene and/or gene–environment interactions that may alter NC specification, migration,
or differentiation. Only some of the anomalies can be addressed surgically. In addition, the NC is known to be important for coor-
dinating head and brain development, since removal of cranial crest results in severe brain abnormalities (Le Douarin et al., 2007).
Some NC-related birth defects have their roots in mutations in transcription factors that play an important role in the NC GRN.
For example, human mutations in the transcription factor AP2, a NC specifier gene, cause severe defects in facial development
(Satoda et al., 2000). Children with this disorder, called Char syndrome, typically have characteristic facial features with a ‘duck-
bill’ appearance resulting from a flattened midface, wide-set eyes, and flat nasal bridge and tip of the nose. Defects in another
NC specifier gene, Ets1, cause a mutation in cardiac NC development that causes a cardiovascular septation defect (Gao et al.,
2010; Ye et al., 2010). And several other mutations in genes affecting the NC cause colonic agangliogenesis or Hirschsprung’s disease
(Obermayr et al., 2013; Iwashita et al., 2003).
A less well-characterized NC disorder is familial dysautonomia, which affects the development and survival of sensory, sympa-
thetic, and some parasympathetic neurons. This causes many debilitating symptoms, including insensitivity to pain, poor growth
and fluctuating blood pressure. The disorder appears to be caused by incomplete development of NC-derived sensory and auto-
nomic neurons (Nordborg et al., 1981; Lee et al., 2009). These are only a few examples of the many birth defects that have their
origin in defects in NC development. Collectively, these are referred to as neurocristopathies.
Perhaps because NC cells are highly migratory and invasive by nature, many of their derivatives are prone to metastasis, giving
rise to several common types of cancer. These include melanoma, neuroblastoma, and neurofibromatosis (Elephant man’s disease).
Mutations in genes associated with EMT within the developing or adult organism often result in tumor development and metastasis.
These same genes are often critical for normal NC development and their successful EMT. Accordingly, genes like Snail2 and Sox10,
which function as important NC specifier genes also, are highly elevated in many types of adult cancer cells (Shakhova et al., 2012;
Chakrabarti et al., 2012). This raises the hopeful prospect that understanding normal NC development may lead to targets of ther-
apeutic intervention to help prevent metastasis of several types of cancers.
NC Stem Cells and the Potential to Treat Disease
Work by several investigators has led to the identification and purification of NC stem cells that have some ability to self-renew and
can also give rise to diverse derivatives. Clonal analysis of migrating cranial NC cells has demonstrated that many of these progen-
itors are multipotent and can give rise to bone, neural, and pigment cell types. On the other hand, NC stem cells can be driven by
environmental factors to adopt specific fates under proper conditions. Neuregulin, for example, mediates development of NC stem
cells into Schwann cells and glia, whereas BMP-2 promotes neuronal differentiation, and TGF-b1 favors development of smooth
muscle cells (Shah et al., 1994; Shah et al., 1996; Shah and Anderson, 1997). Transient activation of Notch also promotes glial
production by NC at the expense of neurogenesis (Morrison et al., 2000).
In addition to the embryo, NC stem cells can be isolated from several NC derivatives including the gut, peripheral nerve, skin,
and ganglia, as well as several craniofacial tissues such as the cornea and dental pulp, from both the fetus and adult (Li et al., 2007;
Nagoshi et al., 2008; Yoshida et al., 2006; Wong et al., 2006; Gronthos et al., 2000; Morrison et al., 1999; Kruger et al., 2002). Under
proper culture conditions, these cells can self-renew and differentiate into neurons, glia, and smooth muscle cells within single colo-
nies, demonstrating that they retain multipotency.
Because many birth defects are caused by abnormal NC development, it is hoped that understanding development of NC stem
cells may allow investigators to ‘replace’ abnormal NC derivatives for potential use in regenerative medicine. A critical first step is to
obtain sufficient numbers of human NC cells that can be differentiated into the appropriate cell type. Embryonic NC cells have the
ability to generate a vast array of distinct cell types, such as bones and cartilage, neurons and glia, melanocytes, as well as endocrine,
connective and adipose tissues. Thus, there has been great interest in characterizing the prospective stem cell properties of human
NC populations and differentiating them into particular derivatives.
NC cells can be derived in tissue culture from human embryonic stem cells (hESCs). The basic process has been to induce hESCs
into neural stem cells in either adherent or suspension culture and further induce them into NC cells using either coculture with
other cell types or defined medium with added factors (Lee et al., 2007, 2010). The cells located at the periphery of these cultures
often express NC specifier genes like Snail2 and Sox10. Because the efficiencies tend to be low, it is often necessary to purify NC cells
by fluorescence-activated cell sorting and/or perform clonal analysis to examine differentiation in detail. Ongoing efforts are
focused on differentiating these into particular types of derivatives like sensory or autonomic neurons, for treatment of diseases
like familial dysautonomia, in which peripheral neurons are defective. NC cells derived from hESC have been shown to differentiate
into a wide range of NC derivatives, including sensory and autonomic neurons, Schwann cells, myofibroblasts, adipocytes, cartilage,
and bone cells (Lee et al., 2007).
Potential problems with hESC are their general lack of availability and the fact that they may be rejected by the patients’ immune
system. With recent advances in stem cell technology, it is now possible to reprogram somatic cells from adult tissue into induced
pluripotent stem (iPS) cells, thus expanding the possibilities of cell therapy. The knowledge obtained from basic research in various
organisms to build the NC GRN can be applied to direct and monitor differentiation of NC cells and their derivatives from both
stem and iPS cells. This opens many interesting new opportunities to establish cell lineages from tissue obtained from patients with
genetic syndromes and it may help elucidate the molecular mechanisms underlying the genetic syndromes affecting NC cells, as well
as helping devise efficient therapies.
Acknowledgments
We thank Dr. Marcos Simões Costa for helpful discussion and for providing the images of avian embryos and help with the diagram of NC derivative
(Figure 3).
References
Batlle, E., Sancho, E., Franci, C., Dominguez, D., Monfar, M., Baulida, J., & Garcia De Herreros, A. (2000). The transcription factor snail is a repressor of E-cadherin gene expression
in epithelial tumour cells. Nat. Cell Biol., 2, 84–89.
Bellmeyer, A., Krase, J., Lindgren, J., & LaBonne, C. (2003). The protooncogene c-myc is an essential regulator of neural crest formation in xenopus. Dev. Cell, 4, 827–839.
Betancur, P., Bronner-Fraser, M., & Sauka-Spengler, T. (2010). Assembling neural crest regulatory circuits into a gene regulatory network. Annu. Rev. Cell Dev. Biol., 26, 581–603.
Bryder, D., Rossi, D. J., & Weissman, I. L. (2006). Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am. J. Pathol., 169, 338–346.
Cano, A., Perez-Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., Del Barrio, M. G., Portillo, F., & Nieto, M. A. (2000). The transcription factor snail controls epithelial-
mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol., 2, 76–83.
Chakrabarti, R., Hwang, J., Andres Blanco, M., Wei, Y., Lukacisin, M., Romano, R. A., Smalley, K., Liu, S., Yang, Q., Ibrahim, T., Mercatali, L., Amadori, D., Haffty, B. G., Sinha, S.,
& Kang, Y. (2012). Elf5 inhibits the epithelial-mesenchymal transition in mammary gland development and breast cancer metastasis by transcriptionally repressing Snail2. Nat.
Cell Biol., 14, 1212–1222.
Collins, C. A., & Partridge, T. A. (2005). Self-renewal of the adult skeletal muscle satellite cell. Cell Cycle, 4, 1338–1341.
Dale, L., Howes, G., Price, B. M., & Smith, J. C. (1992). Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development. Development, 115, 573–585.
Endo, Y., Osumi, N., & Wakamatsu, Y. (2002). Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development.
Development, 129, 863–873.
Gao, Z., Kim, G. H., Mackinnon, A. C., Flagg, A. E., Bassett, B., Earley, J. U., & Svensson, E. C. (2010). Ets1 is required for proper migration and differentiation of the cardiac neural
crest. Development, 137, 1543–1551.
Garcia-Castro, M. I., Marcelle, C., & Bronner-Fraser, M. (2002). Ectodermal Wnt function as a neural crest inducer. Science, 297, 848–851.
Glavic, A., Silva, F., Aybar, M. J., Bastidas, F., & Mayor, R. (2004). Interplay between Notch signaling and the homeoprotein Xiro1 is required for neural crest induction in Xenopus
embryos. Development, 131, 347–359.
Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., & Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A., 97, 13625–
13630.
Hanna, L. A., Foreman, R. K., Tarasenko, I. A., Kessler, D. S., & Labosky, P. A. (2002). Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes
Dev., 16, 2650–2661.
Hemmati-Brivanlou, A., Kelly, O. G., & Melton, D. A. (1994). Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell,
77, 283–295.
Ikenouchi, J., Matsuda, M., Furuse, M., & Tsukita, S. (2003). Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of
claudins/occludin by Snail. J. Cell Sci., 116, 1959–1967.
Iwashita, T., Kruger, G. M., Pardal, R., Kiel, M. J., & Morrison, S. J. (2003). Hirschsprung disease is linked to defects in neural crest stem cell function. Science, 301, 972–976.
Kaartinen, V., Voncken, J. W., Shuler, C., Warburton, D., Bu, D., Heisterkamp, N., & Groffen, J. (1995). Abnormal lung development and cleft palate in mice lacking TGF-beta 3
indicates defects of epithelial-mesenchymal interaction. Nat. Genet., 11, 415–421.
Kee, Y., & Bronner-Fraser, M. (2005). To proliferate or to die: role of Id3 in cell cycle progression and survival of neural crest progenitors. Genes Dev., 19, 744–755.
Khudyakov, J., & Bronner-Fraser, M. (2009). Comprehensive spatiotemporal analysis of early chick neural crest network genes. Dev. Dyn., 238, 716–723.
Knecht, A. K., Good, P. J., Dawid, I. B., & Harland, R. M. (1995). Dorsal-ventral patterning and differentiation of noggin-induced neural tissue in the absence of mesoderm.
Development, 121, 1927–1935.
Kruger, G. M., Mosher, J. T., Bixby, S., Joseph, N., Iwashita, T., & Morrison, S. J. (2002). Neural crest stem cells persist in the adult gut but undergo changes in self-renewal,
neuronal subtype potential, and factor responsiveness. Neuron, 35, 657–669.
LaBonne, C., & Bronner-Fraser, M. (1998). Neural crest induction in Xenopus: evidence for a two-signal model. Development, 125, 2403–2414.
LaBonne, C., & Bronner-Fraser, M. (1999). Molecular mechanisms of neural crest formation. Annu. Rev. Cell Dev. Biol., 15, 81–112.
Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. N., Stahl, N., Yancopolous, G. D., & Harland, R. M. (1993). Neural induction by the secreted polypeptide
noggin. Science, 262, 713–718.
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L. Z., Cai, C. L., Lu, M. M., Reth, M., Platoshyn, O., Yuan, J. X., Evans, S., & Chien, K. R. (2005).
Postnatal isl1þ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature, 433, 647–653.
Le Douarin, N. (1982). The Neural Crest. Cambridge: Cambridge University Press.
Le Douarin, N., & Kalcheim, C. (1999). The Neural Crest. New York: Cambridge University Press.
Le Douarin, N. M., Brito, J. M., & Creuzet, S. (2007). Role of the neural crest in face and brain development. Brain Res. Rev., 55, 237–247.
Lee, G., Chambers, S. M., Tomishima, M. J., & Studer, L. (2010). Derivation of neural crest cells from human pluripotent stem cells. Nat. Protoc., 5, 688–701.
Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Tabar, V., & Studer, L. (2007). Isolation and directed differentiation of neural crest stem cells derived from
human embryonic stem cells. Nat. Biotechnol., 25, 1468–1475.
Lee, G., Papapetrou, E. P., Kim, H., Chambers, S. M., Tomishima, M. J., Fasano, C. A., Ganat, Y. M., Menon, J., Shimizu, F., Viale, A., Tabar, V., Sadelain, M., & Studer, L. (2009).
Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature, 461, 402–406.
Lefebvre, V., Huang, W., Harley, V. R., Goodfellow, P. N., & De Crombrugghe, B. (1997). SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II)
collagen gene. Mol. Cell. Biol., 17, 2336–2346.
Lewis, J. L., Bonner, J., Modrell, M., Ragland, J. W., Moon, R. T., Dorsky, R. I., & Raible, D. W. (2004). Reiterated Wnt signaling during zebrafish neural crest development.
Development, 131, 1299–1308.
Li, H. Y., Say, E. H., & Zhou, X. F. (2007). Isolation and characterization of neural crest progenitors from adult dorsal root ganglia. Stem Cells, 25, 2053–2065.
Liem, K. F., Jr., Tremml, G., Roelink, H., & Jessell, T. M. (1995). Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell, 82,
969–979.
Light, W., Vernon, A. E., Lasorella, A., Iavarone, A., & LaBonne, C. (2005). Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells.
Development, 132, 1831–1841.
Ludwig, A., Rehberg, S., & Wegner, M. (2004). Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf
transcription factors. FEBS Lett., 556, 236–244.
Marchant, L., Linker, C., Ruiz, P., Guerrero, N., & Mayor, R. (1998). The inductive properties of mesoderm suggest that the neural crest cells are specified by a BMP gradient. Dev.
Biol., 198, 319–329.
Mayor, R., Guerrero, N., & Martinez, C. (1997). Role of FGF and noggin in neural crest induction. Dev. Biol., 189, 1–12.
Mayor, R., Morgan, R., & Sargent, M. G. (1995). Induction of the prospective neural crest of Xenopus. Development, 121, 767–777.
Meulemans, D., & Bronner-Fraser, M. (2004). Gene-regulatory interactions in neural crest evolution and development. Dev. Cell, 7, 291–299.
Monsoro-Burq, A. H., Fletcher, R. B., & Harland, R. M. (2003). Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals. Development, 130,
3111–3124.
Monsoro-Burq, A. H., Wang, E., & Harland, R. (2005). Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction. Dev. Cell, 8, 167–178.
Morrison, S. J., Perez, S. E., Qiao, Z., Verdi, J. M., Hicks, C., Weinmaster, G., & Anderson, D. J. (2000). Transient Notch activation initiates an irreversible switch from neurogenesis
to gliogenesis by neural crest stem cells. Cell, 101, 499–510.
Morrison, S. J., White, P. M., Zock, C., & Anderson, D. J. (1999). Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural
crest stem cells. Cell, 96, 737–749.
Mundell, N. A., & Labosky, P. A. (2011). Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates. Development, 138,
641–652.
Nagoshi, N., Shibata, S., Kubota, Y., Nakamura, M., Nagai, Y., Satoh, E., Morikawa, S., Okada, Y., Mabuchi, Y., Katoh, H., Okada, S., Fukuda, K., Suda, T., Matsuzaki, Y.,
Toyama, Y., & Okano, H. (2008). Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell, 2,
392–403.
Nieto, M. A. (2002). The snail superfamily of zinc-finger transcription factors. Nat. Rev. Mol. Cell Biol., 3, 155–166.
Nikitina, N., Sauka-Spengler, T., & Bronner-Fraser, M. (2008). Dissecting early regulatory relationships in the lamprey neural crest gene network. Proc. Natl. Acad. Sci. U.S.A., 105,
20083–20088.
Nordborg, C., Conradi, N., Sourander, P., & Westerberg, B. (1981). A new type of non-progressive sensory neuropathy in children with atypical dysautonomia. Acta Neuropathol., 55,
135–141.
Obermayr, F., Hotta, R., Enomoto, H., & Young, H. M. (2013). Development and developmental disorders of the enteric nervous system. Nat. Rev. Gastroenterol. Hepatol., 10,
43–57.
Piccolo, S., Sasai, Y., Lu, B., & De Robertis, E. M. (1996). Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell, 86, 589–598.
Proetzel, G., Pawlowski, S. A., Wiles, M. V., Yin, M., Boivin, G. P., Howles, P. N., Ding, J., Ferguson, M. W., & Doetschman, T. (1995). Transforming growth factor-beta 3 is required
for secondary palate fusion. Nat. Genet., 11, 409–414.
Sanford, L. P., Ormsby, I., Gittenberger-De Groot, A. C., Sariola, H., Friedman, R., Boivin, G. P., Cardell, E. L., & Doetschman, T. (1997). TGFbeta2 knockout mice have multiple
developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development, 124, 2659–2670.
Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K., & De Robertis, E. M. (1994). Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes.
Cell, 79, 779–790.
Sato, T., Sasai, N., & Sasai, Y. (2005). Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm. Development, 132, 2355–2363.
Satoda, M., Zhao, F., Diaz, G. A., Burn, J., Goodship, J., Davidson, H. R., Pierpont, M. E., & Gelb, B. D. (2000). Mutations in TFAP2B cause Char syndrome, a familial form of patent
ductus arteriosus. Nat. Genet., 25, 42–46.
Sauka-Spengler, T., & Bronner-Fraser, M. (2008). A gene regulatory network orchestrates neural crest formation. Nat. Rev. Mol. Cell Biol., 9, 557–568.
Sauka-Spengler, T., Meulemans, D., Jones, M., & Bronner-Fraser, M. (2007). Ancient evolutionary origin of the neural crest gene regulatory network. Dev. Cell, 13, 405–420.
Shah, N. M., & Anderson, D. J. (1997). Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness. Proc.
Natl. Acad. Sci. U.S.A., 94, 11369–11374.
Shah, N. M., Groves, A. K., & Anderson, D. J. (1996). Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members. Cell, 85, 331–343.
Shah, N. M., Marchionni, M. A., Isaacs, I., Stroobant, P., & Anderson, D. J. (1994). Glial growth factor restricts mammalian neural crest stem cells to a glial fate. Cell, 77, 349–360.
Shakhova, O., Zingg, D., Schaefer, S. M., Hari, L., Civenni, G., Blunschi, J., Claudinot, S., Okoniewski, M., Beermann, F., Mihic-Probst, D., Moch, H., Wegner, M., Dummer, R.,
Barrandon, Y., Cinelli, P., & Sommer, L. (2012). Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma. Nat. Cell Biol., 14, 882–890.
Streit, A., Lee, K. J., Woo, I., Roberts, C., Jessell, T. M., & Stern, C. D. (1998). Chordin regulates primitive streak development and the stability of induced neural cells, but is not
sufficient for neural induction in the chick embryo. Development, 125, 507–519.
Taneyhill, L. A., Coles, E. G., & Bronner-Fraser, M. (2007). Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest. Development, 134,
1481–1490.
Teng, L., Mundell, N. A., Frist, A. Y., Wang, Q., & Labosky, P. A. (2008). Requirement for Foxd3 in the maintenance of neural crest progenitors. Development, 135, 1615–1624.
Tompers, D. M., Foreman, R. K., Wang, Q., Kumanova, M., & Labosky, P. A. (2005). Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo. Dev. Biol.,
285, 126–137.
Trentin, A., Glavieux-Pardanaud, C., Le Douarin, N. M., & Dupin, E. (2004). Self-renewal capacity is a widespread property of various types of neural crest precursor cells. Proc. Natl.
Acad. Sci. U.S.A., 101, 4495–4500.
Tribulo, C., Aybar, M. J., Nguyen, V. H., Mullins, M. C., & Mayor, R. (2003). Regulation of Msx genes by a Bmp gradient is essential for neural crest specification. Development, 130,
6441–6452.
Wilson, P. A., & Hemmati-Brivanlou, A. (1995). Induction of epidermis and inhibition of neural fate by Bmp-4. Nature, 376, 331–333.
Wong, C. E., Paratore, C., Dours-Zimmermann, M. T., Rochat, A., Pietri, T., Suter, U., Zimmermann, D. R., Dufour, S., Thiery, J. P., Meijer, D., Beermann, F., Barrandon, Y., &
Sommer, L. (2006). Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J. Cell Biol., 175, 1005–1015.
Ye, M., Coldren, C., Liang, X., Mattina, T., Goldmuntz, E., Benson, D. W., Ivy, D., Perryman, M. B., Garrett-Sinha, L. A., & Grossfeld, P. (2010). Deletion of ETS-1, a gene in the
Jacobsen syndrome critical region, causes ventricular septal defects and abnormal ventricular morphology in mice. Hum. Mol. Genet., 19, 648–656.
Yoshida, S., Shimmura, S., Nagoshi, N., Fukuda, K., Matsuzaki, Y., Okano, H., & Tsubota, K. (2006). Isolation of multipotent neural crest-derived stem cells from the adult mouse
cornea. Stem Cells, 24, 2714–2722.
Zammit, P. S., Partridge, T. A., & Yablonka-Reuveni, Z. (2006). The skeletal muscle satellite cell: the stem cell that came in from the cold. J. Histochem. Cytochem., 54,
1177–1191.
Further Reading
Achilleos, A., & Trainor, P. A. (2012). Neural crest stem cells: discovery, properties and potential for therapy. Cell Res., 22, 288–304.
Bailey, C. M., Morrison, J. A., & Kulesa, P. M. (2012). Melanoma revives an embryonic migration program to promote plasticity and invasion. Pigment Cell Melanoma Res., 25,
573–583.
Bellin, M., Marchetto, M. C., Gage, F. H., & Mummery, C. L. (2012). Induced pluripotent stem cells: the new patient? Nat. Rev. Mol. Cell Biol., 13, 713–726.
Bronner, M. E., & Le Douarin, N. M. (2012). Development and evolution of the neural crest: an overview. Dev. Biol., 366, 2–9.
Dupin, E., & Sommer, L. (2012). Neural crest progenitors and stem cells: from early development to adulthood. Dev. Biol., 366, 83–95.
Hall, B. K., & Gillis, J. A. (2013). Incremental evolution of the neural crest, neural crest cells and neural crest-derived skeletal tissues. J. Anat., 222(1), 19–31.
Le Douarin, N. M., & Dupin, E. (2012). The neural crest in vertebrate evolution. Curr. Opin. Genet. Dev., 22, 381–389.
Milet, C., & Monsoro-Burq, A. H. (2012). Embryonic stem cell strategies to explore neural crest development in human embryos. Dev. Biol., 366, 96–99.
Osteoarthritis at the Cellular Level: Mechanisms, Clinical Perspectives, and
Insights From Development
Melanie Fisher, Tyler Ackley, Kelsey Richard, Bridget Oei, and Caroline N Dealy, UConn Health, Farmington, CT, United States
Introduction 660
The Osteoarthritis Epidemic 660
Articular Cartilage Structure 661
Articular Cartilage is Built to Last 662
Mechanisms of Osteoarthritis 662
Multiple OA Phenotypes 662
OA Is a “Whole-Joint” Disease 662
Anabolic Versus Catabolic Phases of OA 663
Chondrocyte Proliferation and Matrix Turnover: Catabolic or Anabolic Signs? 663
Chondrocyte Hypertrophy in OA 664
Articular Cartilage Development 664
Adult Articular Cartilage Progenitor Cells 665
Cell-Based Osteoarthritis Treatments 666
Cartilage Grafts 666
Autologous Chondrocyte Implantation 666
Microfracture 667
MSC Implants 667
MSC Injection 668
Donor Versus Host? 668
Challenges in MSC-Based OA Interventions 669
Alternative Cell-Based Strategies 670
Future Directions 670
References 672
Further Reading 676
Introduction
Osteoarthritis is a painful and debilitating degenerative joint disease present in epidemic proportions worldwide. Osteoarthritis
occurs when the articular cartilage of the joint surfaces degrades and is lost. Although historically, articular cartilage loss has
been attributed to passive wear-and-tear, research informs us that osteoarthritis is an active disease, triggered by injury, inflam-
mation, metabolic disorder, or cellular senescence; and leading to a complex and dynamic series of events that disrupts homeo-
stasis of the entire joint, and ultimately results in net destructive loss of the articular cartilage. Because widespread articular
cartilage loss is likely irreversible, the current clinical focus is to repair focal loss before it progresses to overt osteoarthritis.
Based on the dogma that articular cartilage cannot repair by itself, clinical approaches have attempted articular cartilage repair
using various kinds of exogenous cells, especially progenitor cells, either implanted directly into focal articular cartilage defects,
or even just injected into the damaged joint. Although clinical efficacy of these approaches is not yet unequivocally demon-
strated, trends towards positive outcomes have prompted mechanistic investigation to understand and ultimately optimize
progenitor cell-based interventions as a way to slow or halt osteoarthritic disease. Surprisingly, these studies are revealing
unsuspected potential for endogenous repair capacity by adult articular cartilage in response to as-yet-undefined signals
provided by exogenous progenitor cells. This review discusses recent insights on cellular mechanisms of osteoarthritis; cell-
based interventions in the clinic; and our current understanding of articular cartilage development which is informing growing
appreciation of its natural potential for progenitor-mediated self-repair. The findings that are discussed emphasize the
continued need for basic research to understand how cartilage and joint tissue responds to the triggers that cause osteoarthritis,
and how these responses might be reversed and re-directed to support cell-mediated cartilage restoration instead of cartilage
destruction.
The Osteoarthritis Epidemic

Osteoarthritis is a painful and disabling degenerative joint disease characterized by progressive loss of the articular cartilage, which
is the layer of connective tissue that covers the ends of the long bones, providing a smooth gliding surface and functional weight

Regenerative Engineering j Osteoarthritis at the Cellular Level 661
bearing (Martel-Pelletier et al., 2016). Osteoarthritis has been a problem for humankind for thousands of years, and examination of
the pre-historic record links OA occurrence to the same risk factors experienced today; namely intense physical activity, age, diet,
and/or genetics (Dequeker and Luyten, 2008; Cheverko and Bartelink, 2017; Zhang et al., 2017). Today, OA is present in epidemic
proportions around the world, with an estimated 27–30 million adults affected with OA in the US alone (Lawrence et al., 2008;
Johnson and Hunter, 2014; Cisternas et al., 2016). The incidence of OA is expected to rise to 67 million by 2030 (Hootman
and Helmick, 2006) due to aging of the population (Holt et al., 2011; Anderson and Loeser, 2010) and increasing prevalence of
obesity (Sridhar et al., 2012). Specific groups at particular high risk for osteoarthritis include women, who have a three times higher
osteoarthritis prevalence than men (Johnson and Hunter 2014), athletes (Gouttebarge et al., 2015) and military service members
(Cameron et al., 2016; Showery et al., 2016), and populations with bias for the disease due to genetic ethnicity (Reynard and
Loughlin, 2013). OA is a major cause of disability and decreased quality of life in adults (Ma et al., 2014). The societal loss of
productivity due to pain and disability, along with the health costs of caring for those affected with OA, is estimated at $67–
185 billion annually in the United States (Kotlarz et al., 2009; Losina et al., 2015). Despite intensive basic research and clinical
investigation, the mechanisms that cause OA are still poorly understood, and an effective treatment has yet to be found, making
osteoarthritis an “intractable disease.”
Articular Cartilage Structure

Articular cartilage has a unique structure and physiology that reflects its physical demands (Fox et al., 2009). Normal human
articular cartilage is 2–4 mm thick (Shepherd and Seedhom, 1999); 10% of the tissue is cells (chondrocytes), 10%–25% is
the extracellular matrix the chondrocytes synthesize (Eyre, 2002; Archer and Francis-West, 2003; Quinn et al., 2013; Martel-
Pelletier et al., 2016), and 65%–80% of the tissue is water (Fox et al., 2009). Articular cartilage matrix is rich in collagen (espe-
cially collagen type II), proteoglycans (especially aggrecan), and glycosaminoglycans, especially hyaluronan, a water-retentive
molecule which is responsible for the high water content of articular cartilage (Leyett et al., 2014). The layered structure of artic-
ular cartilage is diagrammed in Fig. 1. The uppermost layer of the articular cartilage is known as the superficial zone, which
comprises about 10%–20% of the total articular cartilage thickness, and contains linearly-arranged collagen fibers interspersed
with occasional flattened chondrocytes. This layer provides a smooth, durable surface for articulation, and also secretes abundant
hyaluronan and lubricin, which are essential for joint lubrication (Seror et al., 2015). The middle-zone comprises 40%–60% of
the articular cartilage. The upper portion of the middle zone contains randomly (isometrically) arranged collagen fibrils, and
rounded chondrocytes that are sparsely but fairly evenly distributed. The lower portion of the middle zone contains linear
collagen fibrils arranged perpendicular to the surface, with rounded chondrocytes arranged in short stacks in alignment with
the fibers (Fox et al., 2009). The extracellular matrix of the middle zone is rich in proteoglycans, especially aggrecan. The
main function of the middle zone is shock absorption. The remaining 30% of the articular cartilage is the deep zone. The
deep zone is divided by the tidemark into an upper hyaline cartilage portion that is continuous with the lower middle zone,
and extends its linear arrangement of collagen fibrils and chondrocyte stacks; and a lower region which is calcified (mineralized).
The mineralized matrix of the deep zone provides a gradual transition in mechanical stiffness of the cartilage tissue as it merges
with the supporting underlying subchondral bone. Hypertrophic chondrocytes, which secrete mineralized matrix, are present in
the deepest region of the deep zone, where it meets the subchondral bone. The junction where cartilage and bone meet is called
the chondro-osseous junction or osteochondral interface. The subchondral bone contains blood vessels and nerves; while the
articular cartilage itself is avascular and aneural (Fox et al., 2009).
Superficial Zone
Middle Zone
Deep Zone
Tidemark
Chondro-osseous Junction
Subchondral Bone
Fig. 1 The structure and cellular features of adult articular cartilage are uniquely designed to create and maintain a tissue with maximal durability
and stability.
662 Regenerative Engineering j Osteoarthritis at the Cellular Level
Articular Cartilage is Built to Last

The natural design of hyaline articular cartilage tissue is optimized for strength, durability, and stability. Some individual structural
components of articular cartilage are so stable that they are essentially permanent. For example, estimates of the half-life of collagen,
the primary structural component of articular cartilage, range from 117 to 400 years (Verzijl et al., 2000; Heinemeier et al., 2016),
and aggrecan, the most abundant proteoglycan in articular cartilage, has a half-life of at least 25 years (Maroudas et al., 1998). The
cross-layered fiber arrangement of articular cartilage gives it great resistance to compressive and torsional forces, while its avascular
nature contributes its very low metabolic state and high overall stability. Clearly, the structure of articular cartilage is meant to
enable it to last the length of a human lifetime, or more. However, it is also apparent that the stability of articular cartilage tissue
comes at the expense of a relative lack of response to insult or injury, since once damaged, articular cartilage has little native ability
to heal itself. The lack of repair response of articular cartilage is what makes osteoarthritis an enormous clinical challenge.
Mechanisms of Osteoarthritis
Multiple OA Phenotypes
It is becoming apparent that osteoarthritis is not a single disease. Multiple clinical OA phenotypes exist, which vary by age at onset,
severity of signs, and rate of degeneration. Idiopathic/primary OA is the classic progressive, chronic disease phenotype associated
with the elderly, and is the most common form of OA, with some estimates placing 70% of the population over the age of 60 as
being affected (Lawrence et al., 2008). Idiopathic/primary OA is the most common form of OA, and has no defined root cause
(hence idiopathic, or “unknown”). Since disease signs appear late in life, and slowly but progressively worsen with time, idio-
pathic/primary OA has been considered an unavoidable consequence of a lifetime of mobility, causing cartilage wear over time.
More likely, idiopathic/primary OA is related to the effects of earlier, un-diagnosed cartilage and joint injury. For instance, in
one study, undiagnosed articular cartilage defects were detected in the majority (60%–67%) of 25,124 patients receiving arthros-
copies, 90% of whom were over age 50 (Widuchowski et al., 2007). The presence of un-diagnosed joint damage in idiopathic/
primary OA is a probable contributing factor to the low-grade, chronic joint inflammation observed in these patients, which is
now recognized as a key factor in OA pathology (Robinson et al., 2016).
A less common but better-understood form of OA is known as post-traumatic osteoarthritis (PTOA) (Anderson et al., 2011). PTOA
comprises about 12% of all OA cases (Brown et al., 2006). PTOA results from a known acute traumatic injury to the joint, typically
sustained in sports, combat or accidents (von Porat et al., 2004). Compared to idiopathic/primary OA, a distinguishing feature of
PTOA is its rapid onset, which is in the range of 10–15 years from injury to severe disease. Another feature of PTOA that distinguishes
it from idiopathic/primary OA is that it occurs in young and otherwise healthy individuals. These features decouple the factor of chro-
nological age as a requisite for joint degeneration. Why then, does PTOA have such a rapid progression? One possibility is that the
rapid onset of PTOA is related to sudden load imbalance caused by the injury, which accelerates wear and leads to faster cartilage
loss (Du et al., 2016; Hsia et al., 2017). Indeed, some cases of PTOA involve discrete injuries to the articular cartilage itself, which serve
as focal sites of further cartilage loss (Martin et al., 2017). However, many PTOA cases can be traced back to ligament and/or meniscal
injury, especially ACL tears. In one study, 80% of patients with traumatic ACL tear progressed to radiographic PTOA within 12–
14 years (Svoboda, 2014). We now know that a major cause of PTOA is the acute inflammatory response that occurs with injury
to the joint (Lieberthal et al., 2015), and that it is likely that the rapidity of disease onset and severity of signs is directly related to
the magnitude of this response. Compelling evidence for inflammation as a key mediator of PTOA is intriguing lack of evidence
that ACL reconstruction after injury improves joint structural outcomes or delays OA onset (Heard et al., 2013; Svoboda, 2014).
What we have learned by comparing the etiology and disease course of idiopathic/primary OA and PTOA is that factors like age
and load-induced wear, which appear to be obvious disease triggers, may not by themselves be directly sufficient to induce OA
disease, nor are they necessarily even required for OA disease to occur. Rather, these factors lead to a change in joint homeostasis
that shifts the balance in the joint towards unstable pathologic phenotypes. This is illustrated when considering the role of obesity
in OA development. At first glance increased load-bearing and accompanying damage to the joints due to weight gain in obese indi-
viduals would logically seem to be responsible for the correlation between rising rates of obesity and rising rates of OA (Sridhar
et al., 2012). However, recent studies suggest that the direct agents of change in obesity-related OA are more likely to be metabolic
disturbances associated with obesity, including metabolic syndrome, diabetes, or even changes in the joint microbiome (Li et al.,
2016a; Mobasheri et al., 2017). In fact, obesity-related OA has been suggested to comprise a third OA phenotype, known as meta-
bolic OA (Kluze et al., 2015). What is common to each of these phenotypes is profound loss of joint homeostasis, causing the cells
of the various tissues of the joint to acquire inappropriate and ultimately harmful behaviors, which eventually converge on irrevers-
ible cartilage loss. It is clear that the modern view of osteoarthritis etiology is much more complex than previously thought, and it
will be necessary to understand how the unique mechanisms underlying each phenotype cause OA, in order to develop successful
approaches to prevent or treat it.
OA Is a “Whole-Joint” Disease
Although osteoarthritis is characterized by degeneration and loss of the articular cartilage (Martel-Pelletier et al., 2016), osteoar-
thritis is a “whole joint” disease, with articular cartilage loss being accompanied by morphological changes to other joint tissues.
For example, after joint injury, the subchondral bone beneath the articular cartilage undergoes rapid changes including increased
resorption leading to loss of trabecular bone, increased vascularization, and transient thinning of the supportive subchondral bone
plate (Botter et al., 2011). The loss of underlying bony support may predispose the articular cartilage to further damage due to
imbalanced load support, and the possible presence of trauma-induced bone microcracks and increased subchondral vasculariza-
tion may facilitate vascular breach of the osteochondral junction, allowing transfer of cartilage-degrading signals from the bone to
the cartilage layer (Goldring, 2012a; Zhen and Cao, 2014; Findlay and Kuliwaba, 2016). In an over-compensatory reaction to the
initial bone loss triggered by joint injury, subsequent changes in bone lead to overall subchondral bone sclerosis (thickening), pres-
ence of subchondral bone cysts (Neogi, 2012) and formation of osteophytes within the joint space (van der Kraan and van den Berg,
2007). Additionally, other joint tissues are also affected in osteoarthritis including mineralization/hardening of ligaments and
menisci (Tsujii et al., 2017); and thickening/hyperplasia of the synovial joint lining (Scanzello and Goldring, 2012). The morpho-
logical changes that characterize OA are mediated by active behaviors on the part of the cells including increased or decreased prolif-
eration or survival; loss of appropriate stable phenotypes, changes in metabolic status, and changes in synthesis of anabolic (tissue
building) or catabolic (tissue destructive) genes and factors (Ramos et al., 2014; Remst et al., 2014; Mueller et al., 2017; Steinberg
et al., 2017). In order to develop rational OA interventions, it will be necessary to understand the nature of these cellular behaviors
during normal joint homeostasis, as well as in response to OA-inducing stimuli, so we can 1 day harness or re-direct these behaviors
towards prevention and/or treatment of OA.
Anabolic Versus Catabolic Phases of OA

Much of our knowledge of OA mechanisms has been gained from studies in animal models of post-traumatic osteoarthritis, in
which disease can be conveniently triggered by a known injury event and followed over a fairly rapid (weeks/months) and predict-
able time course (Little and Hunter, 2013). Many of these models involve surgical transection of the anterior cruciate ligament and/
or other ligaments of the knee, which can be imposed on small animals like mice or rats, or large animals including goats, sheep,
dogs, minipigs or horses (Moran et al., 2016). In rodents, non-invasive, non-surgical, mechanical load-induced ACL rupture or artic-
ular fracture (Christiansen et al., 2015) models are also available. Studies in these PTOA models have revealed a phasic progression
of cell-mediated catabolic (tissue-destructive) and anabolic (tissue-building) events that occur after joint trauma and during disease
progression (Anderson et al., 2011). The first phase of PT-OA, is a transient early and acute catabolic phase, occurring in the days
following joint injury, and characterized by pro-inflammatory macrophage infiltration of the synovium and joint, causing synovial
hyperplasia and release of inflammatory cytokines, and increased expression of degradative enzymes (MMPs) by joint tissues that
mediate cartilage cell death and matrix breakdown. The tissues in the joint that secrete the highest levels of destructive enzymes are
not the articular cartilage itself, but rather the ligaments, tendons, menisci, and synovium (Hausler et al., 2013), emphasizing the
need to consider the whole joint when trying to understand and therapeutically-manipulate OA phenotypes/outcomes. The second
phase in PTOA is a transient anabolic phase in which articular chondrocytes and/or chondroprogenitors proliferate and increase
synthesis of cartilage matrix proteins. The thickness of articular cartilage as a whole even after ACL or ligament disruption increases
slightly and transiently during this phase (Anderson et al., 2011). Unfortunately, however, the anabolic response and increased
articular cartilage thickness it causes is not sufficient to reverse OA progression, and after a few weeks is overcome by a prolonged
and catabolic third phase in which continued joint inflammation and cartilage degradation leads to late-stage progressive articular
cartilage loss accompanied by development of other associated abnormalities such as osteophytes and subchondral bone changes
(Anderson et al., 2011). It is important to note that this phasic disease progression was validated in PTOA models, thus it is not
known to what extent phasic progression of cellular behavior changes occurs in other OA phenotypes. Nonetheless, interventions
that may be common to all phenotypes may include ways to inhibit catabolic responses in the joint (such as blocking acute or
chronic inflammation), or ways to promote anabolic responses (such as stimulating cartilage growth and matrix synthesis).
Chondrocyte Proliferation and Matrix Turnover: Catabolic or Anabolic Signs?

While increased articular cartilage thickness is an obvious beneficial outcome of the transient anabolic phase of PTOA, the cellular
behaviors that precede cartilage thickeningdwhich include cell proliferation and matrix turnoverdhave not always been viewed as
beneficial, or even anabolic. Chondrocyte proliferation is a necessary part of cartilage growth, but in OA, cell proliferation is known
as “chondrocyte cloning” and is considered a morphological hallmark of disease. Chondrocyte cloning is the presence of numerous
clusters of daughter cells, typically located in the middle zone of the degenerating articular cartilage, which are each surrounded by
an immediately-localized region of territorial cartilage matrix synthesized by the dividing cells. Although chondrocyte proliferation
and new matrix synthesis are anabolic cartilage behaviors, aspects of these responses are not exclusively beneficial in articular carti-
lage. For instance, the territorial matrix synthesized in OA chondrocyte clones is different in composition than newly-synthesized
territorial matrix in young, healthy, growing cartilage, in that it is rich in collagen types III and VI, which are otherwise not abundant
articular cartilage collagens (Pullig et al., 1999; Hosseininia et al., 2016). Paradoxically, chondrocyte clones also produce copious
amounts of matrix degradative enzymes such as matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with
Thrombospondin Motifs (ADAMTS), which are known to be major effectors of cartilage matrix degradation (Li et al., 2017a; Yang
et al., 2017). Together, these observations indicate that chondrocyte clones are sites of active, localized, matrix remodeling in OA.
Indeed, the progressive destruction of articular cartilage in OA has been suggested to involve continual catabolic matrix degradation,
coupled with simultaneous but ultimately insufficient anabolic matrix synthesis (Goldring, 2012b).
Chondrocyte Hypertrophy in OA
Another cellular characteristic of OA cartilage is an increased number of hypertrophic chondrocytes in the deepest cartilage layer.
Chondrocyte hypertrophy is a normal chondrocyte fate in the growth plate, where hypertrophic cells carry out the essential func-
tion of synthesizing the mineralized cartilage that will become the substrate for bone deposition by osteoblasts (Sun and Beier,
2014). Growth plate hypertrophic chondrocytes also secrete factors that stimulate vascular entry, which brings clasts/osteoblasts
into the region to remodel the mineralized matrix and replace it with bone. Hypertrophic chondrocytes may even directly differ-
entiate into osteoblasts (Zhou et al., 2014). The role of hypertrophic chondrocytes in articular cartilage, and the role of increased
hypertrophy in OA, is not yet clear (van der Kraan and van den Berg, 2012; Pesesse et al., 2013). In healthy as well as OA articular
cartilage, hypertrophic chondrocytes are present beneath the tidemark, the distinct line that demarcates the upper non-
mineralized cartilage layers from the deepest, mineralized cartilage layer (Fox et al., 2009). One of the characteristics of idio-
pathic/primary OA is that the tidemark line is closer to the surface, which has been attributed to increased thickening of the
underlying mineralized layer and/or increased thickening of the subchondral bone (Deng et al., 2016), both of which are consis-
tent with the presence of greater numbers of hypertrophic chondrocytes below the tidemark. As the function of the mineralized
cartilage layer is to transition mechanical strength in a gradual fashion from relatively flexible cartilage to the stiff underlying
bone, increased tissue mineralization could exacerbate cartilage damage caused by loading (Schultz et al., 2015). Excessive chon-
drocyte hypertrophy in OA might also facilitate the abnormal neovascularization that occurs across the chondro-osseous junc-
tion (Pesesse et al., 2013).
While it is not yet clear if it is a consequence, or a cause, the tendency of chondrocytes within the injured or osteoarthritic joint
to assume unstable hypertrophic phenotypes is a key pathologic feature of articular cartilage disease (Caron et al., 2015; Guidotti
et al., 2015; Yahara et al., 2016). Indeed, a classic morphological sign of OA is formation of osteophytes (bone spurs), which are
preceded by localized regions of inappropriate chondrocyte hypertrophy and subsequent endochondral ossification (Gelse et al.,
2003; van der Kraan and van den Berg, 2007). Inappropriate chondrocyte hypertrophy in OA is not limited to the articular carti-
lage. The fibrocartilage of the menisci also commonly hypertrophies and mineralizes in end-stage idiopathic/primary OA
(Abraham et al., 2014) and in PTOA models (Sun and Mauerhan, 2012). The fibrocartilage of the joint ligament entheses
also undergoes hypertrophy and mineralizes in response to joint destabilizing injury, and the former fibrocartilage cells eventu-
ally make major contributions to formation of osteophytes as OA progresses (Dyment et al., 2015). The presence of excessive
hypertrophic chondrocytes in OA has been suggested to reflect an overall shift from a stable, permanent articular cartilage pheno-
type, to an unstable phenotype which favors inappropriate formation of mineralized tissues and bone within the joint via endo-
chondral ossification.
Articular Cartilage Development
Since chondrocyte hypertrophy, a developmental phenotype, is a key phenotypic switch in OA disease, better understanding of
how joint tissues, including the articular cartilage, form during development may help us decipher the cellular mechanisms that
go awry in osteoarthritis, and may even inform future cellular approaches to repair or regenerate damaged articular cartilage
tissue (Iwamoto et al., 2013). Studies using cell lineage tracing in transgenic mice have revealed that the tissues of the adult joint,
including articular cartilage, tendon, ligament, synovium and menisci, all originate from one embryonic mesenchyme cell pop-
ulation that is collectively known as the “joint interzone” (Archer et al., 2003). The joint interzone comprises the localized region
of mesenchyme in the developing limb that is found in the space between the ends of the early skeletal elements (Archer et al.,
2003). Until recently it was thought that the joint interzone formed as a result of localized cartilage de-differentiation occurring at
sites of presumptive joints along the cartilage models (Hyde et al., 2007). The de-differentiated interzone cells re-acquired
distinct joint tissue fates based on their location within the interzone region, with cells close to the cartilage elements contrib-
uting to articular cartilage, and cells in the middle of the interzone contributing to the rest of the joint tissues (Koyama et al.,
2008). More recent studies have re-explored the process of joint development using sophisticated lineage-tracking approaches,
which have refined our understanding of this complex process and shed new insight on the origin and fate of the cells that
comprise the different joint tissues (Li et al., 2017a,b; Shwartz et al., 2017). While there remains agreement that there is
a common lineage to all of the structures of the joint that is represented by their embryonic shared expression of the marker
GDF5, we now know that the cells that contribute to joint do not predominantly arise from the cartilage model. Rather, the inter-
zone region appears to be continuously populated by cells that migrate into the area from an adjacent, as yet undetermined
source, and which then transit out of the interzone to form the different joint tissues (Shwartz et al., 2017). By pulsing the inter-
zone cells with genetic lineage label at different times, it was shown that early transiting interzone cells contributed mainly to
menisci, ligaments and the epiphyseal cartilage at the ends of the cartilage models (destined to become growth plate, not articular
cartilage). Cells that transited through the interzone later on contributed to articular cartilage, as well as menisci and ligaments,
but not to the epiphysis (Shwartz et al., 2017). Thus, the articular cartilage in the embryo appears to form from a mixture of
growth plate and joint progenitor cells (Shwartz et al., 2017). Surprisingly however, only descendants of the joint progenitor cells
are retained in adult articular cartilage. This was shown by tracing the fate of individual cells in the superficial zone of the devel-
oping articular cartilage (Li et al., 2017a,b). These cells express lubricin, the product of the Prg4 gene, and an essential protein for
lubrication of the joint surfaces (Rhee et al., 2005). The progeny of the Prg4-expressing cells in Prg4-GFP mice all ended up either
in the superficial zone or in the deeper layers of the adult articular cartilage, but none of the cells were found in the adult growth
plate (Li et al., 2017a,b). Conversely, Col2-lineage tagged cells, which were initially co-mingled with Prg-4 expressing cells in the
superficial zone of the articular cartilage, were not found in any region of the adult articular cartilage (Li et al., 2017a,b). The
restriction of growth plate versus articular cartilage lineages may involve constraints provided by local sources of growth factors,
such as BMPs or Wnt (Ray et al., 2015). Intriguingly, close examination of the pattern of cell division by the Prg4-expressing
superficial zone cells indicated that most daughter cells from each division populated the deeper cartilage layers, but some
daughter cells were retained within the superficial zone itself (Li et al., 2017a,b). The behavior of articular cartilage superficial
cells is an example of population asymmetry, where some stem cells can renew themselves symmetrically (one daughter cell
becomes a replacement stem cell, and the other becomes a differentiated cell) and others are lost through asymmetric differen-
tiation (in which both cells become differentiated cells). This behavior is typical for stem cell niches in adult organisms, and
suggests the superficial zone could be a self-renewing articular cartilage stem cell niche (Li et al., 2017a,b). Consistent with
this possibility, the surface zone cells are a slow-cycling population, which is another characteristic of stem cell niches (Hsu
and Fuchs, 2012).
Adult Articular Cartilage Progenitor Cells

The observation that the superficial zone of adult articular cartilage may be a stem cell niche is important, as it suggests that during
adulthood, the superficial zone might be able to supply new chondrocytes to participate in and/or augment cartilage repair.
Whether or not superficial zone cells participate in articular cartilage repair in vivo is not yet clear (Chagin and Medvedeva,
2017). Evidence that superficial zone cells might have capacity to repair articular cartilage is suggested by their ability to readily
undergo chondrogenesis in vitro (Dowthwaite et al., 2004; Hattori et al., 2007; Yu et al., 2014) and their ability to migrate to sites
of cartilage injury (Soel et al., 2012). This was shown in an in vitro study, in which the surface zone progenitors of bovine articular
cartilage explants were fluorescently-labeled prior to subjecting the explant to a cutting injury (Soel et al., 2012). Remarkably, the
labeled superficial zone cells were found to be surprisingly mobile, responding to the injury by undergoing directed chemotaxis
towards the site of damage (Soel et al., 2012). However, in a transgenic Prg4-reporter mouse model, articular cartilage defects
made in the trochlear groove were observed to be filled 7 days after surgery with cells that originated from the synovium, rather
than from the adjacent superficial zone articular cartilage (Decker et al., 2017). Furthermore, surprisingly, ablation of the superficial
zone cells by Prg4-Cre-directed expression of diphtheria toxin in a mouse model was found to improve, rather than exacerbate,
subsequent surgically-induced PTOA, suggesting that in response to injury, superficial zone cells acquire harmful catabolic activities,
rather than helpful anabolic ones (Zhang et al., 2016). Further studies are necessary to define the role, participation, and behavior of
superficial zone cells in articular cartilage repair.
Progenitor-like cells have also been found in other regions of the articular cartilage besides the superficial zone (Alsalameh
et al., 2004; Grogan et al., 2009; Pretzel et al., 2011). These cells typically display mesenchymal stem cell (MSC)dlike features
like multipotency (the ability to differentiate into cartilage, bone or fat lineages); clonicity (an indicator of proliferative
ability) and migratory capacity. For example, the middle (and superficial) zone contains chondrocytes that express the mesen-
chymal stem cell markers CD105 and CD166 (Pretzel et al., 2011). FACS-isolated CD105/CD166 þ cells have been isolated
from both healthy and OA human articular cartilage (Alsalameh et al., 2004; Pretzel et al., 2011), and were found to be
surprisingly abundant, comprising from 5% to 15% of all chondrocytes (Alsalameh et al., 2004; Pretzel et al., 2011). The
CD105/CD166 þ cells migrated on matrices in vitro and displayed enhanced chondrogenic potential in in vitro assays (Alsa-
lameh et al., 2004; Pretzel et al., 2011). Indeed, articular chondrogenic progenitors isolated from equine cartilage not only
had enhanced in vitro chondrogenic capability compared to bone marrow-derived MSC, but also underwent chondrogenesis
without progression to hypertrophy (McCarthy et al., 2012). A population of stem-cell like progenitors has also been isolated
from the deep zone of human osteoarthritic articular cartilage (Koelling et al., 2009). This multipotent population was iso-
lated by its ability to migrate out of OA explants in vitro, and in intact tissue, the cells were observed to enter the articular
cartilage deep zone via vascular breach from the subchondral bone (Koelling et al., 2009). The location of these progenitors in
the deep zone, and the association of their presence with vascularization of this layer during disease, suggests that these
progenitors were likely bone marrow stem cells originating from the subchondral bone, rather than chondroprogenitors orig-
inating from the superficial or middle zones.
Although one explanation for the relative inability of articular cartilage to repair itself could be a lack of a sufficient population of
chondrogenic progenitor cells, several studies have reported that the number of progenitor cells in osteoarthritic cartilage is actually
increased (Alsalameh et al., 2004; Pretzel et al., 2011). However, a subset of the progenitors in osteoarthritic cartilage has been
found to display characteristics of enhanced senescence (aging) including telomere shortening (Fellows et al., 2017), which could
suggest that their regenerative ability is impaired (Fellows et al., 2017). Chondrocyte senescence may result from cumulative oxida-
tive stress causing DNA damage or from disruption of cellular processes involving mitochondrial function which can lead to
apoptosis (Carames et al., 2010). Consistent with this possibility, chondrocytes with senescent characteristics and disrupted mito-
chondrial function accumulate in the articular cartilage of aged mice, consistent with generally decreased regenerative potential of
tissues as aging occurs (Jeon et al., 2017). Adding to the complexity of the role of cellular senescence in articular cartilage degen-
eration is the observation that senescent cells also accumulate in the articular cartilage of the joints of mice after ACL
transection-induced post-traumatic OA, demonstrating that chondrocyte senescence is not just an aging response, but also is an
injury response (Jeon et al., 2017). The complication of senescence among chondroprogenitor cells in osteoarthritis may mean
that selective removal of these cells, and/or suppression of their activity, may be necessary to facilitate repair by endogenous progen-
itors that still retain chondrogenic potential.
There is excitement in the possibility that endogenous progenitor cells naturally present in the joint and/or articular cartilage will
1 day prove to be suitable sources for repair of articular cartilage damage and disease. More research is needed to understand the
activity and nature of these progenitor cells, and the signals that influence them, in order to realize this potential. Meanwhile, since it
is clear that endogenous progenitor cells are not by themselves sufficient to repair articular cartilage damage, current clinical
approaches are focusing on strategies to replace damaged or lost articular cartilage with exogenously-supplied cartilage cells or
progenitors.
Cell-Based Osteoarthritis Treatments
There are several approaches in use and/or under investigation, in which replacement of lost cartilage is attempted with cartilage
grafts or cells. One of the challenges of these approaches is that often, it is only at the end-stage of OA when articular cartilage
loss encompasses large areas of the joint surfaces that patients report to their doctors, who have little to offer at that point other
than total joint replacement with a prosthetic joint. Because the prosthetics have a limited lifespan and revision surgeries tend
to have poor outcomes, patients are encouraged to wait as long as possible before having joint replacement surgery, meanwhile
enduring pain and disability that compromises their quality of life. Accordingly, a way to prevent widespread articular cartilage
loss by correcting it before it spreads would be an important clinical advance. Unfortunately, there are no available biomarker assays
that are validated to detect early cartilage damage. Cell-based repair of focal articular cartilage defects as an approach to prevent or
slow OA progression is therefore only feasible at the present time for patients who have experienced a known traumatic joint injury
and are at risk for PTOA.
Cartilage Grafts
Various kinds of approaches have been developed for articular cartilage repair that use cartilage tissue grafts. In these approaches,
to ensure that the inserted graft does not fall out, the defect in the patient’s articular cartilage is extended by the surgeon down
into the subchondral bone, which provides a better anchoring site than the articular cartilage. The grafted plug consists of both
articular cartilage and underlying bone (known as an “osteochondral graft”), and is inserted and press-fit into the defect. Osteo-
chondral Allograft Transfer System (OATS) is the only procedure available for attempted repair of both large and small articular
cartilage defects. In this procedure, the donor osteochondral plugs are obtained from a cadaver and inserted into the milled defect
in the patient’s articular cartilage. Reports of success of OATS are variable, with a 2015 systematic review of 11 clinical trials of
allografts reporting graft survival of nearly 90% at 5 years, with return to activity and overall good functional outcomes (De Caro
et al., 2015). Problems with the approach include risk of disease transmission from the donor, difficulty in finding matching
histocompatible grafts to minimize tissue rejection, and lack of integration of the cartilage portion of the graft with the adjacent
articular cartilage (De Caro et al., 2015). In the best of cases in which patient follow-up histology was performed, integration with
the articular cartilage was only via a layer of fibrocartilage “grouting”; in the worst cases, there was no cartilage integration at all
(De Caro et al., 2015). In contrast, integration of the bone was typically robust (De Caro et al., 2015). The failure of articular
cartilage integration in these clinical reports is consistent with animal studies showing that osteochondral autografts in sheep
and goats remain as inert structures within the cartilage defects and fail to induce integrative cartilage repair (Lane et al.,
2004; Gelse et al., 2014).
Autologous Chondrocyte Implantation

Because of the problems with graft tissue source and lack of integration at the graft/articular cartilage interface, alternatives to osteo-
chondral grafts have been explored including the use of dissociated articular chondrocytes obtained from the patient’s own carti-
lage, which are implanted into the defect region. This procedure is known as autologous chondrocyte implantation (ACI) and was
approved in 1998 under the name Carticel for the repair of relatively small focal defects in young adult patients. In this approach,
a small biopsy of articular cartilage is harvested arthroscopically from a non-load bearing region of the patient’s own joint, and
digested to release the chondrocytes within, which are then expanded in vitro for 6 weeks, implanted in an open surgery into
the prepared defect, and covered with a periosteal membrane (Madeira et al., 2015). While initial reports following the Carticel
procedure were favorable and histological biopsy suggested formation of hyaline-like cartilage in some cases (McCarthy and Rob-
erts, 2013), issues with durability and persistence of repair have been frequently reported and large, randomized, double bind trials
assessing outcome 14–15 years after ACI have been systematically reviewed with the conclusion that Carticel was not effective in
half of the patients with OA, and in fact more patients in the ACI group ended up needing joint replacement than controls (Knutsen
et al., 2016). Carticel was replaced in 2017 by MACI (Matrix-assisted ACI), a modified procedure in which the chondrocytes are
cultured in a hydrogel sponge, which is then implanted into the defect site using a membrane to secure the cells in place. While
functional improvement after MACI was noted in small trials at 5 years (Eber et al., 2017) and at 15 years (Gille et al., 2016), it
is concerning that in a study examining 150 biopsies taken within 1½ years of the procedure, nearly 28% contained hypertrophic
cells, revealing formation of inappropriate growth plate like cartilage instead of articular cartilage (Eber et al., 2015). A systematic
review of 15 MACI trials noted variations in surgical procedures and post-surgical therapy, but concluded overall that any improve-
ment in function tended to stop improving and decline at 24 months (Shaikh et al., 2017). The clinical disappointment of Carticel/
MACI has been attributed to the propensity of the harvested articular chondrocytes to de-differentiate during in vitro expansion,
even when maintained in the 3D sponge, losing their articular cartilage chondrogenic characteristics, so that they subsequently
form fibrocartilage in vivo, which is structurally weaker than hyaline cartilage, or growth plate cartilage, which tends to form bone.
Microfracture
The ineffectiveness of adult articular chondrocytes as a cell source for cartilage defect repair has prompted a search for alternate cell
sources, with particular interest in progenitor cells with chondrogenic potential, especially mesenchymal stem cells (MSCs). The
most commonly-used source of MSCs is adult bone marrow, and the most direct way in which MSCs have been used for articular
cartilage defect repair is through a procedure known as microfracture (Mithoefer et al., 2009). In this procedure, which can be done
arthroscopically, small holes are punched through the patient’s damaged articular cartilage down into the subchondral bone, allow-
ing blood and bone marrow (containing the MSCs) to flow into the joint, pooling in the holes and eventually filling them with
tissue. Systematic review of 28 clinical studies suggested microfracture offers short term improvement in function for patients
(Mithoefer et al., 2009), however, as is the case with Carticel/MACI, the tissue formed in microfracture is not hyaline cartilage,
but is instead the less-durable fibrocartilage (Mithoefer et al., 2009). Fibrocartilage lacks the proteoglycan-rich matrix characteristic
of hyaline/articular cartilage, and contains high amounts of collagen type I, rather than collagen type II. In the human knee joint,
fibrocartilage is found in the tendons and menisci, but is not normally found in the articular cartilage (Fox et al., 2009). The fibro-
cartilage nature of microfracture repair has been conclusively shown using T2-MRI mapping, which detects the zonal architecture of
collagen fibers (Welsch et al., 2008). Microfracture is still commonly performed in the clinic, and there are reports that encourage its
use (Davatchi et al., 2016; Soler et al., 2016) particularly on a cost: benefit basis (Schrock et al., 2017); however, randomized trials
do not support long-term benefit of the procedure (Knutsen et al., 2016). A recent systematic meta-analysis comparing 3–6 clinical
trials each of OATS, ACI, and microfracture, with a total of 765 patients, at 2-year follow-up, concluded there is no significant differ-
ence in functional outcomes among any of the treatment or control groups (Mundi et al., 2016). A 2017 study compared clinical
effectiveness of ACI compared to microfracture (Mistry et al., 2017) in four randomized, controlled trials. The trials compared
included large studies such as ACTIVE (Autologous Chondrocyte Implantation/Transplantation Versus Existing Treatment); and
SUMMIT (Superiority of Matrix Induced ACI versus MF for treatment of symptomatic articular cartilage defects); with 5-year follow
ups. In one study, MACI gave better outcomes than MF, but in another, there was no difference (Mistry et al., 2017). These examples
illustrate how rigorous statistical comparisons through systematic meta-analysis of large, randomized, double blind, multi-site,
trials make it clear that none of the current clinical approaches (microfracture, OATS and ACI/MACI) are as effective as once believed
(Mistry et al., 2017).
MSC Implants
Although bone marrow is the most commonly-used source of MSCs for clinical use, it actually contains very few MSCsda 1995
study calculated that only 0.01% of cells in a bone marrow aspirate are MSCs (Jones et al., 2002). The relative paucity of MSCs
in bone marrow may be one reason that microfracture forms fibrocartilage instead of hyaline cartilage. To address this, protocols
have been developed to enrich for MSCs using cell surface markers and FACS, or through their predilection to adhere to tissue
culture plastic. Adipose tissue, which is readily obtained and contains more MSCs than bone marrow (Ruetze and Richter,
2014) has been considered as an alternate MSC source to bone marrow. In a recent study, patient outcomes were compared after
microfracture with and without addition of adipose-derived MSCs (Koh et al., 2016). At 2-year follow-up, there were improved
clinical scores (patient-reported pain, mobility) with the adipose-MSC-supplemented group, and a slight improvement in repair
by second-look arthroscopy, and in morphology by tissue biopsy (Koh et al., 2016). Infrapatellar fat pad and cord-blood are
also being investigated as MSC sources (do Amaral et al., 2017); cord blood appear particularly promising but is not that easy
to obtain (Zhang et al., 2011). Another source of MSCs that is gaining attention is the joint synovium (De Bari et al., 2001;
Sakaguchi et al., 2005). Synovial cells are highly chondrogenic (Huang et al., 2017), and since they can be harvested fairly easily
through arthroscopy from the patient’s joint without donor site morbidity, they may offer a clinically-feasible autologous MSC
source for articular cartilage repair (De Sousa et al., 2014)
Hundreds of studies have been carried out testing the repair ability of MSCs obtained from various sources, and implanted into
articular cartilage defects in small animals like rats, mice, and rodents; and in large animals such as the horse, dog, minipig, and
sheep (e.g., Wilke et al., 2007; Ha et al., 2015; Zorzi et al., 2015; Kazemi et al., 2017). Clinical efficacy of MSC-mediated articular
cartilage defect repair has also been investigated in patients, mostly using implants of bone-marrow or adipose-derived MSCs, but
more recently also using synovial-derived MSCs (Bornes et al., 2014; Sekiya et al., 2015). Systematic reviews have attempted to
collate the multiplicity of these studies. In Pastides et al. (2013), 36 preclinical studies including 21 small animal and 15 large
animal, and 15 human clinical trials, were compared, which overall displayed relatively positive outcomes, including positive func-
tional outcomes reported at 12–48 months after MSC implantation in articular cartilage defects in large animals and humans
(Pastides et al., 2013). However, in Bornes et al. (2014), systematic review of 11 human clinical trials produced only limited
evidence showing benefit of MSC implantation in articular cartilage defects in humans (Bornes et al., 2014). Both studies noted
wide variation in cell preparation, surgical implant methodology, presence or absence of scaffolds or growth factors, study design,
follow-ups and criteria for functional outcomes reporting; and called for standardization of approaches so that efficacy of MSC
implants for articular cartilage repair can be clearly examined.
MSC Injection
While implantation of cells directly into articular cartilage defects may offer a therapeutic approach for repair of localized damage,
this approach is not feasible in cases of late stage OA, where cartilage loss is widespread throughout the joint. Interest in developing
a non-invasive (non-surgical) means for articular cartilage restoration in overt OA has prompted consideration of MSCs as an inject-
able therapy delivered directly into the joint space. Some of the first use of intra-articular MSC injections were carried out in veter-
inary practice, where the approach is regularly used on horses and dogs (Cryanoski, 2013). Mostly anecdotal outcomes suggested
benefit of the approach in terms of reducing pain and increasing mobility, encouraging consideration of intra-articular autologous
MSC injections in human OA patients. To date, several clinical trials have been carried out to examine the safety and efficacy of knee
MSC intra-articular injections (Koh et al., 2013; Jo et al., 2014; Afiza and Hu, 2016; Davatchi et al., 2016; Soler et al., 2016; Jo et al.,
2017; Cui et al., 2016), finding them generally safe and leading to short-term improved patient scores on subjective criteria and on
cartilage morphology as assessed by MRI or biopsy. In Koh et al. (2013), 18 OA patients received intra-articular injections of autol-
ogous MSC derived from infrapatellar fat pad, and were followed up at an average of 24 months later, reporting improved pain and
function scores (Koh et al., 2013). In 2016 a study was reported with 15 patients who each received a single injection of bone
marrow derived MSC (Soler et al., 2016). At 6 months there was significant patient reported reductions in pain, and increased func-
tional scores, which persisted for up to 12 months, at which time T2 MRI mapping also revealed signs of cartilage thickening (Soler
et al., 2016). In another 2016 study of four patients with moderate to severe OA, improvement in mobility was noted 6 months after
autologous MSC intra-articular injection, but this improvement declined thereafter, and by 5 years, there was no improvement in
range of motion between the injected or non-injected knee (Davatchi et al., 2016). The only study that employed biopsy as an
outcome was Jo et al. (2014, 2017), in which 18 OA patients with a mean age of 61 years received a single injection of autologous
MSC derived from buttock adipose tissue, at either a high, medium or low dose, and were followed for 2 years. There was significant
improvement in functional scores at 6 months for the high dose, which was paralleled by MRI structural outcome, and biopsies
taken from the region of damage before and after MSC injection showed thickening of the articular cartilage and synthesis of
new matrix following the MSC treatment (Jo et al., 2014). While promising, the biopsies also revealed increased collagen type I
production consistent with potential fibrocartilage formation (Jo et al., 2014). At 12 month follow-up, functional outcomes
declined in the lower dose groups, and improved pain and function scores in the high dose group plateaued until final follow-
up at 24 months. Disturbingly, the size of the defects increased regardless of the treatment dose, some by as much as 78% by 2 years
(Jo et al., 2017), raising concerns over durability of the repair tissue. Since this study had no control group, it is impossible to deter-
mine if the defect size also increased in the absence of MSC treatment (Jo et al., 2017), but this study emphasizes the need for long-
term follow-up and tissue biopsy as monitoring assays
Systematic mega-analyses of studies reporting effects of MSC intra-articular injections for OA treatment have been carried out to
attempt to obtain meaningful consensus on efficacy outcomes (Afiza and Hu, 2016; Cui et al., 2016; Goldberg et al., 2017). A meta-
analysis of 18 clinical studies concluded that MSC treatment generally ameliorated overall outcomes of patients with knee OA,
including pain and functional evaluations, particularly at 12 and 24 months follow-up (Cui et al., 2016). Another systematic review
of clinical studies concluded that safety of MSC intra-articular injections is clearly established (Afiza and Hu, 2016), suggesting that
the focus should now be on optimizing MSC efficacy for OA treatment. However, the largest systematic review to date, which
analyzed 252 studies (100 in vitro studies; 111 animal studies, and 31 clinical studies) on the use of MSCs for cartilage repair
and regeneration as either implant or intra-articular injection, found tremendous variability in the source of MSCs used, their prep-
aration and dose, and choice of allogenic or autologous cells, which combined to make drawing definitive conclusions about the
efficacy of MSC treatment for cartilage restoration difficult, calling for a return to basic science and better communication among
pre-clinical and clinical stakeholders in order to move the field forward in a meaningful way (Goldberg et al., 2017).
Donor Versus Host?

An example of how basic science can (and should) inform the clinic is highlighted by studies in which MSCs that were injected into
the osteoarthritic joints of animals were tracked to determine where they end up and how long they persist. Although this is a logical
question to ask before injecting MSCs into the joints of patients, in actuality it was mainly after results started to surface in the clinic
that analysis of fluorescently or otherwise labeled MSC, which can be tracked in the joints of animals, was rigorously pursued. Some
studies reported robust colonization of the joint following MSC intra-articular injection. For example, in the Harley spontaneous
OA guinea pig model, labeled MSCs in a hyaluronan gel were injected into the diseased joints, and donor MSCs were detected histo-
logically 3 weeks later in the upper and middle zones of the articular cartilage, where they appeared to integrate and make new carti-
lage matrix (Sato et al., 2012). Another study examined distribution of fluorescently-labeled human adipose-derived MSCs injected
into the joints of rats following OA-inducing surgical disruption of the knee ligaments, using IVIS, and found strong signal present
in the joints at 35 days, which diminished markedly thereafter, although histologically, some labeled cells persisted in the menisci
and cartilage for up to 10 weeks (Li et al., 2016a,b). These studies suggested that injected MSCs do have the potential to engraft after
inter-articular injection, however, most studies revealed surprisingly weak engraftment of intra-articularly-injected MSCs, especially
considering that the studies uniformly reported improved articular cartilage healing. For example, in a rat model in which OA was
induced by injection of mono-iodoacetate, signal from radiolabeled MSCs decreased just 3 days after injection, and was virtually
absent 3 weeks later (van Buul et al., 2014). Moreover, in another study, synovial-derived GFP-labeled MSCs were injected into
the joints of a mouse surgical OA model, and were detected in only isolated locations at the injury site at 2 weeks, and not at
all at 4 weeks (Mak et al., 2016). Similarly, dye-labeled MSCs injected into the joints of a mouse surgical OA model were virtually
absent when examined histologically at day 3 and day 7, despite impressive cartilage restoration when examined 21 days later
(Diekman et al., 2013). It is particularly interesting that in most cases, if engraftment of intra-articularly-injected MSCs occurred,
it was most likely to be in non-cartilage joint tissue such as synovium, menisci or ligament. For example in the study by Diekman
et al. (2013), injected MSC appeared only in the synovium, ligaments and menisci, not cartilage; and similarly, in another study,
labeled MSCs injected in the joints of a sheep OA model were found in the synovial lining, dorsal fat pad, and meniscus, but not in
cartilage or bone (Delling et al., 2015).
How then, do we reconcile the inconsistent engraftment of intra-articularly-injected MSCs, with consistent clinical reports of
improved joint function, and in animal studies, even improved cartilage morphology? One possible explanation is that the
injected MSCs are acting as a general suppressor of inflammation. MSCs have long been known as immunomodulatory
(Contreras et al., 2016), and release of anti-inflammatory compounds like IL-10 may serve to suppress the inflammation that
occurs coincident with OA signs, restoring homeostasis within the joint, and establishing an environment which is conducive
to anabolic articular cartilage responses. This activity is consistent with the “whole joint” view of OA disease, and the idea
that tissues other than the articular cartilage are responsible for secreting the harmful signals and factors that create the toxic envi-
ronment within the diseased joint. Indeed, Hausler et al. (2013) showed that ligaments, menisci and especially synovium were
the source of 80% of the degradative enzymes produced in the joint in response to trauma. It is probably not a coincidence that in
the cases where MSC integration is observed in the OA joint after injection, it is within these tissues, raising the possibility that the
MSCs actively “home” to these regions in response to the pro-inflammatory chemokines the tissue express. Consistent with this
possibility, in the study of Li et al. (2016a,b), MSC engraftment after intra-articular injection was weaker in joints that did not
experience surgery, suggesting injury stimulated the engraftment response (Li et al., 2016a,b). Remarkably, a recent study using
a non-human primate (monkey) model suggests MSCs may even be able to home to the surgically-manipulated joint from the
bloodstream (Fernandez-Pernas et al., 2017).
Perhaps the most surprising studies correlating MSC engraftment with cartilage outcome are those that have examined the persis-
tence of donor MSC’s after direct implantation into articular cartilage defects, where homing or retention in the joint are not at issue.
In Ostrander et al. (2001), using presence of the Y-chromosome in male cells as a marker, female rabbit perichondrial-derived MSCs
were implanted into an articular cartilage defect in a male animal, and followed over time. After 28 days, the defect region showed
some healing, but less than 15% of the original population of implanted male donor cells remained (Ostrander et al., 2001). In
another study, rabbit MSCs were labeled with a fluorescent dye and implanted into a full thickness defect within a polymer scaffold.
Formation of new collagen type II-containing tissue was initiated, which contained labeled donor cells, but this tissue was slowly
replaced over a 2-month period by tissue that did not contain labeled donor cells (Tatebe et al., 2005). A similar result was reported
by Niemietz et al. (2014) using human articular chondrocytes implanted into minipig articular cartilage defects, which were 95%
gone after 6 weeks despite repair of the defect region by host cells (Niemietz et al., 2014). Using a sophisticated double transgenic
labeling system, Zwolanek et al. (2017) demonstrated that healing of articular cartilage defects in the joints of rats 6 months
following intra-articular injection of immune-compatible MSCs was exclusively the result of host cells, not donor cells. Thus,
MSC implantation into articular cartilage defects suggest a conundrum similar to that presented by MSC intra-articular injection
into the OA joint: some healing occurs, but few repair cells are present in the healed cartilage tissue. The interpretation is that
the introduced cells are providing a source of factor(s) that somehow facilitates the ability of the host to repair damaged tissue.
In the case of intra-articularly injected MSCs, it is possible that this factor could be related to the immune-suppressive activity of
MSCs (Contreras et al., 2016) which is likely responsible for their chondroprotective activity when injected into the chronically-
osteoarthritic joint. In the case of implanted MSCs, the factor(s) in question could be some type of growth factor that acts in a para-
crine fashion (Xu et al., 2016) to locally recruit and/or simulate cells in the injured region, which subsequently carry out repair.
Challenges in MSC-Based OA Interventions

While MSC-based approaches for future articular cartilage repair or treatment of advanced OA may offer potential, evidence for their
efficacy in the clinic or the lab remains inconsistent, and inconclusive. This is due in part to the inability to systematically compare
published studies in a meaningful way because of tremendous variability in study design, rigor and/or reporting. Some of these
variabilities include the species being studied (human, or large or small animal, and if animal, what model? -spontaneous OA,
ACL rupture or transection, or osteochondral defect); the OA condition (idiopathic/primary OA or injury-induced PTOA); the
delivery approach (intra-articular injection, grafts, scaffolds, gels); and the outcomes (qualitative patient scores, MRI, histology).
Studies examining clinical outcomes tend to use small cohorts which may not have controls, and typically are only a few years
in follow-up. Greater rigor in the form of large, controlled, randomized, multi-center, double-blind clinical trials are needed to
make definitive conclusions. Long-term follow-up over 10–15 years is required to satisfactorily assess durability. A particular chal-
lenge is the need for second-look arthroscopy and tissue biopsy, invasive procedures which are difficult to justify (particularly
biopsy), but which are essential to track and monitor early stages in potential repair that might dictate future directions. Only biopsy
can provide information about the nature of the tissue structure and its molecular profile, in order to conclusively show that the
repair tissue made is comparable to native articular cartilage.
Additional confounders in experimental and clinical studies using MSCs as a stem-cell based OA intervention relate to the nature
and biology of MSCs themselves. Consensus has not emerged on which is the optimal MSC tissue sourcedbone-marrow, adipose
tissue, cord-blood, or synoviumdand each has advantages and disadvantages in terms of chondrogenic potential and availability.
MSC are relatively rare cells, and enrichment is likely necessary to obtain sufficient MSCs from these sources, but selection protocols
use different cell surface markers (if MSC are isolated using FACS) or different passage number (if MSC are isolated via adherence to
tissue culture plastic). The age and health status of the donor is also a factor. MSCs tend to proliferate poorly in vitro, especially
when obtained from older patients (Payne et al., 2010), making it difficult to expand autologous cells to a number sufficient for
clinical use, and the culture conditions used for expansion vary greatly (e.g., different growth factors, cell density, etc.). These issues
result in highly variable MSC preparations from patient to patient, or even from the same patient. A major disadvantage of MSCs is
their inherently unstable nature, and their tendency to undergo fibrochondrogenesis and/or terminal hypertrophic differentiation,
which leads to formation of repair tissue that lacks the necessary durability of true articular cartilage. Ideally, in the future, a source
of MSCs for cartilage repair or OA treatment will be developed that enables generation of large numbers of homogeneous progen-
itors with enhanced chondrogenic potential, uniformly characterized for dose and activity, that could be used by physicians as an
“off-the-shelf” product, perhaps supplied from an allogenic cell bank.
Alternative Cell-Based Strategies

Because of their unlimited capacity for self-renewal, human pluripotent stem cells (embryonic stem cells, ESC, or induced plurip-
otent stem cells, iPSC) could provide a readily obtainable allogenic cell source for articular cartilage repair or OA treatment
(Mobasheri et al., 2014; Lietman, 2016; Murphy et al., 2017). Estimates are surprisingly low of the number of cell lines ( 200)
needed to provide allogenic cells to suit most of world’s MHC haploptypes (Taylor et al., 2005; Nakajima et al., 2007; Lin et al.,
2009). Moreover, protocols have been developed for efficient generation of chondroprogenitor cells from pluripotent stem cells,
including some that create cells with enhanced potential for forming stable cartilage tissue (Gong et al., 2010; Oldershaw et al.,
2010; Sternberg et al., 2012; Koyama et al., 2013; Toh et al., 2010; Yamashita et al., 2015; Guzzo et al., 2014; Craft et al., 2015;
Nam et al., 2017). Preclinical studies in small animals have begun to assess the safety and efficacy of implanting pluripotent-
derived MSCs or chondroprogenitors into articular cartilage defects, and/or injecting them into the joints of OA models, finding
the cells do not form tumors and generally there are reported improvements in cartilage tissue histology (Toh et al., 2010; Uto
et al., 2013; Cheng et al., 2014; Yamashita et al., 2015; Gibson et al., 2016). Several studies have examined persistence of the
human-derived cells, with variable but often little long-term persistence within the joint (Toh et al., 2010; Cheng et al., 2014;
Yamashita et al., 2015; Gibson et al., 2016). This suggests ESC-derived cells may act via a similar paracrine mechanism of action
as suspected for native allogenic or autologous MSCs. Pluripotent-derived chondroprogenitor cells may offer a readily-
obtainable, infinitely-expandable exogenous cell source for OA intervention, with unique advantages including the potential to
differentiate the cells directly into articular-chondrocyte progenitors, and/or to screen and select cell lines with exceptional chon-
drogenic ability. These advantages emphasize that pluripotent-based cell sources, as compared to native MSCs, should continue
to be considered as an alternativedor even potentially preferabledcell source for OA intervention.
Future Directions
As no therapy yet exists to treat widespread, overt OA, the key to OA management will be prevention, by restoring local articular
cartilage defects before they progress to irreversible disease. Current clinical methods for articular cartilage defect repair (OATS, ACI/
MACI, MF) have not met this need. Approaches using progenitor cells such as MSCs, or MSC-derived chondroprogenitors have
some promise, but there is a lack of consistency in reported efficacy studies which makes it difficult to determine which of the
many different approaches is most likely to succeed. The concept of using the body’s own articular chondrocytes to regenerate
lost articular cartilage, rather than just repair it, is exciting; however, it is clear that endogenous cells cannot effectively heal articular
cartilage on their own, and that some stimulating signal(s) is required. The complexity of cellular behaviors suggests that multiple
signals will likely be needed to stimulate native chondroprogenitors to a sufficient extent that a successful regenerative response is
obtained. Until the identity of each of the necessary signals is known, the best way to provide these signals to the articular chon-
droprogenitors in situ may well be through paracrine activity from locally-delivered MSCs, implanted or injected into the joint. In
this regard, it is also essential to consider the environment of the joint and the participation of other joint tissues including syno-
vium, menisci and ligaments as either hostile or nurturing to articular neochondrogenesis. Modifying agents will likely be needed to
restore joint homeostasis before any approach for articular cartilage defect repair or regeneration will be able to proceed successfully.
Delivery of such agents will need to be precisely timed to achieve desired effect, with consideration of the natural anabolic and cata-
bolic phases of OA progression after injury. Until this complex “interactome” of factors, stimuli and modifiers is understood and
defined, there will be a role for exogenous cells to supply the requisite pro-reparative cues. However, it will be necessary to find an
exogenous cell source that is reproducible and predictable in its behavior, so that it can be safely dosed and controlled; as well as
readily obtained, so that costs can be minimized; and a uniform protocol for handling and delivering the cells will need to be estab-
lished and validated, so that the therapy can be “off-the-shelf” and available to all patients.
Serious gaps in our knowledge of articular cartilage biology and development, and the mechanisms by which cartilage
responds to injury, have contributed to establishment of dogmas that have misdirected progress in cartilage repair and
regeneration strategies. Historically, the anecdotal success of MSC injections into the joints of large animals, especially in
veterinary practice, led to trials of autologous MSC-based injections or implantations in human patients even prior to vali-
dation of the approach at the histological level in small animals. The observation that articular cartilage morphology
appeared to be improved after MSC injection or implantation in human or animal studies led to the assumption that the
exogenous MSCs formed the new cartilage tissue, reinforcing a dogma that articular cartilage cannot heal itself, and that
accordingly, any viable cartilage repair treatment would require exogenous cells. It was years before this dogma was ques-
tioned as the result of cell-tracking studies in small animal models that definitively showed that exogenously-introduced cells,
even when implanted directly into the articular cartilage, eventually disappear and are replaced by host cells. This observation
prompted a re-examination of articular cartilage development which in turn revealed unexpected potential lability in the
superficial zone of adult articular cartilagedthereby launching an entire new research direction examining native chondropro-
genitor self-healing. This history illustrates the hazards of pre-conceived bias in mis-guiding and delaying research progress,
and emphasizes the importance of mechanistic understanding and validation gained by basic science, prior to human clinical
testing.
Going forward, and as diagrammed in Fig. 2, continued studies in genomics, development, immunology and cartilage
biology will be needed to fully understand the cellular mechanisms that underlie OA disease. Then, to translate this basic infor-
mation into feasible therapeutic interventions that can be tested in preclinical studies, input from the field of bioengineering
will be needed to develop scaffolds and materials for support or delivery of cells and/or co-factors, and to better monitor and
evaluate cartilage restoration and tissue integrity using biomechanical testing. Careful and rigorous pre-clinical studies will be
essential to test the most promising interventions, with standardization of methods and outcomes so that studies can be mean-
ingfully compared. In particular, there is a need for discussion of what doesn’t work, which is just as important as what
doesdbut often is not reported. Most importantly, in order to efficiently translate research advances to the clinic, better
communication among all stakeholders is needed to integrate knowledge in basic biology and applied engineering towards
the common goal of a preventative therapy, or even a lasting cure, for the many individuals at risk for, or already afflicted
by, osteoarthritis.
RESEARCH
CLINIC
Fig. 2 Input from fields of molecular biology, cartilage biology, and developmental biology are needed to inform research in normal joint homeo-
stasis, osteoarthritis disease mechanisms, and cartilage injury responses, in order to successfully and rationally translate discovery to clinical cures.
References
Abraham, A. V., Pauly, H. M., & Donahue, T. L. H. (2014). Deleterious effects of osteoarthritis on the structure and function of the meniscal enthesis. Osteoarthritis and Cartilage, 22,
275–283.
Afiza, H., & Hu, J. H. P. (2016). Mesenchymal stem cell therapy for osteoarthritis. Journal of Clinical Orthopaedics and Trauma, 7, 177–182.
Alsalameh, S., Amin, R., Gemba, T., & Lotz, M. (2004). Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis and
Rheumatism, 50, 1522–1532.
Anderson, S., & Loeser, R. F. (2010). Why is osteoarthritis an aging disease? Best Practice & Research. Clinical Rheumatology, 24, 15–26.
Anderson, D. D., Chubinskaya, S., Guilak, F., Martin, J. A., Oegema, T. R., Olson, S. A., & Buckwalter, J. A. (2011). Post-traumatic osteoarthritis: Improved understanding and
opportunities for early intervention. Journal of Orthopaedic Research, 29, 802–809.
Archer, C. W., & Francis-West, P. (2003). The chondrocyte. International Journal of Biochemistry and Cell Biology, 35, 401–404.
Archer, C. W., Dowthwaithe, G. P., & Francis-West, P. (2003). Development of synovial joints. Birth Defects Research. Part C, Embryo Today, 69, 144–155.
Bornes, T. D., Adesida, A. B., & Jomha, N. M. (2014). Mesenchymal stem cells in the treatment of traumatic articular cartilage defects: A comprehensive review. Arthritis Research
& Therapy, 16, 432.
Botter, S. M., van Osch, G. J., Clockaerts, S., Waarsing, J. H., Weinans, H., & van Leeuwen, J. P. (2011). Osteoarthritis induction leads to early and temporal subchondral plate
porosity in the tibial plateau of mice: An in vivo microfocal computed tomography study. Arthritis and Rheumatism, 63, 2690–2699.
Brown, T. D., Johnston, R. C., Saltzman, C. L., Marsh, J. L., & Buckwalter, J. A. (2006). Posttraumatic osteoarthritis: A first estimate of incidence, prevalence, and burden of
disease. Journal of Orthopaedic Trauma, 20, 739–744.
Cameron, K. L., Driban, J. B., & Svoboda, S. J. (2016). Osteoarthritis and the tactical athlete: A systematic review. Journal of Athletic Training, 51, 952–961.
Carames, B., Taniquchi, N., Otsuki, S., Blanco, F. J., & Lotz, M. (2010). Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death
and osteoarthritis. Arthritis and Rheumatism, 62, 791–801.
Caron, M. M., Emans, P. J., Surtel, D. A., van der Kraan, P. M., van Rhijn, L. W., & Welting, T. J. (2015). BAPX-1/NKX-3.2 acts as a chondrocyte hypertrophy molecular switch in
osteoarthritis. Arthritis and Rheumatism, 67, 2944–2956.
Chagin, A. S., & Medvedeva, E. V. (2017). Regenerative medicine: Cartilage stem cells identified, but can they heal? Nature Reviews Rheumatology, 13, 522–524.
Cheng, A., Kapacee, Z., Peng, J., Lu, S., Lucas, R. J., Hardingham, T. E., & Kimber, S. J. (2014). Cartilage repair using human embryonic stem cell-derived chondroprogenitors.
Stem Cells Translational Medicine, 3, 1287–1294.
Cheverko, C. M., & Bartelink, E. J. (2017). Resource intensification and osteoarthritis patterns: Changes in activity in the prehistoric Sacramento-San Joaquin Delta region. American
Journal of Physical Anthropology, 164, 331–342.
Christiansen, B. A., Guilak, F., Lockwood, K. A., Olson, S. A., Pitsillides, A. A., Sandell, L. J., Silva, M. J., van der Meulen, M. C., & Haudenschild, D. R. (2015). Non-invasive mouse
models of post-traumatic osteoarthritis. Osteoarthritis and Cartilage, 23, 1627–1638.
Cisternas, M. G., Murphy, L., Sacks, J. J., Solomon, D. H., Pasta, D. J., & Helmick, C. G. (2016). Methods for defining osteoarthritis and the impact on estimating prevalence in a us
population-based survey. Arthritis Care and Research, 68, 574–580.
Contreras, R. A., Figueroa, F. E., Djouad, F., & Luz-Crawford, P. (2016). Mesenchymal stem cells regulate the innate and adaptive immune responses dampening arthritis
progression. Stem Cells International, 2016, 3162743.
Craft, A. M., Rockel, J. S., Nartiss, Y., Kandel, R. A., Alman, B. A., & Keller, G. M. (2015). Generation of articular chondrocytes from human pluripotent stem cells. Nature
Biotechnology, 33, 638–645.
Cryanoski, D. (2013). Stem cells boom in vet clinics. Nature, 496, 148–150.
Cui, G.-H., Wang, Y. Y., Li, C.-J., Shi, C.-H., & Wang, W.-S. (2016). Efficacy of mesenchymal stem cells in treating patients with osteoarthritis of the knee: A meta-analysis.
Experimental and Therapeutic Medicine, 12, 3390–3400.
Davatchi, F., Sadeghi Abdollahi, B., Mohyeddin, M., & Nikbin, B. (2016). Mesenchymal stem cell therapy for knee osteoarthritis: 5 years follow-up of three patients. International
Journal of Rheumatic Diseases, 19, 219–225.
De Bari, C., Dell’ Accio, F., Tylzanowski, P., & Luyten, F. P. (2001). Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis and Rheumatism, 44,
1928e42.
De Caro, F., Bisicchia, S., Amendola, A., & Ding, L. (2015). Large fresh osteochondral allografts of the knee: A systematic clinical and basic science review of the literature. The
Journal of Arthroscopic and Related Surgery, 31, 757–765.
De Sousa, E., Casado, P. L., Neto, V. M., Duarte, M. E. L., & Aguiar, D. P. (2014). Synovial fluid and synovial membrane mesenchymal stem cells: Latest discoveries and therapeutic
perspectives. Stem Cell Research & Therapy, 5, 112.
Decker, R. S., Um, H. B., Dyment, N. A., Cottingham, N., Usami, Y., Enomoto-Iwamoto, M., Kronenberg, M. S., Rowe, D., Maye, P., Koyama, E., & Pacifici, M. (2017). Cell origin,
volume and arrangement are drivers in articular cartilage formation, morphogenesis and response to injury. Developmental Biology, 426, 56–68.
Delling, U., Brehm, W., Metzger, M., Ludewig, E., Winter, K., & Julke, H. (2015). In vivo tracking and fate of intra-articularly injected superparamagnetic iron oxide particle-labeled
multipotent stromal cells in an ovine model of osteoarthritis. Cell Transplantation, 24, 2379–2390.
Deng, B., Wang, F., Yin, L., Chen, C., Guo, L., Chen, H., Gong, X., Li, Y., & Yang, L. (2016). Quantitative study on morphology of calcified cartilage zone in OARSI 0 4 cartilage
from osteoarthritic knees. Current Research in Translational Medicine, 64, 149–154.
Dequeker, J., & Luyten, F. P. (2008). The history of osteoarthritis-osteoarthrosis. Annals of the Rheumatic Diseases, 67, 5–10.
Diekman, B. O., Wu, C. L., Louer, C. R., Furman, B. D., Huebner, J. L., Kraus, V. B., Oslon, S. A., & Guilak, F. (2013). Intra-articular delivery of purified mesenchymal stem cells
from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis. Cell Transplantation, 22, 1395–1408.
do Amaral, R. J. F. C., Almeida, H. V., Kelly, D. J., O’Brien, F. J., & Kearney, C. J. (2017). Infrapatellar fat pad stem cells: From developmental biology to cell therapy. Stem Cells
International, 2017, 6843727.
Dowthwaite, G. P., Bishop, J. C., Redman, S. N., Khan, L. M., Rooney, P., Evans, D. J., Laughton, I., Bayram, Z., Boyer, S., Thomson, B., Wolfe, M. S., & Archer, C. W. (2004). The
surface of articular cartilage contains a progenitor cell population. Journal of Cell Science, 117, 889–897.
Du, G., Zhan, H., Ding, D., Wang, S., Wei, X., Wei, F., Zhang, J., Bilgen, B., Reginato, A. M., Fleming, B. C., Deng, J., & Wei, L. (2016). Abnormal mechanical loading induces
cartilage degeneration by accelerating meniscus hypertrophy and mineralization after ACL injuries in vivo. American Journal of Sports Medicine, 44, 652–663.
Dyment, N. A., Hagiwara, Y., Jiang, X., Huang, J., Adams, D. J., & Rowe, D. W. (2015). Response of knee fibrocartilage to joint destabilization. Osteoarthritis and Cartilage, 23,
996–1006.
Eber, J. R., Smith, A., Fallon, M., Butler, R., Nairn, R., Breidahl, W., & Wood, D. J. (2015). Incidence, degree, and development of graft hypertrophy 24 months after matrix-induced
autologous chondrocyte implantation: Association with clinical outcomes. American Journal of Sports Medicine, 43, 2208–2215.
Eber, J. R., Fallon, M., Wood, D. J., & Janes, G. C. (2017). A prospective clinical and radiological evaluation at 5 years after arthroscopic matrix-induced autologous chondrocyte
implantation. American Journal of Sports Medicine, 45, 59–69.
Eyre, D. (2002). Collagen of articular cartilage. Arthritis Research, 4, 30–35.
Fellows, C. E., Wiliams, R., Davies, I. R., Gohil, K., Baird, D. M., Fairclough, J., Rooney, P., Archer, C. W., & Khan, I. M. (2017). Characterization of a divergent progenitor cell sub-
populations in human osteoarthritic cartilage: The role of telomere erosion and replicative senescence. Science Reports, 7, 41421.
Fernandez-Pernas, P., Rodriguez-Lesende, I., de la Fuente, A., Mateos, J., Fuentes, I., De Toro, J., Blanco, F. J., & Arufe, M. C. (2017). CD105þ-mesenchymal stem cells migrate
into osteoarthritis joint: An animal model. PLoS One, 12, e0188072.
Findlay, D. M., & Kuliwaba, J. S. (2016). Bone-cartilage crosstalk: A conversation for understanding osteoarthritis. Bone Research, 4, 16028.
Fox, A. J. S., Bedi, A., & Rodeo, S. A. (2009). The basic science of articular cartilage: Structure, composition, and function. Sports Health, 1, 461–468.
Gelse, K., Soder, S., Eger, W., Diemtar, T., & Aigner, T. (2003). Osteophyte development-molecular characterization of differentiation stages. Osteoarthritis and Cartilage, 11,
141–148.
Gelse, K., Riedel, D., Pachowsky, M., Hennig, F. F., Trattnig, S., & Welsch, G. H. (2014). Limited integrative repair capacity of native cartilage autografts within defects in a sheep
model. Journal of Orthopaedic Research, 33, 390–397.
Gibson, J. D., O’Sullivan, M. B., Guzzo, R. M., & Drissi, H. (2016). Regeneration of articular cartilage by human ESC-derived mesenchymal progenitors treated sequentially with
BMP-2 and Wnt5a. Stem Cells Translational Medicine, 6, 40–50.
Gille, J., Behrens, P., Schulz, A. P., Oheim, R., & Kienast, B. (2016). Matrix-associated autologous chondrocyte implantation: A clinical follow-up at 15 years. Cartilage, 7, 309–315.
Goldberg, A., Mitchell, K., Soans, J., Kim, L., & Zaidi, R. (2017). The use of mesenchymal stem cells for cartilage repair and regeneration: A systematic review. Journal of
Orthopaedic Surgery and Research, 12, 39.
Goldring, S. R. (2012a). Alterations in periarticular bone and cross talk between subchondral bone and articular cartilage in osteoarthritis. Therapeutic Advances in Musculoskeletal
Disease, 4, 249–258.
Goldring, M. J. (2012b). Articular cartilage degradation in osteoarthritis. Hospital for Special Surgery Journal, 8, 7–9.
Gong, G., Ferrari, D., Dealy, C. N., & Kosher, R. A. (2010). Direct and progressive differentiation of human embryonic stem cells into the chondrogenic lineage. Journal of Cellular
Physiology, 224, 664–671.
Gouttebarge, V., Inklaar, H., Backx, F., & Kerkhoffs, G. (2015). Prevalence of osteoarthritis in former elite athletes: A systematic overview of the recent literature. Rheumatology
International, 35, 405–418.
Grogan, S. P., Miyaki, S., Asahara, H., D’Lima, D., & Lotz, M. K. (2009). Mesenchymal progenitor cell markers in human articular cartilage: Normal distribution and changes in
osteoarthritis. Arthritis Research & Therapy, 11, R85.
Guidotti, S., Minguzzi, M., Platano, D., Cattini, L., Trisolino, G., Mariani, E., & Borzì, R. M. (2015). Lithium chloride dependent glycogen synthase kinase 3 inactivation links
oxidative DNA damage, hypertrophy, and senescence in human articular chondrocytes and reproduces chondrocyte phenotype of obese osteoarthritis patients. PLoS One,
10, e0143865.
Guzzo, R. M., Scanlon, V., Sanjay, A., Xu, R. H., & Drissi, H. (2014). Establishment of human cell type-specific iPS cells with enhanced chondrogenic potential. Stem Cell Reviews,
10, 820–829.
Ha, C.-W., Park, Y.-B., Chung, J.-Y., & Park, Y.-G. (2015). Cartilage repair using composites of human umbilical cord blood-derived mesenchymal stem cells and hyaluronic acid
hydrogel in a minipig model. Stem Cells Translational Medicine, 4, 1044–1051.
Hattori, S., Oxford, C., & Reddi, H. (2007). Identification of superficial zone articular chondrocytes stem/progenitor cells. Biochemical and Biophysical Research Communications,
358, 99–103.
Hausler, C. M., Elsaid, K. A., Fleming, B. C., Proffen, B. L., Johnson, V. M., & Murray, M. M. (2013). Loss of extracellular matrix from articular cartilage is mediated by the synovium
and ligaments after anterior cruciate ligament injury. Osteoarthritis and Cartilage, 21, 1950–1957.
Heard, B. J., Solbak, N. M., Achari, Y., Chung, M., Hart, D. A., Shrive, N. G., & Frank, C. B. (2013). Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL
reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism. Osteoarthritis and Cartilage, 21, 1942–1949.
Heinemeier, K. M., Schjerling, P., Heinemeier, J., Moller, M. B., Krogsgaard, M. R., Grum-Schwensen, T., Petersen, M. M., & Kjaer, M. (2016). Radiocarbon dating reveals minimal
collagen turnover in both healthy and osteoarthritic human cartilage. Science Translational Medicine, 8, 346ra90.
Holt, H. L., Katz, J. N., Reichmann, W. M., Gerlovin, H., Wright, E. A., Hunter, D. J., Jordan, J. M., Kessler, C. L., & Losina, E. A. (2011). Forecasting the burden of advanced knee
osteoarthritis over a 10-year period in a cohort of 60–64 year old US adults. Osteoarthritis and Cartilage, 16, 44–50.
Hootman, J. M., & Helmick, C. G. (2006). Projections of us prevalence of arthritis and associated activity limitations. Arthritis & Rhematology, 54, 226–229.
Hosseininia, S., Weis, M. A., Rai, J., Kim, L., Funk, S., Dahlberg, L. E., & Eyre, D. E. (2016). Evidence for enhanced type III collagen deposition focally in the articular cartilage.
Osteoarthritis and Cartilage, 24, 1029–1035.
Hsia, A. W., Anderson, M. J., Heffner, M. A., Legmav, E. P., Zavodovskaya, R., & Christiansen, B. A. (2017). Osteophyte formation after ACL rupture in mice is associated with joint
restabilization and loss of range of motion. Journal of Orthopaedic Research, 35, 466–473.
Hsu, Y.-C., & Fuchs, E. (2012). A family business: Stem cell progeny join the niche to regulate homeostasis. Nature Reviews Molecular and Cell Biology, 13, 103–114.
Huang, Y.-Z., Xie, H.-Q., Silini, A., Parolini, O., Zhang, Y., Deng, L., & Huang, Y.-C. (2017). Mesenchymal stem/progenitor cells derived from articular cartilage, synovial membrane
and synovial fluid for cartilage regeneration: Current status and future perspectives. Stem Cell Reviews and Reports, 13, 575.
Hyde, G., Dover, S., Aszodi, A., Wallis, G. A., & Boot-Handford, R. P. (2007). Lineage tracing using matrilin-1 gene expression reveals that articular chondrocytes exist as the joint
interzone forms. Developmental Biology, 304, 825–833.
Iwamoto, M., Ohta, Y., Larmour, C., & Enomoto-Iwamoto, E. (2013). Towards regeneration of articular cartilage. Birth Defects Research. Part C, Embryo Today, 99, 192–202.
Jeon, Q., Kim, C., Laberge, R. M., Demaria, M., Rathod, S., Vasserot, A. P., Chung, J.-W., Kim, D.-H., Poon, Y., David, N., Baker, D. J., van deursen, J. M., Campisi, J., & Elisieff, J.
(2017). Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nature Medicine, 23,
775–781.
Jo, C. H., Lee, Y. G., Shin, W. H., Kim, H., Chai, J. W., Jeong, E. C., Kim, J. E., Shim, H., Shin, J. S., Shin, I. S., Ra, J. C., Oh, S., & Yoon, K. S. (2014). Intra-articular injection of
mesenchymal stem cells for the treatment of osteoarthritis of the knee: A proof-of-concept clinical trial. Stem Cells, 32, 1254–1266.
Jo, C. H., Chai, J. W., Jeong, E. C., Oh, S., Shin, J. S., Hackjoon, S., & Yoon, K. S. (2017). Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of
the knee. A two-year follow up story. American Journal of Sports Medicine, 45, 2774–2783.
Johnson, V. L., & Hunter, D. J. (2014). The epidemiology of osteoarthritis. Best Practice & Research. Clinical Rheumatology, 28, 5–15.
Jones, E. A., Kinsey, S. E., English, A., Jones, R. A., Straszynski, L., Meredith, D. M., Markham, A. F., Jack, A., Emery, P., & McGonagle, D. (2002). Isolation and characterization of
bone marrow multipotential mesenchymal progenitor cells. Arthritis and Rheumatism, 46, 3349–3360.
Kazemi, D., Sham, S., Asenjan, K., Dehdilani, N., & Parsa, H. (2017). Canine articular cartilage regeneration using mesenchymal stem on platelet rich fibrin. Bone & Joint Research,
6, 98–107.
Kluze, S., Newton, J. L., & Arden, N. K. (2015). Is osteoarthritis a metabolic disorder? British Medical Bulletin, 115, 111–121.
Knutsen, G., Drogset, J. O., Engebretsen, L., Grontvedt, T., Ludvigsen, T. C., Loken, S., Solheim, E., Strand, T., & Johansen, O. (2016). A randomized multicenter trial comparing
autologous chondrocyte implantations with microfracture: Long-term follow up at 14–15 years. The Journal of Bone and Joint Surgery. American Volume, 98, 1332–1339.
Koelling, S., Kruegel, J., Irmer, M., Path, J. R., Sadowsky, B., Miro, X., & Miosge, N. (2009). Migratory chondrogenic progenitor cells from repair tissue during the later stages of
human osteoarthritis. Cell Stem Cell, 4, 329–335.
Koh, Y.-G., Jo, S.-B., Kwon, O.-R., Suh, D.-S., Lee, S.-W., Park, S.-H., & Choi, Y.-J. (2013). Mesenchymal stem cell injections improve symptoms of knee osteoarthritis.
Arthroscopy, 29, 748–755.
Koh, Y.-G., Kwon, O.-R., Kim, Y.-S., Choi, Y.-J., & Tak, D.-H. (2016). Adipose-derived mesenchymal stem cells with microfracture versus microfracture alone: 2-year follow-up of
a prospective randomized trial. Arthroscopy, 32, 97–109.
Kotlarz, H., Gunnarsson, C. L., Fang, H., & Rizzo, J. A. (2009). Insurer and out-of-pocket costs of osteoarthritis in the US: Evidence from national survey data. Arthritis and
Rheumatism, 60, 3546–5335.
Koyama, E., Shibukawa, Y., Nagayama, M., Sugito, H., Young, B., Yuasa, T., Okabe, T., Ochiai, T., Kamiya, N., Rountree, R. B., Kingsley, D. M., Iwamoto, M., Enomoto-Iwamoto, M.,
& Pacifici, M. (2008). A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis. Developmental Biology,
316, 62–73.
Koyama, N., Miura, M., Nakao, K., Kondo, E., Fujii, T., Taura, D., Kanamoto, N., Sone, M., Yasoda, A., & Arai, H. (2013). Human induced pluripotent stem cells differentiated into
chondrogenic lineage via generation of mesenchymal progenitor cells. Stem Cells and Development, 22, 102–113.
Lane, J. G., Massie, J. B., Ball, S. T., Amiel, M. E., Chen, A. C., Bae, W. C., Sah, R. L., & Amiel, D. (2004). Follow-up of osteochondral plug transfers in a goat model. American
Journal of Sports Medicine, 32, 1440–1450.
Lawrence, R. C., Felson, D. T., Helmick, C. G., Arnold, L. M., Choi, H., Deyo, R. A., Gabriel, S., Hirsch, R., Hochberg, M. C., Hunder, C. G., Jordan, J. M., Katz, J. N.,
Kremers, H. M., Wolfe, F., & National Arthritis Data Workgroup. (2008). Estimates of the prevalence of arthritis and other rheumatic conditions in the Unites States. Part II.
Arthritis and Rheumatism, 58, 26–35.
Leyett, P. A., Hutmacher, D. W., Malda, J., & Klein, T. J. (2014). Hyaluronic acid enhances the mechanical properties of tissue-engineered cartilage constructs. PLoS One, 9,
e113216.
Li, Y., Luo, W., Deng, Z., & Lei, G. (2016a). Diet-intestinal microbiota axis in osteoarthritis: A possible role. Mediators of Inflammation, 2016, 3495173.
Li, M., Luo, X., Ly, X., Liu, V., Zhao, G., Zhang, X., Cao, W., Wang, R., & Wang, W. (2016b). In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular
delivery in a rat osteoarthritis model. Stem Cell Research & Therapy, 7, 160.
Li, H., Wang, D., Yuan, Y., & Min, J. (2017a). New insights on the MMP-13 regulatory network in the pathogenesis of early osteoarthritis. Arthritis Research & Therapy, 19, 248.
Li, L., Newton, P., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qiang, H., Dyachuj, V., Lassar, A. B., Warman, M. L.,
Barenius, B., Adameyko, I., & Chagrin, A. S. (2017b). Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. FASEB
Journal, 31, 1067–1084.
Lieberthal, J., Sambamurthy, N., & Scanzello, C. R. (2015). Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis and Cartilage, 23, 1825–1834.
Lietman, S. A. (2016). Induced pluripotent stem cells in cartilage repair. World Journal of Orthopedics, 7, 149–155.
Lin, G., Xie, Y., Ouvang, Q., Xie, P., Zhou, X., et al. (2009). HLA-matching potential of an established human embryonic stem cell bank in China. Cell Stem Cell, 5, 461–465.
Little, C. B., & Hunter, D. J. (2013). Post-traumatic osteoarthritis: From mouse models to clinical trials. Nature Reviews Rheumatology, 9, 485–497.
Losina, E., Paltiel, A. D., Weinstein, A. M., Yelin, E., Hunter, D. J., Chen, S. P., Klara, K., Suter, L. G., Solomon, D. H., Burbine, S. A., Walensky, R. P., & Katz, J. N. (2015). Lifetime
medical costs of knee osteoarthritis management in the united states: Impact of extending indications for total knee arthroplasty. Arthritis Care and Research, 67, 203–215.
Ma, V. Y., Chan, L., & Carruthers, K. J. (2014). Incidence, prevalence, costs, and impact on disability of common conditions requiring rehabilitation in the United States: Stroke,
spinal cord injury, traumatic brain injury, multiple sclerosis, osteoarthritis, rheumatoid arthritis, limb loss, and back pain. Archives of Physical Medicine and Rehabilitation, 95,
986–995.
Madeira, C., Santhagunam, A., Salguez, J. B., & Cabra, J. M. S. (2015). Advanced cell therapies for articular cartilage regeneration. Trends in Biotechnology, 33, 35–40.
Mak, J., Jablonski, C. L., Leonard, C. A., Dunn, J. F., Raharjo, E., Matyas, J. R., Biernaskie, J., & Krawetz, R. J. (2016). Intra-articular injection of synovial mesenchymal stem cells
improves cartilage repair in a mouse injury model. Scientific Reports, 6, 23076.
Maroudas, A., Bayliss, T., Uchitel-Kaushansky, N., Schniedermann, R., & Gilav, E. (1998). Aggrecan turnover in human articular cartilage: Use of aspartic acid racemization as
a marker of molecular age. Archives of Biochemistry and Biophysics, 350, 61–71.
Martel-Pelletier, J., Barr, A. J., Cicuttini, F. M., Conaghan, P. G., Cooper, C., Goldring, M. B., Goldring, S. R., Jones, G., Teichtahl, A. J., & Pelletier, J. P. (2016). Osteoarthritis.
Nature Reviews. Disease Primers, 2, 16072.
Martin, J. A., Anderson, D. D., Goetz, J. E., Fredericks, D., Pedersen, D. R., Avati, B. P., Marsh, J. L., & Buckwalter, J. A. (2017). Complementary models reveal cellular responses
to contact stresses that contribute to post-traumatic osteoarthritis. Journal of Orthopaedic Research, 35, 515–523.
McCarthy, H. S., & Roberts, S. (2013). A histological comparison of the repair tissue formed when using either Chondrogide or periosteum during autologous chondrocyte
implantation. Osteoarthritis and Cartilage, 21, 2048–2057.
McCarthy, H. E., Bara, J. J., Brakspear, K., Singhrao, S. K., & Archer, C. W. (2012). The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal
cells as potential cell sources for cartilage repair in the horse. The Veterinary Journal, 192, 345–351.
Mistry, H., Connock, M., Pink, J., Shyangdan, D., Clar, C., Royle, P., Court, R., Biant, L. C., Metcalfe, A., & Waugh, N. (2017). Autologous chondrocyte implantation in the knee:
Systematic review and economic evaluation. Health Technology Assessment, 21, 1–294.
Mithoefer, K., McAdams, T., Williams, R. J., Kreuz, P. C., & Mandelbaum, B. R. (2009). Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: An
evidence-based systematic analysis. American Journal of Sports Medicine, 37, 2053–2063.
Mobasheri, A., Kalamegam, G., Musumeci, G., & Batt, M. E. (2014). Chondrocyte and mesenchymal stem cell-based therapies for cartilage repair in osteoarthritis and related
orthopaedic conditions. Maturitas, 78, 188–198.
Mobasheri, A., Rayman, M. P., Gualillo, O., Sellam, J., van der Kraan, P., & Fearon, U. (2017). The role of metabolism in the pathogenesis of osteoarthritis. Nature Reviews
Rheumatology, 13, 302–311.
Moran, C. J., Ramesh, A., Brama, P. A., O’Brien, F. J., & Levingstone, T. J. (2016). The benefits and limitations of animal models for translational research in cartilage repair.
Journal of Experimental Orthopedics, 3, 1.
Mueller, A. J., Canty-Laird, E. G., Clegg, P. D., & Tew, S. R. (2017). Cross-species gene modules emerge from a systems biology approach to osteoarthritis. NPJ Systems Biology
and Applications, 3, 13.
Mundi, R., Bedi, A., Chow, L., Crouch, S., Simunovic, N., Sibilski, E. E., & Ayeno, O. R. (2016). Cartilage restoration in the knee: A simultaneous level 1 studies. American Journal of
Sports Medicine, 44, 1888–1895.
Murphy, C., Mobasheri, A., Tancos, Z., Kobolak, J., & Dinnyes, A. (2017). The potency of induced pluripotent stem cells in cartilage regeneration and osteoarthritis treatment.
Advances in Experimental Medicine and Biology, 22, 1–14. https://doi.org/10.1007/5584_2017_141.
Nakajima, F., Tokunaga, K., & Nakatsuji, N. (2007). Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese
population for use in cell transplantation therapy. Stem Cells, 25, 983–985.
Nam, Y., Rim, Y. A., Jung, S. M., & Hyeon, J. (2017). Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration. Stem Cell
Research & Therapy, 8, 16.
Neogi, T. (2012). Clinical significance of bone changes in osteoarthritis. Arthritis Research & Therapy, 14, S2.
Niemietz, T., Zass, G., Hagmann, S., Diederichs, S., Gotterbarm, T., & Richter, W. (2014). Xenogeneic transplantation of articular chondrocytes into full-thickness articular cartilage
defects in minipigs: Fate of cells and the role of macrophages. Cell and Tissue Research, 358, 749–761.
Oldershaw, R. A., Baxter, M. A., Lowe, E. T., Bates, N., Grady, L. M., Soncin, F., Brison, D. R., Hardingham, T. E., & Kimber, S. J. (2010). Directed differentiation of human
embryonic stem cells toward chondrocytes. Nature Biotechnology, 28, 1187–1194.
Ostrander, R. V., Goomer, R. S., Tontz, W. L., Khatod, M., Harwood, F., Maris, T. M., & Amiel, D. (2001). Donor cell fate in tissue engineering for articular cartilage repair. Clinical
Pastides, P., Chimutengwende-Gordon, M., Maffuli, N., & Khan, W. (2013). Stem cell therapy for human cartilage repair. Osteoarthritis and Cartilage, 21, 646–654.
Payne, K. A., Didiano, D. M., & Chu, C. R. (2010). Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cells. Osteoarthritis and Cartilage,
18, 705–713.
Pesesse, L., Sanchez, C., Delcour, J. P., Bellahcène, A., Baudouin, C., Msika, P., & Henrotin, Y. (2013). Consequences of chondrocyte hypertrophy on osteoarthritis cartilage:
Potential effect on angiogenesis. Osteoarthritis and Cartilage, 21, 1913–1923.
Pretzel, D., Linss, S., Rochler, S., Endres, M., Kaps, C., Alsalameh, S., & Kinee, R. W. (2011). Relative percentage and zonal distribution of mesenchymal progenitor cells in human
osteoarthritic and normal cartilage. Arthritis Research and Therapy, 13, R64.
Pullig, O., Weseloh, G., & Swoboda, B. (1999). Expression of type VI collagen in normal and osteoarthritic human cartilage. Osteoarthritis and Cartilage, 7, 191–202.
Quinn, T. M., Hauselmann, H.-J., Shintani, N., & Hunziker, E. B. (2013). Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-
associated adaptations to biomechanical and anatomical roles. Osteoarthritis and Cartilage, 21, 1904–1912.
Ramos, Y. F. M., den Hollander, W., Bovée, J. V. M. G., Bomer, N., van der Breggen, R., Lakenberg, N., Keurentjes, J. C., Goemann, J. J., Slagboom, P. E., Nelission, R. G. H. H.,
Bos, S. D., & Meulenbelt, I. (2014). Genes involved in the osteoarthritis process identified through genome wide expression analysis in articular cartilage; the RAAK Study. PLoS
One, 9, e103056.
Ray, A., Singh, P. N. R., Sohaskey, M. L., Harland, R. M., & A, B. (2015). Precise spatial restriction of BMP signaling is essential for articular cartilage differentiation. Development,
142, 1169–1179.
Remst, D. F., Blom, A. B., Vitters, E. L., Bank, R. A., van den Berg, W. B., Blaney Davidson, E. N., & van der Kraan, P. M. (2014). Gene expression analysis of murine and human
osteoarthritis synovium reveals elevation of transforming growth factor b-responsive genes in osteoarthritis-related fibrosis. Arthritis and Rheumatism, 66, 647–656.
Reynard, L. N., & Loughlin, J. (2013). Insights from human genetic studies into the pathways involved in osteoarthritis. Nature Reviews Rheumatology, 9, 573–583.
Rhee, D. K., Marcelino, J., Baker, M., Gong, Y., Smits, P., Lefebvre, V., Jay, G. D., Stewart, M., Wang, H., Warman, M. L., & Carpten, J. D. (2005). The secreted glycoprotein
lubricin protects cartilage surfaces and inhibits synovial cell overgrowth. Journal of Clinical Investigation, 115, 622–631.
Robinson, W. H., Lepus, C. M., Wang, Q., Raghu, H., Mao, R., Lindstrom, T. M., & Sokolove, J. (2016). Low-grade inflammation as a key mediator of the pathogenesis of
osteoarthritis. Nature Reviews Rheumatology, 12, 580–592.
Ruetze, M., & Richter, W. (2014). Adipose-derived stromal cells for osteoarticular repair: Trophic function versus stem cell activity. Expert Reviews in Molecular Medicine, 16, e9.
Sakaguchi, Y., Sekiya, I., & Yagishita, M. T. (2005). Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source. Arthritis
and Rheumatism, 52, 2521–2529.
Sato, M., Uchida, K., Nakajima, H., Miyazaki, T., Watanabe, R. S., & Baba, H. (2012). Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea
pig with spontaneous osteoarthritis. Arthritis Research & Therapy, 14, R31.
Scanzello, C. R., & Goldring, S. R. (2012). The role of synovitis in osteoarthritis pathogenesis. Bone, 51, 249–257.
Schrock, J. B., Kraeutler, M. J., Houck, D. A., McQueen, M. B., & McCarty, E. C. (2017). A cost-effectiveness analysis of surgical treatment modalities for chondral lesions of the
knee: Microfracture, osteochondral autograft transplantation, and autologous chondrocyte implantation. Orthopedics, 5, 2325967117704634.
Schultz, M., Molligan, J., Schon, L., & Zhang, Z. (2015). Pathology of the calcified zone of articular cartilage in post-traumatic osteoarthritis in rat knees. PLoS One, 10,
e0120949.
Sekiya, I., Muneta, T., Horie, M., & Koga, H. (2015). Arthroscopic transplantation of synovial stem cells improves clinical outcomes in knees with cartilage defects. Clinical
Seror, J., Zhu, L., Goldberg, R., Day, A. J., & Klein, J. (2015). Supramolecular synergy in the boundary lubrication of synovial joints. Nature Communications, 6, 6497.
Shaikh, N., Seah, M. K. T., & Khan, W. S. (2017). Systematic review on the use of autologous matrix-induced Chondrogenesis for the repair of articular cartilage defects in patients.
World Journal of Orthopedics, 8, 588.
Shepherd, D. E. T., & Seedhom, B. B. (1999). Thickness of human articular cartilage in joints of the lower limb. Annals of the Rheumatic Diseases, 58, 27–34.
Showery, J. E., Kusnezov, N. A., Dunn, J. C., Bader, J. O., Belmont, P. J., Jr., & Waterman, B. R. (2016). The rising incidence of degenerative and posttraumatic osteoarthritis of the
knee in the United States military. Journal of Arthroplasty, 31, 2108–2114.
Shwartz, Y., Viukov, S., Krief, S., & Zelzer, E. (2017). Joint development involves a continuous influx of GDF5-positive cells. Cell Reports, 15, 2577–2587.
Soel, D., McCabe, D. J., Choe, H., Zheng, H., Yu, Y., Jang, K., Walter, M. W., Lehman, A. D., Ding, L., Buckwalter, J. A., & Martin, J. A. (2012). Chondrogenic progenitor cells
respond to cartilage injury. Arthritis and Rheumatism, 64, 3626–3637.
Soler, R., Orozco, L., Munar, A., Huguet, M., López, R., Vives, J., Coll, R., Codinach, M., & Garcia-Lopez, J. (2016). Final results of a phase I-II trial using ex vivo expanded
autologous Mesenchymal Stromal Cells for the treatment of osteoarthritis of the knee confirming safety and suggesting cartilage regeneration. The Knee, 23, 647–654.
Sridhar, M. S., Jarrett, C. D., Xerogeanes, J. W., & Labib, S. A. (2012). Obesity and symptomatic osteoarthritis of the knee. The Journal of Bone and Joint Surgery. British Volume,
94, 433–440.
Steinberg, J., Ritchie, G. R. S., Roumeliotis, T. I., Jayasu, R. L., Clark, M. J., Brooks, R. A., Binch, A. L. A., Shah, K. M., Coyle, R., Pardo, M., Le Maitre, C. L., Ramos, Y. F. M.,
Nelissen, R. G. H. H., Meulenbelt, I., McCaskie, A. W., Choudhary, J. S., Wilkinson, J. M., & Zeggini, E. (2017). Integrative epigenomics, transcriptomics and proteomics of
patient chondrocytes reveal genes and pathways involved in osteoarthritis. Scientific Reports, 7(1), 8935.
Sternberg, H., Murai, J. T., Erickson, I. E., Funk, W. D., Das, S., Wang, Q., Snyder, E., Chapman, K. B., Vangsness, C. T., Jr., & West, M. D. (2012). A human embryonic stem cell-
derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regenerative Medicine, 7, 481–501.
Sun, M. M., & Beier, F. (2014). Chondrocyte hypertrophy in skeletal development, growth, and disease. Birth Defects Research. Part C, Embryo Today, 102, 74–82.
Sun, Y., & Mauerhan, D. R. (2012). Meniscal calcification, pathogenesis and implications. Current Opinion in Rheumatology, 24, 152–157.
Svoboda, S. J. (2014). ACL injury and posttraumatic osteoarthritis. Clinics in Sports Medicine, 33, 633–640.
Tatebe, M., Nakamura, R., Kagami, H., Okada, K., & Ueda, M. (2005). Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy,
7, 520–530.
Taylor, C. J., Bolton, E. M., Pocock, S., Sharples, L. D., Pedersen, R. A., & Bradley, J. A. (2005). Banking on human embryonic stem cells: Estimating the number of donor cell lines
needed for HLA matching. Lancet, 366, 2019–2025.
Toh, W. S., Lee, E. H., Gyo, X. M., Chan, J. K., Yeow, C. H., Choo, A. B., & Cao, T. (2010). Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-
derived chondrogenic cells. Biomaterials, 31, 6968–6980.
Tsujii, A., Nakamura, N., & Horibe, T. (2017). Age-related changes in the knee meniscus. The Knee, 24, 1262–1270.
Uto, S., Nishizawa, S., Takasawa, Y., Asawa, Y., Fujihara, Y., Takato, T., & Hoshi, K. (2013). Bone and cartilage repair by transplantation of induced pluipotent stem cells in murine
joint defect model. Biomedical Research, 34, 281–288.
van Buul, G. M., Siebelt, M., Leijs, M. J., Bos, P. K., Waarsing, J. H., Kops, N., Weinans, H., Verhaar, J. A., Bernsen, M. R., & van Osch, G. J. (2014). Mesenchymal stem cells
reduce pain but not degenerative changes in a mono-iodoacetate rat model of osteoarthritis. Journal of Orthopaedic Research, 32, 1167–1174.
van der Kraan, P. M., & van den Berg, W. B. (2007). Osteophytes: Relevance and biology. Osteoarthritis and Cartilage, 15, 237–244.
van der Kraan, P. M., & van den Berg, W. B. (2012). Chondrocyte hypertrophy and osteoarthritis: Role in initiation and progression of cartilage degeneration. Osteoarthritis and
Cartilage, 20, 223–232.
Verzijl, N., DeGroot, J., Thorpe, S. R., Bank, R. A., Shaw, N., Lyons, T. J., Bijlsma, J. W., Lafeber, F. P. J. G., Baynes, J. W., & TeKoppele, J. M. (2000). Effect of collagen turnover
on the accumulation of advanced glycation end products. Journal of Biological Chemistry, 275, 39027–39031.
von Porat, A., Roose, E. M., & Roose, H. (2004). High prevalence of osteoarthritis 14 years after an anterior cruciate ligament tear in male soccer players: A study of radiographic
and patient relevant outcomes. Annals of the Rheumatic Diseases, 63, 269–273.
Welsch, G. H., Mamisch, T. C., Domayer, S. E., Dorotka, R., KutschaLissberg, F., Marlovits, S., White, L. M., & Trattnig, S. (2008). Cartilage T2 assessment at 3-TMR imaging:
In vivo differentiation of normal hyaline cartilage from reparative tissue after two cartilage repair procedures–initial experience. Radiology, 247, 154–161.
Widuchowski, W., Widuchowski, J., & Trzaska, T. (2007). Articular cartilage defects: Study of 25,124 knee arthroscopies. The Knee, 14, 177–182.
Wilke, M. M., Nydam, D. V., & Nixon, A. J. (2007). Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine
model. Journal of Orthopaedic Research, 25, 913–925.
Xu, L., Wu, Y., Xiong Z Zhou, Y., Ye, Z., & Tan, W.-S. (2016). Mesenchymal stem cells reshape and provoke proliferation of articular chondrocytes by paracrine secretion. Scientific
Reports, 6, 2705.
Yahara, Y., Takemori, H., Okada, M., Kosai, A., Yamashita, A., Kobayashi, T., Fujita, K., Itoh, Y., Nakamura, M., Fuchino, H., Kawahara, N., Fukui, N., Watanabe, A., Kimura, T., &
Tsumaki, N. (2016). Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3. Nature Communications, 7, 10959.
Yamashita, A., Morioka, M., Yahara, Y., Okada, M., Kobayashi, T., Kuriyama, S., Matsuda, S., & Tsumaki, N. (2015). Generation of scaffoldless hyaline cartilaginous tissue from
human iPSCs. Stem Cell Reports, 4, 404–418.
Yang, C. Y., Chanalaris, A., & Troeberg, L. (2017). ADAMTS and ADAM metalloproteinases in osteoarthritisdLooking beyond the “usual suspects”. Osteoarthritis and Cartilage, 25,
1000–1009.
Yu, Y., Zheng, H., Buckwalter, J. A., & Martin, J. A. (2014). Single cell sorting identifies progenitor cell population from full thickness bovine articular cartilage. Osteoarthritis and
Cartilage, 22, 1318e26.
Zhang, X., Hirai, M., Cantero, S., Ciubotariu, R., Dobrila, L., Hirsh, A., Igura, K., Sato, H., Yokomi, I., Nishimura, T., Yamaguchi, S., Yoshimura, K., Rubinstein, P., & Takahashi, T.
(2011). Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: Reevaluation of critical factors for successful isolation and high ability to
proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. Journal of Cellular Biochemistry, 112, 1206–1218.
Zhang, M., Mani, S. B., He, Y., Hall, A. M., Xu, L., Li, Y., Zurakkowski, D., Jay, G. D., & Warman, M. L. (2016). Induced superficial chondrocyte death reduces catabolic cartilage
damage in murine posttraumatic osteoarthritis. Journal of Clinical Investigation, 126, 2893–2902.
Zhang, H., Merrett, D. C., Jing, Z., Tang, J., He, Y., Yue, H., Yue, Z., & Yang, D. Y. (2017). Osteoarthritis, labor division, and occupational specialization of the late Shang
ChinadInsights from Yinxu (ca. 1250-1046 B.C.). PLoS One, 12, e0176329.
Zhen, G., & Cao, X. (2014). Targeting TGF-beta signaling in subchondral bone and articular cartilage homeostasis. Trends in Pharmacological Sciences, 35, 227–236.
Zhou, X., von der Mark, K., Henry, S., Norton, W., Adams, H., & de Crombrugghe, B. (2014). Chondrocytes transdifferentiate into osteoblasts in endochondral bone during
development, postnatal growth and fracture healing in mice. PLoS Genetics, 10, e1004820.
Zorzi, A., Amstalden, E. M. I., Plepis, A. M. G., Martins, V. C. A., Ferretti, M., Antonioli, E., Duarte, A. S. S., Luzo, A. C. M., & Miranda, J. B. (2015). Effect of human adipose tissue
mesenchymal stem cells on the regeneration of ovine articular cartilage. International Journal of Molecular Sciences, 16, 26813–26831.
Zwolanek, D., Satue, M., Proell, V., Godoy, J. R., Odofir, K. I., Flicker, M., Hoffmann, S. C., Rulikce, T., & Erbin, R. G. (2017). Tracking mesenchymal stem cell contributions to
regeneration in an immunocompetent cartilage regeneration model. Journal of Clinical Investigation Insight, 2(20), 87322.
Further Reading
Goldberg, A., Mitchell, K., Soans, J., Kim, L., & Zaidi, R. (2017). The use of mesenchymal stem cells for cartilage repair and regeneration: A systematic review. Journal of
Orthopedic Surgery and Research, 12, 39.
Li, L., Newton, P., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qiang, H., Dyachuj, V., Lassar, A. B., Warman, M. L.,
Barenius, B., Adameyko, I., & Chagrin, A. S. (2017). Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice. FASEB
Journal, 31, 1067–1084.
Jo, C. H., Lee, Y. G., Shin, W. H., Kim, H., Chai, J. W., Jeong, E. C., Kim, J. E., Shim, H., Shin, J. S., Shin, I. S., Ra, J. C., Oh, S., & Yoon, K. S. (2014). Intra-articular injection of
mesenchymal stem cells for the treatment of osteoarthritis of the knee: A proof-of-concept clinical trial. Stem Cells, 32, 1254–1266.
Jo, C. H., Chai, J. W., Jeong, E. C., Oh, S., Shin, J. S., Hackjoon, S., & Yoon, K. S. (2017). Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of
the knee. A two-year follow up story. American Journal of Sports Medicine, 45, 2774–2783.
Carbone, A., & Rodeo, S. (2017). Review of current understanding of post-traumatic osteoarthritis resulting from sports injuries. Journal of Orthopaedic Research, 35, 397–405.
Courties, A., Sellam, J., & Berenbaum, F. (2017). Metabolic syndrome-associated osteoarthritis. Current Opinion in Rheumatology, 29, 214–222.
Feng, C., Luo, X., Xia, H., Lv, X., Zhang, X., Li, D., Wang, F., He, J., Zhang, L., Lin, X., Lin, L., Yin, H., He, J., Wang, J., Cao, W., Wang, R., Zhou, G., & Wang, W. (2017). Efficacy
and persistence of allogeneic adipose-derived mesenchymal stem cells combined with hyaluronic acid in osteoarthritis after intra-articular injection in a sheep model. Tissue
Engineering Part A, 24, 219–233. https://doi.org/10.1089/ten.tea.
Jiang, Y., Cai, Y., Zhang, W., Yin, Z., Hu, C., Tong, T., Lu, P., Zhang, S., Neculai, D., Tuan, R. S., & Ouyeang, H. W. (2016). Human cartilage-derived progenitor cells from
committed chondrocytes for efficient cartilage repair and regeneration. Stem Cells Translational Medicine, 5, 733–744.
Mirando, A. J., Liu, Z., Moore, T., Lang, A., Kohn, A., Osinski, A. M., OKeefe, R. J., Mooney, R. A., Zuscik, M. J., & Hilton, M. J. (2013). RBPjk-dependent notch signaling is
required for articular cartilage and joint maintenance. Arthritis and Rheumatism, 65, 2623–2633.
Ozturk, A., Ozdemir, M. R., & Ozkan, Y. (2006). Osteochondral autografting (mosaicplasty) in grade IV cartilage defects in the knee joint: 2- to 7-year results. International
Orthopaedics, 30, 200–204.
Toh, W. S., & Cao, T. (2014). Derivation of chondrogenic cells from human embryonic stem cells for cartilage tissue engineering. In K. Turksen (Ed.), Methods in molecular biology:
vol. 1307. Human embryonic stem cell protocols. New York, NY: Humana Press.
Reproductive Technologies, Assisted
D Pergament, Case Western Reserve University School of Law, Cleveland, OH, USA; and Children’s Law Group, LLC, Chicago, IL,
USA
Religious Bioethics and ARTs 679

Roman Catholicism 679
Protestantism 679
Judaism 679
Islam 679
Greek Orthodoxy 679
Access to ARTs 680
Access to ARTs for Gays, Lesbians, and Unmarried Persons 680
Access to IVF for Postmenopausal Women 681
Gamete Donation 681
Donor Anonymity 681
Embryo Donation 682
Three-Person IVF 682
Surrogacy 682
Intrafamilial Collaborative Reproduction 683
Fertility Preservation 683
Posthumous Collection and Use of Reproductive Tissues 683
Risks of ARTs 684
Multifetal Pregnancies 684
Birth Defects 684
Preimplantation Genetic Diagnosis 684
References 685
Glossary
Artificial insemination (AI) The deliberate introduction of semen into a female vagina or oviduct for the purpose of achieving
a pregnancy through fertilization by means other than natural insemination.
Assisted reproductive technologies (ARTs) Methods to achieve pregnancy by artificial or partially artificial means.
Autonomy, reproductive Autonomy is a moral, political, and bioethical philosophical construct. Within the context of human
reproduction, it is the capacity of a rational individual to make an informed, uncoerced decision.
Beneficence Beneficence is action that is done for the benefit of others.
Collaborative reproduction Achieving pregnancy with the help of a third party to provide gametes or the uterus; reproduction
involving more than two biogenetic parents.
Cross-border reproductive care The movement of people across national borders in search of fertility and related treatments in
other jurisdictions to obtain services often at lower costs or to circumvent legal restrictions on the treatment being sought. The
terms transnational reproduction, reproductive travel, and procreative tourism are also used.
Cryopreservation Freezing at very low temperatures, such as in liquid nitrogen (196 C), to keep embryos, oocytes, or sperm
viable.
Fertility preservation Fertility preservation is the effort to help patients undergoing treatment with gonadotoxic agents retain
their fertility or ability to procreate. Fertility preservation is also used by women choosing to delay childbearing for social
reasons to extend their fertility.
Gestational carrier or gestational surrogate A woman who carries a pregnancy for another couple or person. The pregnancy is
derived from the egg and sperm of the couple or gamete donors. Although she carries the pregnancy to term, she does not have
a genetic relationship with the resulting child.
Heterologous insemination Refers to artificial insemination in which semen is used from a donor.
Homologous insemination Refers to artificial insemination in which semen is used from the woman’s husband or partner,
also called therapeutic donor insemination.

678 Regenerative Engineering j Reproductive Technologies, Assisted
Human leukocyte antigen (HLA) The major histocompatibility complex in humans. In some diseases treated by
hematopoietic stem cell transplantation, preimplantation genetic diagnosis may be used to assess embryos in an attempt to
conceive a child with matching HLA to serve as donor for a sibling.
Intended parents People who use assisted reproduction technologies to create a child whom they intend to parent, whether or
not they have a genetic or biological relationship to the child; also called contracting or commissioning parents.
Intrafamilial collaborative reproduction Achieving pregnancy with the help of a third party to provide gametes or the uterus
provided by a family member; also called intrafamilial medically assisted reproduction.
Intracytoplasmic sperm injection (ICSI) A micromanipulation procedure in which a single sperm is injected directly into an
oocyte to attempt fertilization, used with male infertility or couples with prior IVF fertilization failure.
In vitro fertilization A process in which an egg and sperm are combined in a laboratory dish to facilitate fertilization. If
fertilized, the resulting embryo is transferred to the uterus.
Microepididymal sperm aspiration (MESA) A microsurgical procedure used to collect sperm in men with blockage of the male
reproductive ducts such as prior vasectomy or absence of the vas deferens. Used in IVF/ICSI procedures.
Multifetal pregnancy reduction A procedure to reduce the number of fetuses in the uterus. This procedure is sometimes
performed on women who are pregnant with multiple fetuses who are at increased risk of late miscarriage or premature labor.
These risks increase with the number of fetuses; also known as selective reduction.
Next-generation DNA sequencing A colloquial term that is used to describe techniques to rapidly compare genetic sequences
among multiple genomes and identify germline and somatic variants of interest, such as single nucleotide polymorphisms
(SNPs), insertions and deletions (indels), copy number variants (CNVs), and other structural variations.
Nonmaleficence Nonmaleficence means to do no harm by refraining from providing ineffective or harmful treatments as
determined by whether the benefits of treatment outweigh the burdens.
Ovarian stimulation The administration of hormone medications that stimulate the ovaries to produce multiple oocytes. This
is sometimes called controlled ovarian hyperstimulation, ovarian induction, or follicular recruitment.
Preeclampsia Is a condition characterized by high blood pressure and significant amounts of protein in the urine of a pregnant
woman. If left untreated, it may develop into eclampsia, a life-threatening occurrence of seizures during pregnancy.
Preimplantation genetic diagnosis (PGD) Refers to the genetic analysis of embryos prior to implantation. It is performed by
an embryologist using a variety of methods for removal of one or two cells from an embryo, which are then screened for genetic
abnormalities or HLA type. PGD is performed in conjunction with IVF.
Surrogacy Is an arrangement in which a woman carries and delivers a child for another couple or person. The surrogate may be
the child’s genetic mother (traditional surrogacy) or she may be genetically unrelated to the child (gestational surrogacy).
Testicular sperm extraction (TESE) Operative removal of testicular tissue in an attempt to collect living sperm for use in an
IVF/ICSI procedure.
Assisted reproductive technologies (ARTs) are methods to achieve pregnancy by artificial or partially artificial means. Although arti-
ficial insemination (AI) had been used in humans since the eighteenth century, infertility was historically an area of medicine with
few treatment options. This dramatically changed on 25 July 1978 with the announcement of the birth in England of a baby girl
conceived through in vitro fertilization (IVF); she is popularly referred to as the world’s first test tube baby. In the decades since,
ARTs have become commonplace technologies. It is estimated that around 5 million babies have been born since the first IVF
baby was born (Sandin et al., 2013). The use of ARTs also enables individuals to extend or to preserve their fertility or single indi-
viduals and same-sex couples to conceive their own biologically related offspring. Other forms of ARTs are used to facilitate preim-
plantation genetic diagnosis (PGD) of embryos.
The development of ARTs raises questions about whether it is proper for science to interfere, and to what degree, with reproduc-
tion. The use of ARTs generates myriad ethical, religious, legal and regulatory, and social issues concerning the nature of human life
and reproduction, justice and equality, as well as genetic and social familial relationships. The use of ARTs separates human concep-
tion from the sexual act, thereby challenging traditional paradigms regarding gender roles and family relationships. The use of
donor gametes and embryos raises questions about family structures and how lineage, inheritance, and citizenship are determined.
In many modern societies, reproductive choices are most often personal decisions that individuals make in accordance with their
own ethics and beliefs. Whether and how ARTs are used in an effort to have a child is affected by a confluence of laws, medical
guidelines, economic factors, and cultural and religious norms. There is wide variability in access to ARTs and the laws regulating
the use of ARTs among different countries and even states/provinces within a single country. In countries with publicly funded
health care systems, reproductive choices, including the use of ARTs, may not be considered purely personal decisions, but may
be viewed as public health issues.
The number and variety of ARTs is remarkable, therefore a detailed discussion of all technologies and the ethical issues that arise
from them is precluded in this article. Accordingly, the focus is on AI, IVF, intracytoplasmic sperm injection (ICSI), cryopreservation,
and PGD.
Regenerative Engineering j Reproductive Technologies, Assisted 679
Religious Bioethics and ARTs
The use of ARTs challenges many religious doctrines governing martial relationships, family formation, and parenthood. The fact
that IVF and other forms of ARTs involve the creation and potential destruction of embryos has also generated discourse on the
moral status of embryos. Religious doctrines play an essential role in considering many bioethical challenges raised by ARTs as these
views play a role in the decisions individuals may make with regard to using ARTs and have influenced legislative and regulatory
responses.
Roman Catholicism
One of the major forces of opposition to ARTs is the Roman Catholic Church. The Catholic Church views embryos as individual
human beings with the right to life and that they should be defended against anything that may threaten their human dignity. The
Catholic Church opposes IVF based on the argument that it separates procreation from the unitive and procreative elements of
reproduction within a marriage. The Catholic Church rejects surrogacy, gamete donation, and posthumous reproduction as contrary
to the values of marriage, the unity of spouses, and the dignity proper to parents and child. The Catholic Church considers homol-
ogous insemination less reprehensible than other ARTs that require the use of donor sperm or a surrogate mother. Yet, even homol-
ogous insemination remains morally unacceptable because of the dissociation of the sexual act from procreation except for
situations in which technical means are not a substitute for the conjugal act but help facilitate conception (Vatican, 1987).
Protestantism
Mainstream Protestant denominations view embryos as having the potential for personhood and therefore deserving of respect. The
Anglican Church supports IVF; however, it takes the position that no embryo should be used, selected against, or destroyed for
trivial reasons. Moreover, the welfare of the conceived child is paramount above the procreative interests of the adults involved.
The major synods of the Lutheran Church generally accept IVF and agree that marriage partners may use IVF to conceive. The Meth-
odist and Presbyterian Churches accept IVF but advocate that embryos should not be created for research or with the intention of
destroying them, but that for those embryos that would have been destroyed, research is permissible. Evangelical and fundamen-
talist Protestants typically believe that embryos are human beings, equate the potential destruction of embryos with abortion, and
advocate for public policies mandating the transfer of excess embryos resulting from IVF to adoptive families.
Judaism
Many rabbinical authorities agree that ARTs are permitted under Jewish law. IVF is widely accepted among Reform, Reconstruc-
tionist, Conservative, and Orthodox branches of Judaism. There are debates among Orthodox rabbinic authorities concerning
whether multifetal pregnancy reduction (MPR) is permitted and when it may be performed (Schenker, 2008). There are also
disputes about gamete donation and surrogacy. Traditional interpretations of Jewish law hold that Jewishness is conferred through
matrilineal descent particularly from the acts of gestating and birthing the baby. Most Orthodox rabbis prefer that non-Jewish donor
sperm be used, to prevent adultery between a Jewish man and a Jewish woman. In Israel, because it is a country with a small pop-
ulation, another concern is preventing genetic incest among the offspring of anonymous donors. This has resulted in widespread use
of sperm imported from other countries. Orthodox authorities who permit surrogacy prefer single Jewish women as surrogates to
avoid the implications of adultery for married surrogate women and to ensure that the baby is Jewish.
Islam
Both Sunni and Shia authorities agree that the use of ARTs should be limited to those with a valid marriage contract. IVF is permitted
using the gametes of a married couple, if the procedure is indicated for a medical reason and performed by an expert physician.
Embryos may be cryopreserved. They are considered the property of the married couple but they can only be transferred to the
woman during the duration of the marital contract. MPR is allowed only if the prospect of carrying a high-order pregnancy is
very small or to protect the mother. The use of homologous AI is accepted as it involves a married couple. There is a significant
difference between Sunni and Shia Islam regarding gamete donation. Sunni authorities prohibit heterologous insemination because
it is outside of the marriage contract. In the late 1990s, some Shia authorities began to permit egg donation under the parameters of
a form of temporary marriage between a man and an unmarried woman that is permissible under some Shia interpretations of
Islamic law (Inhorn, 2006).
Greek Orthodoxy
The Greek Orthodox Church posits that the exact moment human life begins is unknown but views embryos as having a human
identity as a person under development, therefore rejecting embryo research and cyropreservation. The Orthodox Church has
reservations about homologous and heterologous AI and IVF, but does not prohibit married infertile couples from using these
techniques. The Orthodox Church rejects surrogacy, gamete donation, selective fetal reduction, posthumous reproduction, the
use of IVF by postmenopausal women, as well as the use of IVF by same-sex couples and single women, and expresses reser-
vations toward ICSI and PGD (Nikolaos, 2008).
Access to ARTs
Some feminist bioethicists argue that because women carry greater burdens with regard to biological and social reproduction,
bioethical issues surrounding ARTs are essentially debates over women’s rights to bodily integrity and reproductive autonomy.
Related concerns also include how the use of ARTs may affect women’s overall health and what impact ARTs have on women’s
efforts to achieve economic and political equality. Several feminists criticize ARTs as another means of subjugating women by
emphasizing their social roles as mothers. Yet, others view ARTs as useful tools in achieving women’s liberation and autonomy
by allowing them to control their reproduction.
Other bioethicists argue that issues surrounding procreation involve human rights, regardless of the gender, because the desire to
have children and to participate in family life is a fundamental biological and social aspect of being human. Reproductive freedom
or procreative liberty is essentially the individual right to have or avoid having children and to have the information and means to
do so. The right to procreate can be construed as a negative or positive right. As a negative right, it is a right against coercive inter-
ference with decisions regarding procreation and reproduction. As a positive right, it provides an entitlement to assistance in
reproduction.
One of the most obvious and long-standing ethical challenges surrounding ARTs is the inequitable access to treatments and
whether high costs interfere with procreative rights that may include access to ARTs. Some argue that the financial and psychological
costs of infertility enable the development of a fertility industry that profits by exploiting desperate patients, but actually limits their
reproductive autonomy by providing what may be ineffectual and sometimes even dangerous treatments at exorbitant costs.
If there are rights to ARTs, how should potentially scarce resources be allocated becomes an essential question; specifically, does
the state have an obligation to mandate insurance coverage for treatment or fund the use of ARTs through the public health system.
Some argue that access to publicly funded ARTs is justified by the social advantages resulting from facilitating reproductive choice. A
related argument is that state funding of ARTs fulfills the state’s obligation to promote good health, including reproductive health.
Another, more practical, argument is that public funding of ARTs will help halt declining fertility rates and promote the addition of
productive members of society. This may be an increasingly acceptable justification for public funding of ARTs in countries expe-
riencing a negative or flat population growth rate and seeking to develop public policies to alleviate the financial dilemmas of an
aging population (Hoorens et al., 2007).
The funding structure for ARTs is highly variable among different countries. Many European countries with generous public
financing of health care tend to restrict access to ARTs to a greater degree by imposing specific requirements governing age limits
and relationship status, and imposing other restrictions such as refusing treatment to smokers or obese patients. In these countries,
patients denied publicly funded treatment may use private facilities or travel abroad to engage in cross-border reproductive care to
find more affordable options. In the United States, 15 states mandate insurance coverage for some forms of infertility diagnosis and
ARTs and federal tax laws allow deductions for some expenses associated with ARTs. The recently enacted Affordable Care Act does
not clearly mandate infertility treatment as an essential health benefit and individual state health exchanges can decide whether or
not to pay for ARTs (Omurtag and Adamson, 2013).
Access to ARTs for Gays, Lesbians, and Unmarried Persons
Whether it is acceptable for individuals or couples to reproduce regardless of their sexual orientation or marital status raises ques-
tions about the reproductive rights and interests of unmarried individuals and same-sex couples and the value of practicing nondis-
crimination. It also involves questions regarding the welfare of any potential offspring. The answers vary widely throughout the
world and often depend on culturally specific norms regarding single motherhood, homosexuality, and nontraditional families.
Access to ARTs may depend on what types of family units are eligible for publicly funded treatments, whether gamete and embryo
donation and gestational surrogacy is permitted, and whether same-sex civil unions or marriages are recognized. The decision to use
ARTs may also be influenced by the legal status of resulting offspring; for example, some countries reject the argument that the citi-
zenship of the intended parents should determine citizenship and deny citizenship to children born to surrogate mothers in other
countries. Other countries or states/regions only recognize one legal parent in same-sex families. In comparison, other countries or
states/regions have created various legal procedures such as second parent adoptions that result in the legal recognition of both
parents.
In the United States, private providers may claim that the exercise of professional autonomy justifies limiting treatment to
married heterosexual couples. However, professional practice guidelines advise that refusing treatment to patients based solely
on sexual orientation and marital status may constitute illegal discrimination and does not have a sound ethical basis. A long-
standing argument against allowing single individuals and same-sex couples to use ARTs was the potential harm to children if
they are raised in single-parent or same-sex households. Professional guidelines advise that there is no pervasive evidence that
children raised by single parents or gays or lesbians are harmed or disadvantaged by that fact alone. The same factors that
would prevent anyone from receiving services, such as serious doubt about the ability to parent for reasons unrelated to sexual
orientation or the fact that the clinic does not offer anyone a particular service, would be the only justification for denying
treatment to gays, lesbians, or single people (ASRM, 2009a).
Access to IVF for Postmenopausal Women
IVF has allowed women in their 50s and 60s to become pregnant using donor oocytes or embryos. In the near future, there may be
greater numbers of women who use their own cryopreserved eggs to become pregnant after they enter menopause. Although life-
style choices and medical care enable many older women to remain fit and active, statistically speaking, an older mother is more
likely to die sooner than a younger mother.
The use of ARTs by postmenopausal women involves the dichotomy between the right of society to exert control over private
behaviors, including actions undertaken by both the mothers and treatment providers. An analysis considering the ethical principles
of beneficence and nonmalfeasance raises additional questions about the rights of potential offspring. The essential question is
whether the woman’s right to reproductive autonomy is outweighed by any potential suffering that her children may experience
from potentially being left motherless at a young age. This also generates questions about the potential cost to society resulting
from the burden on public funds if the children become dependent on government resources after the death of their primary care-
taker. However, a consideration of the balance of personal rights and costs to society must also address issues related to gender
equality and justice. Some argue that it is not equal or just to deny postmenopausal women access to ARTs if men of the same
age are able to father biological offspring, given the sole difference in their access to parenting results from biological and not social
factors.
Some countries limit public funding of IVF based on age. However, such limits may not resolve any ethical challenges or avoid
the potential burden on public resources created by any resulting and later dependent offspring. These patients may simply turn to
private providers or cross-border reproductive care to obtain IVF and then return to their home countries. In the United States, physi-
cians are permitted to decide whether to offer treatment to postmenopausal patients based on personal judgment. Some may decide
that a healthy and legally competent patient, regardless of age, has an absolute right to reproductive autonomy and that she should
determine the best interest of any of her children. Other treatment providers may use the ethical principles of beneficence and non-
malfeasance to evaluate the risks, burdens, and benefits of all parties including potential offspring.
Gamete Donation
Both men and women choose to donate gametes for many reasons including compensation, altruism, or wanting biological
offspring without the responsibility of raising them. The commodification of human gametes is considered by some as an inher-
ently immoral result of ARTs. This is a position that has been advocated by influential political, religious, and social institutions
that view embryos not as property but human life. While some legislative enactments reflect these arguments, Western jurisprudence
has largely embraced the view that gametes are property.
The use of donor sperm predates the advent of the modern ART revolution by almost a century, as the use of human donor sperm
was reported in the nineteenth century. In the 1980s and the 1990s, innovations in ovulation stimulation and oocyte retrieval made
egg donation possible. There is little controversy over whether men should be compensated as sperm donors; paying small amounts
of compensation to these donors is a long-standing practice. In sharp contrast, the morality of egg donation especially when donors
are paid remains controversial. Both feminists and social conservatives, who usually have diametrically opposite opinions about
reproductive autonomy, have argued that allowing women to profit from their bodies through egg donation is exploitative and
tantamount to prostitution.
A less politically charged explanation for the controversy is that biological differences make oocyte donation riskier. Women
who donate oocytes must undergo IVF and are exposed to the risks of ovarian stimulation and oocyte retrieval. It is a fundamental
ethical prerequisite that oocyte donors participate voluntarily and without coercion or undue influence. Donors may proceed
against their own best interests, given the risks involved, because of the potential for lucrative payments. Although the immediate
risks are well established, information about the long-term physical risks and the emotional benefits and risks of donation is still
being developed. In the absence of robust data regarding the long-term health consequences of IVF, the debate will continue over
whether it is ethical, just, and fair to place young fertile donors at risk for the benefit of older, infertile women.
Donor Anonymity
The ethical consideration of gamete donor anonymity requires an examination of the distinct and sometimes competing interests
and rights of donors, recipients, and offspring. Recipients are interested in having healthy offspring and control over the resulting
childrearing. This includes choosing the gametes that will be used and receiving health information about the donor. They may also
want protection from later involvement with the donor or the ability to control the parameters of that contact. Offspring may be
interested in basic health issues or have more complex concerns about personal identity as information about genetic history is
universally important in the formation of self-identity. Gamete donors are interested in their ability to donate, being protected
during that process, being treated fairly if interests occur, and not having obligations imposed upon them without their consent.
Donors also have an interest in having or not having contact with offspring (Ethics Committee ASRM, 2009b).
Arguments for allowing either donors or their offspring to break anonymity include the potential advantages of sharing medical
information with their genetic offspring, in the case of the donor, or learning about their genetic history directly, in the case of the
offspring. Other arguments include the inherent right or even emotional need to meet and to develop a relationship with a biolog-
ical relative.
The jurisprudence and legislation addressing these issues vary from country to country and parameters governing donor
anonymity are continuing to evolve. Some states and countries have laws that mandate donor registries and create formal systems
of third-party intermediaries. Moreover, as the use of ARTs becomes more prevalent and more children are born from donor
gametes, social conventions regarding the disclosure of donor information and the formation of relationships among recipients,
donors, and offspring will also evolve and may resolve many of these debates.
Embryo Donation
There are four possible approaches to the disposition of surplus embryos that often result from IVF cycles: thawing and discarding,
donating to research, indefinite storage, and donating the embryos for transfer to another woman. All these approaches have
attracted staunch supporters and detractors resulting from long-standing debates over the moral and legal status of embryos.
The use of embryos for research purposes, particularly as it relates to human stem cells and possible cloning, has been the source
of intense debate and has resulted in substantial regulation that varies from nation to nation. Embryo donation is recognized and
regulated in some countries as part of the comprehensive system that regulates all aspects of ARTs. In the United States, the Food and
Drug Administration oversees the process through regulations that apply to all types of donated human tissue.
Three-Person IVF
Researchers are now perfecting mitochondrial transfer (popularly called ‘three-person IVF’) to prevent mitochondrial disease. These
techniques inject DNA from the intended parents into a donor egg with normal mitochondria (mitochondrial DNA (mtDNA)) that
has had its nucleus removed. The resulting embryo carries the DNA from the intended parents and healthy mtDNA from the donor.
Ethical and legal guidelines are currently being developed in the United Kingdom and debated in other countries. Ethical concerns
include the unknown effects of germline DNA alteration on subsequent generations and whether the technique will lead to other
forms of genetic modification (Parliamentary Office of Science and Technology, 2013).
Surrogacy
Although sometimes used as interchangeable terms, there is an important distinction between traditional surrogacy and gestational
surrogacy. Surrogacy is a broad term used to describe situations when a woman agrees to carry a pregnancy for someone else with
the intent of giving custody over to the intended parents. In a traditional surrogacy arrangement, the surrogate is the child’s genetic
mother as she agreed to conceive through AI and deliver a child for the intended parents. In gestational surrogacy, an embryo is
transferred to a woman who has agreed to carry the pregnancy and deliver the child. The embryo may have been created from sperm
and/or oocytes from the intended parents or donor gametes.
Surrogacy arrangements are characterized as altruistic surrogacy or commercial surrogacy. Altruistic surrogacy arrangements
often occur between relatives or friends such as when a mother or a sister carries a pregnancy. Commercial surrogacy is often
arranged by a fertility clinic or another private resource and typically involves the formation of a legal contract between the women
and intended parents. Both surrogates and gestational carriers are subject to medical and emotional risks from carrying a pregnancy
and undergoing a delivery. Commercial and traditional surrogacies have generated the most heated controversy.
Some opponents focus on psychosocial concerns and take issue with the entire concept of surrogacy as contrary to the definition
of motherhood, which they believe cannot be altered by a legal contract. They argue that a legal contract cannot undermine the
biological and psychological attachments formed during pregnancy. The woman carrying the fetus is the mother regardless of
whether donor gametes or embryos were used.
There are some feminist scholars who are proponents of surrogacy believing that it is the ultimate exercise of reproductive
autonomy, but only if the woman retains the right to terminate the pregnancy and revoke any agreement regarding the pregnancy.
These arguments reject the idea that women should be compelled to carry out specific performance elements of any surrogacy
contract such as allowing the intended parents to decide whether a pregnancy should continue or not or whether the woman should
undergo multifetal reduction. This view also rejects prohibitions on surrogacy as sexist and paternalistic.
Others argue that commercial surrogacy results in the commodification of the body and the commercialization of parenthood.
Surrogacy is compared to prostitution without the stigma of prostitution because there is no sexual act. Under this rubric, it is argued
that men will inevitably control the surrogacy process and use it solely for their own economic interests. A corollary argument
focuses on the potential harms to children viewing commercial surrogacy as a form of human trafficking, disrespectful of their
inherent moral value as individual human beings. Another less extreme argument addresses issues of children’s best interests. These
include the harm that may come to a child subject to a custody dispute or continued uncertainty about identity and origin because
of the potential dividing of the genetic, biological, and social roles of mother among three different women.
Regulation of surrogacy varies widely among different countries and even between states or within regions of individual coun-
tries. Some laws facilitate these practices. Other jurisdictions refuse to enforce them, and if a contractual or custody dispute arises,
they will recognize the surrogate as a legal parent. The compensation paid to surrogates also varies throughout the world; some
surrogates are only allowed to receive payments for their medical care and basic living expenses, while other surrogates are also
compensated for carrying the pregnancy.
The differences in regulations and costs have resulted in the development of international surrogacy. Such arrangements typically
involve intended parents from the developed countries and gestational carriers in developing countries. This form of cross-border
reproductive care has intensified already difficult questions about exploitation, commodification, stigmatization, and coercion and
informed consent. These questions are particularly challenging because of significant differences in wealth and bargaining power
between surrogates and intended parents and the ostracism that surrogates may experience in socially conservative cultures that
consider reproduction as only acceptable within marriage.
Intrafamilial Collaborative Reproduction
Intrafamilial collaborative reproduction is when a donor or surrogate is related. Some countries categorically prohibit collaborative
reproduction or impose the ‘anonymity rule’ in the context of collaborative reproduction, meaning that any collaborator should be
unknown to the prospective parents. Therefore, these arrangements are illegal (ASRM, 2012).
In countries that permit such arrangements, the ethical questions that must be addressed include whether there is undue pressure
on family members to participate as a result of emotional or financial dependency. Another concern is the potential that the child
may have identity problems because of growing up in an unconventional familial environment or when there is role confusion or
conflict between genetic and social parents. Other issues include questions about apparent incest or consanguinity. In the United
States, professional guidelines advise that gamete arrangements in which the child would have the same genetic relationship to the
participants as would children of incestuous or consanguineous unions between first-degree relatives (including adopted and step-
children) are considered unethical (ASRM, 2012).
Fertility Preservation
Exposure to gonadotoxic agents during chemotherapy or other medical treatment can jeopardize the prospects of genetic parent-
hood. Patients undergoing these treatments may be offered the chance to preserve their gametes with cryopreservation. This is called
fertility preservation (FP). FP for patients with life-threatening illnesses raises the question whether it is ethical to delay treatment to
obtain reproductive tissues safely. Patients must also confront questions about the disposition of stored gametes, embryos, or
gonadal tissue in the event of death.
Ethical questions also surround the use of FP for pediatric patients because they cannot legally consent to treatment and would
normally not face reproductive decisions until they reach adulthood. Current ethical guidelines recommend that treatment
providers discuss these issues and involve children in deciding on FP. Because only a parent or guardian can consent, they must
determine what is in the child’s best interests related to current treatment and their future as adults. Guidelines also suggest that
parents should consider their child’s opinion, the details of the procedures involved, and whether such procedures are proven or
experimental (ASRM, 2005).
With increasing numbers of women delaying parenthood until their late 30s and 40s, healthy women wishing to guard against
age-related infertility and having the option of delaying childbearing to pursue education and career goals or until they find the right
partner with whom they want to share their life are undergoing FP. This has been termed social egg freezing. The contraceptive tech-
nology revolution gave women unprecedented ability to prevent pregnancies and resulted in many changes in women’s social roles.
The use of FP may similarly challenge existing gender conventions, social structures, and even economic systems. However, FP is
currently only available to women able to pay for IVF and long-term cryopreservation. This will likely result in future ethical
and social issues related to the inequalities in access to this ART.
Posthumous Collection and Use of Reproductive Tissues
Posthumous reproduction typically involves the use of sperm after a man’s death to initiate a pregnancy in his surviving female
partner. Currently, techniques such as stimulated ejaculation, microepididymal sperm aspiration, or testicular sperm extraction
can be used to procure sperm from a dead or brain-dead individual. Physicians have also been asked to harvest oocytes and ovarian
tissue from comatose and brain-dead women for later transfer to a gestational surrogate. These practices raise questions about
whether it is ethical to initiate a pregnancy using reproductive materials from a person who may not have consented to its harvesting
or use.
Posthumous reproductive treatment generates a conflict between the rights of reproductive autonomy of the deceased person
and surviving partner. In the United States, professional guidelines advise that posthumous gamete (sperm or oocyte) procurement
and reproduction are ethically justifiable if written documentation from the deceased authorizing the procedure is available or
when such requests are initiated by the surviving spouse or life partner (ASRM, 2009b). Others reject the right of the spouse to
initiate posthumous reproduction without written documentation stating the deceased person’s wishes even if the posthumous
reproduction will use gametes that the deceased had preserved before death. While the existence of cryopreserved gametes or
embryos demonstrates intent to reproduce, it does clearly demonstrate that the deceased may have wished posthumous reproduc-
tion to occur.
Posthumous reproduction also raises ethical concerns about potential offspring. These questions include whether the circum-
stances surrounding their birth will have any long-term negative consequences for the resulting children’s psychosocial develop-
ment. The number of children who have been conceived in this manner is few in number and many are still in early childhood,
limiting the ability to research this issue; it may be some time before their experiences are fully appreciated.
More practical concerns surround questions about the resulting children’s legal status with regard to lineage, citizenship, rights of
inheritance, and entitlements to government benefit programs. In 2012, the United States Supreme Court addressed some of these
issues in Astrue v. Capato. The Court determined that twins conceived and born after their genetic father’s death were not entitled to
Social Security Survivor benefits based upon an interpretation of a Florida law that did not recognize the children as legal heirs as
they were conceived after his death. This decision is applicable to children conceived from oocytes used from deceased women.
Some states have made changes to their inheritance laws, but there is no uniformity with regard to the rights of posthumously
conceived children.
Risks of ARTs
Multifetal Pregnancies
A major risk of IVF is multifetal pregnancies. They often lead to premature delivery and its accompanying problems, cerebral palsy
and other disabilities, lung and gastrointestinal problems, and even neonatal death. The mother is at risk for hypertension,
preeclampsia, gestational diabetes, and death. To reduce the incidence of multiple gestations, some countries have passed laws
about the number of embryos that can be transferred during IVF cycles. This protocol is called single embryo transfer (SET). In
the United States, there is no legal requirement to use SET and professional guidelines emphasize the right to reproductive
autonomy. Therefore, it is for patients to decide whether to use SET based on consultations with their doctors.
Some women choose to transfer multiple embryos believing that becoming pregnant outweighs the risks or they choose to
undergo MPR. Others reject this procedure because of opposition to abortion. Many argue that either one of these decisions is
a reasonable exercise of reproductive choice. However, even among physicians who perform MPR, controversy surrounds the reduc-
tion of twins. Some believe that twin reduction is unethical because it involves selecting one fetus over another when either one is
equally wanted. In addition, twin reductions are viewed as crossing the ethical line between doing the procedure for medical indi-
cations versus social indications such as to avoid the financial and social stresses of caring for two or more same-aged children.
Birth Defects
There is a general consensus that IVF creates a small but statistically significant increased risk for congenital abnormalities including
rare epigenetic and anatomical abnormalities (Brezina and Zhao, 2012; Hansen et al., 2013). There is also evidence that IVF is asso-
ciated with a small but statistically significant risk of mental retardation and IVF with ICSI performed because of paternal infertility
is associated with a small increase in the relative risk for autism (Sandin et al., 2013). Questions remain as to whether any possible
increased risk is related to the parents’ underlying infertility or is caused by the actual process of IVF (Davies et al., 2012). The exam-
ination of the lasting effects that ARTs may have on early development will continue as the population of people born through IVF
increases and ages.
The uncertainties that continue to surround the use of ARTs will likely not dissuade patients from utilizing them in their efforts to
become parents. Therefore, physicians and patients will continue to struggle with balancing patient autonomy and other ethical
principles such as beneficence and nonmaleficence when deciding to use ARTs. Each will have to consider whether the chance to
parent outweighs the risk of having a child who may have disability, at least in part, because of the means used to achieve the preg-
nancy. The risks associated with ARTs may also generate debate about the public health implications including whether the benefits
of widespread access to ARTs outweigh the potential costs associated with children who may be born with disabilities as a result of
the use of ARTs.
Preimplantation Genetic Diagnosis
PGD is a fusion of genomics and ARTs. It allows would-be parents to screen and select the genetic characteristics of their potential
offspring, to a limited but growing degree. Objections to PGD echo debates over the moral status of embryos. People who believe
that the embryo or fetus is a person also object to creating and destroying embryos including those involved in PGD. Others believe
embryos are too rudimentary in development to have interests or rights but they are entitled to special respect as the first stage
toward human development. Under this view, PGD is ethically acceptable when done for such reasons as preventing offspring
with serious genetic disease or for obviating the need for selective abortion to prevent those diseases.
Other ethical concerns include the use of PGD to screen for adult-onset conditions such as breast cancer that may be life limiting
but might not develop until middle age. Another concern is the use of PGD for sex selection of embryos unrelated to diagnosis of X-
linked genetic disorders but for gender preference or family balancing. Several countries prohibit gender selection for reasons other
than the detection of disease and professional guidelines in the United States discourage the use of PGD solely for nondiagnostic sex
selection. While some ethicists argue that such bans are unwarranted intrusions on reproductive autonomy, others argue that the
principles of equal justice and the need to promote gender equality and ensure a gender balanced population ratio mandate such
restrictions.
Another ethical concern is the use of PGD for the deliberate selection of embryos with disabling conditions (e.g., genetic forms of
deafness or dwarfism) that the parents would share with their offspring and they believe forms a part of their family or cultural
identity. Some ethicists argue that despite the principle of reproductive autonomy, children possess a unique class of rights called
rights in trust – rights that they cannot yet exercise, but which they will be able to exercise when they reach maturity. Based on this
principle, it is unethical for parents to take deliberate steps to create children with disabilities because doing so denies the child’s
right to an open future, which is the right to have available as an adult the widest variety of life choices possible.
Another controversial use of PGD is for the creation of the so-called ‘savior siblings.’ In these cases, the aim is to create embryos
without the same disorder that are human leukocyte antigen matches for an existing child that requires stem cell or bone marrow
transplant. Ethical issues include whether it is ethical to create a child not just for himself or herself but with the responsibility to
save a sibling. There is the related concern that the parents may prioritize the interests of the first child over the second and allow that
child to suffer physically and/or emotionally.
Some argue that PGD for ‘savior siblings’ creates a slide toward the acceptance of screening embryos for nonmedical traits with
the aim of selecting better children. Some believe that emerging technologies such as the recent introduction of next-generation
DNA sequencing for PGD could result in screening using whole genome sequencing of all IVF embryos prior to transfer. This raises
issues about what are the consequences of ARTs that permit parents not only to choose to have children but also to choose the kind
of children they will have. These developments will further challenge the principles of reproductive autonomy and the right of the
child to an open future, and may increase the responsibility of clinicians toward the welfare of the future child.
References
American Society for Reproductive Medicine. (2005). Fertility preservation and reproduction in cancer patients. Fertil. Steril., 83, 1622–1628.
American Society for Reproductive Medicine. (2009a). Access to fertility treatment by gays, lesbians, and unmarried persons. Fertil. Steril., 92, 1190–1193.
American Society for Reproductive Medicine. (2009b). Interests, obligations, and rights of the donor in gamete donation. Fertil. Steril., 91, 22–27.
American Society for Reproductive Medicine. (2012). Using family members as gamete donors or surrogates. Fertil. Steril., 98, 797–803.
Astrue v. Capato, 566 U.S. _______ 2012.
Brezina, P. R., & Zhao, Y. (2012). The ethical, legal, and social issues impacted by modern assisted reproductive technologies. Obstet. Gynecol. Int., 2012, 1–7.
Davies, M. J., Moore, V. M., Wilson, K. J., et al. (2012). Reproductive technologies and the risk of birth defects. N. Engl. J. Med., 366, 1803–1813.
Hansen, M., Kurinczuk, J. J., Milne, E., de Klerk, N., & Bower, C. (2013). Assisted reproductive technology and birth defects: a systematic review and meta-analysis. Hum. Reprod.
Update, 19, 330–353.
Hoorens, S., Gallo, F., Cave, J. A. K., & Grant, J. C. (2007). Can assisted reproductive technologies help to offset population ageing? an assessment of the demographic and
economic impact of ART in Denmark and UK. Hum. Reprod., 22, 2471–2475.
Inhorn, M. C. (2006). Making Muslim babies: IVF and gamete donation in Sunni versus Shi’a Islam. Cult. Med. Psychiatry, 30, 427–450.
Nikolaos, M. (2008). The Greek orthodox position on the ethics of assisted reproduction. Reprod. Biomed. Online, 17, 25–33.
Omurtag, K., & Adamson, G. D. (2013). The Affordable Care Act’s impact on fertility care. Fertil. Steril., 99, 652–655.
Parliamentary Office of Science and Technology. (2013). Preventing mitochondrial disease. PostNote, 431, 1–4.
Sandin, S., Nygren, K. G., Iliadou, A., Hultman, C. M., & Reichenberg, A. (2013). Autism and mental retardation among offspring born after in vitro fertilization. JAMA, 310, 75–84.
Schenker, J. G. (2008). The beginning of human life: status of embryo. perspectives in Halakha (Jewish religious law). J. Assist. Reprod. Genet., 25, 271–276.
Vatican. (1987). Sacred congregation for the doctrine of the faith. In Lawful and Illicit Uses of New Techniques in Human Embryology.
Relevant Websites
www.asrm.org – American Society for Reproductive Medicine.

www.cdc.gov/art – Centers for Disease Control and Prevention.
www.eshre.eu – European Society of Human Reproduction and Embryology.
www.resolve.org – Resolve: The National Infertility Association.
www.sart.org – Society of Assisted Reproductive Technology.
Tooth Regenerative Therapy: Tooth Tissue Repair and Whole Tooth Replacement
M Oshima, K Ishida, R Morita, and M Saito, Tokyo University of Science, Noda, Chiba, Japan
T Tsuji, Tokyo University of Science, Noda, Chiba, Japan; and Organ Technologies Inc., Tokyo, Japan
Introduction to and Concepts for Tooth Regenerative Therapy 686

Tooth Development 687
Tissue Repair Using Tooth Tissue-Derived Stem Cells and Cytokines 688
The Use of Apical Papilla Stem Cells for Dental Pulp Regeneration 688
Periodontal Tissue-Derived and Dental Follicle Stem Cells and Their Application to Periodontal Tissue Regeneration 689
Bioengineered Root Regeneration Using Dental Stem Cell Populations and Tissue Engineering Technology 690
Whole Tooth Regeneration as a Future Organ Replacement Regenerative Therapy 690
Generation of a Bioengineered Tooth Germ Using a Biodegradable Scaffold 691
Bioengineering of Tooth Germ Using Cell Aggregation Methods 691
Novel Three-Dimensional Cell Manipulation Methods for Bioengineered Tooth Germ: The ‘Organ Germ Method’ 691
Functional Tooth Replacement Therapy 691
Transplantation of Bioengineered Tooth Germ or Bioengineered Mature Tooth Unit as a Tooth Replacement Strategy 692
Bioengineered Tooth Response to Mechanical Stress 693
Perceptive Neuronal Potential of Bioengineered Teeth 693
Future Perspectives for the Tooth Regeneration 694
References 694
Glossary
Bioengineered tooth germ Regenerated tooth germ reconstituted by the organ germ method between tooth-germ-derived
epithelial and mesenchymal cells can develop correctly, both orthotopically and ectopically.
Bioengineered tooth unit They are composed of bioengineered mature tooth, periodontal ligament, and alveolar bone and are
generated by ectopic transplantation. This unit can be transplanted into a properly sized hole in the alveolar bone through
bone integration.
Epithelial–mesenchymal interaction Tooth development is regulated by sequential and reciprocal interactions between
epithelial and mesenchymal tissues. These interactions are mediated by multigene signal families, such as bone morphogenetic
protein (BMP), fibroblast growth factor (FGF), sonic hedgehog (Shh), and Wnt.
Organ germ method The organ germ method was developed as a technology to generate bioengineered organ germ that
properly reproduces the three-dimensional cell processes that occur during embryonic development to yield bioengineered
organ germ in vitro (Nakao et al., 2007).
Tooth germ All organs develop from organ germs that are generated during the embryonic period. The organ germ of tooth is
referred to as tooth germ, which is composed of two tissues: the oral epithelium and neural-crest-derived mesenchyme.
Introduction to and Concepts for Tooth Regenerative Therapy
Oral functions such as enunciation, mastication, and occlusion are important components of a healthy life. The tooth is an ecto-
dermal organ regulated by reciprocal epithelial–mesenchymal interactions (Thesleff, 2003) and has distinctive hard tissues that
include enamel, dentin, cementum, and alveolar bone. Teeth also have soft connective tissue such as pulp and periodontal ligament
that contain nerve fibers and blood vessels to maintain tooth homeostasis (Avery, 2002). Damage, loss, and disease in teeth,
including dental caries and periodontal disease, cause fundamental problems for oral function and are associated with a number
of health issues (Proffit et al., 2004). Conventionally, the restoration of tooth functions in these circumstances involves replacement
with dentures or dental implants. Although these artificial therapies are very effective, there have been recent improvements that
enhance the biological functions underlying tooth movement through bone remodeling (Huang et al., 2009).
The current advances in future regenerative therapies have been influenced by many previous studies of embryonic development,
stem cell biology, and tissue engineering technologies (Brockes and Kumar, 2005). To restore the partial loss of organ functions and
to repair damaged tissues, attractive regenerative therapy concepts include stem cell transplantation into various tissues and organs

Regenerative Engineering j Tooth Regenerative Therapy: Tooth Tissue Repair and Whole Tooth Replacement 687
Figure 1 Concepts for tooth regenerative therapy. Approaches to developing technologies for tooth regenerative therapy have included tissue repair,
tissue engineering, and whole tooth organ replacement.
and cytokine therapy to activate tissue stem/progenitor cells. Tooth tissue stem cells and the cytokine network that regulates tooth
development have been well characterized at the molecular level (Thesleff, 2003). These advances can be applied to the repair of
dental pulp and periodontal tissues, including the alveolar bone (Figure 1).
The ultimate goal of regenerative therapy is to develop fully functional bioengineered tissues that can replace lost or damaged
organs following disease, injury, or aging (Ikeda and Tsuji, 2008). In the dental field, this therapy would involve replacement of
a lost or damaged tooth with a bioengineered tooth built with stem cells that has the capacity to become a functional unit, including
the whole tooth and periodontal tissue surrounding alveolar bone (Yen and Sharpe, 2006). It is expected that regenerative tooth
replacement therapy will be established in the near future as a novel and successful biological treatment that will provide essential
functional recovery of lost teeth to satisfy esthetic and physiological requirements (Sharpe and Young, 2005). Many approaches to
functionally replace missing teeth have been evaluated in the past three decades, including three-dimensionally bioengineered teeth
and tooth germ generation using biodegradable materials and cell aggregation methods (Duailibi et al., 2006; Ikeda and Tsuji,
2008; Yen and Sharpe, 2006). The first reports of fully functioning bioengineered tooth replacement with correct tissue orientation,
masticatory potential, responsiveness to mechanical stress, and perceptive potential following transplantation into a lost tooth
region were recently published (Nakao et al., 2007; Ikeda et al., 2009). In this article, the most recent findings and technologies
for tooth tissue repair and whole tooth replacement, also known as tooth regenerative therapy, which have the potential to provide
essential functional recovery and ultimately replace currently used artificial materials, are discussed.
Tooth Development
Organs such as hair, glands, kidneys, and teeth arise from their respective germs, which are induced by reciprocal interactions
between epithelial and mesenchymal tissues in the developing embryo (Thesleff, 2003). The principle mechanisms of tooth
organogenesis are also regulated by reciprocal epithelial and mesenchymal interactions, particularly those involved in stem
cell, signaling molecule, and transcription factor pathways (Figure 2). In tooth germ development, the dental lamina first
thickens (lamina stage), followed by epithelial thickening (placode stage) at the sites of future teeth and subsequent epithelial
budding to the underlying neural crest-derived ectomesenchyme. Tooth germ formation is initiated on embryonic days (ED) 10–
11 in mice by epithelial signals such as fibroblast growth factor 8 (FGF8), bone morphogenetic protein 4 (BMP4), sonic
hedgehog (Shh), tumor necrosis factor, and Wnt10b. These signals induce the expression of transcription factors such as
Barx1, Dlx1/2, Lhx6, Lhx7 Msx1, Pax9, and Gli in the dental mesenchyme, which then condense around the developing epithelial
bud (bud stage) (Thesleff, 2003). At ED14 (cap stage), a transient epithelial signaling center known as an enamel knot, which
expresses several signaling molecules, including Shh, BMP family molecules, FGFs, and Wnts, is thought to regulate individual
cell fates and epithelial–mesenchymal interactions. The terminal differentiation of dental epithelial and dental mesenchyme cells
into ameloblasts and odontoblasts, respectively, occurs during ED16–18 (bell stage) to perinatal stage. These differentiated cells
secrete enamel proteins or a collagenous extracellular matrix that mineralizes into the enamel and dentin matrix at the epithe-
lium–mesenchyme interface. A portion of the dental mesenchymal cells forms the apical papilla, which is capable of differenti-
ating odontoblasts and pulp cells following their migration to the root apex side (Sonoyama et al., 2006). The outer
mesenchymal cells outside of the tooth germ, which comprises an epithelium and condensed mesenchymal cells, forms the
dental follicle that can generate periodontal tissue, including cementum, periodontal ligament, and alveolar bone. After tooth
development, immature cells seem to be maintained as adult tissue stem cells that are thought to act as a self-repair system
for dental tissues and supply a wide variety of dental cell types, both under steady-state conditions and after dental tissue injury.
Tooth morphogenesis begins with cusp formation in the early bell stage to form the tooth crown. Tooth size and shape are
thought to be regulated by signaling molecules such as the BMPs and FGF4 emanating from the secondary enamel knots, which
regulate the cusp pattern of the mature natural tooth (Thesleff, 2003). Tooth root formation is initiated after tooth crown forma-
tion and is followed by tooth eruption in the oral cavity.
688 Regenerative Engineering j Tooth Regenerative Therapy: Tooth Tissue Repair and Whole Tooth Replacement
Figure 2 Schematic of tooth germ development. Tooth germ development begins at the lamina stage and proceeds to the bud stage, during which
the dental epithelial bud and neural-crest-derived ectomesenchyme are formed. Subsequent morphogenesis occurs at the cap stage during the devel-
opment of dental epithelium and dental mesenchyme, which can later diverge into the papilla and the follicle. Tooth crown is formed from the early
bell stage to the late bell stage. During tooth eruption, the root is developed and dental follicle cells differentiate into periodontal tissue to attach the
tooth root and jawbone (adult tooth).
Tissue Repair Using Tooth Tissue-Derived Stem Cells and Cytokines
Recent studies of stem/progenitor cells and organogenesis have provided considerable new insights that have furthered our under-
standing of tooth tissue-derived stem cells, which can differentiate into various dental cell lineages such as odontoblasts, pulp cells,
periodontal ligament cells, cementoblasts, and osteoblasts (Huang et al., 2009). Dental stem cells will be useful for developing stem
cell transplantation therapy, one of the more promising concepts in regenerative therapy, to restore the partial loss of organ function
by replacing enriched and purified stem cells and thereby achieving dental tissue repair (Figure 3(a)).
The Use of Apical Papilla Stem Cells for Dental Pulp Regeneration
Dental pulp is composed of connective tissue, blood vessels, nerves, fibroblasts, and odontoblasts and develops from the dental
papillae after being encased by dentin tissue (Huang et al., 2009). Dental pulp stem cells (DPSCs) and stem cells from human exfo-
liated deciduous teeth (SHED) have been isolated from the dental pulp of human permanent third molars and exfoliated deciduous
teeth, respectively. DPSCs and SHED express CD146/STRO-1 and are thought to be tooth tissue-derived stem cells with high prolif-
erative capacities and sufficient potency to develop into odontoblasts, adipocytes, and neurallike cells. Therefore, these stem cells
may be a good resource for stem cell-mediated tissue repair, including dentin or pulp regeneration. As the origin of root and pulp
development, the dental papilla is located apically to the developing pulp and is thus known as the apical papilla, which is less
vascular and contains cellular, gelatinous soft tissue. Apical papilla contain unique stem cells, known as stem cells from apical
papilla (SCAP), which have a high proliferative potential that is reflected by high levels of telomerase activity and multipotent differ-
entiation capability that produces odontoblasts and adipocytes (Sonoyama et al., 2006). SCAP cells can also generate typical dentin
structures after transplantation in vivo and may offer a promising avenue for cell-based therapies for tissue repair and tissue
engineering.
Dental decay is the most commonly observed tooth pathology, and the current standard treatment involves the substitution of
the natural/physiological dental tissue with artificial material. A molecular medical approach using growth factors involved in tooth
development and based on epithelial–mesenchymal interactions is anticipated to enable tooth tissue repair, such as dentin regen-
eration via the addition of BMPs and transforming growth factor (TGF)-b1 (Huang et al., 2009). Tooth tissue-derived stem cells,
such as DPSCs and pulp stem cell subfractions, CD31/CD146 side population (SP) cells and CD105þ cells, which can generate
pulp tissue, may also be useful for tooth tissue repair and dental pulp regeneration. It is feasible that growth factors and tooth tissue-
derived stem cells will one day be used to repair damaged dental pulp tissue.
Figure 3 Dental tissue repair and engineering. (a) Dental tissue treatment against caries, pulp injury, and periodontal disease. Tooth regenerative
therapy, stem cell transplantation, and cytokine therapies are regarded as attractive approaches for repairing tissue damaged by dental caries and
periodontal disease. For dental caries and pulp injury, the transplantation of dental stem cells including DPSCs, SHED, and SCAP, which can differen-
tiate into odontogenic progenitors and pulp cells, has been examined (right panel). Cytokines that have the potential to activate and differentiate
dental stem cells such as BMPs, TGF-b, NGF, VEGF, GDNF, and BDNF are also expected to mediate tissue repair. For periodontal tissue repair, the
application of PDLSCs and DFSCs for stem cell transplantation and cytokines including PDGF, IGF, BDNF, bFGF, EMP, and ADAMTSL6b have the
potential to regenerate periodontal tissue (left panel). (b) Bioengineered tooth root regeneration by tissue engineering. The bioengineered root struc-
ture is generated using a root-shaped HA/TCP carrier loaded with SCAP that is covered with gelfoam containing PDLSCs and is inserted onto a porce-
lain crown (left panel). The HA/SCAP-gelfoam/PDLSC implant successfully regenerates dentin and periodontal ligament tissue on the outside of the
implant after transplantation into the alveolar bone, resulting in normal tooth function (right panel). PDLSCs, periodontal ligament stem cells; DFSCs,
dental follicle stem cells; PDGF, platelet-derived growth factor; IGFs, insulinlike growth factors; BDNF, brain-derived neurotrophic factor; bFGF, basic
fibroblast growth factor; EMP, enamel matrix protein; ADAMTSL6b, a disintegrin-like metalloprotease domain with thrombospondin type I motifs like
6b; DPSCs, dental pulp stem cells; SHED, stem cells from human exfoliated deciduous teeth; SCAP, stem cells from apical papilla; BMPs, bone
morphogenetic proteins; TGF-b, transforming growth factor-b; NGF, nerve growth factor; VEGF, vascular endothelial growth factor; GDNF, glial cell
line-derived neurotrophic factor.
Periodontal Tissue-Derived and Dental Follicle Stem Cells and Their Application to Periodontal Tissue Regeneration
Periodontal tissue is a tooth-supporting connective tissue that acts as a shock absorber for occlusal force. Periodontal tissue struc-
ture, including the periodontal ligaments, cementum, and alveolar bone, can be irreversibly damaged by periodontitis, a chronic
inflammatory disease. No reliable treatment for regenerating periodontium has been established. Periodontal tissues, including
cementoblasts, periodontal ligaments, and osteoblasts, are derived from dental follicle stem cells (DFSCs) (Yen and Sharpe,
2006), which migrate onto tooth root surfaces to form periodontal tissue during the tooth root-forming stages. Periodontal
ligament-derived mesenchymal stem cells (PDLSCs), which have also been identified in adult human periodontal ligaments and
can be cultured as stem cells in vitro, can differentiate into all periodontal cell types after transplantation in vivo. DFSCs have the
ability to reproduce periodontal formations and are thought to be stored as PDLSCs in adult periodontal ligaments after tooth
development. Using cell sheet engineering in conjunction with the stem cell transplantation therapies described above, stem cell
sheets are now being developed for clinical use in periodontal tissue regeneration. Recently developed molecular treatments can
be administered via the local application of human recombinant cytokines such as platelet-derived growth factor, insulinlike growth
factor-I, brain-derived neurotropic factor, and basic FGF to accelerate periodontal regeneration. Furthermore, periodontal tissue is
composed of fibrillar extracellular matrices such as collagen fibrils and microfibrils, which play a critical role in periodontal tissue
formation and in the onset of severe periodontitis. Recently, it was reported that the local administration of fibrillin-1-associated
protein ADAMTSL6b effectively accelerates wound healing in periodontal tissues by restoring microfibrils (Saito et al., 2011). This
molecular medical therapy involving microfibril reinforcement is a novel approach to periodontal tissue regeneration, in addition
to stem cell transplantation and cytokine treatment.
Bioengineered Root Regeneration Using Dental Stem Cell Populations and Tissue Engineering Technology
Dental implants, which require direct integration into the alveolar bone, have recently gained momentum as a valid therapy for
replacing missing teeth and an alternative to fixed or removable dentures. Although dental implants have been the mainstay of
dental therapy over the past few decades, they are associated with an absence of periodontal ligaments, and are therefore deficient
in essential tooth function and natural structural relationship with the tooth root and alveolar bone. To regenerate the tooth root
and its associated periodontal tissues, both of which are necessary for maintaining physiological tooth function, a tissue engineering
application has been anticipated for stem cell-based root regeneration (Figure 3(b)). A unique approach for tooth root regeneration
employing a root-shaped hydroxyapatite/tricalcium phosphate carrier loaded with gelfoam/PDLSC-covered SCAP cells has been
reported to form a rootlike structure to which a porcelain crown can be attached, resulting in normal tooth function (Sonoyama
et al., 2006). This tissue engineering technology produced a bioengineered root by using a combination of root and periodontal
tissues and may underlie the next generation of regenerative medical technologies that integrate stem cell-mediated tissue regener-
ation strategies, engineered materials as structural components, and current dental crown technologies. The root regeneration
concept also has the potential for earlier clinical application compared to whole tooth regeneration.
Whole Tooth Regeneration as a Future Organ Replacement Regenerative Therapy
The current approach to generating ectodermal organs such as teeth, hair follicles, and salivary glands is to recreate organogenesis
through epithelial–mesenchymal interactions that occur in the developing embryo and thereby develop fully functioning bio-
engineered organs from bioengineered organ germ generated from immature stem cells via three-dimensional cell manipulation
in vitro (Ikeda and Tsuji, 2008; Sharpe and Young, 2005). For tooth regeneration, a concept has been proposed in which a bio-
engineered tooth germ will be transplanted into recipient jaw and develop into a functional mature tooth (Figure 4, upper panel).
It is also expected that it will be possible to transplant a bioengineered tooth unit that includes mature tooth, periodontal ligament,
and alveolar bone, which will achieve engraftment through the physiological bone integration of the recipient’s jaw (Figure 4, lower
panel; Oshima et al., 2011). To realize whole tooth replacement, the first major issue is to develop a three-dimensional cell
Figure 4 Strategies for whole tooth replacement via regenerative therapies. Functional teeth can now be regenerated by transplanting bioengineered
tooth germ reconstituted from epithelial and mesenchymal cells via the organ germ method or by transplanting bioengineered tooth units with peri-
odontal ligament and alveolar bone developed from bioengineered tooth germ.
manipulation technology using completely dissociated epithelial and mesenchymal cells in vitro. Two conventional approaches and
a novel cell manipulation method are currently being investigated for the purpose of generating bioengineered tooth germ or
mature tooth and are described below:
Generation of a Bioengineered Tooth Germ Using a Biodegradable Scaffold

The scaffold technology has proven useful in three-dimensional tissue engineering methods to regenerate suitably shaped tissues by
seeding single cells onto degradable materials fabricated from either natural ingredients or synthetic polymers. This technique can
be used to grow tissues with a uniform cellular distribution and has been applied in clinical bone and cartilage regenerative ther-
apies. Several previous studies that employed polyglycolic acid and poly-L-lactate-co-glycolide copolymer or a collagen sponge as
scaffolding materials reported that a tooth-shaped scaffold onto which epithelial and mesenchymal cells isolated from porcine
unerupted third molars or a rat tooth bud were seeded could generate small tooth structures containing all of the correct tooth tissue
components, including pulp, dentin, and enamel, but not periodontal tissue (Sharpe and Young, 2005; Yen and Sharpe, 2006).
Although the scaffold method could be useful for producing the desired tooth shape and size, it has critical limitations, including
tooth formation frequency, appropriate replication of tooth tissue structures such as enamel–dentin complex formation, and proper
arrangements of ameloblast and odontoblast cell lineages, which are achieved through epithelial–mesenchymal interactions that
mimic natural tooth development (Avery, 2002; Thesleff, 2003).
Bioengineering of Tooth Germ Using Cell Aggregation Methods

Cell aggregation is a typical bioengineering method that aims to reconstitute bioengineered organ germ that will properly reproduce
epithelial–mesenchymal interactions and organogenesis (Sharpe and Young, 2005; Yen and Sharpe, 2006). Previously, transplan-
tations of bioengineered cell aggregates using hair follicles and mammary-gland-derived stem cells have shown promise for regen-
erating correct organ structures with proper arrangements of different cell types in vivo. Artificial tooth germ created from precipitates
separated into dental epithelial and mesenchymal cells with cellular centrifugation has also been shown to be capable of generating
the appropriate tooth formation. Mixed cell aggregates of dissociated epithelial and mesenchymal cells isolated from molar tooth
germ also have a demonstrated capacity to develop into a tooth with the correct structure, following epithelial cell sorting and subse-
quent epithelial and mesenchymal cell self-reorganization. Although this technique replicated organogenesis, the frequency of
tooth development and correct tissue formation was insufficient.
Novel Three-Dimensional Cell Manipulation Methods for Bioengineered Tooth Germ: The ‘Organ Germ Method’
To achieve large-scale replication of organogenesis, an in vitro three-dimensional novel cell manipulation method designated as
a bioengineered organ germ method has been developed. This method is accomplished with cell compartmentalization between
epithelial and mesenchymal cells at a high cell density in type I collagen gel (Figure 5(a)). Bioengineered tooth germ created by
this technique could allow for large-scale organ development and mimics multicellular assembly and epithelial–mesenchymal
interactions, as well as natural tooth development. The bioengineered tooth germ generates a structurally correct tooth after trans-
plantation in an organ culture in vitro but also following placement into a subrenal capsule in vivo. The isolated single bioengineered
tooth germ also developed in the oral cavity and formed the correct tooth structure (Nakao et al., 2007). Furthermore, there is
a unique technology that can successfully generate a size-controlled bioengineered tooth unit comprising mature tooth, periodontal
ligament, and alveolar bone that can also be generated by transplantation into the subrenal capsule (Figure 5(b); Oshima et al.,
2011). These technologies have the potential to be adapted for successful functional tooth replacement in vivo and are expected
to represent an innovative advance in bioengineered organ replacement regenerative therapies.
Functional Tooth Replacement Therapy
Organ replacement regenerative therapy holds great promise for the future replacement of dysfunctional organs with a bio-
engineered reconstruction created using three-dimensional cell manipulation in vitro. Regenerated organs should be able to
complete organ-intrinsic functions in cooperation with the surrounding environment in the recipient. In previous reports, however,
artificial organs constructed with various cells and artificial materials could not restore functionality and thus were not viable
options for long-term organ replacement in vivo. However, bioengineered organs can be grown in vivo in amphibian models;
activin-treated cell aggregates could form a secondary heart with pumping function and also regenerate eyes that were light respon-
sive and connected with the host nervous system. Oral functions such as mastication, pronunciation, and facial esthetics have an
important influence on the quality of life because they facilitate both oral communication and nutritional intake (Proffit et al.,
2004). These functions are achieved with the teeth, masticatory muscles, and the temporomandibular joint, under control of the
central nervous system. For the realization of tooth replacement regenerative therapy, a tooth developed from bioengineered
germ or a transplanted bioengineered mature tooth unit must be capable of properly engrafting in the lost tooth region in an adult
oral environment and acquiring full functionality, including sufficient masticatory performance, biochemical cooperation with
periodontal tissues, and afferent responsiveness to noxious stimulations via neurons in the maxillofacial region (Proffit et al., 2004).
Figure 5 The organ germ method: a three-dimensional cell processing system. (a) A high density of dissociated mesenchymal cells are injected
into the center of a collagen drop (left panel). Dissociated tooth germ-derived epithelial cells are subsequently injected into the drop adjacent to the
mesenchymal cell aggregate (right panel). (b) Transplanting bioengineered tooth germ into a subrenal capsule for 30 days can yield a bioengineered
tooth unit composed of a mature tooth, periodontal ligament, and alveolar bone with the correct structural components, such as enamel (c), dentin
(d), periodontal ligament (PDL), and alveolar bone (AB).
Transplantation of Bioengineered Tooth Germ or Bioengineered Mature Tooth Unit as a Tooth Replacement Strategy
A successful tooth regenerative therapy via the transplantation of bioengineered tooth germ into the lost tooth region requires that
the transplanted germ erupt and properly occlude with the opposing tooth in an adult jawbone (Figure 4, Upper panel). Tooth
eruption is a corporative regulatory mechanism that involves the tooth germ cell component and the surrounding alveolar/
jawbone. It has been shown that follicle cells migrate from near the surface of the enamel organs and dental papillae to give rise
to the cementum, periodontal ligament, and alveolar bone. Because these cells affect overlying bone resorption and can cause enzy-
matic degeneration in growth and bodily movement of the teeth, they most likely play a role in tooth eruption. Follicle cells may
contribute to the formation of tissues surrounding and underlying the teeth during tooth eruption. So far, it has been demonstrated
that transplanted natural tooth germ erupts in a murine toothless diastema region. Furthermore, bioengineered tooth germ can only
develop correct tooth structure in an oral cavity (Nakao et al., 2007) and can successfully erupt 37 days after transplantation. The
bioengineered tooth subsequently reaches the occlusal plane and achieves and maintains occlusion with the opposing tooth from
49 days onward (Figure 6(a) and 6(b); Ikeda et al., 2009).
In the case of a transplanted bioengineered mature tooth unit comprising a mature tooth, periodontal ligament, and alveolar
bone, the most critical consideration is whether the unit can be engrafted into the tooth loss region through bone integration, which
involves natural bone remodeling in the recipient. A bioengineered tooth unit transplanted at a position reaching the occlusal plane
with the opposing upper first molar was successfully engrafted at 40 days and thereafter maintained the periodontal ligament orig-
inated from the bioengineered tooth unit through successful bone integration (Figure 6(b); Oshima et al., 2011). The enamel and
dentin hardness of bioengineered tooth components were in the normal range when analyzed by the Knoop hardness test (Ikeda
Figure 6 Bioengineered tooth regeneration by the transplantation of bioengineered tooth germ or a tooth unit. (a) A transplanted bioengineered
tooth germ erupted and reached the occlusal plane with the opposing tooth 49 days after transplantation (left panels). The GFP-labeled tooth
erupted in an adult oral environment (right panel). (b) The erupted bioengineered tooth occluded with the opposing natural tooth (left panels).
The bioengineered tooth unit was engrafted by bone integration at a position reaching the occlusal plane 40 days post transplantation (right panels).
:: Bioengineered tooth.
et al., 2009; Oshima et al., 2011). These approaches demonstrate the potential to successfully recover masticatory performance and
natural tooth tissue through new technologies.
Bioengineered Tooth Response to Mechanical Stress

Loss of teeth and functional disorders in periodontal ligaments cause fundamental problems for oral functions, including pronun-
ciation, mastication, occlusion, and associated health issues (Proffit et al., 2004). The periodontal ligament plays important roles in
the pathogenic and physiologic responses of teeth to extreme mechanical forces from bone remodeling accompanied by ortho-
dontic tooth movement. Autologous tooth transplantations following a traumatic dental injury have indicated that natural peri-
odontal tissue remaining on the root could successfully restore physiological tooth function, including bone remodeling, and
effectively prevent ankylosis. In contrast, the absence of a periodontal ligament in dental implants is associated with deficiencies
in essential tooth functions and in the natural structural relationship with the root and alveolar bone (Huang et al., 2009). The
periodontal ligaments of bioengineered teeth that erupted following the transplantation of bioengineered tooth germ and mature
tooth units achieved functional tooth movement as well as that of natural tooth. They successfully reproduced bone remodeling via
the proper localization of osteoclasts and osteoblasts in response to mechanical stress, indicating that a bioengineered tooth can
replicate critical dental functions through the restoration and reestablishment of cooperation with the surrounding jawbone (Ikeda
et al., 2009; Oshima et al., 2011).
Perceptive Neuronal Potential of Bioengineered Teeth

The peripheral nervous system is established by the growth of axons that navigate and establish connections with developing target
organs during embryogenesis. The perceptive potential for noxious stimulation, including mechanical stress and pain, is important
for proper organ function. It is also thought that the recovery of the nervous system, which is associated with the reentry of nerve
fibers following organ transplantation, is critical for organ function reconstitution. Teeth are a peripheral target organ for the sensory
trigeminal and sympathetic nerves, both of which play important roles in tooth function and protection. It is anticipated that tooth
regenerative therapies will be able to recover the neuronal perceptive ability of mechanical forces that are lacking in implant
patients. Sensory and sympathetic nerve fibers innervate both the pulp and periodontal ligament of a bioengineered tooth following
its eruption (Ikeda et al., 2009). These bioengineered teeth display appropriate perceptive potentials for nociceptive pain stimula-
tions, such as pulp stimulation and orthodontic treatment, and they can properly transduce these events to the central nervous
system through c-Fos immunoreactive neurons (Ikeda et al., 2009; Oshima et al., 2011). In this way, bioengineered teeth can indeed
restore the perceptive potential for noxious stimuli in cooperation with the maxillofacial region.
Future Perspectives for the Tooth Regeneration
There are several problems that must be solved before bioengineered teeth become feasible. To fully realize the practical clinical
application of tooth regenerative, suitable cell sources must be identified. Ideally, tooth regenerative therapy should employ the
patient’s own cells to avoid immunological rejection (Ikeda and Tsuji, 2008). Recent studies of stem cells and organogenesis
have led to considerable advances in our understanding of the potential of cell sources for tissue repair and organ reconstitution,
including tooth regenerative therapy. Tooth tissue-derived stem cells such as DPSCs, SHED, SCAP, PDLSCs, and DFSCs can differ-
entiate into dental cell lineages and contribute to the turnover and supply of various cell populations.
These lineages will be useful cell sources for stem cell transplantation therapy for dental tissue repair of dental caries and peri-
odontal disease (Ikeda and Tsuji, 2008; Huang et al., 2009), but human teeth do not regenerate like hair or skin, and there are no
stem cell niches in teeth or surrounding tissue that maintain progenitors for whole tooth reproduction. Current whole tooth regen-
erative therapy research is attempting to induce bioengineered tooth germ to develop a fully functioning tooth using embryonic
tooth germ-derived epithelial and mesenchymal cells via the organ germ method (Nakao et al., 2007; Ikeda et al., 2009; Oshima
et al., 2011). In the future, it will be important to identify cell sources from somatic dental and nondental tissue-derived stem cell
populations isolated from patients that have the potential to reproduce the epithelial and mesenchymal interactions that occur
during organogenesis, as well as tooth-forming ability. It will also be critical to recognize inductive genes with the potential to
initiate tooth organogenesis, reconstitute bioengineered tooth germ, and ultimately to develop a functional bioengineered tooth.
Candidate cell sources for whole tooth regeneration also include embryonic stem cells and induced pluripotent stem (iPS) cells,
which are capable of differentiating into endoderm, ectoderm, and mesoderm (Takahashi and Yamanaka, 2006). Recently, iPS cells
have been established from various oral tissues. However, iPS cells could be applied with reprogrammed progenitor cell lines; iPS
programming procedures are required to establish dental epithelium and mesenchyme fates. Although there are multiple candidates
for tooth developmental genes that promote significant expression of dental epithelial and mesenchymal cells, the master genes
responsible for tooth development, remain to be discovered. The important task for determining a future tooth regenerative cell
source is the identification of specific combinations of factors capable of reprogramming nondental cells to dental epithelium
and mesenchyme.
Tooth types such as incisors, canines, premolars, and molars have unique morphological features that are programmed at pre-
determined sites in the oral cavity during tooth development. Several studies have proposed molecular mechanisms for tooth
morphology regulation (Ishida et al., 2011). Tooth size as well as crown and root shape are important considerations when gener-
ating a bioengineered regenerated tooth with proper functional occlusion and esthetics.
Concluding Remarks
The progress of regenerative technology is remarkable, and many patients look forward to the implementation of tooth regenerative
therapy. Further studies are required to establish bioengineering technologies that can control tooth morphology, including tissue
engineering using scaffolds, the identity of morphogenesis-related genes, and appropriate cytokines to use to guide morphogenesis.
Tooth regenerative therapy is now regarded as a crucial study model for future replacement regenerative therapies that can be
applied to more complex organs and will contribute substantially to the knowledge and technology required to regenerate organs
(Ikeda and Tsuji, 2008; Sharpe and Young, 2005).
References
Avery, J. K. (2002). Oral Development and Histology. New York: Thieme Press.
Brockes, J. P., & Kumar, A. (2005). Appendage regeneration in adult vertebrates and implications for regenerative medicine. Science, 310, 1919–1923.
Duailibi, S. E., Duaibili, M. T., Vacanti, J. P., et al. (2006). Prospects for tooth regeneration. Periodontol., 2000(41), 177–187.
Huang, G. T., Gronthos, S., & Shi, S. (2009). Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.
J. Dent. Res., 88, 792–806.
Ikeda, E., & Tsuji, T. (2008). Growing bioengineered teeth from single cells: potential for dental regenerative medicine. Expert Opin. Biol. Ther., 8, 735–744.
Ikeda, E., Morita, R., Nakao, K., et al. (2009). Fully functional bioengineered tooth replacement as an organ replacement therapy. Proc. Natl. Acad. Sci. U.S.A., 106, 13475–13480.
Ishida, K., Murofushi, M., Nakao, K., et al. (2011). The regulation of tooth morphologenesis is associated with epithelial cell proliferation and the expression of Sonic hedgehog
through epithelial-mesenchymal interactions. Biochemi. Biophys. Res. Commun., 405, 455–461.
Nakao, K., Morita, R., Saji, Y., et al. (2007). The development of a bioengineered organ germ method. Nat. Methods, 4, 227–230.
Oshima, M., Mizuno, M., Imamura, A., et al. (2011). Functional tooth regeneration using a bioengineered tooth unit as a mature organ replacement regenerative therapy. PloS One,
6, e21531.
Proffit, W. R., Fields, H. W., & Sarver, D. M., Jr. (2004). Contemporary Orthodontics. St. Louis, MO: Mosby Press.
Saito, M., Kurokawa, M., Oda, M., et al. (2011). ADAMTSL6b rescues fibrillin-1 microfibril disorder in Marfan syndrome mouse model through the promotion of fibrillin-1 assembly.
J. Biol. Chem., 286(44), 38602–38613.
Sharpe, P. T., & Young, C. S. (2005). Test-tube teeth. Sci. Am., 293, 34–41.
Sonoyama, W., Liu, Y., Fang, D., et al. (2006). Mesenchymal stem cell-mediated functional tooth regeneration in swine. PloS One, 1, e79.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–676.
Thesleff, I. (2003). Epithelial-mesenchymal signalling regulating tooth morphogenesis. J.Cell Sci., 116, 1647–1648.
Yen, A. H., & Sharpe, P. T. (2006). Regeneration of teeth using stem cell-based tissue engineering. Expert Opin. Biol. Ther., 6, 9–16.
Vascularized Tissue Regenerative Engineering Using 3D Bioprinting Technology
Sungwoo Kim, Arnaud Bruyas, Chi-Chun Pan, Alexander Martin Stahl, and Yunzhi Yang, Stanford University, Stanford, CA,
United States
The Importance of Prevascularization in Tissue Engineering 696

Processes by Which Implants Vascularize In Vivo 697
Strategies to Create an In Vitro Vascularization Model System 698
Biomaterials for Vascularization 698
In Vitro Endothelialization 699
Angiogenic Growth Factors and Biomolecules 699
Mechanobiology 700
Perfusion Systems 700
Vascular Maturation in Coculture Systems 700
3D Printing Methods for Prevascularized Tissue Constructs 701
General Process of Forming a 3D Printed Scaffold 701
Forming acellular scaffolds for supporting the vascularized scaffold 701
Manufacturing the cell-laden constructs 702
Challenges of 3D Printed Tissue Construct 703
Applications in Vascularization in Bone and Cardiac Tissue Engineering 703
Applications in Bone Tissue Engineering 704
Challenges for engineered bone grafts 704
Strategies for Developing Vascularized Bone Grafts 704
Applications in Cardiac Tissue Engineering 705
Challenges for engineered cardiac grafts 705
Strategies for developing vascularized cardiac tissues 705
Summary 706
Further Reading 706
Glossary
Additive manufacturing (AM) A processes used to synthesize a three-dimensional object in which successive layers of material
are formed under computer control to create an object.
Free flap A term used to describe the transplantation of tissue with intact vascular bed from one site of the body to another in
order to reconstruct an existing defect with the circulation in the tissue reestablished by anastomosis of arteries and veins.
Lumen The inside space of a tubular structure in which blood flows.
Matrix metalloproteinases (MMPs) The calcium-dependent zinc-containing endopeptidases. These enzymes are capable of
degrading all kinds of extracellular matrix proteins, but also can process a number of bioactive molecules.
Mechanobiology An emerging field of science at the interface of biology and engineering that focuses on how physical forces
and changes in the mechanical properties of cells and tissues contribute to development, cell differentiation, physiology, and
disease.
Neovascularization The natural formation of new blood vessels, usually in the form of functional microvascular networks,
capable of perfusion by red blood cells.
Osteoinductivity A property of graft material in which it induces de novo bone growth with biomimetic substances, such as
bone morphogenetic proteins.
Photo-crosslinking The photo-induced formation of a covalent bond between two macromolecules or between two different
parts of one macromolecule.
The Importance of Prevascularization in Tissue Engineering
Nearly all metabolically active tissues in the body contain a hierarchical network of blood vessels that is essential for maintaining
tissue viability and function. These vascular networks are responsible for delivering oxygen and nutrients to the cells that make up
tissues and for removing the byproducts of cellular metabolism. In damaged tissues, reestablishment of a functional vascular system

Regenerative Engineering j Vascularized Tissue Regenerative Engineering Using 3D Bioprinting Technology 697
is an essential step for wound healing and in the event a tissue graft is required, achieving integration of the implant with the
surrounding host tissues depends on the formation of a functional vasculature across the graft. Insufficient blood flow to a tissue
or implant leads to hypoxia, which in turn leads to cell death in the affected region if not resolved quickly enough. Providing blood
flow throughout an engineered tissue in a timely manner is thus essential for preventing graft failure and depends on the successful
formation of a vascular network within the implant as well as the connection of such a network to the surrounding host circulation.
Currently, the challenge of achieving complete and timely vascularization across large-scale tissue constructs limits the maximum
volume at which engineered tissues can remain viable and impedes their translation to clinical practice.
A functional vascular bed must furnish an entire tissue with sufficient oxygen and nutrients at the cellular level. In order to meet
these demands, the native vasculature is structured hierarchically, with large arteries carrying the necessary volume of blood to the
target region and then branching into multiple smaller arterioles in order to achieve more even distribution throughout the tissue.
Venules collect the deoxygenated blood and converge into large veins that carry the blood away from the tissue. Capillaries, which
bridge the space between arterioles and venules, constitute the smallest blood vessels in the vascular system with an average diam-
eter of just 8–10 mm. In most metabolically active tissues, the distance between capillaries and their target cells is restricted to 100–
200 mm in order to meet the high cellular demands for oxygen, which imposes a need for extremely dense capillary networks. Due to
the difficulty of reproducing the inherent complexity of native biological tissues and their vasculature, tissue-engineered grafts often
exhibit simplified structures, with the expectation that more complete integration and remodeling by the host will follow. As such,
the majority of current scaffolds used in tissue engineering lack a functioning vascular network prior to implantation, relying instead
on the surrounding host vasculature to invade the graft. Unfortunately, in many cases, the extent of vascular ingrowth by host vessels
into implanted tissue constructs is insufficient, leading to failure of the grafts. Achieving complete revascularization is a key step in
the implementation of engineered tissue constructs in clinical settings. The following review will introduce the biological processes
involved in vascularization in vivo and strategies to reproduce these processes in vitro, followed by a description of current 3D
printing technologies and their potential application for engineering vascularized tissue grafts, with particular focus on applications
in bone and cardiac tissue engineering.
Processes by Which Implants Vascularize In Vivo

The restoration of a functional vascular network within engineered tissue constructs is known as vascularization. There are two
processes by which the formation of new blood vessels can occur: vasculogenesis and angiogenesis.
Vasculogenesis is the process of forming de novo blood vessels through the differentiation of progenitor cells to endothelial cells
(ECs) and their organization into a capillary network within previously avascular tissue (Fig. 1A). Vasculogenesis primarily occurs
during prenatal development, where a primitive capillary network initially forms prior to being remodeled into the more complex
hierarchical networks of the mature vasculature. In adults, vasculogenic processes have been observed to contribute to the revascu-
larization of damaged tissues: cues such as hypoxia, growth factors, and cytokines recruit endothelial progenitor cells from the bone
marrow, augmenting the population of circulating endothelial progenitor cells. These cells home to the site of vascularization by
integrin interactions and engage in tissue healing through a combination of self-assembly into new vessels and growth factor
production.
Angiogenesis refers to the sprouting or splitting of new capillaries from existing blood vessels and represents the chief mecha-
nism by which new blood vessels form in adults (Fig. 1B). Angiogenic sprouting is the primary route for vascular formation in
implanted tissue grafts, with new capillaries growing from surrounding blood vessels to invade nearby engineered tissue constructs.
(A) (B) (C)
Fig. 1 The different routes to vascularization in vivo (A) vasculogenesis; (B) angiogenesis; (C) vasculogenesis and angiogenesis.
698 Regenerative Engineering j Vascularized Tissue Regenerative Engineering Using 3D Bioprinting Technology
The process is initiated by the release of vascular signaling factors from parenchymal cells in poorly perfused tissues in response to
hypoxic conditions. In response to these signalling molecules, certain endothelial cells in the surrounding tissues transition into tip
cells, which put forth long cellular processes called filopodia to detect gradients in the angiogenic factors and guide endothelial
migration in the direction of the hypoxic tissues. The tip cell secretes proteolytic enzymes to clear a path through the surrounding
extracellular matrix (ECM) and endothelial stalk cells proliferate behind the tip cell, allowing the developing sprout to extend
toward the hypoxic region. The stalk cells produce internal vacuoles that fuse to form a central lumen hollowing out the growing
capillary. A host vascular network will generally invade an implanted tissue graft by angiogenic sprouting as a natural response to
the presence of the foreign construct. However, due to the slow progression of the tip cells and proliferating stalk cells, in larger
implants complete vascularization can take weeks, if it occurs at all.
Vasculogenesis and angiogenesis are not mutually exclusive modes of vascularization. Endothelial progenitor cells are often
implicated in both processes and might adopt one of several complimentary roles in the formation of new vascular networks within
regenerating tissues (Fig. 1C). A single developing microvascular network might be simultaneously expanded by the vasculogenic
differentiation and assembly of endothelial progenitor cells which interconnect and merge with the angiogenic sprouting of existing
vessels. Once an initial capillary network forms, mural cells (MCs) are recruited to support the developing vasculature. For capil-
laries, these cells are called pericytes. In larger vessels, smooth muscle cells perform this role. The presence of MCs is essential
for stabilizing the newly formed vessel beds, preventing vascular regression and allowing for further capillary reorganization and
maturation into the native hierarchical vascular framework.
The invasion of ECs and their supporting cells into an engineered tissue construct to form a functional vasculature can take
days to weeks, as the rate of capillary ingrowth is generally limited to several tenths of micrometers per day. While this may be
sufficient to meet the demand for oxygen and nutrients in thin or avascular tissue grafts such as skin and cartilage, large-scale
grafts for most engineered tissue constructs are at high risk of nutrient deficiency and hypoxia, with cells at the center of such
implants typically experiencing extreme anoxic stress that can lead to graft necrosis. Some strategies to overcome this challenge
seek to include preformed channel networks within the scaffolds in an attempt to guide endothelial invasion either with or
without direct surgical attachment to the host vasculature. Other strategies seed grafts with ECs or endothelial progenitor cells
in order to eliminate the need for slow vascular invasion via angiogenic sprouting. Preseeding constructs with ECs additionally
enable primitive endothelial network formation in vitro so that a provisional microvasculature already exists within the scaffold
prior to implantation.
Strategies to Create an In Vitro Vascularization Model System
Prevascularization aims to form functional vascular networks within an engineered tissue construct in vitro under controlled condi-
tions prior to implantation. This can improve cell survival and functionality in vivo via rapid establishment of perfusion by inte-
gration with the host vasculature, which can generate more physiologically relevant tissues. There are numerous approaches to
engineer tissue constructs with vascular networks. Each strategy has unique advantages and disadvantages. Generally, the
capillary-like networks must be self-assembled and self-organized by inclusion of relevant cell types because they are too small
to be fabricated. Thus, the strategy of vascularization in vitro needs to mimic the several stages of the biological mechanisms
involved in vascularization in vivo including cell–cell interaction, elongation, network formation, and maturation. Below, we
will discuss general strategies for creating in vitro model systems of vascularization.
Biomaterials for Vascularization

The selection of appropriate biomaterials is the first important step for the prevascularization of the tissue construct. A variety of
biomaterials have been studied to mimic structural and functional properties of the natural ECM. Whether natural or synthetic,
the ideal material should contain several key properties such as biocompatibility, biodegradability, tunable mechanical strength
and swelling behavior, and binding sites that interact with cells to enable attachment and growth. The biomaterials must not be
toxic and should provide a 3D matrix for cells to grow, proliferate, and function normally. Any degraded by-products should
not interrupt cell behavior or tissue regeneration. The degradation rate should also be controllable so that cells can move freely
and organize themselves within the three-dimensional (3D) matrix. In this regard, hydrogel-based materials have been widely
used to create a 3D microenvironment that allows ECs to self-assemble and organize into functional vascular networks. Although
ECs can form cord-like structures in 2D culture, they can only form functional tubular structures with lumens when cultured in a 3D
microenvironment. However, cells in a 3D environment will not lead to capillary-like tube formation unless the matrix has binding
or cleavage sites for cell attachment and degradation, respectively. Many natural materials have RGD (Arg-Gly-Asp) binding and
matrix metalloproteinase (MMP)-degradable sites, but synthetic materials need to be incorporated with binding and cleavable sites.
Natural materials that have been used to provide a matrix for prevascularization of tissue constructs include collagen, fibrin, matri-
gel, decellularized extracellular matrix, silk, and so on. Among them, collagen is the most abundant protein in mammalian extra-
cellular matrix, and has been widely used for prevascularization. It degrades faster than synthetic materials, and its physicochemical
properties can be readily manipulated to promote angiogenesis by mixing with other biomaterials such as chitosan, fibrin, and hya-
luronic acid. Fibrin plays important roles in blood clotting and wound healing and provides binding for cell adhesion and growth
factor attachment. Fibrin can also promote angiogenesis by incorporation of growth factors or coculturing ECs with MCs. Matrigel is
one of the most widely used natural materials extracted from mouse sarcoma cells. It is pro-angiogenic, but is challenging to control
batch-to-batch reproducibility with regard to chemical composition that makes it difficult to apply to clinical practice.
Synthetic materials, on the other hand, can be made via controllable chemical reactions from different precursors, and are easily
reproduced at large scales. They have tunable mechanical properties, degradation rates, and porosity. However, they do not have cell
binding sites, and thus require additional chemical modification to promote cell–material interactions. Many synthetic materials
that have been used for the prevascularization of tissue constructs include polyethylene glycol (PEG), polylactic acid (PLA), poly-
glycolic acid (PGA), polylactic-glycolic acid (PLGA), polycaprolactone (PCL), polyurethane, and so on. Among them, PEG-based
hydrogels have been widely studied for vascularization applications. The PEG terminal hydroxyl groups can be modified with
various biomolecules to improve cell adhesion, growth, and functionality. The degradation of PEG, which naturally occurs very
slowly, can be accelerated by incorporating other materials such as collagen, fibrin, and MMP-sensitive molecules. Its mechanical
properties can be easily modulated by using different concentrations, molecular weights, and cross-linking densities. Appropriate
biomaterial selection plays an important role in the success of prevascularized tissue constructs. Ideal biomaterials need to maintain
the mechanical integrity of the tissue construct without interfering with preformed vascular networks during the remodeling
process.
In Vitro Endothelialization
The next important issue to be discussed is the alignment and organization of ECs within an in vitro system. There are various cell
sources for prevascularized tissue constructs, including autologous circulating endothelial progenitor cells (EPCs), postnatal stem
cells, and induced pluripotent stem cells (iPSC) that can act as precursors to ECs or other vascular cell phenotypes. In numerous
studies, the addition of ECs or EPCs into engineered tissue constructs has yielded increased vascularization and perfusion both
in vitro and in vivo. ECs are a key cell type that plays an important role in formation of structural and functional blood vessels.
EPCs have also showed angiogenic potential with enhanced proliferation and survival rate. However, randomly organized ECs
or EPCs in vitro will induce uncontrolled vascular networks that may not supply larger constructs with sufficient nutrients or oxygen
after implantation. This is because the randomly formed in vitro networks may not be primed for spontaneous anastomosis, leading
to a delay or failure of blood perfusion in vivo. Generally, the in vitro formation of vascular networks in hydrogels takes time, and
regression after a few days of culture is common. Thus, the in vitro organization of ECs needs to be predesigned and controlled by
patterning the vasculature within the hydrogels. One strategy is to culture ECs in hollow microchannels to avoid initial capillary
formation. The EC lined channels are then used as a template for further vessel formation. In addition, different cell types can
be seeded separately in the surrounding biomaterial to encourage vascular maturation. There are various approaches to create
the hollow microchannels and patterns using laser drills, silicon molds, sacrificial materials, and so on. However, formation of
highly organized networks is usually limited by the resolution of the channel-forming methods, and nondegradable dense hydrogel
layers in in vitro model systems restrain further vascular remodeling. Another strategy is to directly pattern the endothelial cells
within a controlled 2D construct under in vitro conditions using PDMS molds, photopatterning, and cell sheets to enhance the
initial EC alignment and promote organization of vascular networks. More complex 3D vascular structures can be obtained by
combining these 2D constructs or using 3D bioprinting technology. 3D bioprinters can deposit bioinks containing cells or cell
aggregates at a designated area with a desired local density, high resolution, and anatomical shape. However, the initial organization
of the vascular structures within the in vitro system is easily changed during vascular remodeling. Therefore, further strategies are
necessary to maintain the long-term functionality of the preformed vascular networks.
Angiogenic Growth Factors and Biomolecules

Sustained and localized release of growth factors is a well-defined chemotactic cue to control vascular organization and angiogen-
esis in engineered tissue constructs. Since vascular organization consists of multiple phases including the initial network formation
and maturation, the patterning of multiple growth factors in space and time is important to control both stages. Vascular endo-
thelial growth factor (VEGF) has proangiogenic functions such as inducing EC elongation, network formation, and branching. The
combination of VEGF with other growth factors such as platelet-derived growth factor (PDGF) and Angiopoietin 1(Ang1) can
improve both vascular structure formation and maturation. PDGF that is released by ECs in the later stage of vessel formation
recruits MCs such as pericytes, and plays an important role in their proliferation and migration toward the preformed vessel
networks. Ang1 released by MCs and ECs–MCs contact significantly affect EC quiescence and stabilization. As such, patterning
and release of multiple growth factors over different time periods is a major challenge to promote both local vascular formation
and stabilization. One approach is to use a dual growth factor delivery system consisting of a polymer scaffold and microspheres.
VEGF can be incorporated into a polymer scaffold and PDGF can be encapsulated into polymer microspheres that are mixed with
the polymer scaffold. Thus, the release of VEGF and PDGF with different kinetics can promote rapid vessel formation via sustained
release of VEGF and vessel maturation by recruited pericytes via release of PDGF. Another approach is to coculture ECs with sup-
porting cells such as fibroblasts, pericytes, and mesenchymal stem cells (MSCs) without additional chemical stimulus. The sup-
porting cells can modulate the expression of multiple angiogenic factors including VEGF, Ang1, basic fibroblast growth factor
(bFGF), transforming growth factor beta (TGF-b), laminins, and integrins to produce biologically regulated cues based on their
current needs. Thus, controlling cross-talk between different cell types is one of important ways to enhance vessel formation
and maturation.
Mechanobiology
Cell behaviors correspond to the mechanical properties of local microenvironments. Thus, another important requirement for
vascular organization is to control local stiffness of the biomaterials as measured by the compression modulus. The optimal matrix
stiffness varies according to the type of biomaterials used for vascular formation, but it is generally agreed that decreasing stiffness of
the materials is likely to induce more EC capillary formation. It has been reported that human umbilical vein endothelial cells
(HUVECs) expressed higher levels of functional VEGF receptor-2 protein when low stiffness collagen (3kPa) was used for the culture
matrix as compared with high stiffness collagen (30kPa). However, optimal stiffness of the materials must often be determined by
taking into consideration conflicting needs in a tissue construct. For example, in designing a prevascularized bone tissue, a soft
matrix is required for vascularization while a rigid scaffold is preferred for bone formation. To reach a suitable compromise, it is
often a promising approach to place different cell types in separate materials with optimal properties for each desired characteristic.
But it is not easy to achieve high spatial resolution between regions of significantly different local stiffness during the creation of
a prevascularized tissue in vitro. It is challenging to find mechanobiology strategies capable of combining materials of different
properties in order to balance the various needs of the model system.
Perfusion Systems
Most cells in the body are known to be located within 100–200 mm from the nearest capillaries that transport nutrients, oxygen, and
waste products to support cell and tissue viability. Similarly, the engineered tissue constructs need to be cultured with appropriate
perfusion systems providing media flow that enables mass transfer of oxygen and soluble factors to promote cell growth and distri-
bution. In addition, the media flow in perfusion systems can be directed through hollow microchannels within the tissue construct
to mimic hemodynamic forces and pressures. The media flow creates shear stress on the surface of the cells, which is an important
signal to guide the vascular organization and maturation of ECs and EPCs. The mechanical cues provided by perfusion play an
important role in stimulating the formation of ECM molecules such as elastin and collagen, regulate EC proliferation, and improve
vascular mechanical properties in vitro.
Vascular Maturation in Coculture Systems

Successful prevascularization of engineered tissue constructs relies on stabilization of the vasculature in vitro prior to implanta-
tion. Previous studies have demonstrated that immature microvessels are not likely to induce anastomosis with the host vessels
and are prone to regression. This is because vascular networks that are not stabilized during vascular remodeling become disor-
ganized and leaky. Coculture with supporting cells such as MCs and MSCs can directly promote stabilization of EC capillary
formations. Pericytes can help maintain newly formed capillary tubes by releasing tissue inhibitor of metalloproteinase 2 and
3 (TIMP-2/3). Pericytes also induce ECs to deposit ECM components including collagen IV, fibronectin, laminin, perlecan, and
so on. In addition, the coculture of mural precursor cells such as embryonic fibroblasts or MSCs with ECs has been shown to
increase the luminal content of the endothelial structures, which is an indicator of the functionalization and maturation of
vascular networks. Additionally, the coculture enhances expression of smooth muscle actin (a-SMA) of MSCs and promotes their
differentiation into MCs.
However, coculturing multiple cell types in the same system is challenging because the optimal culture conditions for ECs
may not be appropriate for other cell types. There are numerous factors that can affect cell behavior in a coculture system
including the cell sources, cell ratios, seeding densities, culture media formulations, cell seeding methods, and the types of
biomaterials used. Balancing all these conditions is difficult and may not be sufficient to achieve the desired functionality of
the end tissue construct. Thus, an in vitro coculture model system should be designed by taking into consideration the purpose
of the study. If the different cell types are mixed simultaneously, then the homogeneous mixture can be distributed throughout
the in vitro model system. This method can enhance cell–cell contact and cell functionality if these cell types are closely located
with one another in the tissue. However, the timing of maturation is also important during in vitro prevascularization because
vessel maturation usually starts by suppressing EC growth. Initiation of the maturation process should occur after ECs have
formed vascular networks that cover the entire tissue construct. But if it is left too late, then the preformed networks will regress.
To avoid this problem, the different cell types can be seeded sequentially. Culturing ECs first will provide enough time to induce
capillary-like network formation, and the subsequent addition of MCs or MSCs will support the preformed networks in terms of
stability and functionality. However, the sequential seeding method is technically challenging, as the second type of cells must be
incorporated into the in vitro model system after capillary-like network has already been formed. In order for this to be achieved,
the model system should include porous structures, hollow microchannels, or provide chemical stimuli to promote ingrowth of
the second type of cells.
The immense complexity of native tissues is challenging to mimic in tissue engineered constructs, which must attempt to harmo-
nize a myriad of biomolecular conditions and micro to macroscale architectural designs using only nontoxic materials that can
remodel and integrate with host tissues. In order to manufacture such advanced model systems and tissue grafts, 3D printing tech-
niques have gained prominence within tissue engineering as a means to achieve high control over construct composition and spatial
organization that would not be possible to replicate by traditional fabrication processes.
3D Printing Methods for Prevascularized Tissue Constructs
3D printing technologies have led to significant advancements in tissue engineering and regenerative medicine. With 3D bio-
fabrication techniques, researchers have generated prevascularzied tissue constructs with complex 3D architectures to mimic the
microenvironment and biological components of native tissues. Compared with traditional manufacturing processes, such as
particulate leaching and solvent casting, 3D printing provides greater precision and automation with higher processing speeds
for engineering the tissue constructs. A variety of 3D printing technologies exist to manufacture different 3D printed tissues with
tailored applications and material properties. Thus, different 3D printing techniques must sometimes be combined to most effec-
tively print specific tissue components within a complex construct. Including a preformed vasculature within 3D printed constructs
necessitates selecting a printing technique suitable for both ECs and any secondary cells required for the tissue of interest. If such
conditions cannot be found, as is often the case, then a combination of 3D printing technologies might be selected to recreate the
various properties of the constituent tissue parts. Several 3D printing techniques are introduced in the following sections, catego-
rized based on whether the printing materials can include cells or not (acellular or cell-laden materials).
General Process of Forming a 3D Printed Scaffold

The 3D printing process can generally be divided into 4 steps: generating 3D computer-aid-designed (CAD) models, preprocessing
preparation, manufacturing, and postprocessing treatment (Fig. 2). The 3D CAD models can be designed by specialized software
such as SolidWorks and AutoCAD, or can be acquired from computed tomography (CT) or magnetic resonance imaging (MRI). The
next step is to convert the CAD model into commands for the 3D printer. The third step is to run the manufacturing process; based
on the preprocessing files, the product is then printed in a layer-by-layer fashion. Sometimes postprocessing modifications to the
printed product might be necessary, for instance, after fused deposition modeling (FDM) production, it may be required to remove
supporting materials, and constructs manufactured by selective laser sintering (SLS) must be baked in order to stabilize the object
through crosslinking.
In tissue engineering, 3D printed scaffolds have been used to provide mechanical support, encapsulate drugs, and position cells
in a 3D environment. 3D printing technologies can be differentiated into two categories, acellular and cell-laden, depending on
whether the incorporation of cells in the scaffold is compatible with the 3D printing process.
Forming acellular scaffolds for supporting the vascularized scaffold

Due to limitations such as high temperatures or extruding pressures during the manufacturing process, acellular scaffold technol-
ogies cannot be used for encapsulating cells. Instead, with high stiffness, these scaffolds are usually used as supporting structures
within engineered tissues. FDM and SLS are the general methods to fabricate acellular scaffolds.
1. Fused deposition modeling (FDM) and syringe-based extrusion
FDM is the most common technology for commercialized 3D printers due to its low cost and the availability of printing
materials. FDM 3D printers mainly consist of three parts including the printing filament, an extruder, and a printing stage
(Fig. 3A). The FDM 3D printer extruder is able to move in the XY plane, and the printing stage moves along the Z-axis. The 3D
printing filament is the raw material that forms the final scaffold, and is typically made of polymer-based materials such as
polycaprolactone (PCL), polylactic acid (PLA), or composite materials such as polycaprolactone-tricalcium phosphate (PCL-
TCP). During the extrusion process, a motor in the extruder drives the filament through the heater at the hot end, the fila-
ment in the heating zone is then melted, and the hot end is thus filled with molten material. Motors in the extruder rotate as
prescribed by the G-codes and drive the cold filament which is stiff to push out the molten material at the hot end. To print
complex shapes of a scaffold such as suspended bridges, supporting structure is required. In addition, when nozzle moves in the
air, the redundant material from the last move is attached and floated with the nozzle; hence some redundant strands are
formed. After the printing process, any redundant strands or supporting materials are removed from the scaffold.
The G-code programming language provides the commands to control the FDM 3D printer. The user is not required to generate
all the G-code by themselves; instead, open source G-code generators such as Slic3r (http://slic3r.org/) can help the users to
G1 ×1 Y1 E0.01
G1 ×1 Y1.2 E0.01
G1 ×1.2 Y1.2 E0.02
G1 ×1.2 Y1.4 E0.01
G1 ×1 Y1.4 E0.01
..
.
3D CAD model Preprocessing Manufacturing 3D printed constructs

Fig. 2 The 3D Printing Process. The first step of 3D printing is designing the 3D CAD model using specialized software, and then translating the
model into commands to the printer during the preprocessing preparation. During the manufacturing process which runs based on the code gener-
ated from the preprocessing preparation, the construct is printed. Some processes might be required for postprocessing modification.
(A) FDM (B) Extrusion-based (C) SLS

Molten
material Strut
Roller Scanner
Heater Laser
source
Powder
Stage Stage
(D) SLA Prepolymer (E) Inkjet-based (F) Laser-based

Build stage solution Laser
source
Vapor
Build vat bubble Heater Glass Laser
absorbing
layer
Light
Light
Biomaterial
Photocrosslinking source
Fig. 3 The 3D printing process. (A) Fused deposition modeling (FDM). (B) Syringe-based extrusion printing. (C) Selective laser sintering (SLS). (D)
Stereolithography or digital light processing-based stereolithography (DLP-SLA). (E) Inkjet-based printing. (F) Laser-based printing.
convert their 3D model into G-code if given the appropriate defined parameters, such as porosity, nozzle diameter, and printing
pattern.
Syringe-based extrusion is a similar 3D printing technique to FDM. While FDM heats up the filament to extrude the strand,
syringe-based extrusion extrudes viscous biomaterials by pneumatic pump (Fig. 3B). Since the material is stored in the syringe
under low temperature, the syringe-based extrusion method can be used for printing cell-laden materials.
2. Selective laser sintering (SLS)
SLS printing mixes powders made of polymer, ceramics, or composites with crosslinkers and uses this mixture to fill a pool
(Fig. 3C). The CAD model is converted to a path based on the desired dimensions and porosity. The manufacturing process starts
with moving the printing stage downward until it is below the level of the of the powder mixture surface by a fixed distance, then
the roller pushes the powder and crosslinker mixture onto the stage. A laser beam runs through the desired paths to initiate the
crosslinking. After a layer is crosslinked, the stage with the powders moves downward again, and the roller fills the printing stage
with powders. The manufacturing process repeats until all the layers are printed. The SLS printed construct, however, must be
further baked during postprocessing to stabilize the crosslinked structures.
Manufacturing the cell-laden constructs

Stereolithography (SLA), Digital-light-processing-based SLA (DLP-SLA), and inkjet-based methods are the most common 3D
printing methods to manufacture cell-laden hydrogels. The printing must be performed under sterile conditions when the cell-
laden hydrogels are printed, and the printing materials should be mostly water-based and biocompatible.
1. Stereolithography (SLA) and digital light-based stereolithography (DLP-SLA)
The SLA and DLP-SLA methods are used to form photo-crosslinked polymer networks when placed under a light source. During
the preprocessing preparation of SLA, the 3D CAD model is sliced into several layers based on the desired layer thickness. When
it starts printing, after the stage moves up a layer in Z-axis, the laser beam runs through the designed paths and forms a photo-
crosslinked hydrogel layer containing cells. After a layer is formed, the stage moves to the next layer, and these processes repeat
until an engineered tissue construct is built. On the other hand, DLP-SLA method projects each cross-section image directly into
the polymer solution to fabricate the tissue constructs at once instead of using a laser beam (Fig. 3D). For engineering cell-laden
tissue constructs, the printing materials for SLA and DLP-SLA should be biocompatible, and can be made from either synthetic or
natural polymers such as polyethylene glycol (PEG), gelatine, and collagen, or the composites such as gelatin methacryloyl
(GelMA). The properties of the printing materials such as porosity, mechanical stiffness, and degradation are easily adjustable by
controlling the light exposure times, the concentrations of the polymers, and the photoinitiators. The longer the exposure time is,
the higher the degree of crosslinking within the engineered tissue construct. Under the same exposure time, with higher
concentration of the polymers or the photoinitiators, the tissue construct becomes denser and stiffer. In order to print
prevascularized tissue constructs, the EC and GFs can be homogeneously mixed in the prepolymer solution to form the structure
by SLA.
2. Inkjet-based deposition
Inkjet-based 3D printers are designed based on the traditional 2D inkjet paper printer (Fig. 3E). The printing material is stored
inside the chamber at the print head under low temperature to maintain low viscosity of the material, such as thermally sensitive
gelatin- or collagen-based polymer solution. When depositing the droplet, the heater heats up the vapor bubble inside the print
head to increase its volume, and this droplet cuts the stream of the material and pushes out the droplet physically. In the
preprocessing preparation, the CAD model is sliced into layers, and each layer is converted into array of droplets. During the
manufacturing process, the print head ejects the droplets onto the print stage under the low temperature, layer by layer. The
printed tissue construct is then moved to an incubator to cure the droplets. As mentioned, the printing material should be
biodegradable and biocompatible so that living cells can be directly involved inside the droplets during the printing process. The
printer can also be programmed to place multiple printing materials and cell types in a specific pattern in order to pattern
prevascular networks within larger constructs.
3. Laser-induced forward transfer (LIFT)
Similar to the inkjet-based 3D printing, LIFT also forms tissue constructs by depositing droplets (Fig. 3F). There are three main
components for the LIFT 3D printer including a laser source, a glass coated with a laser absorbing layer, and a printing material.
To deposit the printing material onto the printing stage, the laser focuses on the desired point at the laser absorbing layer, then
the focused region is heated and increases its volume to push a droplet of material onto the stage. The construct can be printed by
repeatedly depositing droplets with desired patterns in a layer-by-layer fashion.
Challenges of 3D Printed Tissue Construct

The acellular scaffolds formed by FDM and SLS are mostly used to provide tissue constructs with higher mechanical stiffness. On the
other hand, the cell-laden hydrogel methods are used to provide the supporting cellular matrix and print living cells or drugs in
a specific pattern. SLA, DLP-SLA, inkjet- and laser-based 3D printing methods are the common 3D printing processes used to
make cell-laden hydrogels. To maintain cell viability, the 3D printing materials should form water-swollen hydrogel networks
that remain insoluble after the 3D printing process. These hydrogels can be formed via photo-, chemical-, and thermal-
crosslinking during 3D printing process. Although 3D printing enables researchers to create tissue constructs more easily, forming
vascular networks within the engineered tissues is still challenging due to limited integration between different materials and cell
types. A combinatory 3D printing platform for FDM and DLP-SLA can be used as one strategy to continuously print rigid, porous
scaffolds supporting soft, biodegradable hydrogels. This hybrid printing platform can print different cell types in separate materials
with different local stiffness at high resolution.
Another challenge is the achievement of permeability throughout the large-scale tissue constructs and the design of perfusable
channel systems that provide sufficient exchange of soluble factors such as oxygen, nutrients, and wastes throughout the entire area.
The diffusion distance is generally restricted within 300 mm in the engineered tissue constructs; thus all embedded cells in a tissue
construct thicker than 300 mm may not have immediate access to the mass exchanges, affecting cell survival and functional tissue
formation. Thus, the engineered tissue should contain porous structures and perfusable channels for sufficient media supply.
Approaches to designing perfusable channel systems use water-soluble sacrificial polymers such as carbohydrate glass, Pluronic
F127, sugar, gelatin, polyvinyl alcohol (PVA), and so on. Using 3D printing techniques, the sacrificial materials can be printed
alongside cell-laden hydrogels and dissolved upon infusion of aqueous media or temperature changes. The formed channels
can provide effective perfusion of culture media and soluble growth factors that enhance cell viability and facilitating vessel forma-
tion. In addition, ECs that are perfused through the channels can attach on the surface of the hydrogels and migrate into the hydro-
gel construct. Different types of cells such as pericytes and fibroblasts can be perfused and cocultured with ECs or preformed vascular
networks in the hydrogels.
Another issue to be considered is 3D printing resolution for manufacturing the small-scale tissue constructs. To form microvas-
cular networks, resolution of 3D printing should be as low as 100 um. With the different 3D printing techniques, there are various
resolutions to form hydrogel-based materials depending on the materials, crosslinking methods, and types of tissue constructs. The
printing resolution of photo-crosslinkable polymers is highly affected by the polymer and photoinitiator concentrations, and expo-
sure times of light sources such as visible or UV lamps during the photo-crosslinking process. The chemicals used as photoinitiators
are generally toxic, yet higher concentrations of the photoinitiator can enable printing at higher resolution. Thus, researchers need to
balance in the tradeoff between the cytotoxicity and the resolution based on the purpose of the study and types of tissue constructs.
The polymer concentration and exposure time also need to be considered regarding mechanical stiffness and material degradation,
as higher polymer concentrations and longer exposure times produce stiffer and more slowly degrading hydrogel matrices, but they
may delay formation of vascular networks in the engineered tissues.
Applications in Vascularization in Bone and Cardiac Tissue Engineering
Strategies for engineering vascularized tissues may vary according to the application of the engineered implant. Functional vascu-
larization enables the development of thicker tissue constructs and allows for longer testing periods. Applications for engineered
vascularized tissues are numerous both in vitro and in vivo. In this section, we use bone and cardiac tissue grafts as two models
for the prevascularization of engineered tissue constructs.
Applications in Bone Tissue Engineering

Challenges for engineered bone grafts
More than half a million patients receive bone grafts annually in the United States. Autograft is the gold-standard procedure, con-
sisting in harvesting bone tissues from one site in the patient and transplanting it to the site of need. However, one of the major
drawbacks is the creation of a second defect at the harvesting site. Allograft, which consists in transplanting bone tissue from a donor,
is the second most common technique. But it presents a reduced osteoinductivity and risks of immune reactions. Thus, there is
a great need to improve the current clinical treatments for bone repair and regeneration using engineered functional bone grafts.
In addition, bone tissues are highly vascularized and present a complex intraosseous vascular network that ensures tissue survival
and plays a key role in the bone’s self-healing capability. But mimicking this vascular network is to date one of the major hurdles
that delays the development of functional bone tissues. Given the potential impact of engineered bone grafts, numerous studies
have been conducted for the development of vascularized bone tissues.
Strategies for Developing Vascularized Bone Grafts

Upon implantation, the main functions of engineered bone grafts are to temporarily provide sufficient structural and mechanical
support to adjacent host tissue and create microenvironments for bone cell development and tissue regeneration. Early studies have
proposed the use of rigid scaffolds with porous structures in order to promote the growth of bone forming cells. The geometry of the
scaffolds displays interconnected open pores, with a combination of macro- and micropores (respectively with sizes over 100 mm
and under 20 mm). These porous structures enable the development of bone cells after implantation, but the graft porosity must be
balanced with its mechanical properties. The porous scaffolds are mostly made from ceramics such as hydroxyapatite (HA) and tri-
calcium phosphate (TCP), and polymers such as polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), and polycaprolactone
(PCL) for biocompatibility and osteoinductivity. Applying these porous scaffolds alone to patients with bone defects may lead
to lengthy times required for healing and regeneration after implantation, and is proven to be of limited interest for large-scale
defects (length over 5 cm). In order to reduce the healing time, scaffolds need to be optimized for bone and blood vessel develop-
ment. To date, the development of a fully functional vasculature inside the scaffolds is still an ongoing process. Many different strat-
egies for in vitro or in vivo prevascularization have been developed to overcome the technical limitations.
The goal of in vitro prevascularization is to improve porous engineered bone scaffolds in order to promote conditions amenable
to rapid vascularization prior to implantation (Fig. 4). The prevascularized bone scaffolds will directly anastomose with host
vessels, reducing the time for ECs to form vascular networks after implantation. Additive manufacturing (AM) techniques are great
tools to advance prevascularized bone scaffolds. In bone tissue engineering, FDM is used to manufacture porous rigid scaffolds with
reproducible microstructure, and syringe-based deposition or SLA is augmentatively used to deposit ECM-like hydrogels containing
different growth factors that support EC sprouting and neovessel formation. Patient-specific bone grafts can be designed using
imaging technologies in order to facilitate the implantation surgery and enhances the graft compatibility after insertion. Growth
factors such as VEGF and bFGF and cells such as ECs and fibroblasts can be directly incorporated into the printing materials and
deposited with a predefined pattern to accelerate vascular network formation. Inkjet- and laser-based methods enable researchers
to print droplets of suitable microscopic size and control the deposit location with biologically relevant patterns. Alternatively, the
growth factors can be encapsulated in particles such as fibers or microspheres that are embedded in the hydrogel constructs. The
rigid scaffold can also be directly coated with polymer solutions containing growth factors that are released simultaneously or
sequentially.
Alternatively, in vivo prevascularization strategies consist in implanting the engineered scaffold into a region of the patient adja-
cent to blood vessels that are suitable for surgical transfer. The in vivo incubation period results in the formation of microvascular
networks inside the scaffold. After several weeks, the construct can be harvested with the accompanying blood vessels as a free flap,
(A) (B) (C)
Rigid porous scaffold
Engineered extracellular
matrix
Neovessel developed
through in vivo or in vitro
prevascularization
Extracellular matrix
with growth factors
Fig. 4 Schematic drawing of engineering prevascularized bone grafts for large-size bone defect. (A) bone defect; (B) engineered bone scaffold;
(C) strategies for constructing prevascularized bone graft.
and implanted in the defect site. Although it requires two surgical procedures, this strategy can induce neovascularization with
direct, instantaneous perfusion through the vascular axis after implantation. Different manufacturing methods have been explored
to control the porosity of the scaffold for blood vessel growth. 3DP such as FDM or SLS has proven to be very effective because it
allows accurate control of the pore size and an excellent reproducibility of microporous geometry. A summary of the different strat-
egies mentioned above is presented in Fig. 4, as applied to large bone defect repair. More than simply providing a rigid porous scaf-
fold, these recent bone grafting strategies aim to develop a vascular network inside the scaffold before implantation, in order to
reduce the healing time after implantation and improve the chances of the grafting success.
Applications in Cardiac Tissue Engineering

Challenges for engineered cardiac grafts
Heart disease and strokes, which are the principal cardiovascular diseases, account for more than 40% of all deaths in the United
States. Following myocardial infarction, macrophages remove dead myocytes as part of an inflammatory response, which after
several weeks leads to the development of thick and stiff granulation tissue that then turns into scar tissue. Such tissues reduce
the contractile ability of the heart and can potentially lead to heart failure. By helping to regenerate cardiac tissues, tissue engineering
could hence play a key role in the recovery of patients after myocardial infarction. Heart walls are composed of a high density of cells
such as myocytes and fibroblasts which experience a high metabolic demand because of their constant contractions. A dense
vascular network is therefore necessary in the heart wall for oxygen supply. To be clinically relevant, an engineered cardiac patch
should present a thickness of several millimeters. Such a thickness implies that the integration of a vascular network in the graft
is essential for the cells to survive. Because of the beating nature of the heart, engineered cardiac grafts should present mechanical,
electrical, and functional integration into the organ architecture, and therefore, the constructs should be contractile, electro-
physiologically stable, mechanically robust yet flexible, and vascularized or at least quickly vascularized after implantation.
Strategies for developing vascularized cardiac tissues

Hydrogels are typically used as the main material of cardiac patches, because they present adapted mechanical properties and
biocompatibility. To manufacture such materials, many different processes are used. 3DP has proven to be very efficient, offering
a high resolution and an important freedom of shape. Syringe-based extrusion, inkjet, and laser deposition can be used for cell and
bioagent deposition. Similar to engineering bone grafts, three major factors can be integrated to guide the establishment of vascular
networks including a preformed vascular structure, ECM-like hydrogels, and ECs. Variability in the integration of these components
prior to implantation leads to the development of different approaches.
One strategy is to create a scaffold with a preformed vascular structure integrated into the scaffold geometry. This strategy is to
decellularize a vascularized biologic tissue in order to obtain a tissue construct with intact 3D geometry and vascular networks. This
construct is then used as a scaffold for repopulation by myocytes and ECs, leading to the formation of a fully contractile cardiac
tissue. Another approach aims at building the scaffold from synthetic materials. The geometry of the vascular networks can be
designed using 3D printing technology to manufacture the cardiac grafts with improved freedom of shape and high resolution.
Sacrificial materials such as carbohydrate glass, sugar, and gelatin are used to create a vascular template within the constructs
and removed during a postprocessing phase. ECs can be incorporated inside the channels to facilitate vascular network formation.
Coculture with fibroblasts can improve EC organization and myocyte proliferation. In vitro incubation can be performed in order to
prevascularize the constructs to enable quick anastomosis of the microvessels and the host coronary artery. The main issue in this
approach remains the ability to culture the three kinds of cells together in vitro. Thus, cell compatible manufacturing processes have
to be employed. Indeed, constraints in terms of temperature, internal stress, and cytotoxicity have to be considered. Syringe-based or
laser-based deposition can be adapted processes, as well as SLA using visible and UV light. Angiogenic growth factors can be incor-
porated into the cardiac grafts to promote vascularization. The concentration and diffusion rates of the growth factors are the key
parameters to control, with the goal being to recreate levels as close as possible to biological values. Some techniques that can be
employed to control those parameters include selecting different kinds of hydrogels with varying densities or degradation rates.
Indeed, a proper use of growth factors can stimulate proliferation of fibroblasts and thereby significantly improve the development
of nearby vascular networks. Fig. 5 summarizes an overview of the different approaches. Even though 3D printing presents advan-
tages for manufacturing cardiac grafts, novel printing processes are still under investigation to increase the potential of this tech-
nology for engineering vascularized tissues. The development of vascularized cardiac grafts is an ongoing process, and the ideal
strategy still remains to be found. It is still not entirely clear which of the components (vascular structure, cells, and growth factors)
is needed early on, and to which extent the vascular network should be developed prior to implantation. Achieving reliable vascu-
larization remains a challenge for cardiac grafts and, indeed, for nearly all large-scale tissue grafts to date. Recent manufacturing
technologies such as 3D printing can construct previously inaccessible implant designs and architectures, which will both advance
our comprehension of the underlying biological mechanisms required for tissue regeneration and permit the fabrication of more
appropriate biomimetic tissue implants in the coming years.
(A) (B) (C)
Scaffold with integrated

vascular structure
Myocytes and fibroblasts

Endothelial cells forming
neovessels through a
prevascularization step
Scaffold with embedded

growth factors
Fig. 5 Schematic drawing of engineering prevascularized cardiac patch. (A) heart with scar tissues after myocardiac infarction; (B) engineered
cardiac patch; (C) strategies for constructing prevascularized cardiac patch.
Summary
Numerous prevascularization strategies have been studied to form functional vascular networks within an engineered tissue
construct in vitro under controlled conditions prior to implantation. However, it is difficult but necessary to balance all the condi-
tions, including material selection, alignment, and organization of ECs within an in vitro system, localized chemotactic cues,
optimal stiffness and degradation of the materials, appropriate perfusion systems, and stabilization of the vasculature with cocul-
ture techniques. These conditions can be incorporated into 3D bioprinting technologies for higher precision and speed. Therefore, it
is important for researchers to understand the interrelationship between all the conditions to design appropriate experimental
in vitro and in vivo approaches for the creation of the desirable vascularized tissue constructs and to further the successful trans-
lation of research into clinical settings.
Further Reading
Auger, F. A., Gibot, L., & Lacroix, D. (2013). The pivotal role of vascularization in tissue engineering. Annual Review of Biomedical Engineering, 15, 177–200.
Bose, S., Roy, M., & Bandyopadhyay, A. (2012). Recent advances in bone tissue engineering scaffolds. Trends in Biotechnology, 30(10), 546–554.
Davis, G. E., Bayless, K. J., & Mavila, A. (2002). Molecular basis of endothelial cell morphogenesis in three-dimensional extracellular matrices. The Anatomical Record, 268(3),
252–275.
Davis, G. E., Stratman, A. N., Sacharidou, A., & Koh, W. (2011). Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting.
International Review of Cell and Molecular Biology, 288, 101–165.
Elomaa, L., & Yang, Y. (2017). Additive manufacturing of vascular grafts and vascularized tissue constructs. Tissue Engineering, Part B: Reviews. https://doi.org/10.1089/
ten.teb.2016.0348.
Lovett, M., Lee, K., Edwards, A., & Kaplan, D. L. (2009). Vascularization strategies for tissue engineering. Tissue Engineering, Part B: Reviews, 15(3), 353–370.
Lutolf, M. P., & Hubbell, J. A. (2005). Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnology, 23,
47–55.
Mistry, A. S., & Mikos, A. G. (2005). Tissue engineering strategies for bone regeneration. Regenerative medicine II. Heidelberg: Springer, 1–22.
Murphy, S. V., & Atala, A. (2014). 3D bioprinting of tissues and organs. Nature Biotechnology, 32(8), 773–785.
Novosel, E. C., Kleinhans, C., & Kluger, P. J. (2011). Vascularization is the key challenge in tissue engineering. Advanced Drug Delivery Reviews, 63(4), 300–311.
Patan, S. (2000). Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. Journal of Neuro-Oncology, 50(1), 1–15.
Ribatti, D., Vacca, A., Nico, B., Roncali, L., & Dammacco, F. (2001). Postnatal vasculogenesis. Mechanisms of Development, 100(2), 157–163.
Shanjani, Y., Pan, C. C., Elomaa, L., & Yang, Y. (2015). A novel bioprinting method and system for forming hybrid tissue engineering constructs. Biofabrication, 7(4), 1–16.
Vunjak-Novakovic, G., Tandon, N., Godier, A., Maidhof, R., Marsano, A., Martens, T. P., & Radisic, M. (2009). Challenges in cardiac tissue engineering. Tissue Engineering, Part B:
Reviews, 16(2), 169–187.
Ucuzian, A. A., & Greisler, H. P. (2007). In vitro models of angiogenesis. World Journal of Surgery, 31(4), 654–663.
Wound Healing and the Host Response in Regenerative Engineering
Daniel Chester, Ethan A Marrow, Michael A Daniele, and Ashley C Brown, North Carolina State University and University of North
Carolina at Chapel Hill, Raleigh, NC, United States; and North Carolina State University, Raleigh, NC, United States
Overview of the Wound Healing Process 708

The Four Phases of Wound Healing 708
Hemostatic Phase 708
Inflammatory Phase 709
Proliferation Phase 709
Remodeling Phase 710
Host Responses in Wound Healing 710
Abnormal Wound Healing 711
Host Responses to Biomaterials 712
Biomaterials Used in Wound Healing 713
Natural Biomaterials 714
Engineered Growth Factors/Growth Factor Delivery Systems 716
Emerging Materials 717
Conclusions 717
Further Reading 717
Glossary
Biomaterials Any material that has been engineered specifically to interact with biological systems in order to treat a disorder,
augment a natural process, or repair/ replace a tissue or function of the body.
Chronic Nonhealing Wounds A wound that shows no improvement after four weeks or does not heal completely in eight
weeks.
Decellularization The process by which the extracellular matrix (ECM) of a tissue is isolated from its inhabiting cells resulting
in a scaffold of the original tissue.
Fibrosis The accumulation of excess fibrous scar tissue at the site of an injury.
Fibrous Encapsulation The layer of fibrous connective tissue that forms around an implant sequestering it from the
surrounding tissue.
Foreign Body Response The host’s response to a foreign material occurring at the end stage of the inflammatory process
involving macrophages, foreign body giant cells, and the fibrous encapsulation of the material.
Integration The full acceptance of a biomaterial into native tissue involving the migration and proliferation of cells into the
biomaterial, with minimal scarring.
Natural Biomaterials Biomaterials derived from natural sources and used for their similar structures and composition to native
tissue.
Synthetic Biomaterials Biomaterials manufactured with specific mechanical and chemical properties to match their intended
use in the body.
Wound Healing The four-stage process responsible for maintaining the homeostatic structure and function of tissues following
injury.
Abbreviations
EGF Epithelial growth factor
FACIT Fibril-associated collagens with interrupted triple helices
FBGC Foreign body giant cells
FGF Fibroblast growth factor
MMP Matrix metalloproteinase
NO Nitric oxide
PDGF Platelet-derived growth factor

708 Regenerative Engineering j Wound Healing and the Host Response in Regenerative Engineering
PlGF Placenta growth factor

TGF-a Transforming growth factor alpha
TGF-b Transforming growth factor beta
TNF-a Tumor necrosis factor alpha
Overview of the Wound Healing Process
The wound healing process is responsible for maintaining and reestablishing the homeostatic structure, function, and properties of
tissues. It is a highly orchestrated, complex process modulated by biochemical signals, such as cytokines, and biophysical stimuli
generated from cell–cell or cell–extra cellular matrix (ECM) interactions. Important cell types involved in the wound healing process
include platelets, leukocytes, fibroblasts, keratinocytes, and endothelial cells. Cellular responses are modulated by key cytokines
including transforming growth factor beta (TGF-b), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-a),
vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF). Cellular responses can also be modulated by the
ECM components fibrin/fibrinogen, fibronectin, and collagen. Together, cytokines and ECM components help to regulate the
inflammatory response and control cell proliferation, migration, differentiation, and ECM production.
The ideal result of the wound healing process is the reestablishment of homeostasis through the replacement of the damaged
tissue with new tissue that is structurally and mechanically similar to the native tissue. This makes the wound healing process
a natural regenerative process. As such, understanding how tissue is naturally regenerated will allow for the development of strat-
egies or materials to mimic or greatly augment the natural regeneration process. This junction of understanding the wound healing
process and developing new techniques or materials to control tissue repair embodies the concept of regenerative engineering. The
end result of the wound healing process can be influenced by the host’s response to the wound environment; therefore, many regen-
erative engineering strategies investigate ways to precisely control the host’s response in order to prevent abnormal or deficient tissue
repair.
The Four Phases of Wound Healing
The wound healing process can be separated into four temporarily overlapping phases, the hemostatic, inflammatory, proliferation,
and remodeling phases, which vary in time-scale from several hours to a few weeks. Each phase involves the coordination of specific
cytokines and ECM interactions in order to direct cellular responses. The end result of the wound healing process, when it proceeds
normally, is new tissue with structural and mechanical properties similar to the native tissue. A schematic of the wound healing
process can be seen in Fig. 1 and each phase is also detailed in subsequent sections.
Hemostatic Phase
The hemostatic phase begins immediately following tissue damage with the initiation of the coagulation cascade. The coagu-
lation cascade results in the accumulation of platelets at the wound site and the formation of a fibrin clot which stems blood
flow in order to limit the amount of blood lost. Platelets bind to the exposed collagen found at the wound site, thereby acti-
vating the platelets and amplifying the recruitment of additional platelets. Activated platelets have a higher affinity for fibrin-
ogen; therefore platelet activation leads to fibrinogen accumulation at the wound site catalyzing the formation of the fibrin
clot. Fibrinogen is then cleaved by a-thrombin and spontaneously forms insoluble fibrin fibers increasing the stability of
the provisional fibrin clot. Thrombin also activates the transglutaminase enzyme factor XIIIa, which crosslinks the fibrin fibers
overtime to further increase clot stability. Factor XIIIa also allows for the covalent incorporation of soluble fibronectin into the
provisional fibrin matrix. The resulting matrix of the fibrin clot then becomes the scaffold on which the remaining phases of the
wound repair process proceed.
Along with acting as the scaffold of the wound healing process, fibrin, fibrinogen, and fibronectin also play an important role in
modulating the wound healing process. Fibrin, fibrinogen, and fibronectin are capable of interacting with platelets, leukocytes,
fibroblasts, epithelial cells, and keratinocytes as well as binding with FGF, PDGF, TGF-b, VEGF, and TNF-a. This makes the provi-
sional matrix proteins an important site for cellular interactions as well as a reservoir for growth factors that can provide additional
cues to direct cell fate throughout the wound healing process.
As the formation of the fibrin clot progresses, the bound, activated platelets will begin to release PDGF and TGF-b. PDGF acts as
a chemotaxis agent and will recruit additional platelets to the site of injury along with neutrophils, macrophages, smooth muscle
cells, and fibroblasts. The accumulation and incorporation of platelets into the fibrin clot is a key component in determining the
extent of fibrin clot collapse. Clot collapse occurs due to the contraction of activated platelets incorporated throughout the fibrin
clot and is an important factor in determining the stability of the resulting clot. TGF-b also aides in the chemotaxis of macrophages,
Regenerative Engineering j Wound Healing and the Host Response in Regenerative Engineering 709
(A) (B)
Fibrin Clot Scab
Epidermis
Dermis
Hypodermis
Platelet Blood Vessel
(D) Neutrophil Newly Healed

(C)
Epidermis
Keratinocyte
Fibroblast
Newly Healed
Dermis
Fig. 1 The four stages of wound healing: The wound healing process is separated into four temporarily overlapping phases, the hemostatic (A),
inflammatory (B), proliferation (C), and remodeling phase (D). (A) The hemostatic phase begins immediately following injury and is responsible for
the creation of a fibrin clot in order to stem blood flow and limit the amount of blood lost. Platelets bind to exposed collagen and begin secreting
PDGF and TGF-b in order to recruit additional platelets, macrophages, and cells to the injury site. (B) Approximately 24 h following injury the inflam-
matory phase begins with the accumulation of neutrophils and macrophages at the wound site. The primary purpose of neutrophils and macro-
phages is the removal of any damaged tissue or foreign materials in the wound site. (C) Two to 3 days after injury, the proliferation phase begins.
The proliferation phase is characterized by the accumulation and proliferation of fibroblasts at the site of injury and the production of fibrous extracel-
lular matrix. (D) The final stage of the wound healing process is the remodeling phase where fibroblasts will begin to remodel the newly deposited
collagen matrix into newly healed tissue. This phase can last for months or years, but when concluded results in the formation of new tissue with
similar structure and mechanics to the native tissue.
fibroblasts, and smooth muscle cells to the wound site along with controlling several other important processes that occur in the
subsequent stages of the wound repair process.
Inflammatory Phase
Approximately 24 h after the initiation of the hemostatic phase, neutrophils begin to accumulate at the wound site due to PDGF
secreted by activated platelets within the fibrin clot. The accumulation of neutrophils into the wound site marks the beginning of the
next phase of the wound healing processdthe inflammatory phase. The primary role of the neutrophil is the removal of foreign
materials or cell debris that resulted from the injury through phagocytosis. Phagocytosis is the process by which neutrophils will
engulf foreign material until they no longer have room for other debris. Neutrophils also secrete VEGF which is an important growth
factor in angiogenesis. Approximately 48 h after injury, macrophages accumulate at the wound site due to chemotaxis from the
PDGF and TGF-b secreted by the activated platelets in the fibrin clot. Upon entering the wound site, macrophages begin to phago-
cytose any remaining debris that was too large for the neutrophils and any completely filled neutrophils. Macrophages are also
responsible for secreting additional PDGF, FGF, TNF-a, and TGF-b which leads to an increase in the number of macrophages at
the wound site and attracts fibroblasts, endothelial cells, and smooth muscle cells to the wound site. Macrophages additionally
produce several reactive radicals including nitric oxide (NO), oxygen, and peroxide which are important antimicrobial agents.
NO can also be produced, although to a lesser extent, by fibroblasts, keratinocytes, and endothelial cells and plays an important
role in the later stages of wound healing. 5 days after injury, T-lymphocytes and B-lymphocytes begin to migrate into the wound
site marking the end of the inflammatory phase. T-lymphocytes are believed to play a role in downregulating the inflammatory
process and in mediating cell proliferation and the degree of collagen crosslinking. The role of B-lymphocytes still remains largely
unknown and more research is needed in order to fully elucidate their role in the wound healing process.
Proliferation Phase
The inflammatory phase starts to wane 2–3 days after injury at which point the proliferation phase begins. During the proliferation
stage, cells will migrate into the fibrin provisional matrix and begin to produce a new ECM. Fibroblasts are recruited into the wound
site by FGF and TGF-b secreted by the macrophages and activated platelets. TGF-b is also responsible for increasing the rate at which
new ECM is produced by upregulating the transcription of several genes that promote the synthesis of collagen, proteoglycans, and
fibronectin. At the same time, TGF-b is also responsible for the downregulation of the matrix metalloproteinase (MMP) family of
enzymes that are responsible for the degradation of ECM proteins. Epithelialization also begins as epithelial cells begin to infiltrate
into the wound site due to chemotaxis controlled by epidermal growth factor (EGF) and transforming growth factor a (TGF-a)
which are produced by platelets and macrophages. The NO produced by macrophages, fibroblasts, keratinocytes, and endothelial
cells also plays an important role at this stage of the wound healing process. NO has been shown to control the rate of epithelial-
ization and the rate of collagen syntheses; the inhibition of NO slows the epithelialization process and decreases the rate of collagen
synthesis resulting in slower wound contraction and lower clot strength.
A process called fibroplasia begins during the proliferation phase of the wound healing process. Fibroplasia starts approximately
5 days after the initial injury and is the process by which fibroblasts begin to produce the collagen matrix backbone found in normal
tissue that eventually replaces the provisional fibrin matrix. There are three different classes of collagen that can be deposited during
this process. These classes are fibrillar type I, II, or III collagen, nonfibrillar type IV collagen, or fibril-associated collagens with inter-
rupted triple helices (FACIT) type VI and VII collagen. Each class of collagen plays a specific role in the formation of new ECM with
fibrillar collagen forming rigid linear networks, nonfibrillar collagen forming complex networks of collagen that are usually found
in basement membranes, and FACIT collagen providing matrix stability by integrating with fibrillar collagen and other ECM
proteins.
Each class of collagen is produced at specific times in the wound healing process. At the onset of fibroplasia, fibroblasts
rapidly produce the fibrillar type I and III collagen with peak production occurring between 7 and 14 days following injury.
The initial deposition of type I and III collagen is important for increasing the tensile strength of newly formed tissue with
the collagen fibers becoming linear strands aligned parallel to the forces felt in the tissue. FACIT type VI collagen peaks one
to two weeks after injury and is believed to play an important role in integrating the new collagen matrix with the native neo-
vasculature. The rate of collagen deposition is also self-regulated by the interactions that occur between keratinocytes and fibro-
blasts and the matrix to which they are attached. For example, keratinocytes will increase the amount of collagenase produced
when in direct contact with collagen fibers. The increased amount of collagenase will, in turn, increase the rate of collagen
degradation which ensures that the amount of collagen in the ECM is kept at a homeostatic level. With fibroblasts, an ECM
made primarily of collagen will result in lower proliferation levels as well as lower collagen deposition rates. The lower prolif-
eration and deposition rates occur due to a negative feedback mechanism initiated by the binding of the a1b1 integrin to
collagen.
The process of angiogenesis occurs concurrently to fibroplasia. Angiogenesis is the complex process by which new blood
vessels form that rely on the proper ECM composition as well as cytokine signaling. FGF, VEGF, and TGF-b are important
growth factors involved in angiogenesis; fibrin has also been shown to induce angiogenesis directly. While fibroblasts are
beginning to deposit collagen into the fibrin provisional matrix, endothelial cells are stimulated by the FGF and VEGF released
by platelets, macrophages, and neutrophils to locally dissolve the basement membrane. PDGF, VEGF, and FGF also increase
endothelial cells’ ability to revascularize tissue allowing for the formation of new blood vessels in the basement membrane.
NO also plays a role in angiogenesis as NO promotes vasodilation which improves local blood flow to the wound site allowing
for the faster recruitment of cells, platelets, neutrophils, and macrophages. The newly formed blood vessels are stabilized by
smooth muscle cells which are recruited to the wound site by PDGF that is released by platelets and endothelial cells. Toward
the end of angiogenesis, TGF-b secreted by endothelial cells acts as in inhibitory agent impeding vascular proliferation bringing
angiogenesis to an end.
Remodeling Phase
The final phase of the wound healing process, the remodeling phase, occurs concurrently with granulation tissue formation.
The primary purpose of the remodeling phase is the formation of new epithelium and scar tissue and this process can take up
to a year or longer to complete. Throughout the remodeling phase, fibroblasts degrade and realign collagen fibers. As fibro-
blasts realign and deposit collagen fibers, the fibril packing density in each fiber is seen to increase which leads to collagen
fibers with larger diameters and higher mechanical properties. The realignment of collagen fibers also transforms the initial,
unorganized collagen matrix into a highly organized collagen matrix whose structure closely mimics that of the native tissue.
The realigned collagen matrix will have the potential to regain up to 80% of the native tissue’s tensile strength, but the orig-
inal strength of the tissue will never fully be achieved. How close the new collagen matrix can match the tensile properties of
the native tissue depends on the location and size of the wound as well as the duration of the repair process. As the remod-
eling phase continues, the number of macrophages and fibroblasts decrease due to apoptosis and angiogenesis ends. At the
end of the remodeling phase, new tissue with a high tensile strength and a minimal number of cells and vascularization is
formed.
Host Responses in Wound Healing
There are many different factors that can impact the wound healing process. Systemically, factors such as age, nutrition, and the
presence of diseases such as diabetes can alter the wound healing process. Age does not immediately impair the wound healing
process, but it has been observed that delayed wound healing is common in older organisms and is believed to be associated
with an altered inflammatory response. Malnutrition can lead to impaired capillary formation, fibroblast proliferation, and collagen
synthesis due to lack in the required proteins or energy needed to perform those specific functions. Diseases that lead to hypoxia,
such as diabetes, and diseases that lead to altered immune responses, such as HIV or lupus, have been linked to impaired wound
healing and chronic wounds.
Locally, factors such as foreign bodies, ischemia, and infection can change wound healing responses. The composition of the
foreign body will greatly affect the wound healing response by changing the length and severity of the inflammatory response.
Oxygen is extremely important in the wound healing process so ischemia can also drastically change wound healing responses.
Ischemia can lead to impaired angiogenesis, fibroblast and keratinocyte proliferation, and reepithelialization all of which will
impede wound healing. Infection can lead to a prolonged immune response causing an increased amount of MMPs at the wound
site causing excess degradation of ECM.
Abnormal Wound Healing
As outlined in previous sections, wound healing is a highly regulated process that requires the fine control of both mechanical
and chemical cues in order for normal healing to occur. Any irregularities in the control of the mechanical and/or chemical cues
involved can lead to abnormal wound healing in the form of excessive scar tissue formation or deficient matrix production.
Excess ECM production is known as fibrosis and has been connected to many life-threatening medical conditions, while deficient
ECM is seen in chronic nonhealing wounds which are most readily seen in ulcers. Fig. 2 shows the relationship between the
amount of ECM deposition and the subsequent effect it has on fibroblast cells as they try to repopulate the wound site. If left
untreated, both fibrosis and chronic nonhealing ulcers can lead to detrimental results, and sometimes death, for the affected
organism.
Fibrosis is characterized as the overproduction of connective tissue and ECM resulting in excess scar tissue formation that can
lead to the disruption of the structure, and in extreme cases the function, of the native tissue or organs. Clinical examples of
fibrotic related conditions include keloids, hypertrophic scars, Crohn’s disease, pulmonary fibrosis, and atrial fibrosis. The under-
lying mechanisms behind many fibrosis-related conditions remain poorly understood and are only just beginning to be eluci-
dated. For example, it has been found that fibroblasts isolated from keloids have increased proliferation rates, produce 2–3
times more ECM, and express higher levels of VEGF, PDGF, and TGF-b when compared to normal fibroblasts. Additionally,
a high density of mast cells has been observed at the site of excess scar tissue deposition found in many fibrotic conditions.
Mast cells contain enzymes responsible for processing procollagen and it is believed that in fibrotic conditions abnormally
high amounts of histamine and renin are produced that upregulate collagen synthesis leading to excess ECM deposition and
scar tissue formation.
Nonhealing wounds occur when an abnormally low amount of connective tissue or ECM is produced in the wound bed. It has
been found that chronic ulcers have reduced levels of PDGF, FGF, EGF, and TGF-b. Chronic ulcers also have increased protease
activity which is believed to result from an overexpression of MMPs caused by increased neutrophil infiltration rates and leads to
the increased degradation of ECM. Clinical examples of nonhealing wounds include venous ulcers, diabetic ulcers, or pressure
ulcers. Venous ulcers occur in the legs and are thought to be caused from venous hypertension leading to ischemia. Diabetic
ulcers are believed to stem from neuropathy which inhibits the perception of pain leading to unnoticed injuries that either
get infected or are continuously reinjured. Pressure ulcers are normally found in people suffering from conditions such as paral-
ysis. Pressure ulcers arise from the ischemia that occurs when the pressure on the tissue is greater than the pressure in the
capillaries.
Amount of ECM Deposition and Cell Morphology in

Normal and Abnormal Wound Healing
Chronic Non-Healing Normal Wound

Fibrosis
Wounds Repair
Fig. 2 ECM deposition and cell morphology in normal and abnormal wound healing: key characteristics of abnormal wound healing are the amount
of ECM deposition and the resulting cell morphologies. In chronic nonhealing wounds, there is an abnormally low amount of ECM found at the injury
site. This results in a wound bed with low mechanical properties leading to senescent fibroblasts. Conversely, in fibrosis there is an overproduction
of ECM resulting in a wound environment that has much higher mechanical properties than normal tissue and highly activated fibroblasts. Under
normal wound healing conditions with normal amounts of ECM deposition, fibroblasts will exhibit appropriate amounts of cell spreading and
balanced migration/contraction responses.
Host Responses to Biomaterials
There are many factors that determine how well a biomaterial performs its intended function once implanted in vivo. Such factors
include the biomaterial’s composition, mechanical and material properties, surface topography, size, and placement in the body.
However, the most important factor that determines the success of the implanted material is the resulting host response that begins
immediately following implantation. The host’s response to the biomaterial is a summation of the tissue damage received during
implantation and the response caused by the material itself. The response caused by the tissue damage resolves quickly as part of the
normal healing process, but the foreign body response caused by the material will last as long as the material is present. The prop-
erties of the material implanted dictate the success of the material and whether or not integration or the fibrous encapsulation of the
biomaterial will occur.
Integration is the ideal response for most implanted biomaterials and involves the incorporation of the biomaterial into the
surrounding tissue. In order for integration to occur, a biomaterial should have the proper surface properties to promote the adhe-
sion of the appropriate proteins and cells. Generally, a combination of high porosity and surface roughness or a hydrogel coating is
used to increase cell attachment to a biomaterial. An overview of design considerations to promote material integration is shown in
Fig. 3. Successful integration of the biomaterial into the surrounding tissue is characterized by cellular ingrowth and ECM deposi-
tion along with generating little to no immune response from the host. In practice, complete integration of the biomaterial into the
tissue rarely occurs and it is more likely for fibrous encapsulation to occur to some extent.
Generally, the end result of the wound healing process in the presence of a biomaterial is fibrous encapsulation. The foreign
body response leading to the fibrous encapsulation of the biomaterial is shown in Fig. 4. Fibrous encapsulation is the formation
of scar tissue on the surface of the biomaterial produced by myofibroblasts and fibrocytes and occurs as a response to frustrated
phagocytosis. Frustrated phagocytosis is the process by which macrophages try to dissolve a foreign body that is too large to inter-
nalize. In order to remove the large foreign body, macrophages will join together to form foreign body giant cells (FBGCs) and begin
to release superoxides and free radicals that lower the pH of the local environment resulting in damage to the biomaterial and
surrounding tissue. Fibrous encapsulation will then occur in order to sequester the biomaterial from the rest of the body and
end frustrated phagocytosis. However, if frustrated phagocytosis persists, a chronic nonhealing wound will begin to form at the
site of the implant and lead to the ultimate failure of the implant.
The scar tissue formed as a result of frustrated phagocytosis acts as a barrier between the biomaterial and the wound site severely
limiting the integration of the biomaterial. Fibrous encapsulation also limits the performance of chemical biosensors, electrical
leads/ electrodes, therapeutic delivery systems, and orthopedic/cardiovascular prostheses. Research into the acute inflammatory
Properties for Promoting the Integration of a Biomaterial
Young’s
Modulus Infection Incorporation of
Material Properties
Preventing Drugs Bioactive Agents
Viscoelasticity
Fatigue Strength Cell Encapsulation

Creep
Similar to Control Cellular Growth
Porosity Native Tissue Responses Factors
Biomaterial
Integration
Minimal Non-toxic
Plasma Enhanced Cell Degradation
Response
Attachment Components
Ceramic
Non-fouling
Surface Hydrogel Surface
Coatings Biocompatible Immune Response
ECM Proteins Material
Composition
Fig. 3 Properties for promoting the integration of a biomaterial: the material properties, surface properties, generated immune response, and incor-
poration of bioactive agents should all be considered when choosing a biomaterial to be used for implantation. The closer the biomaterials properties
are to that of the native tissue, the more likely the biomaterial will be incorporated into the native tissue. Properties such as the Young’s modulus,
viscoelasticity, fatigue strength, creep, and porosity should all be considered when creating or choosing a biomaterial for a specific application. It is
highly unlikely that all of the biomaterial properties will be identical to native tissue so, therefore, it is important to prioritize which properties are the
most critical for the specific application. Surface coatings can also be used to increase the rate of cellular attachment to a biomaterial. Plasma
coating, ceramic coatings, hydrogel coatings, and ECM proteins can all be used to increase cellular attachment rates. Controlling the immune
response generated by the biomaterial is also important to the successful integration of the biomaterial. With a low immune response, the foreign
body process will be limited, thereby enhancing the integration of the material. Finally, bioactive agents can be incorporated into the biomaterial in
order to direct and control cellular responses. Antibacterial/antimicrobial drugs can be used to prevent infections and lower immune responses, cells
can be encapsulated into the material in order to provide naturally derived cues to promote cellular interactions with the biomaterial, and growth
factors can also be incorporated in order to direct various cellular responses.
Inflammation Response Foreign Body Giant Cell (FBGC)

Macrophage Adhesion Fibrous Encapsulation
and Protein Adsorption Formation
Neutrophil
Monocyte
Fibroblasts
New ECM
Production
FBGC
Macrophage
Biomaterial
Fig. 4 Host foreign body response to implanted biomaterials: the host response to a biomaterial begins with protein adsorption onto the surface of
that material and the immune response. Due to the large size of biomaterials, neutrophils are unable to phagocytose the implant, begin to undergo
frustrated phagocytosis, and recruit monocytes which then differentiate into macrophages at the implant. Due to the proteins adsorbed to the surface,
macrophages begin to adhere to the surface of the implant while continuing to perform frustrated phagocytosis. The adhered macrophages will then
join together to form foreign body giant cells (FBGCs) which help to recruit fibroblasts to the biomaterial. The recruited fibroblasts will then begin to
synthesize new ECM on the surface of the implant resulting in the fibrous encapsulation of the implant.
responses to implanted biomaterials has shown that the adsorption of fibrinogen onto the surface results in integrin-mediated
leukocyte recruitment and adhesion. The adhered leukocytes will then secrete growth factors that recruit other cell types leading
to fibrous tissue deposition. However, how chronic inflammation mediates fibrous encapsulation and the mechanisms behind it
remain poorly understood.
Biomaterials Used in Wound Healing
Biomaterials can be classified into two main groups: synthetic or natural biomaterials. Synthetic biomaterials consist of metals,
ceramics, and polymers while natural biomaterials consist of protein-based biomaterials, polysaccharide-based biomaterials, and
decellularized-based biomaterials. Both types of biomaterials can be characterized by their surface properties, mechanical proper-
ties, chemical properties, biological properties, and implantation lifetime resulting in many similarities and differences between
both types of materials.
A key difference between synthetic and natural biomaterials is seen in their applications. A division of synthetic biomaterials
consists of metal alloys and ceramics which have Young’s moduli on a similar order of magnitude as bone. This makes metals
and alloys perfect candidates for bone or joint replacements, but not for soft tissues and/or wound healing applications. As
such, metals and ceramics are outside of the scope of this book chapter. On the other hand, synthetic polymers have diverse
and tunable properties making them ideal for soft tissue and wound healing applications and will be discussed below. Since natural
biomaterials are derived from tissues and organs, they are already prime candidates for uses in wound healing applications due to
their similar structure and composition to native tissue. However, naturally derived biomaterials can have limitations in certain
applications due to lack of mechanical robustness and immune rejection adding additional concerns.
The decision of whether to use a synthetic polymer or natural biomaterial for each wound healing application is based on the
properties of the specific biomaterial and the properties that are required to elicit the desired biological response. To this end,
natural biomaterials are more advantageous to use in applications where the structure of the implant needs to be similar to the
environment. Another advantage that natural biomaterials afford is their built-in bioactivity due to being composed of similar
macromolecules as native ECM allowing for interactions between the material and cells. Examples of naturally derived biomaterials
include silk, chitin, ECM components, and decellularized ECM. Additionally, naturally derived decellularized ECM materials also
have growth factors bound to them naturally which can be released to promote specific cellular processes as cells infiltrate and
remodel the scaffold. However, a predominant limitation of many naturally derived biomaterials is that they usually have poor
mechanical properties. Furthermore, naturally derived biomaterials can have a high degree of variability in material properties from
batch to batch. Also, since naturally derived biomaterials have such a similar structure and composition to native tissue, these types
of biomaterials are more likely to cause an immune response which may or may not be desirable depending on the specific
application.
The main advantages for using synthetic polymers in wound healing applications stem from the ability to control their mechan-
ical and chemical properties. Since synthetic polymers are fabricated in controlled settings, it is possible to change the materials
composition during its synthesis such that the desired mechanical or chemical properties are obtained. This also results in relatively
low batch-to-batch variability compared to natural biomaterials. However, the manufactured structures of the synthetic polymer
scaffolds often lack the complexity required to accurately mimic native tissue resulting in reduced levels of cellular infiltration
and bioactivity. Another disadvantage of synthetic polymers is that their biocompatibility is unknown and has to be tested. Since
synthetic polymers are man-made and not normally found in nature, how the host will respond to the material is a serious consid-
eration that needs to be thoroughly tested in order to ensure the success of the material.
Natural Biomaterials
As their name suggests, naturally derived biomaterials are commonly found in nature as part of the tissues and organs of various
organisms. Naturally derived biomaterials are commonly used to repair or replace damaged tissue or organs since they already
contain the required structure and biological composition needed to mimic the native tissue. Because they are naturally derived,
these materials have high degrees of biocompatibility, biodegradability, and are subject to high degrees of remodeling by resident
cells after implantation; all these features further allow for the integration of the biomaterial into the native tissue. Commonly used
examples natural biomaterials that are used in augmenting the wound healing process include silk, chitin, collagen, fibrin, and
various decellularized ECM components.
Silk is a natural fiber composed of hydrophobic fibroin and hydrophilic sericin. Sericin is composed of 25%–30% of the silk
protein and it envelopes the fibroin fibers gluing them together. Most of the sericin needs to be removed before implantation in
the body since the combination of the two proteins has been seen to cause allergic responses. Raw silk can be harvested from several
natural sources such as silkworms and spiders making it an abundantly available material. In its processed form, silk has a high
tensile strength, is biocompatible and biodegradable, and does not generate a significant immune response making it an ideal
candidate for scaffolds and wound dressings. Silk can also be electrospun into nanofibers that have a high specific surface area
and improved thermal and electrical properties compared to nonelectrospun silk which further increases the number of applica-
tions for which it can be used. Clinically, silk is used in sutures for ligament replacement, nonwoven mats for use as wound dress-
ings, and as porous sponge scaffolds for healing bone defects.
Chitin is one of the most abundant biopolymers found on Earth. Chitin is a complex polysaccharide of N-acetyl-glucosamine
and glucosamine, and is predominately found in the exoskeletons of crustaceans and the scales of fish. The degree of deacetylation
and the molecular weight of the polymer can severely impact the material properties and the resulting performance of the chitin
polymer. Chitin is used in wound healing dressings due to its antimicrobial properties and the ability for N-acetyl-glucosamine
to accelerate the rate of tissue repair and prevent the formation of scars. The original use of chitin was as a powder, but more recently
it has been incorporated into films, membranes, and woven/nonwoven dressings. Wound dressing made with chitin was found to
adhere well to wound sites as well as allow for the permeability of oxygen into the wound site.
One of the most abundant ECM components in the body is collagen and it has been widely applied to many different in vitro
and in vivo applications. Clinically, collagen is used because of its nonimmunogenicity and its ability to provide the structural
support needed for tissue regeneration. Collagen is capable of being prepared as a crosslinked solid or into un-crosslinked gels
allowing for versatility in its application. Collagen’s main use as a biomaterial is in the treatment of burns in the form of a wound
dressing or as a bone filling material. Collagen wound dressings have been shown to improve the spreading and growth of chon-
drocytes, fibroblasts, and keratinocytes while collagen bone fillings have been shown to increase bone regeneration rates. Hyalur-
onic acid is another naturally derived ECM component that is commonly used in biomaterials. It is a linear polysaccharide of
glucuronic acid and N-acetyl glucosamine-glucuronic acid disaccharides. Hyaluronic acid is mainly used as a biodegradable hydro-
gel used to deliver drugs or growth factors into the wound environment and has been used to increase cell adhesion and prolifer-
ation rates onto scaffolds. Finally, fibrinogen, a soluble plasma glycoprotein that plays an important role in blood coagulation, is
also commonly used as a biomaterial. The ability for fibrinogen to promote cell adhesion and migration, along with its biocom-
patibility and nonimmunogenicity make it popular for use in tissue scaffolds and wound dressings. Fibrinogen can be electrospun
to form porous, fibrous scaffolds and when combined with thrombin, produce a biodegradable mesh. Fibrin gels are also used clin-
ically with the most popular application being fibrin glues which are formulated by combining fibrinogen and thrombin directly at
the surgical site in high concentrations. Fibrin-based glues function by reproducing the fibrin clot found during the normal wound
healing process. These types of glues are mainly used as hemostatic agents for bleeding surfaces. The microstructure of a fibrin
hydrogel is shown in Fig. 5.
Gelatin-based glues are another type of naturally derived tissue glues that are commonly used clinically. Gelatin is obtained by
controlling the hydrolysis of collagen extracted from animal tissues resulting in a mixture of polypeptides dispersed based off of size
and chemical reactivity. The properties of gelatin are largely based off the tissue and animal it was extracted from and the method of
hydrolysis. The collagen content of gelatin glues result in a resorbable material with a greater bonding strength than fibrin glues
10 µm JEOL 6/14/2016
x 2,000 5.0kV SEI SEM WD 7.0mm 10:49:07
Fig. 5 Network structure of a natural biomaterial gel: a cryo-scanning electron microscopy (SEM) image at 2000 showing the structure of a 3D
fibrin gel composed of 2 mg/mL fibrinogen and 0.1 U/mL thrombin. Image courtesy of Ms. Seema Nandi. Authors acknowledge Dr. Elaine Zhou and
the Analytical Instrumentation Facility at North Carolina State University for their assistance with sample preparation and acquisition.
making them useful for sealing larger tissues. Tissue glues can be used instead of sutures and have been used to treat burns and
adhere skin grafts.
Naturally derived ECM can also be obtained from the decellularization of various tissues and organs. Decellularization aims to
remove all of the cells from their ECM while keeping the ECM intact. With the removal of the cells, a large number of the antigens
that would normally cause an immune response are removed, resulting in a scaffold that has the mechanics and structure of native
tissue with a limited immune response. While the decellularization process does remove cells, many of the growth factors bound
naturally to the ECM remain intact. The bound growth factors can then be used to direct specific cellular responses as they are
released from the ECM by cell-mediated interactions. Common tissues that are decellularized and used as ECM scaffolds commer-
cially include the small intestine submucosa, the urinary bladder matrix, human/porcine/bovine skin, and horse/bovine/porcine
pericardium. These matrices have been used to facilitate the regeneration of skin, tendons, and ligaments. Whole organs are also
being investigated for use as scaffolds and current research on efficiently decellularizing hearts, lungs, and livers for use as organ
replacements is ongoing. Entirely decellularized organs would allow for the exact replication of the organ’s highly complex archi-
tecture which is critical for the function of the organ and would normally be impossible to replicate. The newly decellularized organs
would then be reseeded with cells from the patient and result in a fully functional organ that is an exact genetic match to the patient.
This would then limit the immune response from the patient to the newly implanted organ and increase the safety, efficacy, and
lifetime of implanted organs.
Unlike natural biomaterials, synthetic biomaterials are not found in nature and can only be manufactured. This makes synthetic
materials easy to obtain and available in large quantities. The manufacturing process also allows for the mechanical and chemical
properties to be tailored to the specific application. It is also possible to incorporate drugs during the manufacturing process of
synthetic materials which adds another degree of functionality of these types of materials. Also, synthetic biomaterials can be
coated, functionalized, or conjugated with different proteins or antibodies to limit the immunogenic response and increase the
amount of bioactivity. For wound healing applications, synthetic polymers are commonly used since it is possible to make scaffolds
with material properties that closely match native tissue. Examples of synthetic polymers used clinically are synthetic skin substi-
tutes, tissue glues, hydrogels, and semisynthetic chitosan.
Skin substitutes are artificial skin replacements that provide a protective barrier when placed over burns or other chronic wounds
in a similar manner to normal skin. The primary objective of skin substitutes is to facilitate the repair, regeneration, and restoration
of the functional properties of skin following a traumatic injury such as second or third degree burns. Properties that make a success-
ful skin substitute include water vapor transmission similar to normal skin, minimal inflammatory response, adherence to the
wound site, controlled degradation, impermeable to bacteria, and appropriate mechanical properties. Commercially available
skin substitutes are made of ultrathin silicone, nylon, and petrolatum gauze. Skin substitutes can be temporary or permanent
with the temporary substitutes acting purely as a biodegradable barrier throughout the wound healing process and the permanent
skin substitutes incorporating fibroblasts or epithelial cells along with a treated dermis layer from a human cadaver.
Tissue glues are used to replace traditional sutures and staples in order to seal a wound following damage or a surgical procedure.
There are three different types of tissue glues commercially available. These are fibrin-based glues, gelatin-based glues, and
cyanoacrylate-based glues. Fibrin- and gelatin-based glues have been described above, but a synthetic alternative to these naturally
derived glues are cyanoacrylate-based glues. These types of glues are limited in their use clinically due to their varying degrees of
cytotoxicity. However, they are still used to close dermal wound sites that are still actively bleeding since fibrin- and gelatin-
based glues are not as effective on wounds that are actively bleeding.
Hydrogels are polymer networks that are insoluble in water and swell to an equilibrium volume while retaining their shape.
Hydrogels have structural similarities to the ECM, have the ability to retain a large quantity of water, are biocompatible, have
a low interfacial tension, and cause minimal frictional or mechanical irritation to the implantation site making them a popular
biomaterial for use in regenerative medicine. The hydrophilicity of the polymer network stems from the presence of chemical resi-
dues such as hydroxylic (eOH) and carboxylic (eCOOH) functional groups found along the polymer backbone. The hydrogel
remains insoluble to its 3D network and the balance between the dispersive forces acting on the hydrated chain and the cohesive
forces that prevent the further penetration of water.
Common polymers that are used in the formation of hydrogel are polyethylene oxide, polypropylene oxide, and poly(N-isopro-
pyacrylamide) just to name a few. The properties of hydrogels make them an attractive material to use in the application of implant-
able biomaterials. Since the structure of hydrogels closely mimics that of the ECM, hydrogels are often used as a coating to promote
cell growth and attachment. For example, hyaluronate hydrogels have been used as a coating on an inorganic bone material in order
to increase cell attachment. Hydrogels can also be chemically crosslinked in order to tune their properties to fit the specific appli-
cation. Chemically crosslinked hydrogels offer scaffolds with greater mechanical properties and slower degradation times which
make them ideal for scaffolds designed to last in the body for longer periods of times.
Chitosan is a semisynthetic biomaterial made from the deacetylated form of chitin, which is found naturally. The deacetylation
process makes it a cationic glycosaminoglycan biopolymer with a degree of deacetylation ranging from 30% to 95% and a molecular
weight of 250 kDa to greater than 1000 kDa. The solubility, viscosity, and biocompatibility are proportional to the degree of deace-
tylation while the bioadsorption of chitosan is inversely proportional to molecular weight. Chitosan has been extensively investi-
gated for its potential use in the field of regenerative medicine. One of the most promising applications of chitosan is the
development of dressings to control and promote wound healing. It has been demonstrated that simple application of chitosan-
based materials with varying degrees of deacetylation to a wound can direct healing and regeneration processes. Accordingly, chi-
tosan and chitosan-derived materials have exhibited promising biocompatibility, mucoadhesive properties, and a broad spectrum
of antimicrobial activity.
To date, chitosan has been used to bind, deliver, or promote the presence of biomacromolecules and growth factors implicated
in wound healing. Chitosan-based materials have been developed to deliver heparin, hyaluronic acid, and other growth factors to
the site of the wound. These materials are designed to have a short lifespan, hydrating on contact, and then dissolving slowly while
releasing their payload. This has been utilized to create stable basic FGF containing films that resulted in rapid wound healing in
diabetic mice. Chitosan has also been covalently modified with the basic FGF and bone morphogenic protein, both of which
showed increased wound healing and osteogenesis, respectively. Similarly, chitosan-based materials have been used to deliver
whole cells to treat full thickness wounds. Lastly, chitosan and chitosan-based materials are used for their broad spectrum antimi-
crobial activity. The charge interactions between chitosan and microbial cell membrane components lead to dysregulation of the
microbial transport mechanism and ultimately the death of the microbe. Several studies have shown that the bactericidal and bacte-
riostatic capabilities of chitosan depend on molecular weight and degree of deacetylation.
Engineered Growth Factors/Growth Factor Delivery Systems
Aside from biomaterials, growth factors and growth factor delivery systems have recently begun to be engineered for wound healing
applications. In a recent study by Martino et al. a method for producing engineered growth factors with a super affinity to the ECM is
described. In this article, it was found that a domain in placenta growth factor-2 (PlGF-2) binds to the ECM with a high affinity. This
domain was then fused with VEGF-A, PDGF-BB, and BMP-2 in order to create a new, engineered growth factor with a significantly
higher binding affinity to the ECM than normal growth factors. This newly engineered growth factor was also seen to augment the
wound healing process. When introduced into mouse models of chronic wounds and bone defects, it was seen that wound healing
was greatly enhanced when compared to the normal growth factors.
Controlling the delivery and release of growth factors is also critically important for augmenting the wound healing process. To
that end, there are various strategies that can be employed to make a biomaterial act more like natural ECM. For example, biophys-
ical properties such as the materials density, porosity, charge, and hydrophobicity can all be changed in order to create a material
with the desired release kinetics. However, those types of biophysical changes are often not ideal for cellular attachment, growth,
and remodeling. Another approach is to slow the release of growth factors by functionalizing the material with growth factor
binding sites isolated from ECM molecules. For example, it has been seen that heparin sulfate proteoglycans are capable of binding
with several different growth factors. As such, biomaterials have been created with modified heparin or heparin sulfate mimetic
molecules to bind and slowly release growth factors such as FGF. Other ECM proteins such a fibronectin, fibrinogen, tenascin C,
and vitronectin also have growth factor binding domains that can be isolated and used to bind a wide variety of growth factors
to biomaterial matrices.
Besides attaching growth factors to the matrix via binding sites, growth factors can also be built into the material. For example,
growth factors can be covalently crosslinked into fibrin matrices by creating a recombinant growth factor containing a sequence
recognized by factor XIIIa. As factor XIIIa naturally polymerizes the fibrin matrix, the recombinant growth factor will also be incor-
porated. The release of the incorporated growth factor is then determined by the degradation rate of the fibrin matrix. Growth factors
can also be incorporated into hyaluronic acid hydrogels by loading the growth factor into the hydrogel prior to creating the scaffold.
Emerging Materials
New biomaterials for augmenting the wound repair process are constantly being developed. One of the more recently emerging
materials for use as a tissue scaffold are wound-interfacing microgels. Microgels are crosslinked polymeric particles that are
composed of water-soluble/swellable polymer chains classifying these types of materials as hydrogels. These types of materials offer
unique advantages compared to other types of hydrogels including: tunable size from a range of nanometers to micrometers, a large
surface area for bioconjugation, increased reaction time to temperature and pH, and tunable mechanical properties. For example,
microgels films created from chemically crosslinked poly(allylamine hydrochloride) were deposited onto surgical sutures in a layer-
by-later fashion and were shown to have the ability to release ibuprofen into the wound site while also accelerating the wound heal-
ing process. Microgels have also been used to create microporous scaffolds that can be injected into wound sites in order to promote
cell migration and tissue regeneration. Biomimetic microgels, which mimic the fibrin binding ability and clot retraction features of
natural platelets, have also been shown to enhance clotting in vivo and enhance clot stability. Collectively, these recent studies
demonstrate that microgels are a useful material strategy for modulation wound responses.
Conclusions
Overall, the wound healing process is a highly ordered, highly controlled process that is modulated by cellular interactions with
ECM components and cytokines. Fibrin, fibrinogen, and collagen provide the scaffold through which the wound healing process
occurs on and modulate cellular adhesion, migration, proliferation, and ECM deposition/remodeling. Cytokines such as TGF-b,
PDGF, TNF-a, VEGF, and FGF are also responsible for myriad of important cellular responses including cellular recruitment through
chemotaxis, proliferation, migration, and reepithelialization. However, an abnormal amount of ECM components or growth factors
can lead to abnormal wound healing as seen as either fibrosis or chronic nonhealing wounds resulting in life-threatening conditions
for the organism.
The presence of a biomaterial can also cause a change in the wound healing process based off the severity of the immune
response of the host. A severe immune response can lead to frustrated phagocytosis resulting in either the start of a chronic non-
healing wound if left uncontrolled or the fibrous encapsulation of the material, both of which are undesirable. Increased rates of the
integration of biomaterials can be influenced by controlling the material properties, the surface properties, the resulting immune
response, and by the addition of bioactive agents such as growth factors. To this end, both naturally derived biomaterials and
synthetically derived biomaterials have advantages and disadvantages when it comes to controlling properties that influence inte-
gration. There are also many types of natural biomaterials and synthetic biomaterials that have a myriad of uses clinically. Newly
emerging materials focus on bridging the gap between natural and synthetic biomaterials by creating a synthesized material that has
highly tunable material properties that accurately mimic biological properties while providing a degree of biomimicry to increase
the degree of the resulting material/wound interface. In summation, biomaterials are important clinical tools for the control and
augmentation of the wound repair process and with further control on material and biological properties, the better and more
substantial the host responses will be.
Further Reading
Alrubaiy, L., & Al-Rubaiy, K. K. (2009). Skin substitutes: A brief review of types and clinical applications. Oman Medical Journal, 24, 4–6.
Anderson, J. M., Rodriguez, A., & Chang, D. T. (2008). Foreign body reaction to biomaterials. Seminars in Immunology, 20, 86–100.
Badylak, S. F. (2015). Host response to biomaterials: The impact of host response on biomaterial selection (1st edn.). Cambridge, MA: Academic Press.
Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., & Tomic-Canic, M. (2008). Growth factors and cytokines in wound healing. Wound Repair and Regeneration, 5, 585–601.
Boateng, J. S., Matthews, K. H., Stevens, H. N. E., & Eccleston, G. M. (2007). Wound healing dressings and drug delivery systems: A review. Journal of Pharmaceutical Sciences,
97, 2892–2923.
Chester, D., & Brown, A. C. (2016). The role of biophysical properties of provisional matrix proteins in wound repair. Matrix Biology, 60–61, 124–140.
Clark, R. (2013). The molecular and cellular biology of wound repair (2nd edn.). New York, NY: Springer Science.
Crapo, P. M., Gilbert, T. W., & Badylak, S. F. (2011). An overview of tissue and whole organ decellularization processes. Biomaterials, 32, 3233–3243.
Elena, I. P., Bazaka, K., & Crawford, R. J. (2013). New functional biomaterials for medicine and healthcare (1st edn.). Cambridge: Elsevier.
Gilbert, T. W., Sellaro, T. L., & Badylak, S. F. (2006). Decellularization of tissues and organs. Biomaterials, 27(19), 3675–3683.
Griffin, D. R., Weaver, W. M., Scumpia, P. O., Di Carlo, D., & Segura, T. (2015). Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed
building blocks. Nature Materials, 14, 737–744.
Ha, T. L. B., Quan, T. M., Vu, D. N., & Si, D. M. (2013). Naturally derived biomaterials: Preparation and application. In J. A. Andrades (Ed.), Regenerative Medicine and Tissue
Engineering. Croatia: InTech.
Halim, A. S., Khoo, T. L., & Mohd. Yussof, S. J. (2010). Biologic and synthetic skin substitutes: An overview. Indian Journal of Plastic Surgery, 43, S23–S28.
Hunt, T. K., Hopf, H., & Hussain, Z. (2000). Physiology of wound healing. Advances in Skin & Wound Care, 13, 6–11.
Kim, J. K., Kim, H. J., Chung, J., et al. (2014). Natural and synthetic biomaterials for controlled drug delivery. Archives of Pharmacal Research, 37, 60–68.
Lutolf, M. P., & Hubbell, J. A. (2008). Advances in Tissue Engineering 255–278. London: World Scientific Publishing Co.
Martino, M. M., Briquez, P. S., Guc, E., et al. (2014). Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science, 343, 885–888.
Mazza, G., Rombouts, K., Hall, A. R., et al. (2015). Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Scientific Reports, 5, 13079.
Mogosanu, G. D., & Grumezescu, A. M. (2014). Natural and synthetic polymers for wounds and burns dressing. International Journal of Pharmaceutics, 2, 127–136.
Morais, J. M., Papadimitrakopoulos, F., & Burgess, D. J. (2010). Biomaterials/tissue interactions: Possible solutions to overcome foreign body response. The AAPS Journal, 2,
188–196.
Mutsaers, S. E., Bishop, J. E., McGrouther, G., & Laurent, G. J. (1997). Mechanisms of tissue repair: From wound healing to fibrosis. The International Journal of Biochemistry &
Cell Biology, 29, 5–17.
Nguyen, H., Qian, J. J., Bhatnagar, R. S., & Li, S. (2003). Enhanced cell attachment and osteoblastic activity by P-15 peptide-coated matrix in hydrogels. Biochemical and
Biophysical Research Communications, 311, 179–186.
Roach, P., Eglin, D., Rohde, K., & Perry, C. C. (2007). Modern biomaterials: A reviewdbulk properties and implications of surface modifications. Journal of Materials Science.
Materials in Medicine, 7, 1263–1277.
Tapias, L. F., & Ott, H. C. (2014). Decellularized scaffolds as a platform for bioengineered organs. Current Opinion in Organ Transplant, 19, 145–152.
Velnar, T., Bailey, T., & Smrkolj, V. (2009). The wound healing process: An overview of the cellular and molecular mechanisms. Journal of International Medical Research, 37,
1528–1542.
ENCYCLOPEDIA OF
ENCYCLOPEDIA OF
EDITOR IN CHIEF
Roger Narayan
VOLUME 2
Section Editors
Christian Hellmich
Diego Mantovani
Alexander Wong
William Z Rymer
Rehabilitation Institute of Chicago, Chicago, IL, United States.
Levi Hargrove
Amsterdam • Boston • Heidelberg • London • New York • Oxford

Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
Copyright Ó 2019 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photo-
copying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to
seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the
Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted
herein).
Notice
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in
research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods,
compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the
safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or
damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.
Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress
British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library
ISBN 978-0-12-804829-0
For information on all publications visit our website

at http://store.elsevier.com
Publisher: Oliver Walter

Acquisition Editor: Blerina Osmanaj
Publishing Services Manager: Beckie Brand
Associate Content Project Manager: Kshitija Iyer
Cover Designer: Greg Harris
Printed and bound in the United States

EDITORIAL BOARD
EDITOR IN CHIEF
Roger Narayan
SECTION EDITORS
Levi Hargrove
Christian Hellmich
Sri Krishnan
Ryerson University, Toronto, ON, Canada
Cato Laurencin
Diego Mantovani
William Z Rymer
Pankaj Vadgama
Queen Mary University of London, London, United Kingdom
Min Wang
Alexander Wong
Xiaojun Yu
v
EDITOR IN CHIEF
Roger Narayan
Dr. Roger Narayan is a professor in the Joint Department of Biomedical Engineering at the
University of North Carolina and North Carolina State University. He is an author of over 200
publications as well as several book chapters on processing, characterization, and modeling of bio-
logical and biomedical materials. Dr. Narayan has edited several books, including Biomedical Mate-
rials, Printed Biomaterials, Computer Aided Biomanufacturing, Diamond-Based Materials for Biomedical
Applications, Medical Biosensors for Point of Care (POC) Applications, Monitoring and Evaluation of Bioma-
terials and their Performance In Vivo, Nanobiomaterials: Nanostructured Materials for Biomedical Applica-
tions, and the ASM Handbook on Materials for Medical Devices. He has previously served as chair of the
Functional Materials Division of The Minerals, Metals & Materials Society and is currently chair-elect
of the Bioceramics Division of American Ceramics Society. Dr. Narayan has received several honors
for his research activities, including the North Carolina State University Alcoa Foundation Engi-
neering Research Achievement Award, the North Carolina State University Sigma Xi Faculty
Research Award, the University of North Carolina Jefferson-Pilot Fellowship in Academic Medicine,
the National Science Faculty Early Career Development Award, the Office of Naval Research Young
Investigator Award, the American Ceramic Society Richard M. Fulrath Award, the Royal Academy of Engineering Distinguished Visiting
Fellowship, and TMS Brimacombe Medal. He has served as Fulbright Scholar at the University of Otago, the National Polytechnic Institute
(Mexico City), and the University of Sao Paulo. He has been elected as Fellow of ASM International, the American Association for the
Advancement of Science, the American Ceramic Society, and the American Institute for Medical and Biological Engineering.
vii
SECTION EDITORS
Levi Hargrove
Dr. Hargrove is currently the Director of the Center for Bionic Medicine and of the Neural Engi-
neering for Prosthetic and Orthotics Laboratory at the Shirley Ryan AbilityLab. He is also an
Associate Professor in the Departments of Physical Medicine and Rehabilitation and the
McCormick School of Engineering at Northwestern University.
A major goal of his research is to develop clinically realizable myoelectric control systems
that can be made available to persons with limb loss in the near future. His research addresses
all levels of amputation and has been published in the Journal of the American Medical Associ-
ation and the New England Journal of Medicine, and multiple patents. Key projects include the
development of advanced and adaptive control systems for prosthetic legs, improving control
of robotic hand prostheses, and intramuscular EMG signal processing. In 2012, Dr. Hargrove
cofounded Coapt, a company to transition advanced rehabilitation technologies from the
research laboratory to patients’ homes.
Christian Hellmich
Dr. Christian Hellmich, Full Professor at the Department of Civil Engineering of the Vienna
University of Technology (TU Wien), is the director of the Institute for Mechanics of Materials
and Structures. At TU Wien, he received his engineering, Ph.D., and habilitation degrees (in
1995, 1999, and 2004, respectively). From 2000 to 2002, he was a Max Kade Postdoctoral
Fellow in the Department of Civil and Environmental Engineering at the Massachusetts Insti-
tute of Technology. His work is strongly focused on well-validated material and (micro)struc-
tural models, in terms of theoretical foundations and applications to concrete, soil, rock,
wood, bone, and biomedical implants, up the structural level (tunnels, pipelines, bridges, bio-
logical organs such as the skeleton)dwith complementary experimental activities if necessary.
He has led several projects for the tunnel, railway, and pipeline industries, as well as interna-
tional research activities sponsored by the European Commission, including the coordination
of the mixed industry-academia consortium “BIO-CT-EXPLOIT” at the crossroads of numer-
ical simulation and computer tomography, or the cross-domain COST action NAMABIO inte-
grating engineers, physicists, (stem) cell biologists, and medical doctors across the European
continent and beyond. He has published more than 130 papers in international refereed
scientific journals in the fields of engineering mechanics, materials science, and theoretical biology, more than 20 book chapters,
and more than 120 papers in refereed conference proceedings. Dr. Hellmich has served as the Chairman of both the Properties of
Materials Committee of the Engineering Mechanics Division of the American Society of Civil Engineers (ASCE), and the Porome-
chanics and Biomechanics Committees of the Engineering Mechanics Institute (EMI), as associate editor of the Journal of Engineering
Mechanics (ASCE), and as Coeditor in Chief of the Journal of Nanomechanics and Micromechanics (ASCE). As community service, he
has (co)chaired and/or supported more than 50 international conferences (including chairmanship of the 2013 Biot Conference on
Poromechanics and the 2015 CONCREEP conference; both EMI-ASCE supported), and he has reviewed for 128 different scientific
journals and 15 science foundations. He was awarded the Kardinal Innitzer Science Award of the Archbishopry of Vienna in 2004
(for his habilitation thesis), the Science Award of the State of Lower Austria in 2005 (for his achievements in the micromechanics of
hierarchical composites), and he was the recipient of the 2008 Zienkiewicz Award for Young Scientists in Computational Engi-
neering Sciences, sponsored by the European Community on Computational Methods in Applied Sciences (ECCOMAS). For further
activities in the multiscale poromicromechanics of bone materials, he received one of the highly prestigious ERC Grants of the
ix
x Section Editors
European Research Council in 2010; and he was elected member of the Young Academy of the Austrian Academy of Sciences in
2011. In 2012, he was rewarded the prestigious Walter L. Huber Research Prize of the ASCE, for his contributions to the micropor-
omechanics of hierarchical geomaterials and biomaterials; he was elected Fellow of EMI in 2014 and was corecipient of the 2017
Kajal Mallick Memorial Award of the Institution of Civil Engineers (United Kingdom).
Sri Krishnan
Sridhar (Sri) Krishnan received B.E. degree in Electronics and Communication Engineering
from the College of Engineering, Guindy, Anna University, Chennai, India, in 1993, and
M.Sc. and Ph.D. degrees (with student fellowship from Alberta Heritage Foundation for
Medical Research) in Electrical and Computer Engineering from The University of Calgary,
Calgary, Alberta, Canada, in 1996 and 1999, respectively. Sri Krishnan joined Ryerson Univer-
sity in July 1999 and is currently a Professor in the Department of Electrical and Computer
Engineering. Since July 2011, he is an Associate Dean (Research, Development and External
Partnerships) for the Faculty of Engineering and Architectural Science. He is also the Codi-
rector of the Institute for Biomedical Engineering, Science and Technology (iBEST) and an
affiliate scientist at the Keenan Research Centre in St. Michael’s Hospital, Toronto.
Since January 2002 Sri Krishnan held various administrative leadership positions in the
Department of Electrical and Computer Engineering and the Faculty of Engineering and
Architectural Science. In 2010–2011, Sri Krishnan held Visiting Appointments in University
of Rennes 1 (France), Grenoble Institute of Technology (France) and Indian Institute of Tech-
nology (Madras). Sri Krishnan is a registered professional engineer in the Province of Ontario and is a senior member of IEEE (EMBS
and SP societies). He was the Founding Chair (2005–2015) of IEEE Signal Processing Society, Toronto Section and Region 7 (Can-
ada), and a Founding Member of the IEEE Engineering in Medicine and Biology Society, Toronto Section. He currently serves as
a Technical Committee Member (Biomedical Signal Processing) of IEEE EMBS.
Sri Krishnan held the Canada Research Chair position (2007–2017) in Biomedical Signal Analysis. Sri Krishnan has successfully
supervised/trained 10 postdoc fellows, 10 Ph.D., 30 Masters (thesis), 9 Masters (project), 42 RAs, and 20 Visiting RAs. Sri Krishnan’s
research interests include adaptive signal representations and analysis and their applications in biomedicine, multimedia (audio),
and biometrics. He has published 295 papers in refereed journals and conferences, filed 10 invention disclosures, and has one US
patent. He has presented keynote/plenary/invited talks in more than 35 international conferences and workshops. Sri Krishnan also
serves as a reviewer, committee member, and chair for many international conferences, journals, and granting bodies. Sri Krishnan’s
academic interests include (interdisciplinary) curriculum design, experiential learning, and innovation. Sri Krishnan serves in the
advisory boards of research institutes, innovation centers, incubator zones, and business organizations.
Sri Krishnan is a recipient/awarded Outstanding Canadian Biomedical Engineer Award 2016; Certificate of Appreciation from
PEO York Chapter 2016; Fellow of Canadian Academy of Engineering in 2014; 2014 Exemplary Service Award from IEEE Toronto
Section; 2014 Certificate of Merit from IEEE Signal Processing Society; 2013 Achievement in Innovation Award from Innovate Cal-
gary; 2011 Sarwan Sahota Distinguished Scholar Award; 2011 Certificate of Appreciation from IEEE Signal Processing Society; 2010
Shastri Visiting Professorship; 2010 French Embassy Visiting Researcher; 2008 Ontario Research Innovation Award from Bio-
discovery Toronto; 2007 Canadian Engineers’ Young Engineer Achievement Award from Engineers’ Canada; 2006 New Pioneers
Award in Science and Technology; 2006 South Asian Community Achiever Award; 2006 IEEE Toronto Section Best Chapter Chair
Award; 2005 IEEE AESS Best Chapter Chair Award; 2005 IEEE Certificate of Appreciation from Six Societies; Six Best Research Paper
Awards coauthored with his graduate students in International Conferences; and 2005 FEAS Research Excellence Award.
Cato Laurencin
Cato T. Laurencin, M.D., Ph.D. is the University Professor at UCONN. He is the eighth desig-
nated in UCONN’s history. He is Professor of Chemical Engineering, Professor of Materials
Science and Engineering, and Professor of Biomedical Engineering, and the Van Dusen Distin-
guished Endowed Professor of Orthopaedic Surgery. He directs the Institute for Regenerative
Engineering and the Raymond and Beverly Sackler Center at the University of Connecticut.
Dr. Laurencin earned his B.S.E. degree in Chemical Engineering from Princeton University.
He earned his Ph.D. in Biochemical Engineering/Biotechnology from the Massachusetts Insti-
tute of Technology where he was named a Hugh Hampton Young Fellow. At the same time, he
earned his M.D., Magna Cum Laude from the Harvard Medical School where he received the
Robinson Award for Surgery.
Dr. Laurencin is an expert in biomaterials, nanotechnology, stem cell science, and, the new
field he has pioneered, Regenerative Engineering. He is a fellow of American Institute of Chemical
Section Editors xi
Engineers and was named one of the 100 Engineers of the Modern Era by the AICHE. He received the Percy Julian Medal from National
Organization of Black Chemists and Chemical Engineers, and the Pierre Galletti Award from the American Institute of Medical and
Biological Engineering. He has received the NIH Director’s Pioneer Award and the National Science Foundation Emerging Frontiers
in Research and Innovation Award for his research in Regenerative Engineering.
Dr. Laurencin is an elected member of the National Academy of Engineering, the National Academy of Medicine, the Indian
National Academy of Engineering, the Indian National Academy of Sciences, and the African Academy of Sciences. He is an acade-
mician and foreign member of the Chinese Academy of Engineering.
Dr. Laurencin has two awards named in his honor. The W. Montague Cobb Institute and the National Medical Association estab-
lished the Cato T. Laurencin Lifetime Research Achievement Award, while the Society for Biomaterials established The Cato T. Lau-
rencin, M.D., Ph.D. Travel Fellowship Award.
Dr. Laurencin received the Presidential Faculty Fellow Award from President Bill Clinton and the Presidential Award for Excel-
lence in Science, Mathematics, and Engineering Mentoring from President Barack Obama. He is the recipient of the National Medal
of Technology and Innovation, America’s highest award for technological achievement from President Barack Obama in ceremonies
at the White House.
Diego Mantovani, Ph.D., FBSE.

Prof. Diego Mantovani is the director of Laboratory for Biomaterials and Bioengineering at
Laval University, in Canada, and senior scientist of the Regenerative Medicine Division of
the Quebec University Hospital Research Centre. He received his doctoral degree jointly
from University of Technology of Compiègne, France, and Laval University in 1999 and his
joint Diploma in Engineering from Politecnico di Milano and the University of Technology
of Compiegne, France, in 1993. After an industrial postdoc (1999), he becomes professor
at Laval University School of Science and Engineering in 2000. Since the beginning he estab-
lished is Laboratory at the University Hospital Research Center in Quebec City. Within his
team, researches focus on surface modifications by plasma, thin polymer functional films,
cell–materials interactions, degradable metals, scaffolds, and bioreactors for the replacement
and regeneration of cardiovascular tissue. He has authored more than 260 original articles,
holds 5 patents, and presented more than 185 keynotes, invited and seminar lectures world-
wide. His H-index is 43 (June 2018), and his works were cited more than 7000 times. He was
President of the Canadian Society for Biomaterials (2008–2009), and Executive Cochair of
the World Biomaterials Congress in 2016 in Montreal, Canada. In 2012, he was elected Fellow of the World Biomaterials Science
and Engineering Society. Since 2012, he is the holder of the Canada Research Chair 1 in Biomaterials and Bioengineering for the
Innovation in Surgery. He was member of ad hoc panels at FDA, ISO, and Health Canada and member of a number of funding,
regulatory and scientific committees worldwide. He is Adjunct Professor at Politecnico di Milano and Universita del Piemonte Ori-
entale in Italy, as well as at the Vellore Institute of Technology, in India. He was invited professor in several universities worldwide,
including Campinas, Brasil (2012–2015), Bologna (2015), Bordeaux (2014), Siao Tong West, China (2012), Cergy-Pontoise
(2012), ParisTech (2011), Buenos Aires (2010), Namur, Belgium (2008), Tor Vergata, Italy (2007), Ankara, Turkey (2006), and
others. He is member of the editorial board of five scientific journals in the field and of the advisory board of three medical devices
consortia worldwide.
William Z Rymer
Professor William Z Rymer is Professor of Physical Medicine and Rehabilitation and Physi-
ology at the Rehabilitation Institute of Chicago, Chicago, IL, United States. His focus of
work includes pathophysiology, stroke, spinal cord injury, spinal circuits, biomedical engi-
neering, and neural signal processing.
xii Section Editors
Pankaj Vadgama
Pankaj Vadgama qualified in Degree in Medicine and then in Chemistry at the University of
Newcastle upon Tyne, United Kingdom, with a First Class Honors BSc. He is a chemical
pathologist, becoming a Fellow of the Royal College of Pathology. He completed his Ph.D.
on medical biosensors as an MRC Fellow at Newcastle, and while there, he was made Director
of the Biosensors Group and later appointed as Professor of Clinical Biochemistry at the
University of Manchester, subsequently becoming Research Dean for the Faculty of Medicine.
He was appointed Director of the Interdisciplinary Research Centre in Biomedical Materials at
Queen Mary, University of London and was, until recently, Head of the Department of Clin-
ical Biochemistry, Barts Health NHS Trust. His main interests are variously biosensors,
applied bioelectrochemistry, point-of-care testing, and membrane technology. He has pub-
lished over 200 papers. He is also Fellow of the Royal Society of Medicine, Institute of Physics,
Royal Society of Chemistry, the Institute of Materials Minerals and Mining, and the Royal
Society of Biology. He was given the Foundation Award of the Association of Clinical
Biochemistry and Laboratory Medicine, has been a Sandoz Lecturer of the British Geriatric
Society, and delivered the Latner lecture at the University of Newcastle. He has served on
various UK Research Council grants award committees and is at present member of the Insti-
tute of Materials Minerals and Mining Smart Materials and Nano Committees and the
Biomedical Materials Application Division. He sits on various BSI committees and was Chair of the ISO subpanel on nanomedicine
nomenclature. He sits on various editorial boards and is Editor in Chief of Bioelectrochemistry. He is Deputy Chair of the Council for
the Frontiers of Science based in Uganda directed at research training in East Africa.
Min Wang
Min Wang is a Full Professor at The University of Hong Kong (HKU), and as Programme
Director (2013–2018), he has led HKU’s Medical Engineering Programme (which is retitled
to “Biomedical Engineering Programme” in 2018). He has worked in universities in the
United Kingdom (1991–1997), Singapore (1997–2002), and Hong Kong (2002–Present)
and has been a Guest Professor or Adjunct Professor of several universities in mainland China
(Shanghai Jiao Tong University, Zhejiang University, Tianjin University, Southwest Jiao tong
University, etc.). He was awarded BSc (1985) and Ph.D. (1991), both in Materials Science and
Engineering, by Shanghai Jiao Tong University and University of London, respectively. He is
a chartered engineer (CEng, 1995; UK) and chartered scientist (CSci, 2005; UK). He is an elec-
ted fellow of professional societies in the United Kingdom, Hong Kong, United States, and
internationally (FIMMM, 2001; FIMechE, 2007; FHKIE, 2010; FBSE, 2011; FAIMBE, 2012;
WAC Academician, 2013). Since 1991, he has been conducting research in biomaterials and
tissue engineering and developing new biomaterials using the composite/hybridization
approach. He was a founding member of UK’s Interdisciplinary Research Centre (IRC) in
Biomedical Materials at the University of London. His biomaterials research has covered
metals, polymers, ceramics, and composites and includes surface modification of materials or scaffolds. In recent years, he has
focused on nanobiomaterials, electrospinning, and 3D printing. He and his research staff/students have won many awards at inter-
national conferences. He has authored a large number of research papers as well as many book chapters. His research has been
widely cited by other researchers around the world. He has given many conference presentations, including more than 150 invited
talks at international conferences. He has also given more than 110 seminars in universities, research institutes, and hospitals in
Europe, North America, Asia, and Australia. He has been Chairman/Organizer of many conferences and has served in committees
of more than 70 international conferences. He is the Founding Series Editor of Springer Series in Biomaterials Science and Engineering
books and has been Editor, Associate Editor, or member of the Editorial Board of 20 international, printed journals, including Inter-
national Materials Reviews, Composites Science and Technology, Surface and Coatings Technology, Journal of Materials Science: Materials in
Medicine, and Journal of the Royal Society Interface. He has acted as a referee for more than 110 international journals in the fields of
materials science and engineering, biomaterials and tissue engineering, physics, chemistry, medicine, dentistry, medical devices,
biofabrication, nanoscience, nanotechnology, and 3D printing. He has been active in professional society activities and has served
in various roles in these societies. He was Chairman of the Biomedical Division of Hong Kong Institution of Engineers (HKIE). He
serves/has served in the Nomination Committee of World Academy of Ceramics (WAC) and the ICF-BSE Steering Committee of the
International College of Fellows of the International Union of Societies for Biomaterials Science and Engineering (IUS-BSE). He has
been an elected Council Member of Chinese Society for Biomaterials, Hong Kong Institution of Engineers, Asian Biomaterials Feder-
ation, World Association for Chinese Biomedical Engineers (WACBE), and Administrative Council of International Federation for
Medical and Biological Engineering (IFMBE). (http://web.hku.hk/memwang/).
Section Editors xiii
Alexander Wong
Alexander Wong, P.Eng., is currently the Canada Research Chair in Artificial Intelligence and
Medical Imaging, Codirector of the Vision and Image Processing Research Group, and an
Associate Professor in the Department of Systems Design Engineering at the University of
Waterloo. He had previously received the B.A.Sc. degree in Computer Engineering from
the University of Waterloo, Waterloo, ON, Canada, in 2005, the M.A.Sc. degree in Electrical
and Computer Engineering from the University of Waterloo, Waterloo, ON, Canada, in
2007, and Ph.D. degree in Systems Design Engineering from the University of Waterloo,
ON, Canada, in 2010. He was also an NSERC postdoctoral research fellow at Sunnybrook
Health Sciences Centre. He has published over 400 refereed journal and conference papers,
as well as patents, in various fields such as computational imaging, artificial intelligence,
computer vision, and medical imaging, and has received numerous awards such as 13 paper
awards at international conference and an Early Researcher Award from the Ministry of
Economic Development and Innovation.
Xiaojun Yu
Dr. Yu is Associate Professor, Biomedical Engineering at Stevens Institute of Technology,
Hoboken, NJ, United States. Dr. Yu’s primary research interests focus on tissue engineering,
polymeric biomaterials and drug delivery. His current research activities include nano- and
microscale functionalization of biomimic three-dimensional scaffolds for neural and
musculoskeletal tissue repair and regeneration, investigation of cell and material interac-
tions in bioreactors, development of controlled release systems for the delivery of growth
factors and drugs, and manipulation of microenvironment for stem cell proliferation and
differentiation.
CONTRIBUTORS TO VOLUME 2
Gursel Alici Luis Cardoso

University of Wollongong, Wollongong, NSW, Australia The Graduate School of The City University of New
York, New York, NY, United States
Robert Amelard
University of Waterloo, Waterloo, ON, Canada Ugo Carraro
IRCCS Fondazione Ospedale San Camillo Venezia-Lido,
Orestis G Andriotis
Venezia, Italy
TU Wien, Vienna, Austria
Francesco Casella
Michel Assad
Ospedale Maggiore della Carità, Novara, Italy
AccelLAB Inc., a Citoxlab Group Company, Boisbriand,
QC, Canada Marta C Catoira
University of Piemonte Orientale, Novara, Italy
Stéphane Avril
Ecole Nationale Supérieure des Mines de Saint-Etienne, Mario Cesarelli
CIS-EMSE, SAINBIOSE, Saint Etienne, France; University of Naples Federico II, Naples, Italy
INSERM, Saint Etienne, France; and Université de
Stéphane Chabaud
Lyon, SAINBIOSE, Saint Etienne, France
Laval University, Québec City, QC, Canada
Eliezer Bernart
Rachel W Chan
Federal University of Rio Grande do Sul, Porto Alegre,
Sunnybrook Research Institute, Toronto, ON, Canada
Brazil
Frédéric Chaubet
Thor Besier
Université Paris 13, Paris, France
University of Auckland, Auckland, New Zealand
Jean Chen
Paolo Bifulco
Rotman Research Institute, Baycrest Centre for Geriatric
University of Naples Federico II, Naples, Italy
Health, Toronto, ON, Canada; and University of
Romane Blanchard Toronto, Toronto, ON, Canada
University of Melbourne, Melbourne, VIC, Australia
Pascale Chevallier
Francesca Boccafoschi Laval University, Quebec, QC, Canada
Michel Boissière KTH Royal Institute of Technology, Stockholm, Sweden
Université de Cergy Pontoise, Neuville-sur-Oise, France
Paul K Chu
Stéphane Bolduc City University of Hong Kong, Hong Kong, China
Marie-Annick Clavel
Carolina Catanio Bortolan Laval University, Quebec City, QC, Canada
Laval University, Quebec, QC, Canada
John G Clement
Gilbert Bruce Pike University of Melbourne, Melbourne, VIC, Australia
University of Calgary, Calgary, AB, Canada
Julien Cohen-Adad
Jennifer Shane Williamson Campbell Polytechnique Montreal, University of Montreal,
McGill University, Montreal, QC, Canada Montreal, QC, Canada
xv
xvi Contributors to Volume 2
Caitlyn J Collins Massimiliano Garzaro

TU Wien, Vienna, Austria Eastern Piedmont University, Novara, Italy
David C Cooper Dario Gastaldi
University of Saskatchewan, Saskatoon, SK, Department of Chemistry, Materials and Chemical
Canada Engineering Giulio Natta, Politecnico di Milano,
Milano, Italy
Nancy Cote
Laval University, Quebec City, QC, Canada Shounak Ghosh
Tien Tuan Dao Vellore Institute of Technology (VIT) University, Vellore,
Sorbonne Universités, Paris, France; and Université de India
Technologie de Compiègne, Compiègne, France Magnus K Gislason
Valeria Dell’Era Reykjavik University, Reykjavik, Iceland
Eastern Piedmont University, Novara, Italy Sara Greenberg
Annalisa De Paolis University of Waterloo, Waterloo, ON, Canada
The Graduate School of The City University of New
Martin Guimond
York, New York, NY, United States
University of Montreal, Montreal, QC, Canada
Thomas E Doyle
Ezequiel Guzzetti
School of Biomedical Engineering, McMaster University,
Hamilton, ON, Canada
Bryce T J Dyer Masoom A Haider
University of Toronto, Toronto, ON, Canada; and
Bournemouth University, Bournemouth, United
Ontario Institute for Cancer Research (OICR), Toronto,
Kingdom
ON, Canada
Mehran Ebrahimi
University of Ontario Institute of Technology (UOIT), Matthew G Hanna
Oshawa, ON, Canada University of Pittsburgh Medical Center, Pittsburgh, PA,
United States
Kyle J Edmunds
Reykjavik University, Reykjavik, Iceland Rita Hardiman
University of Melbourne, Melbourne, VIC, Australia
Luca Esposito
University of Naples Federico II, Naples, Italy Christian Hellmich
Vienna University of Technology, Vienna, Austria
Justin Fernandez
University of Auckland, Auckland, New Zealand Wendy Hill
University of New Brunswick, Fredericton, NB, Canada;
Daniel P Ferris
and Atlantic Clinic for Upper Limb Prosthetics,
University of Florida, Gainesville, FL, United States
Fredericton, NB, Canada
Eliezer Soares Flores
Marie-Christine Ho Ba Tho
Federal University of Rio Grande do Sul, Porto Alegre,
Sorbonne Universités, Paris, France; and Université de
Brazil
Technologie de Compiègne, Compiègne, France
Massimiliano Fraldi
Caroline D Hoemann
University of Naples Federico II, Naples, Italy
George Mason University, Fairfax, VA, USA
Martin Frank
Nicolette Jackson
TU Wien, Vienna, Austria
AccelLAB Inc., a Citoxlab Group Company, Boisbriand,
Luca Fusaro QC, Canada
Weihong Jin
Ming Gan City University of Hong Kong, Hong Kong, China
Purdue University, West Lafayette, IN, United States
Halldór Jónsson
Paolo Gargiulo University of Iceland, Reykjavík, Iceland; and
Reykjavik University, Reykjavik, Iceland Landspitali University Hospital, Reykjavík, Iceland
Contributors to Volume 2 xvii
Picard Julien Sergio Loffredo

AKKA Life Science, Lyon, France Laval University, Quebec City, QC, Canada
Sumanta Kar Andrea Malandrino
North Dakota State University, Fargo, ND, United Institute for Bioengineering of Catalonia, Barcelona,
States Spain; and Massachusetts Institute of Technology,
Cambridge, MA, United States
Orestis L Katsamenis
University of Southampton, Southampton, United Geetha Manivasagam
Kingdom VIT University, Vellore, India
Dinesh R Katti Diego Mantovani

North Dakota State University, Fargo, ND, United Laval University, Quebec City, QC, Canada
States Michele Marino
Kalpana S Katti Leibniz Universität Hannover, Hannover, Germany
North Dakota State University, Fargo, ND, United Pietro Mascheroni
States University of Padova, Padova, Italy
Farnoud Kazemzadeh Carmelo Mastrandrea
University of Waterloo, Waterloo, ON, Canada; and Jean Monnet University, Saint-Étienne, France; and
Elucid Labs Inc., Waterloo, ON, Canada Lyon University, Lyon, France
Farzad Khalvati Mathew T Mathew
University of Toronto, Toronto, ON, Canada; and UIC School of Medicine at Rockford, UIC, Rockford, IL,
Lunenfeld-Tanenbaum Research Institute, Toronto, ON, United States
Canada
John McPhee
David A Koff University of Waterloo, Waterloo, ON, Canada
McMaster University, Hamilton, ON, Canada; and
Emad Moeendarbary
Diagnostic Imaging, Hamilton Health Sciences,
Massachusetts Institute of Technology, Cambridge, MA,
Hamilton, ON, Canada United States; and University College London, London,
Abbas Z Kouzani United Kingdom
Deakin University, Geelong, VIC, Australia Shahjahan Molla
Witold Krasny North Dakota State University, Fargo, ND, United
Ecole Nationale Supérieure des Mines de Saint-Etienne, States
CIS-EMSE, SAINBIOSE, Saint Etienne, France; Vanessa Montaño-Machado
INSERM, Saint Etienne, France; Université de Lyon, Laval University, Quebec, QC, Canada
SAINBIOSE, Saint Etienne, France; and Université de
Lyon, Ecole Centrale Lyon, France Claire Morin
Ecole Nationale Supérieure des Mines de Saint-Etienne,
Hyock Ju Kwon CIS-EMSE, SAINBIOSE, Saint Etienne, France;
University of Waterloo, Waterloo, ON, Canada INSERM, Saint Etienne, France; and Université de
Lyon, SAINBIOSE, Saint Etienne, France
Wilfred W Lam
Sunnybrook Research Institute, Toronto, ON, Canada Ehsan Mostaed
Politecnico di Milano, Milan, Italy
Angus Z Lau
Sunnybrook Research Institute, Toronto, ON, Canada; Roger J Narayan
and University of Toronto, Toronto, ON, Canada UNC/NCSU Joint Department of Biomedical
Engineering, Raleigh, NC, United States
Justin Y C Lau
Sunnybrook Research Institute, Toronto, ON, Canada; Sunita Nayak
and University of Toronto, Toronto, ON, Canada VIT University, Vellore, India
Hantao Liu Vedran Nedelkovski
Cardiff University, Cardiff, United Kingdom TU Wien, Vienna, Austria
xviii Contributors to Volume 2
Alexander K Nguyen Erwan Salaun

UNC/NCSU Joint Department of Biomedical Laval University, Quebec City, QC, Canada
Engineering, Raleigh, NC, United States
Raffaella Santagiuliana
Ko Okumura University of Padova, Padova, Italy
Department of Physics and Soft Matter Center,
Ochanomizu University, Tokyo, Japan
University of Aveiro, Aveiro, Portugal
Hazem Orabi Jacob Scharcanski
Laval University, Québec City, QC, Canada Federal University of Rio Grande do Sul, Porto Alegre,
Miao-Jung Y Ou Brazil
University of Delaware, Newark, DE, United States Stefan Scheiner
Liron Pantanowitz Vienna University of Technology, Vienna, Austria
University of Pittsburgh Medical Center, Pittsburgh, PA, Bryan R Schlink
United States University of Florida, Gainesville, FL, United States
Maria-Ioana Pastrama Marco Schneider
KU Leuven, Leuven, Belgium University of Auckland, Auckland, New Zealand
Carlo Paternoster Bernhard Schrefler
Laval University, Quebec City, QC, Canada Technical University of Munich, Garching bei München,
Germany; and Houston Methodist Research Institute,
Philippe Pibarot
Houston, TX, United States
Giraudier Sébastien
Jonathan Pitocchi
Voisin Consulting, Boulogne, France
Reykjavik University, Reykjavik, Iceland
Dwaipayan Sen
Peter Pivonka
Vellore Institute of Technology (VIT) University, Vellore,
Queensland University of Technology, Brisbane, QLD,
India
Australia
Jonathon W Sensinger
Dejan B Popovic
University of New Brunswick, Fredericton, NB, Canada
Serbian Academy of Sciences and Arts, Belgrade, Serbia;
and Aalborg University, Aalborg, Denmark Ashkan Shafiee
Wake Forest School of Medicine, Winston-Salem, NC,
Asokami Rajamanikam
United States
Tamil Nadu Academy of Sciences, Chennai, India
Bonghun Shin
Martina Ramella
Malgorzata Sikora-Jasinska
Sophie Ramsay
Politecnico di Milano, Milan, Italy; and Laval
University, Québec City, Canada
Reza Sharif Razavian
Roy V Sillitoe
Baylor College of Medicine, Houston, TX, United States;
Aakash Reddy and Texas Children’s Hospital, Houston, TX, United
VIT University, Vellore, India States
Violeta Rodriguez-Ruiz Alex Swee
Université de Cergy Pontoise, Neuville-sur-Oise, France University of Auckland, Auckland, New Zealand
Christian J Roth Michelle Sybring
Technical University of Munich, Munich, Germany University of New Brunswick, Fredericton, NB, Canada
Ingrid Saba C David L Thomas
Laval University, Québec City, QC, Canada University of Melbourne, Melbourne, VIC, Australia
Contributors to Volume 2 xix
Philipp J Thurner Wolfgang A Wall

TU Wien, Vienna, Austria Technical University of Munich, Munich, Germany
Vikas Tomar Zhou Wang
Purdue University, West Lafayette, IN, United States University of Waterloo, Waterloo, ON, Canada
Paolo Aluffi Valletti Alexander Wong
Eastern Piedmont University, Novara, Italy University of Waterloo, Waterloo, ON, Canada; and
Hans Van Oosterwyck Elucid Labs Inc., Waterloo, ON, Canada
Biomechanics Section, KU Leuven, Heverlee, Belgium Shasha Yeung
Diego A Vargas University of Auckland, Auckland, New Zealand
Biomechanics Section, KU Leuven, Heverlee, Belgium Lena Yoshihara
Maurizio Vedani Technical University of Munich, Munich, Germany
Politecnico di Milano, Milan, Italy
Aaron J Young
Diego Velasquez Georgia Institute of Technology, Atlanta, GA, United
Universidad CES, Medellin, Colombia States
Pasquale Vena Anne-Sophie Zenses
Department of Chemistry, Materials and Chemical Laval University, Quebec City, QC, Canada; and Aix-
Engineering Giulio Natta, Politecnico di Milano, Marseille University, Marseille, France
Milano, Italy
Ju Zhang
Katari Venkatesh University of Auckland, Auckland, New Zealand
Vellore Institute of Technology (VIT) University, Vellore,
India Yang Zhang
Purdue University, West Lafayette, IN, United States
Larreta-Garde Véronique
University of Cergy-Pontoise, Pontoise, France Yucheng Zhang
University of Toronto, Toronto, ON, Canada; and
Laurence Vico Lunenfeld-Tanenbaum Research Institute, Toronto, ON,
Jean Monnet University, Saint-Étienne, France; and Canada
Lyon University, Lyon, France
CONTENTS OF VOLUME 2
Editorial Board v
Editor in Chief vii
Section Editors ix
Contents of All Volumes xxv
Preface xxxv
Biomechanics
Philipp J Thurner
Michele Marino
Ko Okumura
xxi
xxii Contents of Volume 2
Peter Pivonka
Medical Devices
Asokami Rajamanikam
Contents of Volume 2 xxiii

and Diego Mantovani
Medical Imaging
Mehran Ebrahimi
xxiv Contents of Volume 2

Radiomics 597

Dejan B Popovic
Bryce T J Dyer
Gursel Alici
CONTENTS OF ALL VOLUMES
VOLUME 1
Bioceramics 16
Paul Frank Gratzer
xxv
xxvi Contents of All Volumes

Titanium Alloys 213
Mitsuo Niinomi

Nicholas P Rhodes

Organs-on-Chips 384
Contents of All Volumes xxvii

Antonio Valdevit
M Bohner
Yusuf Khan
Z Master
P Rajan
xxviii Contents of All Volumes

D Pergament
VOLUME 2
Biomechanics
Philipp J Thurner
Michele Marino
Ko Okumura
Contents of All Volumes xxix

Peter Pivonka
Medical Devices
Asokami Rajamanikam
xxx Contents of All Volumes

and Diego Mantovani
Medical Imaging
Mehran Ebrahimi
Contents of All Volumes xxxi

Radiomics 597

Dejan B Popovic
Bryce T J Dyer
Gursel Alici
VOLUME 3
Mathematical Techniques in Biomedical Engineering

Cardiac Modeling 1
Edward Vigmond and Gernot Plank
Mathematical Approaches for Medical Image Registration 21
Barbara Zitova
Mathematical Modeling of Gene Networks 33
Lakshmi Sugavaneswaran
Mathematical Modeling Tools and Software for BME Applications 56
Fred J Vermolen and Amit Gefen
Mathematical Techniques for Biomedical Image Segmentation 64
Roberto Rodríguez and Juan H Sossa
Mathematical Techniques for Circulatory Systems 79
Jason Carson, Raoul Van Loon, and Perumal Nithiarasu
Mathematical Techniques for Noninvasive Muscle Signal Analysis and Interpretation 95
Roberto Merletti, Ales Holobar, and Dario Farina
Optimization Techniques in BME 112
Jeevan Kumar Pant
xxxii Contents of All Volumes
Single-Cell-Based In Silico Models: A Tool for Dissecting Tumor Heterogeneity 130

Aleksandra Karolak, Saharsh Agrawal, Samantha Lee, and Katarzyna A Rejniak
Spectrotemporal Modeling of Biomedical Signals: Theoretical Foundation and Applications 144
Raymundo Cassani and Tiago H Falk
Statistical Modeling in Biomedical Engineering 164
Yunfeng Wu and Pinnan Chen
Time–Frequency Distributions in Biomedical Signal Processing 177
Yashodhan Athavale and Sridhar Krishnan
Wavelets in Biomedical Signal Processing and Analysis 193
Babak Azmoudeh and Dean Cvetkovic
Bioinstrumentation and Bioinformatics

A Systematic Workflow for Design and Computational Analysis of Protein Microarrays 213
Jonatan Fernández-García, Rodrigo García-Valiente, Javier Carabias-Sánchez, Alicia Landeira-Viñuela,
Rafael Góngora, María Gonzalez-Gonzalez, and Manuel Fuentes
Ambulatory EEG Monitoring 223
Bernard Grundlehner and Vojkan Mihajlovic
Automated EEG Analysis for Neonatal Intensive Care 240
Nathan Stevenson and Anton Tokariev
Big Data Calls for Machine Learning 258
Andreas Holzinger
Bioimpedance Spectroscopy Processing and Applications 265
Herschel Caytak, Alistair Boyle, Andy Adler, and Miodrag Bolic
Bioinformatics in Design of Antiviral Vaccines 280
Ashesh Nandy and Subhash C Basak
Bioinformatics in Disease Classification 291
Miguel Ángel Medina
Biopotential Monitoring 296
Julián Oreggioni, Angel A Caputi, and Fernando Silveira
Blood Gas Analysis and Instrumentation 305
Rebecca Symons, Robindro Chatterji, Kirsty Whenan, Rita Horvath, and Paul S Thomas
Computational Approaches in microRNA Biology 317
Ulf Schmitz, Shailendra K Gupta, Julio Vera, and Olaf Wolkenhauer
Detection and Classification of Breast Lesions Using Ultrasound-Based Imaging Modalities 331
Md Kamrul Hasan and Sharmin R Ara
DNA Microarrays: Fundamentals, Data Integration and Applications 349
Eduardo Valente and Miguel Rocha
ECG Monitoring: Present Status and Future Trend 363
Saurabh Pal
Genetic Algorithms for Breast Cancer Diagnostics 380
Florin Gorunescu and Smaranda Belciug
Contents of All Volumes xxxiii
Machine Learning in Biomedical Informatics 389

Carlos Fernandez-Lozano, Adrián Carballal, Cristian R Munteanu, Marcos Gestal, Víctor Maojo,
and Alejandro Pazos
Matrix Assisted Laser Desorption/Ionization as a New Cancer Diagnostic Tool 400
Bozena Hosnedlova, Marta Kepinska, Branislav Ruttkay-Nedecky, Carlos Fernandez, Tomas Parak,
Halina Milnerowicz, Jiri Sochor, Geir Bjørklund, and Rene Kizek
Medical Utility of NIR Monitoring 415
Zuzana Kovacsova, Gemma Bale, and Ilias Tachtsidis
Metabolomics in Biomaterial Research 432
Ana M Gil, Maria H Fernandes, and Iola F Duarte
Nucleic-Acid Sequencing 443
G Dorado, S Gálvez, H Budak, T Unver, and P Hernández
Optical Techniques for Blood and Tissue Oxygenation 461
Panayiotis Kyriacou, Karthik Budidha, and Tomas Y Abay
Polymerase Chain Reaction (PCR) 473
G Dorado, G Besnard, T Unver, and P Hernández
Single-Photon Emission Computed Tomography: Principles and Applications 493
Yong Du and Habib Zaidi
Translational Bioinformatics: Informatics, Medicine, and -Omics 507
Sergio Paraiso-Medina, David Perez-Rey, Raul Alonso-Calvo, Cristian R Munteanu, Alejandro Pazos,
Casimir A Kulikowski, and Victor Maojo
Ultrasonic Imaging in Biomedical Applications 515
Roman Gr Maev and Fedar M Severin
Index 523
PREFACE
The use by man of available technology to treat damaged or diseased tissue is older than the written historical
record. For example, the Mayan people created artificial teeth out of nacre, which were shown to be fused to the
bone (Bobbio, 1972; Westbroek & Marin, 1998). Giovanni Borelli’s studies of the cardiovascular system (e.g.,
the elasticity of arteries), which were published in De Motu Animalium (On Animal Motion), can be considered
as one of the foundations of the field of biomechanics (Parker, 2009). The hypothesis of an intrinsic ’animal
electricity’ by Luigi Galvani in the 18th century led to the development of the field of electrophysiology (Pic-
colino, 1997). In the 19th century, the development of the antiseptic approach to surgical procedures by Joseph
Lister made implantation of medical devices without certain postoperative infection possible; for example,
Lister described the use of silver wire for treatment of a fractured patella (Worboys, 2013). The discovery of X-
rays by Roentgen at the end of the 19th century was rapidly translated for medical imaging (Rowland, 1896;
Schuster, 1896). In our lifetime, the work by W. T. Green on generating new cartilage by seeding of chondrocytes
as well as by John Burke and Ioannis Yannas on generating skin substitutes is recognized as the birth of the field
of regenerative engineering (Vacanti, 2006).
At the beginning of the 21st century, the American Institute for Medical and Biological Engineering
identified several research areas for the field of biomedical engineering. These include: (a) functional geno-
mics and proteomics, (b) nanotechnology, (c) targeted drug delivery, (d) tissue engineering, and (e) the
development of new types of medical instrumentation (Hendee, Chien, Maynard, & Dean, 2002). As some of
these research areas have matured, others have become more prominent. Over the past few years, the use of
3D printing and bioprinting technologies to create medical devices has become more prominent. One benefit
of utilizing 3D printing and bioprinting for patient care is that medical imaging data (e.g., magnetic resonance
imaging and computed tomography data) may be employed to fashion prostheses or artificial tissues with
submicroscale features that conform with the requirements of the patient (Narayan, Doraiswamy, Chrisey, &
Chichkov, 2010; Skoog & Narayan, 2013). Another technology that will likely transform the field of
biomedical engineering over the coming decades involves the use of clustered regularly interspaced short
palindromic repeats (CRISPR)/Cas9 for engineering of the human genome. The interface between biomedical
engineering and the new field of genome engineering has already spawned research into new technologies for
delivery of genome editing tools into the body; the synergy between these fields will only grow over time
(Wright, Nuñez, & Doudna, 2016).
The goal of the Encyclopedia of Biomedical Engineering is to consider the principles and technologies that
underlie the field of biomedical engineering. The encyclopedia is divided into three volumes. The first volume
contains a section on biomaterials, which was edited by Min Wang at the University of Hong Kong, and
a section on regenerative engineering, which was edited by Cato Laurencin at the University of Connecticut and
Xiaojun Yu at the Stevens Institute of Technology. The second volume contains a section on rehabilitation
engineering and integrative technologies, which was edited by William Rymer and Levi Hargrove at North-
western University, a section on biomechanics, which was edited by Christian Hellmich at the Vienna University
of Technology, a section on medical devices, which was edited by Diego Mantovani at the University of Laval,
and a section on medical imaging, which was edited by Alexander Wong at the University of Waterloo. The third
volume contains a section on mathematical techniques in biomedical engineering, which was edited by Sri
Krishnan at Ryerson University, and a section on bioinstrumentation and bioinformatics, which was edited by
Pankaj Vadgama at Queen Mary University of London.
I would like express my sincere appreciation to the section editors and authors for all of their efforts on the
encyclopedia. I would also like thank Beckie Brand, Susan Dennis, Becky Gelson, Ginny Mills, Blerina Osmanaj,
xxxv
xxxvi Preface
Laura Escalante Santos, and Will Smaldon at Elsevier for their outstanding efforts to bring the encyclopedia to
publication. I hope that this work serves the biomedical engineering community by providing a resource that
considers topics at the interface of the biological sciences and engineering.
Roger J Narayan, M.D., Ph.D.

UNC/NCSU Joint Department of Biomedical Engineering.
References
Bobbio, A. (1972). The first endosseous alloplastic implant in the history of man. Bull. Hist. Dent, 20, 1–6.
Hendee, W. R., Chien, S., Maynard, C. D., & Dean, D. J. (2002). The National Institute of biomedical imaging and Bioengineering: history, status, and potential impact. Annals of
Narayan, R. J., Doraiswamy, A., Chrisey, D. B., & Chichkov, B. N. (2010). Medical prototyping using two photon polymerization. Materials Today, 13, 44–50.
Parker, K. H. (February 2009). A brief history of arterial wave mechanics. Medical & Biological Engineering & Computing, 47(2), 111–118.
Piccolino, M. (October 1997). Luigi Galvani and animal electricity: two centuries after the foundation of electrophysiology. Trends in Neurosciences, 20(10), 443–448.
Rowland, S. (March 7, 1896). Report on the Application of the new Photography to medicine and surgery. Br Med J, 1(1836), 620–622.
Schuster, A. (January 18, 1896). On the new Kind of Radiation. Br Med J, 1(1829), 172–173.
Skoog, S. A., & Narayan, R. J. (2013). Stereolithography in medical device fabrication. Advanced Materials & Processes, 171, 32–36.
Vacanti, C. A. (July 2006). The history of tissue engineering. J Cell Mol Med, 10(3), 569–576.
Westbroek, P., & Marin, F. (1998). A marriage of bone and nacre. Nature, 392, 861–862.
Worboys, M. (September 20, 2013). Joseph Lister and the performance of antiseptic surgery. Notes and Records of the Royal Society of London, 67(3), 199–209.
Wright, A. V., Nuñez, J. K., & Doudna, J. A. (January 14, 2016). Biology and Applications of CRISPR systems: Harnessing Nature’s Toolbox for genome engineering. Cell, 164(1–2),
29–44.
PERMISSION ACKNOWLEDGMENTS
The following material is reproduced with kind permission of Taylor & Francis

www.taylorandfrancisgroup.com
The following material is reproduced with kind permission of American Association for the Advancement of
Science

www.aaas.org
The following material is reproduced with kind permission of Oxford University Press
Figure 1 Translational Bioinformatics for Personalised Medicine

www.oup.com
The following material is reproduced with kind permission of Nature Publishing Group
Figure 4b Cell Mechanics and Cell Adhesion - Basic Principles and Computational Modeling
Figure 9 Diffusion Tensor Imaging
Figure 8 Corrosion of Biomaterials
Figure 6 Nanotechnology for Regenerative Engineering
i
ii Permission Acknowledgments

Figure 5 Drug and Gene Delivery for Regenerative Engineering
Figure 3 Holographic Microscopy
Figure 4 Radiomics
http://www.nature.com
BIOMECHANICS
Biomechanics of Cells as Potential Biomarkers for Diseases: A New Tool

in Mechanobiology
Dinesh R Katti, Kalpana S Katti, Shahjahan Molla, and Sumanta Kar, North Dakota State University, Fargo, ND, United States
Introduction 1
Theory 2
Theory of AFM Force Curves 2
Operational modes of AFM 4
Theory of Nanoindentation 4
Modes of nanoindentation 6
Examples of Nanomechanical Testing of Cells 7
AFM 7
Nanoindentation 12
Perspectives 16
Some Current Challenges in Use of AFM 16
Current Challenges in Nanoindentation of Live Cells 16
Acknowledgements 17
References 17
Abbreviations
nanoDMA Nano dynamic mechanical analysis
Introduction
Living cells in the human body, as physical entities, possess structural and physical properties and are constantly subjected to various
mechanical stimulations. Studies in the evaluation of mechanics of single cells, and also various cellular components have indeed
grown in importance due to their potential impact on medicine and human health (Suresh, 2007a,b; Suresh et al., 2005; Bao and
Suresh, 2003). It has been shown that cellular functions such as growth, differentiation, migration, apoptosis, motility, gene expres-
sion, signal transduction, etc. are influenced by cellular biophysics and biomechanics (Chen et al., 1997; Boudreau and Bissell, 1998;
Huang and Ingber, 1999; Schwartz and Ginsberg, 2002; Suresh et al., 2015). Any changes to the biophysical or biomechanical prop-
erties of human cells may interrupt regular physiological functions of cells and may lead to diseases. For example, when red blood
cells are affected by malaria-causing bacteria, plasmodium falciparum, the molecular and structural properties of red blood cells are
significantly compromised (Fedosov et al., 2011; Suresh et al., 2015; Cooke et al., 2001; Bannister and Mitchell, 2003). Biomechan-
ical properties of individual cells can influence the physio-structural properties of the whole tissue by interacting with the extracellular
matrix. Also, mechanical stress on the tissue is transmitted to the cellular level, compromising their physiological functions signifi-
cantly (Guilak and Mow, 2000). Other than disease causing virus and bacteria, many chemicals are also known to have an effect on
mechanical properties of human cells. For instance, latrunculin B cytochalasin D have an adverse effect on the cytoskeletal structure of
the cells (Nagayama et al., 2006; Wakatsuki et al., 2001; Sato et al., 1990). Chemotactic agent fMLP significantly increases the stiffness
of neutrophils (Zahalak et al., 1990; Worthen et al., 1989). The primary structural element of the cytoskeleton, the actin filament and
microtubules are affected by colchicine (Tsai et al., 1998; Borisy and Taylor, 1967; Imazio et al., 2014).
The past few decades has seen a substantial growth of the research in the field of biomechanical properties of cells. Various tech-
niques have been applied to determine the biophysical properties of cells (Stamenovic et al., 1996; Lim et al., 2004; Stamenovic and
Ingber, 2002). However, it is still challenging to evaluate the nanomechanics of a single cell, considering the dynamic nature of cells
during progression of different diseases (Bao and Suresh, 2003; Zhu et al., 2000; Fung and Liu, 1993).

2 Biomechanics j Biomechanics of Cells as Potential Biomarkers for Diseases: A New Tool in Mechanobiology
With the advent of nanotechnology, it has now become feasible to probe cellular mechanics at single cell level. For instance,
biophysical techniques such as micropipette aspiration (Evans and Yeung, 1989; Guo et al., 2012; Sato et al., 1987, 1990, 1996;
Thoumine et al., 1999; Tsai et al., 1998), optical tweezers (Balland et al., 2006; Coceano et al., 2016; Henon et al., 1999), cell
stretchers (Harris et al., 2013; Katsumi et al., 2002; Krishnan et al., 2009; Steward et al., 2011), flow rheometry (Gossett et al.,
2012; Guo et al., 2012; Lu et al., 2004; Steward et al., 2011), magnetic bead cytometry (Deng et al., 2006; Fabry et al., 2001), traction
force microscopy (TFM) (Munevar et al., 2001; Legant et al., 2010), atomic force microscopy (AFM) (Ketene et al., 2012; Kuznetsova
et al., 2007; Lekka, 2016; Nematbakhsh and Lim, 2015), micropillar arrays (Fu et al., 2010), and nanoindentation (Chen et al.,
2010; Duan and Chen, 2015; Khanna et al., 2011, 2012; McDaniel et al., 2007; Yang et al., 2010; Miyamoto et al., 2015), have
been used extensively in the past decade to probe mechanical properties of various cell types. Of these above-mentioned techniques,
a majority have been employed to apply controlled force to the cells (micropipette aspiration, optical tweezers, cell stretchers, flow
rheometry, magnetic bead cytometry, AFM, and nanoindentation), while the rest are used to monitor the cells’ ability to deform
itself by intracellular forces (TFM, and micropillar arrays) to obtain their mechanical properties.
The connection between human disease and biophysics of cells has become a subject of intense scientific research recently. Cellular
nanomechanics is strongly connected to the molecular and physiological changes introduced by the progression of certain disease and
invasion by various external organisms such as a virus, bacteria or other parasites (Chaffey et al., 2003; Boal and Boal, 2012; Miller
et al., 2002; Cooke et al., 2001). Cellular pathology and pathophysiology are heavily influenced by these changes in elastic and visco-
elastic properties of cells (Miller et al., 2002; Cooke et al., 2001; Bao and Suresh, 2003). In the past two decades, advancement in the
field of tissue engineering and bioengineering has enabled us to investigate the real-time biomechanical changes during the progres-
sion of certain diseases. The nanomechanical properties of living cells such as elastic modulus or stiffness can either increase or decrease
in a pathogenic progression depending on the biomolecular and biochemical restructure (Suwanarusk et al., 2004).
It has been demonstrated that the mechanics of cells play a critical role in cellular growth and disease progression such as cancer,
cardiovascular diseases, liver diseases, renal glomerular diseases etc. (Engler et al., 2007; Yallapu et al., 2015; Chaturvedi et al., 2010;
Georges et al., 2007; Wells, 2008; Wyss et al., 2011).
Several studies showed that the stiffness of the substrate in the in vitro studies hugely affects the growth and differentiation of
cells. For instance, it is shown that when cortical brain cells are cultured in a softer substrate (0.15–0.30 kPa), neurons grow selec-
tively. On the other hand, when same cells were cultured in a stiffer substrate (2 kPa), the neurons attach to glial cells, and astrocytes
proliferate (Lu et al., 2006; Georges et al., 2006). The elastic modulus of some of the tissues such as liver, lung, breast, kidney has
been reported in the range of 0.2–4.0 kPa (Levental et al., 2007). The variance in elastic modulus of cells within the same tissue has
been reported to be within a 10%–15% range, and under normal physiological conditions, this controlled mechanical property of
cells helps them to maintain homeostatic cell-cycle progression (Georges et al., 2007). Changes in elastic properties > 12 kPa leads
to abnormal cell cycle progression which may cause irregular cell growth and differentiation (Kumar and Weaver, 2009; Assoian and
Klein, 2008; Klein et al., 2009; Levental et al., 2009). It has been reported that live breast, liver and pancreatic cancer cells obtained
from a body fluid of patients possess very different nanomechanical properties than their normal counterparts although cancerous
and normal cells exhibit similar morphologies (Cross et al., 2008). Baker et al. (2009) demonstrated that cancerous breast cancer
cells are stiffer (4 kPa) than the normal breast tissue (0.2 kPa) (Baker et al., 2009; Levental et al., 2007). It has been reported that
dense and stiffer breast tissue is more likely to develop cancer. In vitro studies suggested that stiffer substrate increases the invasive
nature of cancer cells by increasing ERK and Rho activity of breast cancer cells (Paszek et al., 2005). Further, normal cells (Hu609
and HCV29) have higher Young’s modulus than their cancerous counterpart (Hu456, T24, BC3726) (Lekka et al., 1999). Nanome-
chanical properties of cells also play a significant role in various cardiac diseases (Engler et al., 2008; Chaturvedi et al., 2010). It has
been reported that in some cardiac-related diseases, heart ventricle is stiffer than the normal which causes heart failure. It was
demonstrated that the muscle tissues of these hearts are stiffer than the regular heart muscle cells (Chaturvedi et al., 2010). Arterial
stiffness is another risk factor for cardiac diseases such as stroke and heart failure (Cecelja and Chowienczyk, 2009; Mitchell et al.,
2010). Arterial stiffness is usually developed by numerous factors such as aging, genetic factors, blood pressure, etc. (DeLoach and
Townsend, 2008; Lacolley et al., 2009). High aortic stiffness leads to irregular mechanical properties of the arterial system, impaired
cardiac performance, and difficulty in supplying blood to the heart and cardiac hypertrophy. Cell mechanics is also associated with
liver diseases (Wells, 2008). The elastic modulus of healthy liver cells (0.4–0.6 kPa) could increase to as much as15 kPa following
an injury (Georges et al., 2007). Different liver cells (stellate cells, hepatocytes, portal fibroblasts) develop changes in differentiation
and proliferative characteristics when subjected to the stiffer matrix (Li et al., 2007). It has been reported that renal diseases are also
associated with reduced stiffness renal glomerular podocyte cells. The glomerular podocytes from a mouse model of HIV-associated
nephropathy are significantly softer than normal podocytes (Tandon et al., 2007).
In this article, we will overview two advanced techniques, namely AFM and nanoindentation, as relevant methodologies for eval-
uation of mechanical properties of cells that will possibly play an important role in medical diagnostics as well as contribute
towards understanding mechanisms of disease progression.
Theory
Theory of AFM Force Curves
In the AFM, the elasticity of living cells is determined from interactions of AFM probes with sample surfaces that are interpreted as
a measure of Young’s modulus. AFM consists of three main parts, namely a cantilever, a system that detects its deflection and
a system that enables scanning and positioning (Fig. 1). The most important part of an AFM is the cantilever. AFM cantilevers
Biomechanics j Biomechanics of Cells as Potential Biomarkers for Diseases: A New Tool in Mechanobiology 3
Fig. 1 Schematic showing different components of atomic force microscopy (AFM).
are made of silicon or silicon nitride with a sharp tip at the end. When the tip comes into proximity of a sample surface, forces
between the sample and the tip cause a deflection of the cantilever. The deflection can easily be detected using an optical system
comprised of a laser and a photodetector. The laser is focused on the free end of the cantilever, and when it is reflected, it goes
directly towards the center of the photodiode. Deflection of the cantilever by interaction with features on the sample surface is
monitored during scanning and is translated into a three-dimensional image of the surface. A force–displacement curve is also ob-
tained from the plot of force (tip/sample) and deflection of the cantilever which can further be used to calculate mechanical prop-
erties of the sample. A typical force–displacement curve has three parts: approach, contact with the sample, and retraction. The
approaching curve provides the information repulsive or attractive forces between tip and sample, whereas the second part sheds
light on mechanical properties of the sample, such as Young’s modulus, relaxation time, etc. Finally, the retracting curve describes
the adhesion forces that exist between tip and sample (Benitez and Toca-Herrera, 2014; Vanlandingham et al., 1997) (Fig. 2).
Young’s modulus (E) of cells from the force–displacement (force-indentation) curve is estimated using Hertz model from contact
mechanics which describes the indentation of two purely elastic spheres (Hertz, 1881). The model has been widely used to deter-
mine the apparent cell membrane elasticity. Two important assumptions of the model are the following: the indentation depth is
less than 10% of the sample thickness, and the indentation depth is greater than 200 nm (Pelling et al., 2007; Rico et al., 2005;
Stolz et al., 2004). The thickness of adherent cells varies from 200 nm at the cell edge to 5–10 mm over the center (nucleus) of
the cell. Consequently, elastic modulus at the cell edge is substantially influenced by substrate’s (tissue culture polystyrene (TCPS)
or glass) modulus and usually appears to be higher than that of the nucleus (Pelling et al., 2007). If the indenter is spherical the
relation between the load force and the indentation depth is given by (Hertz, 1881):
4 1 3
F ¼ E0 R2 d2 (1)
3
where F is the load force, d is the indentation depth, and R is the radius of the spherical indenter, and E0 is the reduced Young’s
modulus of the sample. The E0 is the reduced Young’s modulus of a sample, expressed by the following equation,
Fig. 2 @Schematic representation of a force–displacement curve.


1 1 m2tip 1 m2sample
¼ þ (2)
E0 Etip Esample
where mtip and msample are the Poisson ratios of the tip and a sample. It ranges from 0 to 0.5. For living cells, the elastic modulus is
much smaller than Young’s modulus of the tip, that is, Ecell Etip; therefore, the reduced Young’s modulus can be expressed as,
Ecell
E0 z (3)
1 m2cell
The value of mcell is assumed as 0.5 for the ease of calculation as cells can be treated as an incompressible material.
Hertz model has been modified by Sneddon (1965), introducing axisymmetric shapes of the indenter (i.e., conical and pyra-
midal) into the relation between the load force and the indentation depth:
Cone:
2
F ¼ E0 tanad2 (4)
p
Pyramid:
E0
F¼ tanad2 (5)
O2
where a is the opening angle of the cone. The Hertz model does not take into account the attractive forces (van der Waals) within
contact. The JKR theory considers the influence of finite surface energy (Johnson et al., 1971). The JKR model is ideal for tips with
large curvature radius and small stiffness:
Ka3
F¼ O6psKa3 (6)
R
where a is contact area radius, s is work of adhesion and K is effective Young’s modulus of the sample and is given by,
" #
1 3 1 mtip 1 m2sample
2
¼ þ (7)
K 4 Etip Esample
The model assumes that adhesion forces (van der Waals) acting along the contact area perimeter results in an additional probe
sample attraction which weakens forces of elastic repulsion (DMT) (Derjaguin et al., 1975). The DMT model is applied to tips with
small curvature radius and high stiffness:
Ka3
F¼ 2pRs (8)
R
Operational modes of AFM

AFM measures the repulsive (hard sphere) or attractive (van der Waals) interaction forces between the atoms at the proximity of
a fine tip and the atoms at the sample surface (Binnig et al., 1986). Different operational modes of AFM are described below. In
contact mode, the repulsive force between tip and sample is kept constant during the raster scan of the sample, providing a topo-
graphical image of the sample. In tapping mode, the cantilever oscillates at its resonant frequency. When the tip is brought into the
proximity of the surface, the tip-sample interactions (involves atomic repulsions) reduce the amplitude of the oscillations (Putman
et al., 1992) In non-contact mode, the cantilever either oscillates at either its resonant frequency (frequency modulation) or just
above (amplitude modulation) where the amplitude of oscillation is typically a few nanometers down to a few picometers (Gross
et al., 2009).
Theory of Nanoindentation
Nanoindentation is a sophisticated technique which involves the application of a controlled load to penetrate an indenter into the
surface of a specimen and uses recorded load–displacement curve to measure the hardness, elastic modulus, and, some other
mechanical properties of the specimen. It is considered as a nondestructive technique as it imprints the sample at very shallow
depths. Besides the elastic modulus, other mechanical properties such as fracture toughness, yield strength, hardening index,
and residual stress, etc. can be measured using nanoindentation technique. It is essential to understand the basic principle of nano-
indentation for an accurate interpretation of the experimental data. There are several review papers and book chapters on the basic
principle of nanoindentation (Bhushan, 2010; Cheng and Cheng, 2004; Fischer-Cripps, 2006, 2011; Sharpe, 2008).
At the later part of the 19th century, Hertz developed elastic equations of contact for contacting spherical surfaces (Hertz, 1881).
To relate contact radius a, to the combined radius R, where R [ a, Hertz equation is:
3 PR
a3 ¼ (9)
4 E
where, P is the applied force and E* is the elastic modulus. Love (1939) formulated an equation to relate the force P, implemented
by the indenter and the resulting penetration depth or displacement h, as follows:
P a 2E tana 2
Pm ¼ 2 cos h1 and P ¼ h (10)
pa r p
Where a denotes the semi-angle of the conical indenter and the combined elastic modulus E*, defined regarding Young modulus E,
and the Poisson ratio n, as follows:
E
E ¼ (11)
1 n2
Sneddon who employed the integral transformation method to derive the same formula as (10) concluded the slope of a load–
displacement curve for a linearly isotropic half space indented by an axisymmetric arbitrary profile as follows (Sneddon, 1965):
dP 4E tana
¼ h (12)
dh p
where penetration depth h is related to contact depth hc as follows:
p
h ¼ hc (13)
2
The relationship between h and hc, the height of the part of the indenter which is in contact with elastic half-space is illustrated in
Fig. 3.
Now for a conical indenter of half-apical angle a, the contact area is given by
4 2
A ¼ pa2 ¼ ph2c tan 2 a ¼ h tan 2 a (14)
p
Now if we introduce Eq. (14) into Eqs. (10) and (12),
P E cot a
¼ (15)
A 2
dP pffiffiffi.
¼ 2E A p (16)
dh
Although the tangent stiffness regarding projected contact area in Eq. (16) is derived for a conical indenter, it is also applicable
for all asymmetric indenters such as cylindrical punches and spherical indenters (Bulychev et al., 1975; Pharr et al., 1992).
However, in the case of the circular flat punch of radius a (Maugis, 2013), the relation between applied load P, and depth of
penetration h, is as follows:
0:5
P r2
Pm ¼ 1 and P ¼ 2aE h (17)
2pa2 a2
where, A ¼ pa2. Now if we consider a rigid spherical indenter of radius R that is pressed into a linearly elastic specimen, then Hertz
formula can be expressed (Johnson, 1985):
0:5
3P r2 4 pffiffiffi
Pm ¼ 1 and P ¼ E Rh3 (18)
2pa2 a2 3
Now if a is expressed as the radius circular contact surface then a2 ¼ Rh and Eq. (18) certainly satisfies Eq. (16).
Fig. 3 Schematic of the nanoindentation of an elastoplastic solid by a conical cone at full load and unload.
Using Eqs. (10) and (12), reduced modulus of a linearly isotropic solid could be easily determined theoretically. However, in
practice, it is very complex to determine elastic modulus using this simple equation from a load–displacement curve.
The indenter with the three-sided geometry, known as Berkovich indenter, is the most popular indenter used to perform nano-
indentation experiments. For a Berkovich indenter the contact area is calculated as follows:
pffiffiffi
A ¼ 3 3h2c tan 2 q (19)
Where hc is the contact depth, and semi-angle q for a Berkovich indenter is 65.27. So from Eq. (14), we can obtain the relationship
between contact area A and contact depth hc for a Berkovich indenter as follows:
A ¼ 24:5 h2c (20)
In most nanoindentation experiments, the indenter is in contact with a semi-infinite half space which makes R as the radius of
the indenter alone but E* as the elastic modulus of the combined contacting bodies. Usually, the indenter is made of diamond
which is strongly resistant to the deformation, and so for most materials, E* is dominated by the elastic properties of the specimen.
This relationship is described in the Eq. (2):
Further the experimental P–h curve is quite different from the parabolic P–h curve that is predicted by the Eq. (10). Fig. 4 shows
the schematic diagram of a P–h curve for an elastoplastic specimen indented by a conical indenter. Here, maximum penetration
(hmax) by the indenter is the combination of elastic penetration (he) and plastic penetration (hp) or hmax ¼ he þ hp.
The equations presented above have some limitations to analyze a P–h curve obtained by a nanoindentation experiments as
these equations do not consider several factors such as the tip has a finite radius, and indenter leaves a residual imprint on the spec-
imen due to plastic deformations. So, Eq. (16) is modified as follows:
rffiffiffi
dP A
rhmax ¼ 2bE (21)
dh p
where b is a dimensionless constant which accounts for the factors in a nanoindentation experiments which are ignored in the above
equations and the value of which, is a debatable issue? Considering the factors of a nanoindentation experiments, Oliver and Pharr
(1992) proposed an equation which is widely used in nanoindentation experiments (Oliver and Pharr, 1992):
dP
hc ¼ hmax bxPmax rhmax (22)
dh
where ¼ 1 is the correction above factor, x z 0.72 for a conical indenter. Although the Oliver–Pharr method is quite popular in
nanoindentation experiments, it has its limitations.
Modes of nanoindentation
Dynamic nanoindentation: Dynamic nanoindentation is a mode of nanoindentation to determine the viscoelastic properties (Ode-
gard et al., 2005; Loubet et al., 2000). It involves applying an oscillatory force to the indenter during static indentation. Dynamic
properties can be determined by monitoring phase lag between applied displacement and measured force. The dynamic
Fig. 4 Schematic of a load–displacement curve corresponding to the nanoindentation.

nanoindentation technique was reported by Loubet et al. for the very first time, who used this technique to determine the storage
and loss modulus of the rubber polyisoprene (Loubet et al., 2000).
Modulus mapping: Modulus mapping is another technique to investigate the loss modulus and storage modulus characteristics of
materials by combining the aspects of nanoDMA and in situ SPM. In this technique, the indenter tip oscillates with a small force to
monitor the phase lag due to material response as a result of displacement. The advantage of this technique is in situ SPM imaging
enables the indenter tip to scan across the specimen to obtain a topographic image of the sample surface. In this way, modulus
mapping allows performing dynamic indentation at each point of the surface. A large number of indentations can be carried
out within a short period to obtain overall material characteristics like loss and storage modulus, loss and storage stiffness, tan delta
maps, etc.
Load control: In this mode of operation, one can specify the force applied to the indenter as a function of time, the displacement is
achieved with that applied force, and the resultant load–displacement curve is used to determine the stiffness, elastic modulus,
hardness and other mechanical properties. In a load control test, the load is increased at a particular rate. In load controlled oper-
ation, force path that includes loading and unloading can be applied. Load controlled nanoindentation operation is preferred for
conducting experiments as one can apply constant or variable load rate and also the load can be held constant for a period of time.
The forces mimicking real conditions can be more closely represented in this operational mode.
Displacement control: In this mode of operation, displacement path is prescribed. At any time, displacement/position of the tip is
known, and the force is determined to plot the force–displacement curve. In this mode, the softening behavior of the material
(beyond the peak load) can be evaluated. This mode of operation is preferred when indentations to certain depths are to be per-
formed. For example, if the thickness of the sample is known and the depth of penetration is to be limited to 10% of sample thick-
ness. Alternatively, in the case where the interface between two materials is to be probed.
Examples of Nanomechanical Testing of Cells

AFM
Table 1 summarizes the results of Young’s modulus measurement for various types of living cells. The magnitudes indicate that the
elastic modulus value of living cells varies over a wide range. It has long been established that external conditions influence the
elasticity of cell or cell membrane. Hence, the first factor that brings about changes in the elasticity of cells is AFM sample handling.
Erythrocytes treated with 5% formalin showed a 10-fold (119.5 15 kPa) increase in their elastic modulus compared to native
erythrocytes (16.05 2.3 kPa) (Mozhanova et al., 2003). The transverse stiffness of cardiomyocytes is also increased by a factor
of 16 after fixing with formalin (Shroff et al., 1995). A study involving AFM to probe leukemia cells placed in microwells demon-
strated the influence of microwells on the elastic modulus of the cell (Rosenbluth et al., 2006). The cell deformation was described
using Hertzian contact mechanics. The second factor is related to the heterogeneity of mechanical properties of cells. There are signif-
icant variations in the values of elastic modulus at different cell regions. The elastic modulus value of human umbilical vein endo-
thelial cells was 7.22 0.46 kPa over the nucleus; 2.97 0.79 kPa over the area away from the nucleus towards cell edge, and
1.27 0.36 kPa at the cell edge (Mathur et al., 2000). A similar study on cardiomyocytes revealed that cells are softer at the nuclear
region, and become stiffer towards the periphery (Shroff et al., 1995). Elastic modulus values of chicken cardiomyocytes’ soft region
between 5 and 30 kPa, with stress fibers favored region showing stiffness value of 100–200 kPa are reported (Hofmann et al., 1997).
The elastic modulus of the surface layer of living astrocytes ranged from 1 to 40 kPa, being influenced by the inner structure of cells
such as nucleus and F-actin. On the other hand, the elastic modulus of fixed cells was relatively uniform (200–700 kPa), irrespective
of the inner structures of cells (Yamane et al., 2000). The third factor influencing elastic modulus measurement relates to cell thick-
ness. Substrate contributions can be neglected if AFM tip does not indent > 10% of the cell thickness (Mathur et al., 2001).
The elastic modulus of cells has also been found to play an important role in delineating functions of various cell types, some
examples of which are presented below. Some of the earliest AFM studies on living aortic endothelial cells were carried out to study
the effects of shear stress on cellular organization and other factors which may influence the mechanical response of cells to flow
(Barbee et al., 1994; Sato et al., 2000). It was reported that the average elastic modulus of bovine pulmonary artery endothelial cells
(BPAECs) in the range 0.2–2.0 kPa (Pesen and Hoh, 2005). A similar study showed the difference in mechanical properties of rabbit
endothelium, that is, cells were found to be stiffer in the medial wall of aortic bifurcation than in the lateral wall (Miyazaki and
Hayashi, 1999). The elastic modulus of activated platelets was found to be in the range of 1–50 kPa using force mapping techniques
(Radmacher et al., 1996). A few AFM studies have also been carried out to delineate the mechanistic mechanism of cell migration
using fibroblasts (Haga et al., 2000; Nagayama et al., 2001). A relation between the local stiffness distribution on cell and cell migra-
tion was reported, that is, stiffness distribution of cell surface was constant until cells started to move, a sharp decrease in the stiff-
ness in the nuclear region of the cells was observed upon movement (Nagayama et al., 2001). It has been reported in previous
studies that mechanical properties of the lamellipodium are important for a deeper understanding of cell migration mechanism.
It was demonstrated that the rigidity of fibroblast cell body was due to the over-condensation of the actin network, and hardening
of the cell cortex (Haga et al., 2000). An identical study on fish epidermal keratocytes showed rigidity of the cell body as a function
of distance from cell edge, that is, rigidity was the highest at the cell edge, and it gradually decreased towards the cell center. Also, the
change in rigidity was not perturbed by the vertical lamellipodia thickness (Laurent et al., 2005).
AFM has also been found to be effective to study cell mechanical properties during cell adhesion. There are some AFM studies
which characterized osteoblast elastic properties during adhesion (Domke et al., 2000; Simon et al., 2003). The effect of substrate on
Table 1 Summary of the AFM based experiments carried out to evaluate elastic modulus for various cell types
Cell type Young’s modulus, E (kPa) References
Rat astrocytes 2–20 Yamane et al. (2000)

Endothelial cells
HUVEC 10–11 Sato et al. (2004)
– 1.3–7.2 Mathur et al. (2000)
BPAEC 0.2–2.0 Pesen and Hoh (2005)

Osteoblasts 0.3–20 Simon et al. (2003)
SaOS2 2.1–8.8 Domke et al. (2000)
Fibroblasts 4–5 Bushell et al. (1999)
L929 4–5 Wu et al. (1998)
HS68 1.86 1.13 Nikkhah et al. (2010)
Epidermal keratocytes 10–55 Laurent et al. (2005)
Blood
Leukemia myeloid cells (HL60) 0.2–1.4 Rosenbluth et al. (2006)
Leukemia lymphoid (Jurkat) cells 0.02–0.08
Neutrophils 0.2–0.07
Lymphocytes 1.24 0.09 Cai et al. (2010)
Leukemia lymphoid Jurkat cells 0.51 0.06
Platelets 1–50 Radmacher et al. (1996)
Erythrocytes 19–33 Dulinska et al. (2006)
Red blood cells (RBCs) 0.1–0.2 Li et al. (2012)
Raji 0.2–0.4
Hut 1–1.4
K-562 0.6–0.7
Skeletal muscle cells
Murine C2C12 myoblasts 11–45 Collinsworth et al. (2002)
– 28–21 Mathur et al. (2001)
Myofibrils 40–45 Yoshikawa et al. (1999)
Cardiocytes 95–100 Mathur et al. (2001)
Chicken 5–200 Hofmann et al. (1997)
Prostate cells
BPH 2.797 0.491 Faria et al. (2008)
LNCaP 1.401 0.162
PC-3 0.287 0.052
PNT2-C2 1.139 0.320
PZHPV-7 3.09 0.84 Lekka et al. (2012)
LNCaP 0.45 0.21
PC-3 1.95 0.47
DU145 1.36 0.42
DU145 0.6 0.4 Efremov et al. (2015)
Breast cells
MCF10A 1.13 0.44 Li et al. (2008)
MCF7 0.63 0.22
184A1 2.26 0.56
MCF7 1.24 0.46
T47D 1.20 0.28
MCF10A 1.75 0.12 Lee et al. (2012)
MCF7 1.3 0.15
MDA-MB-231 0.8 0.05
MCF10A 1.13 0.84 Nikkhah et al. (2010)
MDA-MB-231 0.51 0.35
Bladder cells
HU609 9.7 3.6 Lekka et al. (1999)
HCV29 7.5 3.6
HU456 0.3 0.2
T24 0.8 0.4
SV-HUC-U1 27.57 Canetta et al. (2014)
MGH-U1 2.46
RT112 5.7 0.45 Abidine et al. (2015)
T24 1.9 0.26
J82 2.7 0.24
Table 1 Summary of the AFM based experiments carried out to evaluate elastic modulus for various cell typesdcont'd
Cell type Young’s modulus, E (kPa) References
Mesothelial cells
Normal 1.97 0.70 Cross et al. (2008)
Cancerous 0.53 0.10
Cervical cells
CRL2614 2.58 0.08 Zhao et al. (2015)
CaSki 2.03 0.14
End1/E6E7 4.8 0.8 Hayashi and Iwata (2015)
HeLa 2.46 0.6
Thyroid cells
S748 2.2–6.8 Prabhune et al. (2012)
S277 1.2–1.4
Ovarian cells
MOSE early 1.097 0.682 Prabhune et al. (2012)
MOSE-intermediate 0.796 0.441
MOSE-late 0.549 0.281
IOSE 2.472 2.048 Xu et al. (2012)
OVCAR4 1.120 0.865
HEY 0.884 0.529
OVCAR3 0.576 0.236
HEYA8 0.494 0.222
Keratinocytes
HaCaT 84.5 Fung et al. (2011)
HaCaT 77–109 Heu et al. (2012)
cell adhesion using osteoblast cell was studied (Domke et al., 2000). Osteoblasts cultured on different substrates (CoCr, Ti, TiV,
glass, and TCPS) exhibited the elastic modulus values ranging from 2 kPa (CoCr and TiV) to 9 kPa (Ti surface). The latter modulus
was found to be in good agreement with the modulus obtained from osteoblasts cultured on TCPS. Another study on the osteo-
blasts demonstrated the influence of cytoskeletal reorganization and state of the cell on the mechanical properties of the cell.
The elastic modulus of osteoblasts was found to be in the range of 0.3–200 kPa depending upon the type of substrate the cells
were cultured on (Simon et al., 2003).
The changes in mechanical properties and cytoskeleton reorganization have been found to correlate well with cell cycle stages in
the following studies (Berdyyeva et al., 2005; Collinsworth et al., 2002; Sato et al., 2004). These results have paved the way for
understanding the mechanism behind cell differentiation and aging. A study on human umbilical vein endothelial cells (HUVEC)
revealed that the culture period influences the mechanical properties of cells (Sato et al., 2004). It was reported that epithelial cells
cultured on type IV collagen for longer than 4 days showed average elasticity values > 10 kPa (Berdyyeva et al., 2005). A similar
study on mouse skeletal myocyte cells hypothesized cell differentiation as a key regulator of transverse elastic properties of the cells.
They observed a significant increase (from 11.5 1.3 to 45.3 4.0 kPa) in elastic modulus of cells upon cell differentiation over
the course of 8 days (Collinsworth et al., 2002). Actin and myosin were found to be major contributors to changes in the transverse
elastic modulus.
AFM studies on single cells have shown that depending on the cell type, either actin filaments or microtubules have a strong
influence on the mechanics of cells. There are a few studies where living cells are subjected to incubation with cytoskeleton-
disrupting drugs to identify the type of cytoskeletal elements that dominate the mechanical response (Cai et al., 2010; Heu
et al., 2012; Hofmann et al., 1997; Lulevich et al., 2010; Rotsch et al., 1997; Rotsch and Radmacher, 2000; Spedden et al., 2012;
Wu et al., 1998). The chemical disassembly of the rat liver macrophages’ actin filaments by applying cytochalasin B and latrunculin
was studied (Rotsch et al., 1997). Treating cells with cytochalasin B resulted in a sevenfold decrease in elastic modulus after 40 min.
On the other hand, latrunculin treatment induced a twofold decrease in the elastic modulus of the perinuclear region after 40 min,
whereas other parts of cell remained intact. The latrunculin-induced disruption of cytoskeleton network of various fibroblast cell
lines was reported by two different groups (Braet et al., 2001; Rotsch and Radmacher, 2000). A decrease in the elastic modulus
of chicken cardiocytes (Hofmann et al., 1997) and L929 cells (Wu et al., 1998) was observed upon treating them with cytochalasin
B and cytochalasin D, respectively. In contrast, treatment of fibroblasts with nocodazole or colcemid resulted in an increase in their
elastic modulus. Another study used cytochalasins B and D, latrunculin A and Jasplakinolide to show disruption of actin filaments
results in a decrease in the elastic modulus of fibroblasts, while reorganization of microtubules does not affect cell elastic modulus
(Rotsch and Radmacher, 2000). Cai et al. (2010) showed that cytochalasin B induced 70% and 60% decrease in the elastic modulus
of lymphocyte and Jurkat cells, respectively. A similar study on keratinocytes reported that latrunculin and nocodazole treatment
not only altered the cell elastic modulus but also treated cells appeared softer than control (Fig. 5). They observed around 50% and
27% decrease in elastic modulus of cells treated with latrunculin and nocodazole, respectively (Lulevich et al., 2010). In a recent
study, glyphosate has been shown to induce cell membrane stiffening and the emergence of cytoskeleton structures at the
Fig. 5 Effect of latrunculin A on keratinocytes, (A) characteristic force versus relative deformation curve for keratinocyte cells treated with latruncu-
lin A (blue, dashed) as compared to control (red, solid), immunocytochemistry for actin (green) in (B) control and (C) latrunculin A-treated keratino-
cyte, (D) and (E) are optical micrographs of a latrunculin A-treated keratinocyte before and after compression, respectively. Reprinted with permission
from Lulevich, V., Yang, H. Y., Isseroff, R. R. and Liu, G. Y. (2010). Single cell mechanics of keratinocyte cells. Ultramicroscopy 110, 1435–1442.
subcellular scale at low concentration (15 mM) (Fig. 6). However, HaCaT cells showed a flattened external cell membrane along
with reduced number of protrusions. Also, quercetin was found to reverse the effects of glyphosate (Heu et al., 2012). Spedden
et al. (2012) showed an increase by > 30% in the elastic modulus of neuronal cell bodies upon Taxol (10 mM) treatment, while
nocodazole (10 nM) did not induce any substantial change in cell body elastic modulus. AFM estimation of cell mechanical prop-
erties has shown the potential for the diagnostics of different pathologies in the past decade as it facilitates the measurement of the
Fig. 6 Effect of glyphosate on HaCaT keratinocytes, (i) topographical changes, (ii) change in mechanical properties, and (iii) induced deformation
for control cells (A) and treated cells (B) at 15 mM glyphosate for a 6 h–incubation. The color scales are (from bottom to top): 0–1 nN, 0–500 kPa,
and 0–250 nm for the peak force error, Young’s modulus and deformation images, respectively. Reprinted with permission from Heu, C., Berquand,
A., Elie-Caille, C. and Nicod, L. (2012). Glyphosate-induced stiffening of HaCaT keratinocytes, a peak force tapping study on living cells. Journal of
Structural Biology 178, 1–7.
influence of various factors on the mechanical properties of the living cells. A comparative study of normal and cancerous human
bladder cells showed that normal cells’ elastic modulus was found to be one order of magnitude higher than cancerous counterparts
(Lekka et al., 1999). In a later study, (Lekka et al., 2001) showed a strong correlation between the reduction of glycolytic activity and
the increase in Young’s modulus values obtained for the bladder cancer cells treated with chitosan. An AFM-based cytomechanical
analysis showed that cancer cell stiffness is 70%–80% less than that of normal cells (Cross et al., 2008) Using AFM and confocal
fluorescence microscope, (Li et al., 2008) demonstrated that MCF-7 (malignant breast) cells had an apparent Young’s modulus
significantly lower (1.4–1.8 times) than that of MCF-10A (normal breast epithelial) cells and their apparent Young’s modulus
increased with loading rate. A similar study on various prostate cells (BPH, LNCaP, and PC-3) revealed that BPH had higher Young’s
modulus among three cell lines examined. Interestingly, the highly invasive PC-3 cells were found to have a higher Young’s
modulus than the non-invasive LNCaP clone FGC cells, showing that there are variations between different types of prostate cancer
cells, a feature which is reflected in their clinical behavior (Faria et al., 2008). The measurement of cell’s elastic modulus using AFM
indentation revealed that HS68 (normal fibroblast cells) cells are significantly stiffer than MCF-10A and MDA-MB-231 (breast
cancer) cells. Upon microtubule disruption with nocodazole, fibroblasts no longer stretched, but adhesion of MCF-10A and
MDA-MB-231 within the etched features remained unaltered (Nikkhah et al., 2010). Recent studies on ovarian cancer cells have
demonstrated cancerous ovarian cells are softer than their normal counterparts but possess higher motility whereas ovarian cancer
cells with lower migratory or invasive potential are almost five times stiffer than the highly invasive ones (Swaminathan et al., 2011;
Xu et al., 2012) Analogous to other cancers, thyroid cancer cells were found to be three to fivefold more deformable than normal
thyroid cells (Prabhune et al., 2012). Lekka et al. (2012) studied both breast and prostate cancer cells using AFM indentation. The
calculated elastic modulus values for the presented results of prostate cancers were 0.45 0.21, 1.36 0.42, and 1.95 0.47 kPa
for LNCaP, Du145, and PC-3, respectively. However, the elastic modulus calculated for PZHPV-7 cells was 3.09 0.84 kPa. For
breast cancers, the results were 1.20 0.28 kPa, 1.24 0.46 kPa, and 2.26 0.56 kPa for T47D, MCF7, and 184A cell lines, respec-
tively. These results showed that the normal cells were characterized by larger Young’s modulus values, irrespective of the cell types
(Fig. 7A). It also indicates their lower ability to deform compared with the normal cells coming from later stages of cancer progres-
sion. Since indentation depth governs cells’ elastic modulus, hence, the dependence of elastic modulus on indentation depth was
studied. For instance, the largest indentation depth resulted in the lowest modulus value for 184A and MCF-7 cells (Fig. 7B). The
loading rate has been known to influence elastic modulus. Therefore, the influence of displacement rate was examined for the
human breast cells (Fig. 7C). It demonstrates an increase in modulus of about 48% and 157% for cancerous MCF7 and normal
184A cells, respectively, for data recorded at 0.5 mm/s as compared with those recorded with a velocity of 10 mm/s. The influence
of prolonged indentation on a single cell was also determined using the modulus measurements obtained in T47D cells (Fig. 7D;
black dots). 232 curves were recorded over an area of 10 10 mm. However, no influence of time over modulus measurements was
observed. When indentation is carried out at the constant position (Fig. 7D; open squares), the continuous indentation influences
the measurements. For instance, for the same T47D breast cancer cells, such experiments ended quickly at the breakdown at the
curve No. 145. AFM and modulated Raman spectroscopy (MRS) have been used to discriminate between normal human urothelial
cells (SV-HUC-1) and bladder cancer cells (MGH-U1). The results demonstrated that MGH-U1 cells were 1.5-fold smaller, 1.7-fold
thicker and 1.4-fold rougher than normal SV-HUC-1 cell and the adhesion energy was 2.6-fold higher in the MGH-U1 cells
compared to normal SV-HUC-1 cells, indicating higher deformability of bladder cancer cells. Also, the elastic modulus of MGH-
U1 cells was found to be 12-fold lower than that of SV-HUC-1 cells (Canetta et al., 2014).
In another study, AFM-based nanomechanics and fluorescence based intracellular calcium dynamics studies were performed on
poorly (LNCaP) and highly (CL-1, CL-2) metastatic human prostate cancer cells. The elastic moduli and calcium dynamics were
found to be greater in CL-1 and CL-2 than LNCaP. The authors suggested that the enhanced elastic moduli and calcium dynamics
were observed due to the intensified tensile stress generated by actin cytoskeletons anchored at more focal adhesion sites (Bastatas
et al., 2012). In a recent study, the effects of actin on the mechanics of normal Vero and prostate cancer cell line DU145 was deter-
mined using AFM before and after treating the cells with specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632
and trypsin. The elastic modulus of Vero cells was found to be around 1.3 0.9 kPa, while the prostate cancer cell DU145 exhibited
significantly lower modulus (0.6 0.4 kPa). Interestingly, cancer cells exhibited diverse viscoelastic behavior and different
responses to actin cytoskeleton reorganization upon drug treatment (Efremov et al., 2015). It is shown that the elastic modulus
values of late-stage mouse ovarian surface epithelial (MOSE) cells (0.549 0.281 kPa) were significantly less than that of their
early-stage ones (1.097 0.632 kPa) (Ketene et al., 2012). Apparent cell viscosity was also found to be reduced significantly
from early (144.7 102.4 Pa s) to late stage (50.74 29.72 Pa s), indicating higher stiffness and viscosity of normal ovarian cells.
The increase in cell deformability was found to correlate directly with the transition of benign phenotype to malignant one. The
decrease in the level of actin in the cytoskeleton and its organization facilitated the changes in cell biomechanical property. Using
spherical probes, (Li et al., 2012) investigated the mechanical properties of RBCs, Raji, HuT, and K562 cells. The elastic modulus was
found to be 0.1 0.2, 0.2 0.4, 1 1.4, and 0.6 0.7 kPa, for RBCs, Raji, HuT, and K562 cells, respectively, indicating aggressive
cancer cells are softer than normal cells. A similar study on HeLa cells (human cervical cancer cell) and End1/E6E7 cells (squamous
epithelial cell from normal human cervix) showed that cancer cells were softer than normal cells, and there were no significant loca-
tional differences in the stiffness of cancer cells between the central and the peripheral regions (Hayashi and Iwata, 2015). A
mechanical and adhesive mismatch between transformed and non-transformed cells in a cell monolayer could trigger enhanced
pulsating migration using various breast cell lines (Lee et al., 2012). They also examined the influence of neighboring stiff epithelial
cells on cancer cells in the early steps of cancer progression. A recent study has shown that the elastic modulus and the transition
frequency can be used as markers of invasiveness for cancer cells (Abidine et al., 2015). The longitudinal elastic modulus
Fig. 7 (A) Comparison of the stiffness of prostate and breast cancer cells, (B) influence of indentation depth, (C) force load on the elastic modulus
of breast cells (184A and MCF7), and (D) effect of prolonged indentation on breast cancer cell (T74D). Reprinted with permission from Lekka, M.,
Gil, D., Pogoda, K., Dulinska-Litewka, J., Jach, R., Gostek, J., Klymenko, O., Prauzner-Bechcicki, S., Stachura, Z. and Wiltowska-Zuber, J. (2012).
Cancer cell detection in tissue sections using AFM. Archives of Biochemistry and Biophysics 518, 151–156.
distribution of human cervical squamous carcinoma cells (CaSki) and normal cervical epithelial cells (CRL2614) was found using
AFM. The results indicated that CaSki cells were less stiff than their normal counterparts, and the nuclear region appeared to be
softer. Also, authors have shown a correlation between heterogenicity in longitudinal elastic modulus and indentation depth.
For instance, CaSki cells, with a modulus of 0.35–0.47 kPa, was found to be located at 237–225 nm; while normal cells showed
an elastic modulus of 1.20–1.32 kPa at 113–128 nm (Zhao et al., 2015).
Nanoindentation
In this section, we discuss some examples of measuring cellular nanomechanics using nanoindentation technique. For the very first
time, mechanical properties of soft tissue using nanoindentation technique was measured by Ebenstein et al. in 2004 (Ebenstein
and Pruitt, 2004). The purpose of the study was to develop a methodology to keep the sample hydrated, select an appropriate
indenter for soft tissue like material indentation, determine an appropriate control material for the development of future inden-
tation protocols and identify a substrate to be used for blunt tip alignment. They developed a hydration system to keep the sample
hydrated for > 8 h without completely submerging the sample. To identify appropriate indenter for the hydrated, soft tissue, they
performed indentation with different types of indenters. Three sided sharp Berkovich indenter with an average radius of curvature of
100–200 nm and conosperical diamond indenters with radius of 10, 50, 100, and 800 mm radius of curvature were used. They
found that conosperical tip with a 100-mm radius of curvature is most suitable for the indentation of the vascular tissue sample
and agarose gel is most effective for the tip alignment. They used Oliver and Pharr method to calculate reduced elastic modulus
and found that artery tissue has an elastic modulus of z 0.73 MPa. In orthopedic research, cartilage repair is one of the main chal-
lenges. To address this challenge they studied the mechanical properties of cartilage repair tissue using nanoindentation experiments
(Ebenstein et al., 2004). They reported that the mechanical properties of cartilage inferior tissue are less than the control cartilage
tissue. In another study, nanoindentation technique to study the mechanical properties of fibrous tissue, blood clots, partially calci-
fied fibrous tissue, and bulk calcifications from human atherosclerotic plaque tissue (Ebenstein et al., 2009). The goal of that study
was to investigate the mechanical changes in plaques tissues during atherosclerotic disease progression as clinical events such as
stroke, and heart attack can be caused by the rupture of atherosclerotic plaques in blood vessel walls. They demonstrated that
the stiffness of the plaque tissue increases with the increase in the mineral content in the tissue.
In bone tissue engineering, the interaction between bone cells and engineered biomaterial is an important area to explore. We
studied the nanomechanical properties of bone cells and bone cell-substrate construct using nanoindentation technique under
physiological condition. Cells were seeded on tissue culture treated polystyrene (TCPS) (Khanna et al., 2011). Once cells are
attached, cell seeded TCPS was placed in a fluid cell submerged in cell culture media and then nanoindentation experiment carried
out by placing the sample on the temperature controlled stage of nanoindentation device. In displacement control mode operation,
cells demonstrated completely elastic load-deformation response (Fig. 8) and cell elastic modulus was found 1.3–12.4 MPa using
Oliver and Pharr method. In another study, we reported a novel in situ nanoindentation technique developed to evaluate the
composite mechanical behavior of cell-biomaterial construct under physiological conditions over the time scale of bone nodule
generation (Khanna et al., 2012). The schematic illustration of cell-substrate indentations on a single cell attached to Chi-PgA-
HAP film deposited onto TCPS substrate is shown in Fig. 9. Nanomechanical properties of cells over the time of adhesion, prolif-
eration, development and bone nodule formation were evaluated (Fig. 10). The study implied that unique interactions between
cells and nanocomposite films provided a favorable mechanical environment for the formation of bone nodules.
In situ quasi-static and dynamic nanoindentation tests on calcified nodules formed by mouse osteoblasts to investigate the
effects of glucocorticoid hormones such as dexamethasone and hydrocortisone on the nanomechanical properties have been con-
ducted (Miyamoto et al., 2015). How dexamethasone affects the hardness, elastic modulus and storage modulus of the calcified
nodules formed by mouse osteoblasts is illustrated in Fig. 11. The proliferation or calcium deposition of the cells were not affected
by dexamethasone, but nodules formed in the presence of dexamethasone were significantly stiffer than the nodules formed in the
absence of dexamethasone. Their result suggested that hormones like dexamethasone are essential for in vitro formation of more
mature calcified nodules by bone cells.
Fig. 8 Representative load–displacement curves obtained on live osteoblast. (A) Cells exhibit complete elastic recovery upon indentation as indi-
cated by linear loading and unloading curves (B). The first portion which is flat shows the cell indentation response and steep loading slope beyond
1250 nm displacement show the stiffer response due to TCPS substrate lying underneath the cell. The flat portion of the loading and unloading curve
in (B) are replotted separately in (C) and (D) respectively. The figure is adapted from Khanna, R., Katti, K. S. and Katti, D. R. (2011). Experiments in
nanomechanical properties of live osteoblast cells and cell–biomaterial interface. Journal of Nanotechnology in Engineering and Medicine 2, 041005 and
reprinted with permission.
Fig. 9 Schematic showing cell-substrate indentations on a single cell attached to Chi-PgA-HAP film deposited onto TCPS substrate and sectional
view of an indenter.
Fig. 10 In situ nanomechanical responses of osteoblast cells and cell-substrates obtained by nanomechanical experiment (A–F). Mechanical
responses of cells and cell-CPH composites are completely reversible as shown by load–displacement curves (A and B).
In an attempt to map the elastic properties of soft tissues, nanoindentation experiments are performed to investigate the micro-
mechanical properties of 5-mm-thick sections of ferret aorta and vena cava and to relate these mechanical properties to the histo-
logical distribution of fluorescent elastic fibers (Akhtar et al., 2009). They reported the elastic modulus of the blood vessel tissues
varies from 8 MPa to 35 MPa depending on a different layer of the tissue using extended Oliver and Pharr method and 10 mm con-
osperical indenter. They demonstrated that it is possible to distinguish the differences in the micromechanical properties of blood
Fig. 11 Nanoindentation technique is used to determine the hardness and elastic modulus of calcified nodules formed by MC3T3-E1 cells in the
presence and absence of dexamethasone. Representative hardness (upper panels) and storage modulus (lower panels) data versus contact depth of
calcified nodules formed in dexamethasone negative medium (A) and dexamethasone positive medium (B). Hardness (C) and elastic modulus
(D) obtained in quasistatic nanoindentation tests and hardness (E) and storage modulus (F) obtained in dynamic nanoindentation tests for calcified
nodules formed in cultures with and without dexamethasone. The figure is adapted from Miyamoto, S., Miyamoto, Y., Shibata, Y., Yoshimura, K.,
Izumida, E., Suzuki, H., Miyazaki, T., Maki, K. and Kamijo, R. (2015). In situ quasi-static and dynamic nanoindentation tests on calcified nodules
formed by osteoblasts: Implication of glucocorticoids responsible for osteoblast calcification. Acta Biomaterialia 12, 216–226 and reprinted with
permission.
vessels, which are related to the tissue microstructure. Continuous depth-sensing nanomechanical characterization of living, fixed
and dehydrated cells attached on a glass substrate using a dynamic contact module are conducted (Yang et al., 2010). The nano-
mechanical characteristics of the cells were reported as the continuous harmonic contact stiffness (HSC). In this study, they tried
to understand how the underlying substrate influences the interpretation of the nanomechanical property of thin soft matter on
a hard substrate (Table 2).
Table 2 Summary of the nanoindentation based nanomechanical experiments carried out to evaluate elastic modulus for different cells and tissues
Cell/tissue Indenter Method Elastic modulus Reference
Mineralized matrix formed on glass coverslips by Berkovich Oliver and Pharr 10–45 GPa Duan and Chen (2015)
immortalized cell line Y201 from human mesenchymal
stem cells
Calcospherulite crystals produced by rat osteoblasts Berkovich Oliver and Pharr 0.41 0.15 GPa Chen et al. (2010)
Cartilage repair tissue 100 mm conosperical Oliver and Pharr Not reported Ebenstein et al. (2004)
Porcine aorta tissue 100 mm conosperical Oliver and Pharr 0.7–0.8 MPa Ebenstein and Pruitt (2004)
Human carotid bifurcation plaque tissue 100 mm conosperical Compliance method 0.05–43.4 MPa Ebenstein et al. (2009)
Osteoblast Berkovich Oliver and Pharr 1.1–12.0 MPa Khanna et al. (2012)
Osteoblast Berkovich Oliver and Pharr 1.3–12.4 MPa Khanna et al. (2011)
Nodule formed by mouse osteoblastic MC3T3-E1 cells Berkovich Oliver and Pharr 10 GPa Miyamoto et al. (2015)
Embedded cross section of aorta and vena 10 mm conosperical Oliver and Pharr 8–35 MPa Akhtar et al. (2009)
Cava tissue
3T3 mouse embryonic fibroblast cells Berkovich Reported as continuous harmonic Yang et al. (2010)
contact stiffness (HCS)
Perspectives
Some Current Challenges in Use of AFM
Here we discuss the fundamental limitations of AFM such as scan range, scan speed, piezoelectric transducer nonlinearity, probe size
and shape, and sample size. An AFM often scans a maximum area of about 150 150 mm and a maximum height on the order of
10–20 mm. On the other hand, scanning electron microscope can image an area on the order of square 1 1 mm with a depth of
field on the order of millimeters. The scanning speed of an AFM is also a limitation. Usually, an AFM requires several minutes for
a typical scan, and the relatively slow rate of scanning often leads to the formation of thermal drift in the image. It makes AFM less
suited for measuring accurate distances between topographical features on the image (Lapshin, 1998, 2004) AFM images can also
get adulterated by nonlinearity, hysteresis, and creep of the piezoelectric material and cross-talk between the x, y, z-axes. Theoret-
ically, piezoelectric transducers allow convenient positioning at or below the nanometer scale. However, the position is not
a single-valued, linear function of the applied voltage, so, if a certain point on the sample is in the center of the image as the
AFM scans in the þ x direction, it may not be at the image center when the AFM scans in the x direction (Lapshin, 1995). Moreover,
scanner response is also subjected to change depending on scan frequency, temperature, and age of the scanner. AFM imaging often
induces the formation of image artifacts by either of the following factors; an incompatible tip, substandard operating environment,
or even by the sample itself (Velegol et al., 2003) Image artifacts are unavoidable. However, their effect on results can be lessened
through various methods. Small sample size seems to be another limitation that AFM suffers from. The overall dimensions of the
samples to be measured by AFM are small so that they can be easily mounted on the sample holder. The sample holder containing
sample is moved in x, y, and z-axes by a piezoelectric transducer. Consequently, even small deflections of a stationary cantilever are
counted as probe signal. Another limitation of AFM is the size and shape of the probe itself. It comes into play when the features to
be imaged have high aspect ratios. For instance, a probe that is large or blunt will easily measure very flat surfaces without much loss
of information, but will not be able to trace the true profile of a surface that includes high aspect ratio features that are sharper, or of
smaller dimensions than the probe.
Current Challenges in Nanoindentation of Live Cells

Challenges of nanoindentation experiments on biological soft samples include problem associated with instrumentation, optimi-
zation of indenter geometry and load–displacement range, an appropriate model for analyzing experimental data, maintaining
hydration state of the biological samples, etc. Commercially available instruments are usually designed for time independent mate-
rials, but the mechanical response of biological materials such as cells is mostly time-dependent. Due to the low elastic moduli of
several biological samples such as cells the evaluation of force–displacement curves for soft samples is challenging. Indentation tip
force is directly related to the tip geometry and material properties of the specimen. If a sharp indenter is used for indentation on soft
tissues, the contact area and the corresponding force is very small for a shallow indentation. It makes it very complicated to deter-
mine when the indenter touches the sample which leads to a zero-point error (Kaufman and Klapperich, 2009). Another nanoin-
dentation instrumentation challenge for biological materials like cells or tissue is maintaining hydration state at the physiological
condition. For this purpose, the Katti research group has used cell culture media in a fluid cell on the temperature controlled stage,
but it is a still a challenge to maintain physiological pH, as no CO2 was supplied to the cell culture media, thus limiting the time for
conducting the tests. (Khanna et al., 2011, 2012). Using fluid to hydrate the sample and temperature controlled stage might intro-
duce further experimental complexities which have not been sufficiently addressed in the literature. Most popular indenter for nano-
indentation experiments is three sided Berkovich tip. The Berkovich indenter has been used for soft tissues and glossy polymers
(VanLandingham, 2003; Habelitz et al., 2001; Rho et al., 1999; Larsson and Carlsson, 1998). Sharp indenters such as Berkovich
can introduce plastic deformation and stress concentration as well as damage the specimen. In an attempt to minimize this
problem, blunt tips such as spherical, conosperical and cylindrical punch have been used (Larsson and Carlsson, 1998; Gerberich
et al., 1998; Ebenstein et al., 2004, 2009; Balooch et al., 1998; Lundkvist et al., 1997). The disadvantage of using flat tips is that it has
a high-stress concentration at the contact perimeter. In addition to the indenter geometry, the size of the indenter needs to be
considered as well. Indenters with small radius can provide nanomechanical properties of a single cell meanwhile indenter with
a larger diameter will provide information on nanomechanical characteristics of the whole tissue including extracellular matrix.
Most of the commercially available nanoindentation instruments are designed with software to analyze experimental data using
Oliver and Pharr method (Oliver and Pharr, 1992). This approach works extremely well for the specimen with a time-independent
mechanical response. However, this method introduces significant errors to calculate elastic modulus for samples with a viscoelastic
time-dependent response like soft tissues as the assumption of elastic unloading is invalidated by the continuing time-dependent
deformation (Oyen and Cook, 2003; Oyen, 2013). Creep or sinking of tip under constant force into the specimen is the most
common effect of viscoelasticity. For viscoelastic specimen, a “nose” is observed on the unloading curve when there is no hold
time which results in a negative slope at the beginning of the unloading curve and makes it very difficult to measure the elastic
modulus (Briscoe et al., 1998). Some correction has been proposed to minimize the creep effect in calculating elastic modulus
of the viscoelastic materials (Tang and Ngan, 2003; Feng and Ngan, 2002; Ngan et al., 2005). Some other models have been devel-
oped to analyze the nanoindentation experimental data obtained from soft tissue like viscoelastic materials (VanLandingham,
2003; Oyen, 2005; Lu et al., 2003; Oyen and Cook, 2003).
Another problem associated with indenting soft tissue samples is the adhesion between the indenter and the sample (Carrillo
et al., 2005, 2006). It has been reported that modulus calculation is significantly affected due to adhesion between the tip and the
soft tissues (Carrillo et al., 2005, 2006). JKR (Johnson et al., 1971) model has been reported to be more accurate to minimize the
contact effect between the soft samples and the indenters while measuring elastic modulus (Ebenstein and Wahl, 2006; Carrillo
et al., 2005, 2006). More sophisticated approaches have also been reported combining JKR model and viscoelastic analysis (Eben-
stein and Wahl, 2006; Greenwood and Johnson, 2006; Wahl et al., 2006).
Future Perspectives
Overall, as instrumentation capabilities improve, including improved and reliable feedback mechanisms, the capability to conduct
AFM force curve experiments and nanoindentation is likely to become routine and thus present itself as a new and novel mecha-
nistic approach to understanding disease progression as well as diagnostics. The combination of experiments and modeling will
also play an important role in improving the accuracy and interpretation of experimental data. Next generation medical treatments
and research will see a large increase in the evaluation of mechanics using these techniques. The critical advances will arise from
making implicit connections between gene expression and other traditional biochemical assays evaluated for disease diagnostics
and mechanisms and mechanics of the cell during the time evolution of the disease state.
Acknowledgements
The authors would like to acknowledge partial support from the NDSU Grand Challenge program for the Center for Engineered Cancer Test-Beds.
References
Abidine, Y., Laurent, V. M., Michel, R., Duperray, A., & Verdier, C. (2015). Local mechanical properties of bladder cancer cells measured by AFM as a signature of metastatic
potential. The European Physical Journal Plus, 130, 202.
Akhtar, R., Schwarzer, N., Sherratt, M. J., Watson, R. E. B., Graham, H. K., Trafford, A. W., Mummery, P. M., & Derby, B. (2009). Nanoindentation of histological specimens:
Mapping the elastic properties of soft tissues. Journal of Materials Research, 24, 638–646.
Assoian, R. K., & Klein, E. A. (2008). Growth control by intracellular tension and extracellular stiffness. Trends in Cell Biology, 18, 347–352.
Baker, E. L., Bonnecaze, R. T., & Zaman, M. H. (2009). Extracellular matrix stiffness and architecture govern intracellular rheology in cancer. Biophysical Journal, 97, 1013–1021.
Balland, M., Desprat, N., Icard, D., Fereol, S., Asnacios, A., Browaeys, J., Henon, S., & Gallet, F. (2006). Power laws in microrheology experiments on living cells: Comparative
analysis and modeling. Physical Review E, 74, 021911.
Balooch, M., Wu-Magidi, I. C., Balazs, A., Lundkvist, A. S., Marshall, S. J., Marshall, G. W., Siekhaus, W. J., & Kinney, J. H. (1998). Viscoelastic properties of demineralized human
dentin measured in water with atomic force microscope (AFM)-based indentation. Journal of Biomedical Materials Research, 40, 539–544.
Bannister, L., & Mitchell, G. (2003). The ins, outs and roundabouts of malaria. Trends in Parasitology, 19, 209–213.
Bao, G., & Suresh, S. (2003). Cell and molecular mechanics of biological materials. Nature Materials, 2, 715–725.
Barbee, K. A., Davies, P. F., & Lal, R. (1994). Shear stress-induced reorganization of the surface-topography of living endothelial-cells imaged by atomic-force microscopy.
Circulation Research, 74, 163–171.
Bastatas, L., Martinez-Marin, D., Matthews, J., Hashem, J., Lee, Y. J., Sennoune, S., Filleur, S., Martinez-Zaguilan, R., & Park, S. (2012). AFM nano-mechanics and calcium
dynamics of prostate cancer cells with distinct metastatic potential. Biochimica et Biophysica Acta-General Subjects, 1820, 1111–1120.
Benitez, R., & Toca-Herrera, J. L. (2014). Looking at cell mechanics with atomic force microscopy: Experiment and theory. Microscopy Research and Technique, 77, 947–958.
Berdyyeva, T. K., Woodworth, C. D., & Sokolov, I. (2005). Human epithelial cells increase their rigidity with ageing in vitro: Direct measurements. Physics in Medicine and Biology,
50, 81–92.
Bhushan, B. (2010). Springer handbook of nanotechnology. Heidelberg: Springer Science and Business Media.
Binnig, G., Quate, C. F., & Gerber, C. (1986). Atomic force microscope. Physical Review Letters, 56, 930.
Boal, D., & Boal, D. H. (2012). Mechanics of the cell. Cambridge: Cambridge University Press.
Borisy, G. G., & Taylor, E. W. (1967). The mechanism of action of colchicine. The Journal of Cell Biology, 34, 525–533.
Boudreau, N., & Bissell, M. J. (1998). Extracellular matrix signaling: Integration of form and function in normal and malignant cells. Current Opinion in Cell Biology, 10, 640–646.
Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., & Wisse, E. (2001). A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells. Journal
of Electron Microscopy, 50, 283–290.
Briscoe, B. J., Fiori, L., & Pelillo, E. (1998). Nano-indentation of polymeric surfaces. Journal of Physics D: Applied Physics, 31, 2395.
Bulychev, S. I., Alekhin, V. P., Shorshorov, M. K., Ternovskij, A. P., & Shnyrev, G. D. (1975). Determination of young modulus by the hardness indentation diagram. Zavodskaya
Laboratoriya, 41, 1137–1140.
Bushell, G. R., Cahill, C., Clarke, F. M., Gibson, C. T., Myhra, S., & Watson, G. S. (1999). Imaging and force-distance analysis of human fibroblasts in vitro by atomic force
microscopy. Cytometry, 36, 254–264.
Cai, X., Xing, X., Cai, J., Chen, Q., Wu, S., & Huang, F. (2010). Connection between biomechanics and cytoskeleton structure of lymphocyte and Jurkat cells: An AFM study. Micron,
41, 257–262.
Canetta, E., Riches, A., Borger, E., Herrington, S., Dholakia, K., & Adya, A. K. (2014). Discrimination of bladder cancer cells from normal urothelial cells with high specificity and
sensitivity: Combined application of atomic force microscopy and modulated Raman spectroscopy. Acta Biomaterialia, 10, 2043–2055.
Carrillo, F., Gupta, S., Balooch, M., Marshall, S. J., Marshall, G. W., Pruitt, L., & Puttlitz, C. M. (2005). Nanoindentation of polydimethylsiloxane elastomers: Effect of crosslinking,
work of adhesion, and fluid environment on elastic modulus. Journal of Materials Research, 20, 2820–2830.
Carrillo, F., Gupta, S., Balooch, M., Marshall, S. J., Marshall, G. W., Pruitt, L., & Puttlitz, C. M. (2006). Erratum: “Nanoindentation of polydimethylsiloxane elastomers: Effect of
crosslinking, work of adhesion, and fluid environment on elastic modulus” [J. Mater. Res. 20, 2820 (2005)]. Journal of Materials Research, 21, 535–537.
Cecelja, M., & Chowienczyk, P. (2009). Dissociation of aortic pulse wave velocity with risk factors for cardiovascular disease other than hypertension: A systematic review.
Hypertension, 54, 1328–1336.
Chaffey, N., Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2003). Molecular biology of the cell (4th edn.). Annals of Botany, 91(3), 401.
Chaturvedi, R. R., Herron, T., Simmons, R., Shore, D., Kumar, P., Sethia, B., Chua, F., Vassiliadis, E., & Kentish, J. C. (2010). Passive stiffness of myocardium from congenital heart
disease and implications for diastole. Circulation, 121, 979–988.
Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., & Ingber, D. E. (1997). Geometric control of cell life and death. Science, 276, 1425.
Chen, J., Birch, M. A., & Bull, S. J. (2010). Nanomechanical characterization of tissue engineered bone grown on titanium alloy in vitro. Journal of Materials Science: Materials in
Medicine, 21, 277–282.
Cheng, Y.-T., & Cheng, C.-M. (2004). Scaling, dimensional analysis, and indentation measurements. Materials Science & Engineering R: Reports, 44, 91–149.
Coceano, G., Yousafzai, M. S., Ma, W., Ndoye, F., Venturelli, L., Hussain, I., Bonin, S., Niemela, J., Scoles, G., Cojoc, D., & Ferrari, E. (2016). Investigation into local cell mechanics
by atomic force microscopy mapping and optical tweezer vertical indentation. Nanotechnology, 27, 065102.
Collinsworth, A. M., Zhang, S., Kraus, W. E., & Truskey, G. A. (2002). Apparent elastic modulus and hysteresis of skeletal muscle cells throughout differentiation. American Journal
of Physiology-Cell Physiology, 283, C1219–C1227.
Cooke, B. M., Mohandas, N., & Coppel, R. L. (2001). The malaria-infected red blood cell: Structural and functional changes. In Advances in parasitology (pp. 1–86). Cambridge, MA:
Academic Press.
Cross, S. E., Jin, Y.-S., Tondre, J., Wong, R., Rao, J., & Gimzewski, J. K. (2008). AFM-based analysis of human metastatic cancer cells. Nanotechnology, 19, 384003.
DeLoach, S. S., & Townsend, R. R. (2008). Vascular stiffness: Its measurement and significance for epidemiologic and outcome studies. Clinical Journal of the American Society of
Nephrology, 3, 184–192.
Deng, L. H., Trepat, X., Butler, J. P., Millet, E., Morgan, K. G., Weitz, D. A., & Fredberg, J. J. (2006). Fast and slow dynamics of the cytoskeleton. Nature Materials, 5,
636–640.
Derjaguin, B. V., Muller, V. M., & Toporov, Y. P. (1975). Effect of contact deformations on the adhesion of particles. Journal of Colloid and Interface Science, 53, 314–326.
Domke, J., Dannohl, S., Parak, W. J., Muller, O., Aicher, W. K., & Radmacher, M. (2000). Substrate dependent differences in morphology and elasticity of living osteoblasts
investigated by atomic force microscopy. Colloids and Surfaces. B, Biointerfaces, 19, 367–379.
Duan, P., & Chen, J. (2015). Nanomechanical and microstructure analysis of extracellular matrix layer of immortalized cell line Y201 from human mesenchymal stem cells. Surface
and Coatings Technology, 284, 417–421.
Dulinska, I., Targosz, M., Strojny, W., Lekka, M., Czuba, P., Balwierz, W., & Szymonski, M. (2006). Stiffness of normal and pathological erythrocytes studied by means of atomic
force microscopy. Journal of Biochemical and Biophysical Methods, 66, 1–11.
Ebenstein, D. M., & Pruitt, L. A. (2004). Nanoindentation of soft hydrated materials for application to vascular tissues. Journal of Biomedical Materials Research. Part A, 69,
222–232.
Ebenstein, D. M., & Wahl, K. J. (2006). A comparison of JKR-based methods to analyze quasi-static and dynamic indentation force curves. Journal of Colloid and Interface Science,
298, 652–662.
Ebenstein, D. M., Kuo, A., Rodrigo, J. J., Reddi, A. H., Ries, M., & Pruitt, L. (2004). A nanoindentation technique for functional evaluation of cartilage repair tissue. Journal of
Ebenstein, D. M., Coughlin, D., Chapman, J., Li, C., & Pruitt, L. A. (2009). Nanomechanical properties of calcification, fibrous tissue, and hematoma from atherosclerotic plaques.
Journal of Biomedical Materials Research. Part A, 91, 1028–1037.
Efremov, Y. M., Dokrunova, A. A., Efremenko, A. V., Kirpichnikov, M. P., Shaitan, K. V., & Sokolova, O. S. (2015). Distinct impact of targeted actin cytoskeleton reorganization on
mechanical properties of normal and malignant cells. Biochimica et Biophysica Acta-Molecular Cell Research, 1853, 3117–3125.
Engler, A. J., Sweeney, H. L., Discher, D. E., & Schwarzbauer, J. E. (2007). Extracellular matrix elasticity directs stem cell differentiation. Journal of Musculoskeletal & Neuronal
Interactions, 7, 335.
Engler, A. J., Carag-Krieger, C., Johnson, C. P., Raab, M., Tang, H. Y., Speicher, D. W., Sanger, J. W., Sanger, J. M., & Discher, D. E. (2008). Embryonic cardiomyocytes beat best
on a matrix with heart-like elasticity: Scar-like rigidity inhibits beating. Journal of Cell Science, 121, 3794–3802.
Evans, E., & Yeung, A. (1989). Apparent Viscosity and Cortical Tension of Blood Granulocytes Determined by Micropipet Aspiration. Biophysical Journal, 56, 151–160.
Fabry, B., Maksym, G. N., Butler, J. P., Glogauer, M., Navajas, D., & Fredberg, J. J. (2001). Scaling the microrheology of living cells. Physical Review Letters, 87(14), 148102.
Faria, E. C., Ma, N., Gazi, E., Gardner, P., Brown, M., Clarke, N. W., & Snooka, R. D. (2008). Measurement of elastic properties of prostate cancer cells using AFM. Analyst, 133,
1498–1500.
Fedosov, D. A., Lei, H., Caswell, B., Suresh, S., & Karniadakis, G. E. (2011). Multiscale modeling of red blood cell mechanics and blood flow in malaria. PLoS Computational
Biology, 7, e1002270.
Feng, G., & Ngan, A. H. W. (2002). Effects of creep and thermal drift on modulus measurement using depth-sensing indentation. Journal of Materials Research, 17, 660–668.
Fischer-Cripps, A. C. (2006). Critical review of analysis and interpretation of nanoindentation test data. Surface and Coatings Technology, 200, 4153–4165.
Fischer-Cripps, A. C. (2011). Nanoindentation testing. In Nanoindentation (pp. 21–37). Berlin/Heidelberg, Germany: Springer.
Fu, J., Wang, Y.-K., Yang, M. T., Desai, R. A., Yu, X., Liu, Z., & Chen, C. S. (2010). Mechanical regulation of cell function with geometrically modulated elastomeric substrates.
Nature Methods, 7, 733–736.
Fung, Y. C., & Liu, S. Q. (1993). Elementary mechanics of the endothelium of blood vessels. Journal of Biomechanical Engineering, 115, 1–12.
Fung, C. K. M., Xi, N., Yang, R. G., Seiffert-Sinha, K., Lai, K. W. C., & Sinha, A. A. (2011). Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy
(AFM). IEEE Transactions on Nanobioscience, 10, 9–15.
Georges, P. C., Miller, W. J., Meaney, D. F., Sawyer, E. S., & Janmey, P. A. (2006). Matrices with compliance comparable to that of brain tissue select neuronal over glial growth in
mixed cortical cultures. Biophysical Journal, 90(8), 3012.
Georges, P. C., Hui, J. J., Gombos, Z., McCormick, M. E., Wang, A. Y., Uemura, M., Mick, R., Janmey, P. A., Furth, E. E., & Wells, R. G. (2007). Increased stiffness of the rat liver
precedes matrix deposition: Implications for fibrosis. American Journal of Physiology. Gastrointestinal and Liver Physiology, 293, G1147–54.
Gerberich, W. W., Yu, W., Kramer, D., Strojny, A., Bahr, D., Lilleodden, E., & Nelson, J. (1998). Elastic loading and elastoplastic unloading from nanometer level indentations for
modulus determinations. Journal of Materials Research, 13, 421–439.
Gossett, D. R., Tse, H. T. K., Lee, S. A., Ying, Y., Lindgren, A. G., Yang, O. O., Rao, J. Y., Clark, A. T., & Di Carlo, D. (2012). Hydrodynamic stretching of single cells for large
population mechanical phenotyping. Proceedings of the National Academy of Sciences of the United States of America, 109, 7630–7635.
Greenwood, J. A., & Johnson, K. L. (2006). Oscillatory loading of a viscoelastic adhesive contact. Journal of Colloid and Interface Science, 296, 284–291.
Gross, L., Mohn, F., Moll, N., Liljeroth, P., & Meyer, G. (2009). The chemical structure of a molecule resolved by atomic force microscopy. Science, 325, 1110–1114.
Guilak, F., & Mow, V. C. (2000). The mechanical environment of the chondrocyte: A biphasic finite element model of cell-matrix interactions in articular cartilage. Journal of
Biomechanics, 33, 1663–1673.
Guo, Q., Park, S., & Ma, H. S. (2012). Microfluidic micropipette aspiration for measuring the deformability of single cells. Lab on a Chip, 12, 2687–2695.
Habelitz, S., S. J. Marshall, G. W. Marshall Jr & M. Balooch (2001) Mechanical properties of human dental enamel on the nanometre scale. Archives of Oral Biology, 46, 173–183.
Haga, H., Sasaki, S., Kawabata, K., Ito, E., Ushiki, T., & Sambongi, T. (2000). Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of the cyto-
skeleton. Ultramicroscopy, 82, 253–258.
Harris, A. R., Bellis, J., Khalilgharibi, N., Wyatt, T., Baum, B., Kabla, A. J., & Charras, G. T. (2013). Generating suspended cell monolayers for mechanobiological studies. Nature
Protocols, 8, 2516–2530.
Hayashi, K., & Iwata, M. (2015). Stiffness of cancer cells measured with an AFM indentation method. Journal of the Mechanical Behavior of Biomedical Materials, 49, 105–111.
Henon, S., Lenormand, G., Richert, A., & Gallet, F. (1999). A new determination of the shear modulus of the human erythrocyte membrane using optical tweezers. Biophysical
Journal, 76, 1145–1151.
Hertz, H. (1881). On the contact of elastic solids. Journal fur die Reine und Angewandte Mathematik, 92, 110.
Heu, C., Berquand, A., Elie-Caille, C., & Nicod, L. (2012). Glyphosate-induced stiffening of HaCaT keratinocytes, a peak force tapping study on living cells. Journal of Structural
Biology, 178, 1–7.
Hofmann, U. G., Rotsch, C., Parak, W. J., & Radmacher, M. (1997). Investigating the cytoskeleton of chicken cardiocytes with the atomic force microscope. Journal of Structural
Biology, 119, 84–91.
Huang, S., & Ingber, D. E. (1999). The structural and mechanical complexity of cell-growth control. Nature Cell Biology, 1, E131–E138.
Imazio, M., Brucato, A., Ferrazzi, P., Pullara, A., Adler, Y., Barosi, A., Caforio, A. L., Cemin, R., Chirillo, F., & Comoglio, C. (2014). Colchicine for prevention of postpericardiotomy
syndrome and postoperative atrial fibrillation: The COPPS-2 randomized clinical trial. JAMA, 312, 1016–1023.
Johnson, K. L. (1985). Contact mechanics (p. 452). New York: Cambridge University.
Johnson, K., Kendall, K., & Roberts, A. (1971). Surface energy and the contact of elastic solids. In Proceedings of the Royal Society of London A: Mathematical, Physical and
Engineering Sciences (pp. 301–313). London, UK: The Royal Society.
Katsumi, A., Milanini, J., Kiosses, W. B., Del Pozo, M. A., Kaunas, R., Chien, S., Hahn, K. M., & Schwartz, M. A. (2002). Effects of cell tension on the small GTPase Rac. The Journal
of Cell Biology, 158, 153–164.
Kaufman, J. D., & Klapperich, C. M. (2009). Surface detection errors cause overestimation of the modulus in nanoindentation on soft materials. Journal of the Mechanical Behavior
of Biomedical Materials, 2, 312–317.
Ketene, A. N., Schmelz, E. M., Roberts, P. C., & Agah, M. (2012). The effects of cancer progression on the viscoelasticity of ovarian cell cytoskeleton structures. Nanomedicine:
Nanotechnology, Biology and Medicine, 8, 93–102.
Khanna, R., Katti, K. S., & Katti, D. R. (2011). Experiments in nanomechanical properties of live osteoblast cells and cell–biomaterial interface. Journal of Nanotechnology in
Engineering and Medicine, 2, 041005.
Khanna, R., Katti, D. R., & Katti, K. S. (2012). AFM and nanoindentation studies of bone nodules on chitosan-polygalacturonic acid-hydroxyapatite nanocomposites. Computer
Modeling in Engineering and Sciences, 87, 530–555.
Klein, E. A., Yin, L., Kothapalli, D., Castagnino, P., Byfield, F. J., Xu, T., Levental, I., Hawthorne, E., Janmey, P. A., & Assoian, R. K. (2009). Cell-cycle control by physiological matrix
elasticity and in vivo tissue stiffening. Current Biology, 19, 1511–1518.
Krishnan, R., Park, C. Y., Lin, Y. C., Mead, J., Jaspers, R. T., Trepat, X., Lenormand, G., Tambe, D., Smolensky, A. V., Knoll, A. H., Butler, J. P., & Fredberg, J. J. (2009).
Reinforcement versus fluidization in cytoskeletal mechanoresponsiveness. PLoS One, 4(5), e5486.
Kumar, S., & Weaver, V. M. (2009). Mechanics, malignancy, and metastasis: The force journey of a tumor cell. Cancer Metastasis Reviews, 28, 113–127.
Kuznetsova, T. G., Starodubtseva, M. N., Yegorenkov, N. I., Chizhik, S. A., & Zhdanov, R. I. (2007). Atomic force microscopy probing of cell elasticity. Micron, 38, 824–833.
Lacolley, P., Challande, P., Osborne-Pellegrin, M., & Regnault, V. (2009). Genetics and pathophysiology of arterial stiffness. Cardiovascular Research, 81, 637–648.
Lapshin, R. V. (1995). Analytical model for the approximation of hysteresis loop and its application to the scanning tunneling microscope. Review of Scientific Instruments, 66,
4718–4730.
Lapshin, R. V. (1998). Automatic lateral calibration of tunneling microscope scanners. Review of Scientific Instruments, 69, 3268–3276.
Lapshin, R. V. (2004). Feature-oriented scanning methodology for probe microscopy and nanotechnology. Nanotechnology, 15, 1135–1151.
Larsson, P. L., & Carlsson, S. (1998). On microindentation of viscoelastic polymers. Polymer Testing, 17, 49–75.
Laurent, V. M., Kasas, S., Yersin, A., Schaffer, T. E., Catsicas, S., Dietler, G., Verkhovsky, A. B., & Meister, J. J. (2005). Gradient of rigidity in the lamellipodia of migrating cells
revealed by atomic force microscopy. Biophysical Journal, 89, 667–675.
Lee, M. H., Wu, P. H., Staunton, J. R., Ros, R., Longmore, G. D., & Wirtz, D. (2012). Mismatch in mechanical and adhesive properties induces pulsating cancer cell migration in
epithelial monolayer. Biophysical Journal, 102, 2731–2741.
Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., & Chen, C. S. (2010). Measurement of mechanical tractions exerted by cells in three-dimensional matrices.
Nature Methods, 7, 969–971.
Lekka, M. (2016). Discrimination between normal and cancerous cells using AFM. Bionanoscience, 6, 65–80.
Lekka, M., Laidler, P., Gil, D., Lekki, J., Stachura, Z., & Hrynkiewicz, A. Z. (1999). Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy.
European Biophysics Journal, 28, 312–316.
Lekka, M., Laidler, P., Ignacak, J., Labedz, M., Lekki, J., Struszczyk, H., Stachura, Z., & Hrynkiewicz, A. Z. (2001). The effect of chitosan on stiffness and glycolytic activity of human
bladder cells. Biochimica et Biophysica Acta-Molecular Cell Research, 1540, 127–136.
Lekka, M., Gil, D., Pogoda, K., Dulinska-Litewka, J., Jach, R., Gostek, J., Klymenko, O., Prauzner-Bechcicki, S., Stachura, Z., & Wiltowska-Zuber, J. (2012). Cancer cell detection in
tissue sections using AFM. Archives of Biochemistry and Biophysics, 518, 151–156.
Levental, I., Georges, P. C., & Janmey, P. A. (2007). Soft biological materials and their impact on cell function. Soft Matter, 3, 299–306.
Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, S. F. T., Csiszar, K., Giaccia, A., & Weninger, W. (2009). Matrix crosslinking forces tumor progression
by enhancing integrin signaling. Cell, 139, 891–906.
Li, Z., Dranoff, J. A., Chan, E. P., Uemura, M., Sévigny, J., & Wells, R. G. (2007). Transforming growth factor-b and substrate stiffness regulate portal fibroblast activation in culture.
Hepatology, 46, 1246–1256.
Li, Q. S., Lee, G. Y. H., Ong, C. N., & Lim, C. T. (2008). AFM indentation study of breast cancer cells. Biochemical and Biophysical Research Communications, 374, 609–613.
Li, M., Liu, L. Q., Xi, N., Wang, Y. C., Dong, Z. L., Xiao, X. B., & Zhang, W. J. (2012). Atomic force microscopy imaging and mechanical properties measurement of red blood cells
and aggressive cancer cells. Science China. Life Sciences, 55, 968–973.
Lim, C. T., Dao, M., Suresh, S., Sow, C. H., & Chew, K. T. (2004). Large deformation of living cells using laser traps. Acta Materialia, 52, 1837–1845.
Loubet, J. L., Oliver, W. C., & Lucas, B. N. (2000). Measurement of the loss tangent of low-density polyethylene with a nanoindentation technique. Journal of Materials Research, 15,
1195–1198.
Love, A. E. H. (1939). Boussinesq’s problem for a rigid cone. The Quarterly Journal of Mathematics, 10, 161–175.
Lu, H., Wang, B., Ma, J., Huang, G., & Viswanathan, H. (2003). Measurement of creep compliance of solid polymers by nanoindentation. Mechanics of Time Dependent Materials, 7,
189–207.
Lu, H., Koo, L. Y., Wang, W. C. M., Lauffenburger, D. A., Griffith, L. G., & Jensen, K. F. (2004). Microfluidic shear devices for quantitative analysis of cell adhesion. Analytical
Chemistry, 76, 5257–5264.
Lu, Y.-B., Franze, K., Seifert, G., Steinhäuser, C., Kirchhoff, F., Wolburg, H., Guck, J., Janmey, P., Wei, E.-Q., & Käs, J. (2006). Viscoelastic properties of individual glial cells and
neurons in the CNS. Proceedings of the National Academy of Sciences, 103, 17759–17764.
Lulevich, V., Yang, H. Y., Isseroff, R. R., & Liu, G. Y. (2010). Single cell mechanics of keratinocyte cells. Ultramicroscopy, 110, 1435–1442.
Lundkvist, A., Lilleodden, E., Siekhaus, W., Kinney, J., Pruitt, L., & Balooch, M. (1997). Viscoelastic properties of healthy human artery measured in saline solution by AFM-based
indentation technique (p. 353). Cambridge: Cambridge University Press.
Mathur, A. B., Truskey, G. A., & Reichert, W. M. (2000). Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells.
Biophysical Journal, 78, 1725–1735.
Mathur, A. B., Collinsworth, A. M., Reichert, W. M., Kraus, W. E., & Truskey, G. A. (2001). Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic
properties as determined by atomic force microscopy. Journal of Biomechanics, 34, 1545–1553.
Maugis, D. (2013). Contact, adhesion and rupture of elastic solids. Berlin/Heidelberg, Germany: Springer Science and Business Media.
McDaniel, D. P., Shaw, G. A., Elliott, J. T., Bhadriraju, K., Meuse, C., Chung, K. H., & Plant, A. L. (2007). The stiffness of collagen fibrils influences vascular smooth muscle cell
phenotype. Biophysical Journal, 92, 1759–1769.
Miller, L. H., Baruch, D. I., Marsh, K., & Doumbo, O. K. (2002). The pathogenic basis of malaria. Nature, 415, 673–679.
Mitchell, G. F., Hwang, S.-J., Vasan, R. S., Larson, M. G., Pencina, M. J., Hamburg, N. M., Vita, J. A., Levy, D., & Benjamin, E. J. (2010). Arterial stiffness and cardiovascular
events. Circulation, 121, 505–511.
Miyamoto, S., Miyamoto, Y., Shibata, Y., Yoshimura, K., Izumida, E., Suzuki, H., Miyazaki, T., Maki, K., & Kamijo, R. (2015). In situ quasi-static and dynamic nanoindentation tests on
calcified nodules formed by osteoblasts: Implication of glucocorticoids responsible for osteoblast calcification. Acta Biomaterialia, 12, 216–226.
Miyazaki, H., & Hayashi, K. (1999). Atomic force microscopic measurement of the mechanical properties of intact endothelial cells in fresh arteries. Medical and Biological
Engineering and Computing, 37, 530–536.
Mozhanova, A., Nurgazizov, N., & Bukharaev, A. (2003). In In: Local elastic properties of biological materials studied by SFMProc. of the SPM-2003, March, 2–5.
Munevar, S., Wang, Y.-L., & Dembo, M. (2001). Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. Biophysical Journal, 80, 1744–1757.
Nagayama, M., Haga, H., & Kawabata, K. (2001). Drastic change of local stiffness distribution correlating to cell migration in living fibroblasts. Cell Motility and the Cytoskeleton, 50,
173–179.
Nagayama, K., Nagano, Y., Sato, M., & Matsumoto, T. (2006). Effect of actin filament distribution on tensile properties of smooth muscle cells obtained from rat thoracic aortas.
Journal of Biomechanics, 39, 293–301.
Nematbakhsh, Y., & Lim, C. T. (2015). Cell biomechanics and its applications in human disease diagnosis. Acta Mechanica Sinica, 31, 268–273.
Ngan, A. H. W., Wang, H. T., Tang, B., & Sze, K. Y. (2005). Correcting power-law viscoelastic effects in elastic modulus measurement using depth-sensing indentation. International
Journal of Solids and Structures, 42, 1831–1846.
Nikkhah, M., Strobl, J. S., De Vita, R., & Agah, M. (2010). The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon
microstructures. Biomaterials, 31, 4552–4561.
Odegard, G. M., Gates, T. S., & Herring, H. M. (2005). Characterization of viscoelastic properties of polymeric materials through nanoindentation. Experimental Mechanics, 45,
130–136.
Oliver, W. C., & Pharr, G. M. (1992). An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of
Oyen, M. L. (2005). Spherical indentation creep following ramp loading. Journal of Materials Research, 20, 2094–2100.
Oyen, M. L. (2013). Nanoindentation of biological and biomimetic materials. Experimental Techniques, 37, 73–87.
Oyen, M. L., & Cook, R. F. (2003). Load–displacement behavior during sharp indentation of viscous–elastic–plastic materials. Journal of Materials Research, 18, 139–150.
Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., Reinhart-King, C. A., Margulies, S. S., Dembo, M., & Boettiger, D. (2005). Tensional homeostasis
and the malignant phenotype. Cancer Cell, 8, 241–254.
Pelling, A. E., Dawson, D. W., Carreon, D. M., Christiansen, J. J., Shen, R. R., Teitell, M. A., & Gimzewski, J. K. (2007). Distinct contributions of microtubule subtypes to cell
membrane shape and stability. Nanomedicine: Nanotechnology, Biology, and Medicine, 3, 43–52.
Pesen, D., & Hoh, J. H. (2005). Micromechanical architecture of the endothelial cell cortex. Biophysical Journal, 88, 670–679.
Pharr, G. M., Oliver, W. C., & Brotzen, F. R. (1992). On the generality of the relationship among contact stiffness, contact area, and elastic modulus during indentation. Journal of
Prabhune, M., Belge, G., Dotzauer, A., Bullerdiek, J., & Radmacher, M. (2012). Comparison of mechanical properties of normal and malignant thyroid cells. Micron, 43,
1267–1272.
Putman, C. A., van der Werf, K. O., de Grooth, B. G., van Hulst, N. F., Greve, J., & Hansma, P. K. (1992). New imaging mode in atomic-force microscopy based on the error signal.
In OE/LASE’92, 198–204. Los Angeles, CA: International Society for Optics and Photonics.
Radmacher, M., Fritz, M., Kacher, C. M., Cleveland, J. P., & Hansma, P. K. (1996). Measuring the viscoelastic properties of human platelets with the atomic force microscope.
Rho, J. Y., Roy, M. E., Tsui, T. Y., & Pharr, G. M. (1999). Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. Journal of
Rico, F., Roca-Cusachs, P., Gavara, N., Farre, R., Rotger, M., & Navajas, D. (2005). Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal
cantilever tips. Physical Review E, 72, 021914.
Rosenbluth, M. J., Lam, W. A., & Fletcher, D. A. (2006). Force microscopy of nonadherent cells: A comparison of leukemia cell deformability. Biophysical Journal, 90, 2994–3003.
Rotsch, C., & Radmacher, M. (2000). Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: An atomic force microscopy study. Biophysical Journal, 78,
520–535.
Rotsch, C., Braet, F., Wisse, E., & Radmacher, M. (1997). AFM imaging and elasticity measurements on living rat liver macrophages. Cell Biology International, 21, 685–696.
Sato, M., Levesque, M. J., & Nerem, R. M. (1987). Micropipette aspiration of cultured bovine aortic endothelial cells exposed to shear stress. Arteriosclerosis, Thrombosis, and
Vascular Biology, 7, 276–286.
Sato, M., Theret, D. P., Wheeler, L. T., Ohshima, N., & Nerem, R. M. (1990). Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell
viscoelastic properties. Journal of Biomechanical Engineering, 112, 263–268.
Sato, M., Ohshima, N., & Nerem, R. M. (1996). Viscoelastic properties of cultured porcine aortic endothelial cells exposed to shear stress. Journal of Biomechanics, 29, 461–467.
Sato, M., Nagayama, K., Kataoka, N., Sasaki, M., & Hane, K. (2000). Local mechanical properties measured by atomic force microscopy for cultured bovine endothelial cells exposed
to shear stress. Journal of Biomechanics, 33, 127–135.
Sato, H., Kataoka, N., Kajiya, F., Katano, M., Takigawa, T., & Masuda, T. (2004). Kinetic study on the elastic change of vascular endothelial cells on collagen matrices by atomic
force microscopy. Colloids and Surfaces. B, Biointerfaces, 34, 141–146.
Schwartz, M. A., & Ginsberg, M. H. (2002). Networks and crosstalk: Integrin signalling spreads. Nature Cell Biology, 4, E65–E68.
Sharpe, W. N. (2008). Springer handbook of experimental solid mechanics. Springer Science and Business Media.
Shroff, S. G., Saner, D. R., & Lal, R. (1995). Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy. American Journal of
Physiology-Cell Physiology, 269, C286–C292.
Simon, A., Cohen-Bouhacina, T., Porte, M. C., Aime, J. P., Amedee, J., Bareille, R., & Baquey, C. (2003). Characterization of dynamic cellular adhesion of osteoblasts using atomic
force microscopy. Cytometry. Part A, 54A, 36–47.
Sneddon, I. N. (1965). The relation between load and penetration in the axisymmetric Boussinesq problem for a punch of arbitrary profile. International Journal of Engineering
Science, 3, 47–57.
Spedden, E., White, J. D., Naumova, E. N., Kaplan, D. L., & Staii, C. (2012). Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy.
Stamenovic, D., & Ingber, D. E. (2002). Models of cytoskeletal mechanics of adherent cells. Biomechanics and Modeling in Mechanobiology, 1, 95–108.
Stamenovic, D., Fredberg, J. J., Wang, N., Butler, J. P., & Ingber, D. E. (1996). A microstructural approach to cytoskeletal mechanics based on tensegrity. Journal of Theoretical
Biology, 181, 125–136.
Steward, R. L., Cheng, C. M., Ye, J. D., Bellin, R. M., & LeDuc, P. R. (2011). Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts.
Scientific Reports, 1, 147.
Stolz, M., Raiteri, R., Daniels, A. U., VanLandingham, M. R., Baschong, W., & Aebi, U. (2004). Dynamic elastic modulus of porcine articular cartilage determined at two different
levels of tissue organization by indentation-type atomic force microscopy. Biophysical Journal, 86, 3269–3283.
Suresh, S. (2007a). Biomechanics and biophysics of cancer cells. Acta Materialia, 55, 3989–4014.
Suresh, S. (2007b). NanomedicinedElastic clues in cancer detection. Nature Nanotechnology, 2, 748–749.
Suresh, S., Spatz, J., Mills, J. P., Micoulet, A., Dao, M., Lim, C. T., Beil, M., & Seufferlein, T. (2005). Connections between single-cell biomechanics and human disease states:
Gastrointestinal cancer and malaria. Acta Biomaterialia, 1, 15–30.
Suresh, S., Spatz, J., Mills, J. P., Micoulet, A., Dao, M., Lim, C. T., Beil, M., & Seufferlein, T. (2015). Reprint of: connections between single-cell biomechanics and human disease
states: Gastrointestinal cancer and malaria. Acta Biomaterialia, 23, S3–S15.
Suwanarusk, R., Cooke, B. M., Dondorp, A. M., Silamut, K., Sattabongkot, J., White, N. J., & Udomsangpetch, R. (2004). The deformability of red blood cells parasitized by
plasmodium falciparum and P. Vivax. The Journal of Infectious Diseases, 189, 190–194.
Swaminathan, V., Mythreye, K., O’Brien, E. T., Berchuck, A., Blobe, G. C., & Superfine, R. (2011). Mechanical stiffness grades metastatic potential in patient tumor cells and in
cancer cell lines. Cancer Research, 71, 5075–5080.
Tandon, R., Levental, I., Huang, C., Byfield, F. J., Ziembicki, J., Schelling, J. R., Bruggeman, L. A., Sedor, J. R., Janmey, P. A., & Miller, R. T. (2007). HIV infection changes
glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties. American Journal of Physiology. Renal Physiology, 292, F701–F710.
Tang, B., & Ngan, A. H. W. (2003). Accurate measurement of tipdSample contact size during nanoindentation of viscoelastic materials. Journal of Materials Research, 18,
1141–1148.
Thoumine, O., Cardoso, O., & Meister, J. J. (1999). Changes in the mechanical properties of fibroblasts during spreading: A micromanipulation study. European Biophysics Journal
with Biophysics Letters, 28, 222–234.
Tsai, M. A., Waugh, R. E., & Keng, P. C. (1998). Passive mechanical behavior of human neutrophils: Effects of colchicine and paclitaxel. Biophysical Journal, 74, 3282–3291.
VanLandingham, M. R. (2003). Review of instrumented indentation. Journal of Research of the National Institute of Standards and Technology, 108, 249.
Vanlandingham, M. R., McKnight, S. H., Palmese, G. R., Eduljee, R. F., Gillespie, J. W., & McCulough, R. L. (1997). Relating elastic modulus to indentation response using atomic
force microscopy. Journal of Materials Science Letters, 16, 117–119.
Velegol, S. B., Pardi, S., Li, X., Velegol, D., & Logan, B. E. (2003). AFM imaging artifacts due to bacterial cell height and AFM tip geometry. Langmuir, 19, 851–857.
Wahl, K. J., Asif, S. A. S., Greenwood, J. A., & Johnson, K. L. (2006). Oscillating adhesive contacts between micron-scale tips and compliant polymers. Journal of Colloid and
Interface Science, 296, 178–188.
Wakatsuki, T., Schwab, B., Thompson, N. C., & Elson, E. L. (2001). Effects of cytochalasin D and latrunculin B on mechanical properties of cells. Journal of Cell Science, 114,
1025–1036.
Wells, R. G. (2008). The role of matrix stiffness in regulating cell behavior. Hepatology, 47, 1394–1400.
Worthen, G. S., Schwab, B., 3rd, Elson, E. L., & Downey, G. P. (1989). Mechanics of stimulated neutrophils: Cell stiffening induces retention in capillaries. Science, 245, 183–186.
Wu, H. W., Kuhn, T., & Moy, V. T. (1998). Mechanical properties of l929 cells measured by atomic force microscopy: Effects of anticytoskeletal drugs and membrane crosslinking.
Scanning, 20, 389–397.
Wyss, H. M., Henderson, J. M., Byfield, F. J., Bruggeman, L. A., Ding, Y., Huang, C., Suh, J. H., Franke, T., Mele, E., Pollak, M. R., Miner, J. H., Janmey, P. A., Weitz, D. A., &
Miller, R. T. (2011). Biophysical properties of normal and diseased renal glomeruli. American Journal of Physiology. Cell Physiology, 300, C397–405.
Xu, W. W., Mezencev, R., Kim, B., Wang, L. J., McDonald, J., & Sulchek, T. (2012). Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells. PLoS One, 7,
e46609.
Yallapu, M. M., Katti, K. S., Katti, D. R., Mishra, S. R., Khan, S., Jaggi, M., & Chauhan, S. C. (2015). The roles of cellular nanomechanics in cancer. Medicinal Research Reviews, 35,
198–223.
Yamane, Y., Shiga, H., Haga, H., Kawabata, K., Abe, K., & Ito, E. (2000). Quantitative analyses of topography and elasticity of living and fixed astrocytes. Journal of Electron
Microscopy, 49, 463–471.
Yang, Y.-T., Lin, C.-C. K., Liao, J.-D., Chang, C.-W., & Ju, M.-S. (2010). Continuous depth-sensing nano-mechanical characterization of living, fixed and dehydrated cells attached
on a glass substrate. Nanotechnology, 21, 285704.
Yoshikawa, Y., Yasuike, T., Yagi, A., & Yamada, T. (1999). Transverse elasticity of myofibrils of rabbit skeletal muscle studied by atomic force microscopy. Biochemical and
Biophysical Research Communications, 256, 13–19.
Zahalak, G. I., McConnaughey, W. B., & Elson, E. L. (1990). Determination of cellular mechanical properties by cell poking, with an application to leukocytes. Journal of
Biomechanical Engineering, 112, 283–294.
Zhao, X. Q., Zhong, Y. X., Ye, T., Wang, D. J., & Mao, B. W. (2015). Discrimination between cervical cancer cells and normal cervical cells based on longitudinal elasticity using
atomic force microscopy. Nanoscale Research Letters, 10, 1–8.
Zhu, C., Bao, G., & Wang, N. (2000). Cell mechanics: Mechanical response, cell adhesion, and molecular deformation. Annual Review of Biomedical Engineering, 2, 189–226.
Bone Micro- and Nanomechanics
Caitlyn J Collins, Orestis G Andriotis, Vedran Nedelkovski, and Martin Frank, TU Wien, Vienna, Austria
Orestis L Katsamenis, University of Southampton, Southampton, United Kingdom
Philipp J Thurner, TU Wien, Vienna, Austria
Bone Structure and Hierarchy 23

Trabecular Bone 24
Mechanical Testing of Trabecular Bone at the Tissue-Level 24
Nanoindentation 25
Micromechanical tests 25
Scanning acoustic microscopy (SAM) 27
Effects of Age and Disease on Micromechanical Properties of Trabecular Bone 27
Cortical Bone 29
Mechanical Testing of Cortical Bone at the Tissue and Osteonal Levels 29
Reference point indentation (RPI) 30
Effects of Age and Disease on the Micromechanical Properties of Cortical Bone 31
Individual Lamellae, Interlamellar Areas, and Cement Lines 31
Mechanical Testing of Cortical Bone at the Lamellar Length Scale 32
Fracture toughness 32
Collagen Fibrils and Nanocrystals: Individual Components of Bone 35
Collagens and Collagen Fibrils 35
Collagen Fibril Structure 35
Collagen Fibril Hydration and Mechanics 36
Mineralized Collagen Fibrils (MCFs) 37
Mineralization of Collagen Fibrils 37
Mechanics of Mineralized Collagen Fibrils (MCFs) 37
Noncollagenous Proteins 38
References 39
Further Reading 44
Glossary
Carbon-substituted hydroxyapatite Calcium phosphate mineral crystals that make up most of the inorganic part of bone.
Collagen Protein family that makes up most of the organic part of bone.
Collagen fibril Sub-mm diameter fibers that are formed from certain collagen types and are one basic building block of bone
and most other skeletal tissues.
Cortical/compact bone A type of bone tissue consisting of closely packed osteons and forms the dense, stiff exterior of bones.
Fracture toughness Measure of susceptibility of a material to resist fracture.
Hierarchical structure Exhibiting structural elements on more than one length scale.
Lamellae A thin layer of tissue made up of a network of mineralized collagen fibers.
Osteon Cylindrical structures made up of concentrically arranged lamellae surrounding individual Haversian canals.
Trabecular/cancellous bone A highly porous type of bone tissue that pervades the interior or cavity of bones with a lattice of
mineralized struts and plates (trabeculae) and intervening spaces filled with marrow or fat.
Transverse isotropic A material with the same physical properties in one plane (e.g., x–y plane) and different physical
properties in the direction normal to this plane (e.g., z-axis). These materials can be described by five independent elastic
constants.
Nomenclature
εkl A dimensionless component of the second-order strain tensor, representing the strain in the l-direction acting on a surface
that is oriented perpendicular to the k-direction
Da Crack extension, measured in mm

Biomechanics j Bone Micro- and Nanomechanics 23
sij A component of the second-order stress tensor, representing the stress in the j-direction acting on a surface that is oriented
perpendicular to the i-direction with dimensions of force per unit area
AGEs Advanced glycation end products, that is, proteins or lipids that become glycated as a result of exposure to sugars)
Cijkl The fourth-order stiffness tensor that is a property of the material and is often dependent on temperature, pressure, and
microstructure. Cijkl is given in units of force per unit area.
FE Finite element
IDI Indentation distance increase, measured in mm
K Stress intensity factor, measured in units of stress * length1/2
Kc Critical stress intensity factor, measured in units of stress * length1/2
NCPs Noncollagenous proteins
NEG Nonenzymatic glycation
OPM Oliver–Pharr method
qBEI Quantitative backscattered electron imaging
R-curves Crack resistance curves generated from plots of fracture energy versus crack length
RPI Reference point indentation
SAM Scanning acoustic microscopy
SEM Scanning electric microscopy
TID Total indentation distance, measured in mm
Bone Structure and Hierarchy
When considering bone, the first structures that come to mind are likely whole bones. At this macroscale or organ level, bones have
complex shapes, which cater to the needs of the human body and its musculoskeletal system, that is, shielding internal organs,
acting as lever arms for load transfer, providing structural stability, and enabling movement and locomotion via muscle attachment
sites. Whole bones also serve as mineral reservoirs and are responsible for the majority of blood cell formation. While bone tissue at
the macroscale generally appears white in color and is considered to be structurally stiff, it is unlike any engineering material. In fact,
bones are complicated composite materials with a distinct hierarchical organization; at the lowest organizational level, bone tissue
consists of collagen in the form of fibrils, carbon-substituted hydroxyapatite mineral in the form of nanocrystals as well as water and
a small amount of noncollagenous proteins (NCPs). A more detailed description of these basic building blocks can be found toward
the end of this article as we will start our description at the macroscale. Here, bone tissue can be examined using a low power light
microscope. Sequentially increasing the microscope magnification and changing imaging modalities, as required, reveals the whole
structural hierarchy of bone. This hierarchy has been well described in the works of Weiner and Wagner as well as Rho et al. (Weiner
and Wagner, 1998; Rho et al., 1998). In fact, seven different structural levels can be isolated (Fig. 1). Earlier works by Katz et al. and
Lakes, focused on the hierarchical organization of cortical bone, differentiated between four and five structural levels (Katz et al.,
1984; Lakes, 1993). Due to this complex structural organization, micro- and nanomechanics of bone are of particular interest as
they characterize specific levels within the hierarchy.
Starting at the macroscale, the bone structural hierarchy begins with the whole bone level, with individual bones being classified
as flat, short, long (femur or tibia) or irregular (vertebrae) (Standring, 2016). Venturing to the next smaller length scale, for example,
cutting open a long bone, the two major types of bone tissue are encountered, namely cortical (or compact) bone and trabecular (or
cancellous) bone (tissue level). These two bone tissue types mainly differ in apparent density as a result of porosity. Cortical bone
has low porosity (several %), while trabecular bone is highly porous and consists of small, sub-mm size plates and rods. Below the
length scale of these two bone tissue types, further structure is observed. The classification of these structures is dependent on which
type of bone tissue is investigated. Osteonal bone is encountered within cortical bone (in humans). So-called osteons are cylindrical
features with a central Haversian canal oriented parallel to the main loading direction, that is, along the long bone axis in long
bones. Typical osteon diameters range from 100 to 400 mm. The equivalent structural feature in trabecular bone at this length scale
is the so-called trabecular bone packet. Both features originate due to the remodeling process in bone and are interfaced into the
surrounding bone via a so-called cement line. Both the bone packet and the osteon consist of bone lamellae with typical thicknesses
of 5–10 mm. At this structural level in osteons, lamellae are arranged in a concentric fashion around the Haversian canal with inter-
spersed boundary layers, so-called interlamellar areas (sometimes also called thin lamellae). Within individual lamellae, mineral-
ized collagen fibrils, the basic building block of bone, are organized into aligned bundles or arrays. Orientation of these fibril arrays
varies across individual lamellae, and there are several models which describe the fibril arrangement within the lamellae of osteons:
the rotated plywood, the twisted plywood model, or spiral twisting model (Giraud-Guille et al., 2003; Wagermaier et al., 2006).
More recently, three-dimensional reconstruction of human lamellar bone has revealed the presence of two distinct inter-
penetrating materials, which contribute to the hierarchical organization at this length scale (Reznikov et al., 2014). Occasionally,
24 Biomechanics j Bone Micro- and Nanomechanics
Fig. 1 Hierarchical structural organization of bone from macro- to sub-nanostructure: whole bone divided into cortical and trabecular (cancellous)
bone; osteons with Haversian systems; lamellae; collagen fiber assemblies of collagen fibrils; individual collagen fibrils; and bone mineral crystals,
collagen molecules, and noncollagenous proteins. Adapted from Rho, J.Y., Kuhn-Spearing, L., and Zioupos, P. (1998). Mechanical properties and the
hierarchical structure of bone. Medical, Engineering and Physics 20, 92–102., Copyright (2017) Elsevier, and Thurner, P.J. (2009). Atomic force
microscopy and indentation force measurement of bone. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 1, 624–649. https://doi.
org/10.1002/wnan.56., Copyright (2017) Wiley.
a local arrangement or order of several fibrils may be called a fiber. As mentioned, fibril arrays consist of a network of mineralized
collagen fibrils. In their pristine form, these fibrils have diameters between 100 and 300 nm. The fibrils themselves consist primarily
of type I collagen, mineral crystals, and water. The mineral crystals are located in the gap regions within the fibrils (intrafibrillar
mineral) (Knapp et al., 2002; Sasaki et al., 2002) as well as decorating the entire fibril surface (extrafibrillar mineral) (Lees et al.,
1984; Prostak and Lees, 1996; Kindt et al., 2007). The attachment of minerals to the collagen is most likely facilitated via NCPs.
The interface between adjacent mineralized fibrils is also thought to consist of NCPs (Thurner, 2009). Here, NCPs have been found
to play both a structural role in terms of controlling crystal size and shape as well as a mechanical role (see Noncollagenous
proteins).
The main body of this article will address the measurement or investigation of mechanical properties of bone at different length
scales ranging from the sub-mm size to the individual components (e.g., NCPs). Evaluation of bone at each length scale is of interest
as potential changes in whole bone tissue mechanics could arise from changes at any hierarchical level.
Trabecular Bone
Trabecular bone is a highly porous variant of bone tissue and is mainly present in the terminal regions of long bones and in the
middle regions of short, flat, and irregular bones such as vertebrae. Trabecular bone is composed of a complex network of intercon-
nected rods and plates, called trabeculae (Lucchinetti et al., 2000). On the microscopic scale, trabeculae are built up of trabecular
bone packets (Jee, 2001), with an average wall thickness of around 50 mm (Lips et al., 1978). These trabecular bone packets consist
of layers of lamellae oriented in slightly different directions, and the packets are separated by cement lines and interstitial lamellae
(Jee, 2001). Individual lamellae consist of mineralized collagen fibrils that are preferentially aligned with the long axis of each
lamella. Within the lamellae and packets, ellipsoid cavities, called lacunae, typically 5–15 mm in cross-section and 25 mm in length,
house osteocytes (Hamed et al., 2012). These cells, which have a higher activity level in trabecular than cortical bone, sense defor-
mation and damage of the bone matrix and trigger bone remodeling and adaptation processes (Oftadeh et al., 2015).
At the component level, trabecular and cortical bone consists mainly of carbon-substituted hydroxyapatite, collagen, and water.
However, trabecular bone has a higher water content (vol. 27%) compared to cortical bone (vol. 23%) (Oftadeh et al., 2015; Gong
et al., 1964). The relative composition of the main bone tissue constituents is highly dependent on age, anatomical site, gender, and
potential pathology (Boskey, 2013). Since water is a major constituent, it may play a significant role in the overall mechanical
behavior of bone. Dehydration has been shown to cause changes in structure as well as mechanical properties of trabecular
bone (Lievers et al., 2010). Further, dehydration results in a transition from ductile buckling to a brittle behavior in compression
loading of individual bone trabeculae (Townsend et al., 2017).
Mechanical Testing of Trabecular Bone at the Tissue-Level

The porous structure of trabecular bone makes the determination of tissue-level mechanical or material properties difficult. At the
trabecular bone tissue-level, measurements at the length scale of individual trabeculae or smaller are of interest. Tissue-level
properties of trabecular bone can be determined in three different ways: with direct measurement via micromechanical tests or
nanoindentation, with scanning acoustic microscopy (SAM), or with indirect assessment from large scale finite element (FE)
models. To remain within the scope of this article, indirect assessment methods are not reviewed.
Nanoindentation
Nanoindentation of bone is usually performed in force-controlled mode (Thurner, 2009). In principle, a tip with defined geometry
is driven into the sample at a specified loading rate until a certain maximum load is reached. Then, this maximum load is usually
held constant for some time (on the order of tens of seconds) to wait for any transient processes to cede. Subsequently, the sample is
unloaded. The elastic modulus and the hardness of the sample are then calculated from the unloading curve using the Oliver–Pharr
method (OPM) (Oliver and Pharr, 1992, 2004), which assumes elastic behavior during the first part of the unloading process. This
method can be seen as the gold standard for data analysis of nanoindentation measurements and is based on several assumptions,
as reviewed in Thurner (2009). While some assumptions are not met for bone, for example, isotropy, the OPM is still commonly
used to extract material properties of cortical and trabecular bone at the microscale. However, the majority of studies reported in the
literature use a variety of test protocols making it difficult to cross compare quantitative results. The elastic modulus of trabecular
bone, determined over the course of several studies, was reported to vary from 6.9 to 23.5 GPa (Thurner, 2009). Comparatively, the
elastic modulus of human cortical bone, excised from the femur midshaft, was reported to vary between 17 and 27 GPa, depending
on the type of lamellae (Thurner, 2009; Zysset et al., 1999; Rho et al., 1999). As yet, no clear distinction has been drawn between the
mechanical properties of cortical and trabecular bone measured via nanoindentation; however, some studies have reported differ-
ences in hardness (Hodgskinson et al., 1989; Weaver, 1966). Exhaustive reviews of bone nanoindentation results can be found in
Thurner (2009) and Lewis and Nyman (2008).
Perhaps the largest limitation of this technique is that nanoindentation is typically conducted on dried samples. Sample holders
and indentation tip assemblies that would allow testing in a liquid environment do exist; however, testing under these conditions
generally renders the operator blind for the choice of indentation location.
Elastic modulus has been shown to be correlated to the mineral content of (calcified) cartilage (Gupta et al., 2005); however,
similar correlations within bone of a single species or across an anatomical location are not clearly present (Spiesz et al., 2013).
This lack of clear correlation between elastic modulus and mineral content is most likely due to the fact that bone is not only hetero-
geneous but also heterogeneously anisotropic. As an exception to this observation, tissue stiffness and mineralization were found to
be highly correlated across transverse sections of individual trabeculae (see Fig. 2). Both variables were found to be higher within the
core compared to the outer surface (Mulder et al., 2007). The distribution of mineralization between the core and the surface of
individual trabeculae stems from the remodeling process in trabecular bone. Remodeling initiates at the surface of the trabeculae
leading to a distinct difference between shell and core regions. In addition to heterogeneity over the trabeculae cross-section, a signif-
icant difference has been reported between the transverse and the longitudinal elastic modulus within individual trabeculae (Rho
et al., 1999).
For the purpose of nanoindentation measurements, bone tissue is often assumed to be isotropic with a Poisson’s ratio of 0.3
(Zysset, 2009). In a study using nanoindentation to evaluate the elastic properties of cortical and trabecular bone lamellae, varying
the Poisson’s ratio from 0.2 to 0.4 was found to only generate relative errors between þ 9.9% to 8.2% (Zysset et al., 1999).
However, bone is inherently anisotropic, and the indentation modulus for anisotropic materials is a function of the axis of inden-
tation and the full elasticity tensor (Zysset, 2009). As such, measurements carried out under the assumption of an isotropic material
model either over- or underestimate the “true” elastic modulus, that is, the elastic constants of the stiffness matrix from the gener-
alized Hooke’s Law (Swadener et al., 2001). Stiffness and hardness have also been shown to be significantly dependent on lamellar
type, anatomical site, and the individual (Zysset et al., 1999). Such dependence might be explained by local differences in the bone
mineral density distribution (Roschger et al., 2008) in addition to differences in mineralization and collagen organization. A sepa-
rate study reported only a weak correlation between the mean degree of mineralization and the indentation modulus and hardness
(Hengsberger et al., 2002). However, the authors mention that other factors such as hypermineralization or a reduction in collagen
cross-links might contribute to the behavior of the extracellular matrix.
The literature is in disagreement with regards to the correlation of nanoindentation results with macroscale mechanical tests.
Hengsberger et al. reported good agreement between nanoindentation and macroscopic tests (Hengsberger et al., 2003), whereas
Silva et al. reported no significant correlation between nanoindentation and three-point-bending tests of macroscopic samples
(Silva et al., 2004).
Micromechanical tests
Due to the aforementioned limitations of nanoindentation (generally dry samples, information only on elastic modulus and hard-
ness), the determination of mechanical or material properties from micromechanical tests of individual trabeculae is still a matter of
ongoing research. However, this faces several challenges, as reviewed by Lucchinetti et al. (2000). Most studies have been conducted
on rodlike trabeculae focusing on the elastic region and only a few studies have investigated postyield behavior (Hodgskinson et al.,
1989; Weaver, 1966; Lewis and Nyman, 2008). Based on the generalized Hooke’s law, the elastic properties of trabecular bone tissue
might be described by sij ¼ Cijklekl, where Cijkl is a fourth-order tensor (the stiffness tensor) that relates stress (s) to strain (ε). This
law is only valid up to the proportionality limit, that is, the point after which stress is no longer proportional to strain, and ceases to
apply past the elastic limit of a material. As such, the generalized Hooke’s law should only be applied in cases where small forces or
displacements are applied. Within this tensor context, trabecular bone is assumed to behave as an orthotropic material, that is, nine
Fig. 2 (A) The tissue stiffness profile from nanoindentation and (B) the degree of mineralization profile across transverse sections of individual
trabeculae from a new born pig. Measurement areas are indicated in black in both images on the right, taken using light microscopy (top) and micro-
computed tomography (mCT) (bottom). Tissue stiffness and degree of mineralization increase toward the central section of the trabeculae. Note that
the two outermost indents are located in the embedding medium. (C) Image of a trabecular cross-section captured using quantitative backscattered
electron imaging (qBEI); tissue lamellae, canaliculi, and the spatial distribution of mineralization across the cross-section can be seen in the image,
where higher brightness indicates higher mineral concentration. (A and B) Reprinted with permission from Mulder, L., Koolstra, J.H., den Toonder,
J.M.J., and van Eijden, T.M.G.J. (2007). Intratrabecular distribution of tissue stiffness and mineralization in developing trabecular bone. Bone 41,
256–265. https://doi.org/10.1016/j.bone.2007.04.188. Copyright (2017) Elsevier. (C) Adapted from Brennan, M.A., Gleeson, J.P., Browne, M.,
O’Brien, F.J., Thurner, P.J., and McNamara, L.M. (2011). Site specific increase in heterogeneity of trabecular bone tissue mineral during estrogen
deficiency. European Cells & Materials 21, 396–406.
independent components fully describe the material behavior. Since single trabeculae tend to have a preferred lamellar orientation
along the longitudinal trabecular axis, they can be considered as transverse isotropic (Lucchinetti et al., 2000). Trabeculae are
primarily subjected to bending, tensile and compressive loads. Thus, the elastic constant/modulus in the longitudinal direction
might be sufficient to describe the elastic behavior. Several approaches have been used to determine the tissue-level elastic modulus
via micromechanical testing, these include: buckling (Townsend et al., 2017; Runkle and Pugh, 1975), three-point bending
(Carretta et al., 2013a, b; Szabó et al., 2011; Busse et al., 2009; Kuhn et al., 1989; Jungmann et al., 2011; Ridha and Thurner,
2013; Szabó and Thurner, 2013), four-point bending (Choi and Goldstein, 1992) and tensile tests (Carretta et al., 2013a, b;
Rho et al., 1993; Bini et al., 2002; Hernandez et al., 2005; Jirousek et al., 2011; Ryan and Williams, 1989; Yamada et al., 2014).
Tissue-level elastic moduli range from 0.8 GPa (Ryan and Williams, 1989) up to 16.2 GPa (Carretta et al., 2013b). Traditionally,
this large variation is attributed to the use of different test setups and their associated boundary conditions. For example, Carretta
et al. tested trabeculae in tension as well as three-point bending and reported significantly higher values for the elastic modulus of
the trabeculae subjected to tensile loading (Carretta et al., 2013a). However, by definition, elasticity is a measure of the energy,
which can be fully transformed into efficient mechanical work without dissipation, and must be independent of both strain distri-
butions and strain rates (Rajagopal and Srinivasa, 2009). Hence, the elastic modulus, which describes material elasticity, should not
differ between experiments. There is a discussion in recent literature on whether bone is an elastic material or if it exhibits elasto-
plastic hardening, thereby necessitating loading–unloading experiments to measure the elastic modulus (Schwiedrzik et al., 2014;
Luczynski et al., 2015).
Most of the studies utilizing micromechanical tests to evaluate individual trabeculae treated them as simple rods with a constant
elliptical cross-section. Carretta et al. argued that true tissue material parameters can only be obtained when combining an exper-
imental approach with FE analysis (Carretta et al., 2013a). Indeed, Frank et al. showed that the curvature of individual trabeculae
Fig. 3 The determined tissue stress–strain curves of trabeculae tested in uniaxial tension, based on an elliptical cross-section (light blue). The bold
line represents the mean tissue stress–strain curve, calculated using the formulas shown in the graph and the mean values of each parameter. The
red crosses and circles illustrate the point of yield and the point of failure, respectively. Reprinted with permission from Frank, M., Marx, D., Pahr,
D.H., Thurner, P.J. (2017) Mechanical properties of individual trabeculae in a physiological environment. Proc. IASTED Int. Conf. Biomed. Eng. (BioMed
2017). https://doi.org/10.2316/P.2017.852-023. Copyright (2017) IEEE.
results in a structural influence on the tissue-level elastic modulus, but an upper and lower boundary can be estimated by simple
geometric assumptions (Frank et al., 2017).
Determination of yield and postyield properties of trabecular bone has been mainly carried out on larger samples (Chang et al.,
1999; Turner, 1989; Kopperdahl and Keaveny, 1998), thereby involving a structural component. Only a few studies have deter-
mined the yield properties of trabecular bone at the tissue-level (Carretta et al., 2013a, b; Frank et al., 2017; Busse et al., 2009;
Hernandez et al., 2005). Carretta et al. reported on the postyield behavior of bovine (Carretta et al., 2013a) and human (Carretta
et al., 2013b) trabeculae under both tension and three-point bending test conditions. In both studies, yield strain, ultimate strain,
and postyield work were reported to be significantly higher in the three-point bending group. Busse et al. subjected osteoporotic and
skeletally intact trabeculae to three-point bending test (Busse et al., 2009); significant differences in yield strength, ultimate stress,
and work to failure were found, highlighting the impact of disease on the postyield behavior of bone. Hernandez et al. demon-
strated that ultimate tensile strain was weakly influenced by nonenzymatic glycation (NEG), also called advanced glycation end
products (AGEs) of type I collagen, a factor associated with aging and diabetes (Hernandez et al., 2005). In this study, individual
trabeculae were hydrated in physiologic salt solution (pH ¼ 7.4) prior to tension testing. The average ultimate tensile strain was
reported to be 8.8% (Hernandez et al., 2005). Carretta et al., in contrast, reported an ultimate tensile strain of 5.1% for individual
trabeculae (Hodgskinson et al., 1989; Weaver, 1966); however, trabeculae in these studies were prepared and tested in a dry envi-
ronment. Frank et al. performed tensile tests on individual trabeculae in a hydrated environment (physiologic salt solution,
pH ¼ 7.4) and reported an average ultimate strain of 9.8% (Frank et al., 2017), thus confirming that hydration leads to significant
postyield deformation (as shown in Fig. 3).
Scanning acoustic microscopy (SAM)

SAM enables nondestructive determination of the elastic mechanical properties of relatively small, stiff samples, such as bone at the
tissue level (Laugier et al., 2013). SAM can be used to determine changes in the longitudinal wave velocity within bone in response
to aging or disease, for example, in individuals before and after menopause (Hasegawa et al., 1995). Bumrerraj and Katz used SAM
to determine the correlation between acoustic microscope brightness and the elastic modulus of known materials. This correlation
was then used to predict the elastic modulus of bone samples subjected to SAM (Bumrerraj and Katz, 2001). The predicted elastic
modulus values were found to have a good correlation with elastic modulus values of cortical and trabecular bone measured using
nanoindentation testing. Turner et al. also used acoustic microscopy and nanoindentation to determine the elastic modulus of
trabecular and cortical bone tissue (Turner et al., 1999). The measured elastic modulus for trabecular bone was reported as
17.5 1.1 GPa using acoustic microscopy and as 18.1 1.7 GPa using nanoindentaion, both were slightly higher than the
measured elastic moduli of cortical bone in the transverse direction (14.9 0.5 and 16.6 0.32, respectively) (Turner et al.,
1999). In a new approach, Litniewski measured the velocity of surface waves to determine elastic modulus. Such an approach
enables evaluation of samples that are only accessible from one side (Litniewski, 2005). A good agreement was found between
the reported elastic modulus and that of a previous study (Turner et al., 1999).
Effects of Age and Disease on Micromechanical Properties of Trabecular Bone

With respect to age and disease, the influence of strain rate on tissue level material properties is of particular interest. Most exper-
iments are carried out at low strain rates in a monotonic fashion. However, the physiological loading experienced by patients is
either cyclical (fatigue) or occurs at high strain rates (falls and fracture). Therefore, a discussion of the micromechanics of aging
trabecular bone under these conditions is important. Unfortunately, very few studies exist that shine light on these questions and, as
such, more research is required to expand the current knowledge and understanding of trabecular bone behavior. Whether the
mechanical properties of individual trabeculae are in fact affected by strain rate is not clear as several conflicting studies exist (Szabó
et al., 2011; Hansen et al., 2008; Ferreira et al., 2006; Currey, 1975). Upon aging, the collagen phase within bone (the main organic
component) can suffer from increased cross-linking via sugars (e.g., present due to diet or disease), known as AGEs. This results in
a tissue which is structurally very different from that generated by the naturally occurring enzymatic cross-linking process. Two
studies that have investigated the effects of AGEs and are briefly discussed below.
Hernandez et al. determined that there was only a weak correlation between AGEs and ductility in individual trabeculae,
although ductility varied tremendously in their study (Hernandez et al., 2005). Ductility was characterized by the strain at
maximum load-carrying capacity and was found to range between roughly 3% and 18%. In a separate study, Tang et al. deter-
mined that AGEs cause stiffness loss and a reduction in damage accumulation within individual trabeculae, thus resulting in
a loss in the ability to dissipate energy (Tang et al., 2007). These studies suggest that the accumulation of sugars and the resultant
cross-linking, that is, AGEs within collagen, are a potential issue, especially in individuals with a high sugar diet, obesity, or
diabetes.
Osteoporosis, one of the most prominent bone diseases, is associated with a significant increase in the incidence of bone fracture
and may be paired with a significant loss of bone mass. One line of thought considers that the properties of bone at the tissue level
might be altered over the course of this disease. In this context, individual trabeculae excised from osteoporotic bone have been
reported to have a lower ultimate strain and postyield work under tensile testing conditions when compared to trabeculae excised
from healthy donors (Carretta et al., 2013b). However, no significant difference was found between the elastic behavior of either
osteoporotic or healthy donors. Similarly, studies using nanoindentation reported no significant difference between osteoporotic
and healthy trabecular bone (Wang et al., 2008; Hu et al., 2015). Wang et al. reported no significant difference between the elastic
properties of trabecular bone measured from patients that had experienced vertebral osteoporotic fracture and those of a control
group (Wang et al., 2008). Hu et al. reported that the elastic modulus and hardness of trabecular bone tissue were unaffected by
an ovariectomy in a murine animal model (rat) (Hu et al., 2015).
Studies investigating effects of bisphosphonate treatment on the microstructure and mechanical strength of bone have reported
a reduced capacity to resist fracture in the treated bone compared to untreated bone (Ma et al., 2017; Acevedo et al., 2015). Although
bisphosphonates have been shown to impede the loss of bone density in osteoporosis patients, the effect of long-term bisphosph-
onate treatment on preyield (elastic) properties is still subject to debate.
Microscopic damage is also thought to be an important determinant of bone fragility (Seref-Ferlengez et al., 2015; Fazzalari et al.,
1998). Two different types of microscopic damage have been identified: linear microcracks, which may run parallel or cross-hatch to
one another, and diffuse damage (Fig. 4). Linear microcracks are sharply defined cracks of roughly 50–100 mm in length that
primarily occur within interstitial bone (Seref-Ferlengez et al., 2015). Diffuse damage is characterized by an accumulation of short
submicroscopic cracks and is preferentially induced inside trabecular bone packets (Vashishth et al., 2000). Interestingly, the pres-
ence of microcracks in trabecular bone of the femoral head have been reported to be significantly higher in old bone from both
healthy and fractured cohorts compared to young bone (Mori et al., 1997). The impact of age and disease on the type of damage
mechanisms found in trabecular bone is a matter of ongoing and future research.
Fig. 4 Micrograph of basic fuchsin stained trabeculae highlighting the morphological differences between microcracks and diffuse damage. The
arrows identify microcracks in linear (left) and cross-hatch (center) configurations as well as diffuse damage with no discernible microcracks (right).
Reprinted with permission from Fazzalari, N.L., Forwood, M.R., Smith, K., Manthey, B.A., Herreen, P. (1998). Assessment of cancellous bone quality
in severe osteoarthrosis: Bone mineral density, mechanics, and microdamage, Bone 22, 381–388. https://doi.org/10.1016/S8756-3282(97)00298-6.
Copyright (2017) Elsevier.
Cortical Bone
Cortical bone is the denser variant of bone tissue and makes up the shells and shafts of long bones as well as the external shells of
short, flat, and irregular bones. The internal microstructure of cortical bone is organized into concentrically arranged cylindrical
structures called osteons (or Haversian systems). This functional unit of human cortical bone is comprised of a central so-called
Haversian canal surrounded by concentric rings of lamellae roughly 5–7 mm in thickness (Gupta et al., 2006a). Haversian canals
house both blood vessels and nerve cells and interconnect via Volkmann’s canals that run transverse to the bone diaphysis. The
average osteon diameter is reported to range from 50 to 500 mm in diameter (Black et al., 1974). Interstitial lamellae, the remnants
of osteons partially resorbed during the remodeling process, occupy the space between individual osteons, that is, interstitial bone.
Osteons and interstitial lamellae are separated by cement lines, zones primarily made up of calcified mucopolysaccharides, lacking
collagen fibrils (Burr et al., 1988), and rich in NCPs (Sodek et al., 2000). Although the degree of cement line mineralization has been
historically controversial, recent work supports the conclusion that cement lines are not poorly mineralized when compared to
surrounding bone (Skedros et al., 2005). Within cortical bone lamellae, osteocyte housing lacunae and canaliculi are found. Canal-
iculi, small channels roughly 0.5 mm in diameter, provide paths for the long dendritic extensions of osteocytes to interconnect with
cells in adjacent lacunae as well as to cells within the Haversian canal.
The spatial organization of osteonal lamellae remains a topic of debate. Under polarized light microscopy, lamellae appear
either light or dark. Models attempting to explain the observed differences in adjacent lamellae can be grouped into either fibril
orientation or fibril density models. In the first, orientation of the mineralized collagen fibril differentiates adjacent layers. In
the second, the collagen is assumed to be homogeneously distributed in adjacent layers and only the relative collagen fibril density
is responsible for the observed differences. A comprehensive review of the existing models of lamellae organization can be found in
Mitchell and van Heteren (2016).
Mechanical Testing of Cortical Bone at the Tissue and Osteonal Levels

At the tissue level, measurements at the length scale of individual osteon or smaller are of interest. A primary argument to attempt
such measurements is that osteons are in fact the product of the bone remodeling process. Therefore, changes in bone mechanics
due to age and disease will be most pronounced at this level. Sub-millimeter specimens of cortical bone allow researchers to isolate
and focus on the desired microstructural features, for example, individual osteons and interstitial lamellae, and access their mechan-
ical behavior under well-controlled conditions. Tissue-level properties of cortical bone can be determined with direct measurement
via micromechanical tests, nanoindentation, or reference point indentation (RPI). Imaging modalities are commonly combined
with mechanical testing protocols to aid in the assessment of postyield and fracture behavior, particularly when evaluating mech-
anisms for energy dissipation at smaller length scales. This section will focus on micromechanical tests and RPI. For detailed reviews
on nanoindentation refer to Thurner (2009) and Zysset (2009).
Rho et al. used microtensile experiments to measure the elastic modulus of cortical bone obtained from the diaphyseal region of
a human tibia as well as individual trabeculae (Rho et al., 1993). The tensile specimens had approximate dimensions of an indi-
vidual trabecula and the measured elastic modulus (18.6 3.5 GPa) was larger than that of single trabeculae (10.4 3.5 GPa). This
study was one of the first to evaluate mineral density versus elastic modulus relationship in cortical and trabecular bone. By mini-
aturizing the cortical specimens to sizes similar to single trabeculae, the authors effectively demonstrated that cortical bone and
trabecular bone have differing material properties.
Ever since, a number of studies have used similar approaches to measure the elastic modulus of miniaturized cortical bone spec-
imens (Reilly and Burstein, 1974, 1975). Other micromechanical testing setups have included cantilever beam bending, three- or
four-point bending, torsion, and compression. The elastic modulus of cortical bone varies depending on the testing method, level of
hydration, porosity, anatomical location as well as orientation of the tissue sample (Currey, 2002). Dry cortical bone was found to
have a 20% higher elastic modulus than hydrated cortical bone (Currey, 2002). Compression tests also revealed that the elastic
modulus of human cortical bone is higher in the longitudinal direction (16–23 GPa) compared to transverse (6–13 GPa) (Rho
et al., 1998). This anisotropy mirrors the primary physiological loading direction.
As early as the 1970s, researchers have sought to understand the mechanical properties of single osteons. The pioneering studies
of Ascenzi and Bonucci which evaluated the tensile (Ascenzi and Bonucci, 1967), compressive (Ascenzi and Bonucci, 1968),
shearing (Ascenzi and Bonucci, 1972), bending (Ascenzi et al., 1990), and torsional (Ascenzi et al., 1994) properties of individual
osteons paved the way for a whole new era of quantitative assessment of bone at and below the microscale. Using basic tools, such
as stereoscopes, microscopes, drills, surgical blades, and impressive manual handling skills, they were able to isolate individual
osteons from bulk cortical bone and subject them to controlled mechanical tests using custom-made apparatuses (Fig. 5). Elastic
moduli of single osteons were reported to range from 2.6 to 11.7 in tension (Ascenzi and Bonucci, 1967), 1.6–9.3 GPa in compres-
sion (Ascenzi and Bonucci, 1968), and 0.9–2.7 GPa in three-point bending (Ascenzi et al., 1990). All moduli were found to be
sensitive to changes in mineral content and the predominant collagen fibril orientation in neighboring lamellae. Osteons at an
initial stage of calcification exhibited greater ductility and reduced stiffness compared to more mature osteons. Further, osteons
with primarily longitudinal collagen fiber alignment were able to support greater stresses in tension and torsion, whereas osteons
with alternating collagen fiber alignment in adjacent lamellae were able to support greater stresses in compression. Notably, the
Fig. 5 (A) Images of (a) an osteon prepared for three-point bending; (b) an “alternate” osteon, with fiber bundles changing orientation in succes-
sive lamellae though an angle of 90 degree, at ultimate bending strength; (c) a “longitudinal” osteon, with longitudinally aligned fiber bundles in adja-
cent lamellae, at ultimate bending strength; and microradiographs of fractured (a) “longitudinal,” (e) “alternate,” and (d) irregular “alternate” osteon
samples. (B) Polarized light microscopy images of (a) an isolated osteon ready to be tested in tension (50 magnification); (b) a “longitudinal”
osteon (100 magnification); and (c) an “alternate” osteon (100 magnification). (C) From left to right: isolated osteon sample; isolated osteon
sample as seen in transmitted light; an end surface of the sample. (A) Reprinted with permission from Ascenzi, A., Baschieri, P., and Benvenuti, A.
(1990). The bending properties of single osteons. Journal of Biomechanics 23, 763–771. https://doi.org/10.1016/0021-9290(90)90023-V. Copyright
(2017) Elsevier. (B) Reprinted with permission from Ascenzi, A. and Bonucci, E. (1967). The tensile properties of single osteons. The Anatomical
Record 158, 375–386. https://doi.org/10.1002/ar.1091580403. Copyright (2017) John Wiley and Sons. (C) Reprinted with permission from Ascenzi,
A. and Bonucci, E. (1968). The compressive properties of single osteons. The Anatomical Record 161, 377–391. Copyright (2017) John Wiley and
Sons.
values reported by Ascenzi and Bonucci are well below elastic modulus values from nanoindentation or other micromechanical
tests. Since there have been no studies reproducing these experiments, it remains unclear why this is the case.
Reference point indentation (RPI)

As noted previously, indentation techniques at various length scales have been used to measure hardness and stiffness of both
trabecular and cortical bone (Thurner, 2009; Zysset, 2009). Given the search for complementary diagnosis of osteoporosis and
age-related bone fracture risk, microindentation has been established as a micromechanical test that can even be conducted in
patients (Diez-Perez et al., 2010). Microindentation via Reference Point Indentation (RPI), which does not require miniaturized
specimens, utilizes a similar approach as nanoindentation (described above), driving an indenter into the bone tissue; although
this technique utilizes larger forces and penetration distances, it can still be considered as a micromechanical technique. Clinically
oriented RPI evaluates the resistance of bone to indenter penetration. Currently, two approaches are used. One is cyclic microinden-
tation, where the indenter makes repeated loading and unloading cycles (Hansma et al., 2006). This approach is predominantly
used in preclinical studies. The other is single load microindentation, where the indenter impacts into bone with a given force
(Bridges et al., 2012). This approach is predominantly used in clinical studies. Both approaches are implemented in RPI devices.
RPI typically uses either a test probe with a spherical tip contained within a hypodermic needle (cyclic RPI) (Fig. 6), or a single probe
and a preload (single load RPI) to establish a reference point. Once a reference point has been established the indentation process
begins. In case of a single load cycle (clinical device), the indentation distance is compared to a PMMA reference material giving the
“bone material strength index” (BMSi). For multiple loading cycles (laboratory device), the indentation distance increase (IDI) and
the total indentation distance (TID) are measured. IDI is the indentation distance between the first and last cycle and TID is the
indentation depth of the last indentation relative to the initial reference point. Since RPI causes microscopic damage to open
and may relate to the separation of mineralized collagen (Diez-Perez et al., 2010), IDI and TID parameters have been assumed
Fig. 6 Reference Point Indentation. (A) Force–distance curves for first and last indentation cycle. (B) Diagram of indentation probe at the first and
last cycle. Adapted from Diez-Perez, A., Güerri, R., Nogues, X., Cáceres, E., Peña, M.J., Mellibovsky, L., Randall, C., Bridges, D., Weaver, J.C.,
Proctor, A., Brimer, D., Koester, K.J., Ritchie, R.O., Hansma, P.K. (2010). Microindentation for in vivo measurement of bone tissue mechanical prop-
erties in humans. Journal of Bone and Mineral Research 25, 1877–1885. https://doi.org/10.1002/jbmr.73. Copyright (2017) John Wiley and Sons.
to be related to fracture toughness. However, the measured parameters in both approaches have been found to have little to no
correlation with a single material property. Instead, they likely relate to multiple material and structural properties of bone such
as fracture mechanics, elastoplastic behavior, and structural properties (porosity and pore proximity) of bone (Jenkins et al.,
2017). While RPI has been used in a number of preclinical and clinical studies (Diez-Perez et al., 2010; Güerri-Fernández et al.,
2013; Poundarik et al., 2015; Sundh et al., 2016; Uppuganti et al., 2017; Rozental et al., 2017; Abraham et al., 2015; Jenkins
et al., 2016), whether or not such approaches can indeed improve diagnosis of bone fracture risk in individuals remains to be shown
(Allen et al., 2015).
Effects of Age and Disease on the Micromechanical Properties of Cortical Bone

Age, anatomical location, sex, and pathology impact the morphology of cortical bone at the macro- and microscale. Cortical thin-
ning and an increase in porosity are associated with aging and various pathologies (Zebaze et al., 2005; Granke et al., 2016; Mirzaali
et al., 2015). Human osteons exhibit distinctive morphological heterogeneity depending on the age of the individual, skeletal site,
and the presence or absence of both local and systemic factors, that is, hormones, cytokines, chemokines, etc. (Ascenzi, 2012). With
age, osteon cross-sectional area and diameter have been shown to decrease (Currey, 2002; Bernhard et al., 2013) while cortical bone
osteon density has been shown to increase (Currey, 2002). One of the important functions of an osteon is to serve as a barrier to
crack propagation, similar to grain boundaries in metals, in order to increase bone toughness. Studies have shown that the ability of
an osteon to inhibit crack propagation significantly decreases with age (Diab and Vashishth, 2005, 2007). Cortical bone specimens
(4 4 48 mm in dimension) from younger donors (38 9 years) were reported to have a significantly longer bending fatigue
life than bone from older donors (82 5 years); moreover, histological analysis revealed that younger bone predominantly formed
diffuse damage as opposed to linear microcracks (Diab and Vashishth, 2005, 2007). Although osteon damage morphology appears
to change with age, the underlying mechanisms for this shift from diffuse damage to microcrack accumulation is still a matter of
ongoing research (Katsamenis et al., 2015; Yeni and Norman, 2000a).
Individual Lamellae, Interlamellar Areas, and Cement Lines
At the several-micron length scale, the structural features of cortical bone observable by light or electron microscopy are the so-called
lamellae, which can be found with varying thicknesses in the range 2–10 mm. Although lamellae are generally characterized as
packed layers of mineralized collagen fibrils with slightly alternating orientation, details regarding composition, arrangement,
and collagen fiber orientation are still poorly understood (Reznikov et al., 2014; Mitchell and van Heteren, 2016). Lamellae are
interfaced with so-called interlamellar areas (also labeled thin lamellae), distinct features that differ from lamellae in mineralization
degree and collagen orientation (Katsamenis et al., 2013a). Lamellae and interlamellar areas are arranged concentrically around
Haversian canals in osteons and are built-up layer by layer during bone remodeling to form secondary osteons. Other types of
lamellae include interstitial lamellae, found in the space between osteons, and those formed in fibrolamellar bone (or plexiform
bone). Fibrolamellar bone is found in fast growing animals where bone tissue needs to be laid down before tissue organization
via bone remodeling can take place (Weiner and Wagner, 1998). At the length scale of the lamella, only osteocyte lacunae and canal-
iculi contribute to cortical bone porosity.
Mechanical Testing of Cortical Bone at the Lamellar Length Scale

The determination of mechanical or material properties at the lamellar level is a challenge since the relevant structural features are
on the order of a few microns. Imaging techniques such as scanning electron microscopy (SEM) and atomic force microscopy (AFM)
have been used as indirect methods for assessing fracture surfaces, that is, the micro-level fracture mechanisms of larger, millimeter
sized cortical bone specimens. In recent years, bone biomechanics researchers have utilized focused ion beam (FIB) milling tech-
niques to isolate well defined micron-sized volumes on the order of a single lamella. Direct measurement of lamellar mechanical
properties can then be achieved by subjecting these isolated volumes to micromechanical tests using AFM or nanoindenters.
Fracture toughness
Fracture toughness is a mechanical property used to describe the ability of a material to resist fracture, typically measured in terms of
stress intensity at the crack tip. A review of how to measure fracture toughness of bone can be found in Ritchie et al. (2008). Within
bone, distinct toughening mechanisms have been identified at each hierarchical level (Fig. 7), each contributing to whole bone frac-
ture resistance (Launey et al., 2010). Intrinsic toughening mechanisms work to inhibit crack initiation, while extrinsic toughening
mechanisms primarily inhibit crack propagation and, to some extent, rupture.
SEM is an imaging technique capable of generating high-resolution images with detailed topographical, morphological, and
compositional information. As such, SEM is commonly used to evaluate the extrinsic toughening mechanisms of bone, which
are observable at length scales from a single micron to hundreds of micrometers. In this context, studies assessing bone crack prop-
agation have been conducted using environmental SEM (Ritchie et al., 2005; Koester et al., 2008, 2011), which does not require
bone samples to be completely dehydrated and coated with a conductive material layer. However, only small scale samples with
sample thicknesses ranging between 1 and 4 mm have been assessed using environmental SEM (Koester et al., 2008; Nalla
et al., 2005a).
Despite this limitation, SEM has been instrumental in the determination of cortical bone fracture toughness, particularly in the
development of full crack resistance curves (R-curves). Fracture toughness testing is typically conducted on small, sub-mm compact
tension (Norman et al., 1995; Yeni and Norman, 2000b) or single-edge notched bending specimens (Katsamenis et al., 2015;
Ritchie et al., 2008) machined from cortical bone in accordance to ASTM standards E399 (ASTM, 1997) and E1820 (ASTM,
2001), respectively. R-curves can then be constructed by tracking the crack propagation, or crack extension, on each loading-step
and plotting it against the stress intensity factor, yielding information on crack initiation and propagation behavior. Crack extension
can be measured directly if tests are conducted within an environmental SEM (Ritchie et al., 2005; Koester et al., 2008, 2011),
inferred by measuring the crack-tip opening displacement, or through the use of high definition videography (Katsamenis et al.,
2013b). Crack extension can also be indirectly measured using standardized load-line compliance calibrations.
Since fracture toughness is a material property, changes in composition and structure due to aging or tissue ultrastructure will
have an effect. Nalla et al. reported a 40% reduction in crack initiation toughness with age from experiments on compact tension
specimens of human cortical bone (donor age: 34–99 years). Cortical bone propagation toughness was also reported to be nearly
eliminated in the older donors (Nalla et al., 2006). Single-edge notched bending tests revealed a link between the energy release rate
(crack extension energy per unit area) and the collagen fiber orientation (Fig. 8); specifically, the energy release rate perpendicular to
the collagen fibrils was reported to be nearly two orders of magnitude higher than in the direction of the collagen fibers (Peterlik
et al., 2006). Osteon orientation further contributes to this observed anisotropy in cortical bone fracture toughness. Crack propa-
gation around osteons has been shown to require significantly less energy than cracking through osteons (Katsamenis et al., 2015,
2013b; Nalla et al., 2005b).
Similar to SEM, crack propagation at the micrometer scale can be captured using AFM, a surface characterization technique
that can be used both for imaging and mechanical assessment via indentation or pulling. For example, AFM imaging of bovine
trabecular bone fracture surfaces revealed that exposed collagen fibrils, as previously hypothesised from SEM images (Braidotti
et al., 1997, 2000), are densely coated with mineral platelets, implying that the nature and the mechanical behavior of the
interface between neighboring mineralized fibers is of significant importance (Kindt et al., 2007). Similarly, AFM analysis of
fractured cortical bone revealed that cement lines and interlamellar areas, apart from providing a crack propagation path of
least resistance (Katsamenis et al., 2013a; Peterlik et al., 2006; Fratzl, 2008), exhibit reduced modulus of elasticity compared
to lamellae (Fig. 8) (Katsamenis et al., 2013a). As such, cement lines and interlamellar areas are thought to positively
contribute to bone toughness.
As mentioned above, FIB milling can be used to prepare microscale specimens for mechanical testing. During FIB milling, a focused
beam of heavy ions (typically Gallium) accelerated by high voltage (in the range of 30 kV) is directed toward a sample. The kinetic
energy of the impinging ions knocks out target atoms, microscopically eroding and milling out material in a controlled manner.
One of the earliest studies using FIB milling on biological mineralized tissue (Chan et al., 2009) employed a dual beam FIB/
SEM to fabricate microscopic cantilever beams with a triangular cross-section from a human primary molar. These beams were
then bent using a conventional nanoindenter and Berkovich tip. Further studies were conducted using micron-sized cantilevers
and micropillars machined to the length scale of a cortical bone lamella.
The FIB only operates under high vacuum conditions, which can lead to substantial dehydration of the material and alteration of
the mechanical behavior of the tissue. Jimenez-Palomar et al. investigated the effect of high vacuum exposure on micron-sized
Fig. 7 The toughness of bone results from a mutual competition between extrinsic (crack-tip shielding) toughening mechanisms and intrinsic
(plastic deformation) toughening mechanisms. Distinct toughening mechanisms occur at each level of bone hierarchy. Molecular uncoiling and inter-
molecular sliding of molecules are observed at the smallest level (see Noncollagenous proteins). Microcracking and fibrillar sliding are observed at
the level of fibril arrays. At higher levels, crack bridging by collagen fibrils combines with the breaking of sacrificial bonds to increase the energy
dissipation capacity of bone at the interface of fibril arrays. At the highest length scales (10–100 mm range), the primary sources of toughening result
from extensive crack deflection and crack bridging by uncracked ligaments, both motivated by the occurrence of microcracking. Reprinted with
permission from Launey, M.E., Buehler, M.J., Ritchie, R.O. (2010). On the mechanistic origins of toughness in bone. Annual Review of Materials
Research 40, 25–53. https://doi.org/10.1146/annurev-matsci-070909-104427. Copyright (2017) Annual Reviews.
cantilever beams (10 2 2 mm in dimension) FIB-milled from a rat femur. Displacement was applied near the end of the beams
using an in situ AFM with a FIB flattened AFM-cantilever tip (Jimenez-Palomar et al., 2012). Microbeams were exposed to and
measured in three different environments, high vacuum, low vacuum and air, and were rehydrated in a vapor chamber between
experiments. Environmental conditions were reported to have no significant effect on the elastic moduli ( 5 GPa) for all
microbeams.
Fig. 8 (Left) The energy required for crack extension in cortical bone is strongly correlated with the collagen fibril orientation angle (g). A signifi-
cant jump in the crack extension energy is observed at an orientation angle of approximately 50 degrees. (Right) Cracks in cortical bone preferentially
propagate through cement lines and interlamellar areas. Inserts i, ii and iii are time-lapsed AFM images of stable crack propagation (scale bar:
20 mm). (Left) Adapted from Peterlik, H., Roschger, P., Klaushofer, K., and Fratzl, P. (2006). From brittle to ductile fracture of bone. Nature Materials
5, 52–55. https://doi.org/10.1038/nmat1545. Copyright (2017) Nature Publishing Group. (Right) Reprinted with permission from Katsamenis, O.L.,
Chong, H.M.H., Andriotis, O.G., and Thurner, P.J. (2013). Load-bearing in cortical bone microstructure: Selective stiffening and heterogeneous strain
distribution at the lamellar level. Journal of the Mechanical Behavior of Biomedical Materials 17, 152–165. https://doi.org/10.1016/j.jmbbm.2012.08.016.
Copyright (2017) Elsevier.
Similar to osteons, collagen fibril orientation and tissue pathology may have an impact on the mechanical response of lamellae.
These effects were investigated in successive studies using the same microbeam bending technique. Here, a range of elastic moduli
from 3.7 to 11.2 GPa was reported, dependent on the alternating collagen fibril orientation within individual microbeams (Fig. 9)
(Jimenez-Palomar et al., 2015a). In a further study, microbeams FIB-milled from femurs of ovariectomized (OVH) rats, an animal
model for osteoporosis, were reported to have lower elastic modulus (1.59 1.26 GPa) but higher strain at failure (10 4.04%)
Fig. 9 (Left) High-resolution SEM micrographs taken after testing and FIB cross-sections showing failure modes encountered in compression tests
of micropillars milled in the axial direction. Micropillars mostly deformed homogeneously and failed in shear by development of a single slip plane
(Left, top). A minority of the axial pillars failed by mushrooming (Left, middle) or axial splitting (Left, bottom), which is a brittle failure mode. (Right)
SEM micrographs showing (a) in situ cantilever beam testing in bending provided by the AFM tip pushing into the free end of the bone microbeam
until (b) failure of the microbeam occurs. (Left) Adapted from Schwiedrzik, J., Raghavan, R., Bürki, A., LeNader, V., Wolfram, U., Michler, J., and
Zysset, P. (2014). In situ micropillar compression reveals superior strength and ductility but an absence of damage in lamellar bone. Nature Materials
13, 740–747. https://doi.org/10.1038/nmat3959. Copyright 2017, Nature Publishing Group. (Right) Reprinted with permission from Jimenez-Palomar,
I., Shipov, A., Shahar, R., Barber, A.H. (2015). Structural orientation dependent sub-lamellar bone mechanics. Journal of the Mechanical Behavior of
Biomedical Materials 52, 63–71. https://doi.org/10.1016/j.jmbbm.2015.02.031. Copyright (2017) Elsevier.
than control mice (2.9 1.45 GPa and 6.3 1.89%) (Jimenez-Palomar et al., 2015b). No difference in strength was reported
between the control and OVH mice. Note that these beams were tested in compression, with the AFM tip applying a displacement
parallel to the long access of the beam.
Bending experiments characterize the behavior of a structural element, that is, beam, subjected to an external load applied
perpendicular to its longitudinal axis. This results in a complex stress state within the beam, dependent on both beam geometry
in a nonlinear fashion as well as material properties. As a result, bending experiments require the use of a beam theory such as
Euler–Bernoulli or Timoshenko, both of which have inherent assumptions, to derive an elastic modulus. Due to these complica-
tions, uniaxial compression tests on FIB-milled samples, offering a much simpler loading case, were also investigated:
Studies utilizing micropillar compression on FIB-milled ovine osteonal lamellae have been conducted in both dry (Schwiedrzik
et al., 2014) and rehydrated (Schwiedrzik et al., 2017) environments (Fig. 9). Micropillars were machined from ovine tibiae in both
axial and transverse directions and compression was performed both monotonically and cyclically beyond the elastic limit. Both dry
and rehydrated micropillars were reported to exhibit anisotropic behavior in the elastic and postyield regime. Axial micropillars
were reported to yield at higher stresses compared to transverse ones and were less likely to exhibit strain hardening postyield
behavior. Rehydration reduced the yield stress anisotropy ratio between the axial and transverse micropillars. High-resolution
SEM micrographs taken after testing revealed that shear via a single slip plane was the most predominant mode of failure; however,
a minority of the axial micropillars exhibited brittle failure modes (Fig. 9) (Schwiedrzik et al., 2014). Note that the fabrication tech-
nique used in these studies results in a tapered cylindrical micropillar that remains attached to the underlying bone. The tapered
geometry results in an inhomogeneous stress distribution over the pillar height and the substrate acts as an elastic half-space impact-
ing the overall mechanical response of the micropillar. Luczynski et al. developed a protocol to mill out, extract, and transfer unta-
pered micropillars with square cross sections from the lamellae of bovine tibia onto a silicon wafer, a much more rigid substrate.
Here, once fixed, the micropillars were subjected to loading and unloading cycles with a flat punch nanoindenter tip (Luczynski
et al., 2015). Elastic moduli, derived from unloading force–deflection curves, were reported to range from 24.1 to 32.2 GPa.
Note that nearly all of the micropillars in the aforementioned studies were FIB-milled from sections of lamellae lacking canaliculi
or lacunae.
Collagen Fibrils and Nanocrystals: Individual Components of Bone
Beyond the individual lamellae, one approaches the individual nanoscale building blocks of bone tissue. Here, collagen, carbon-
substituted calcium phosphate nanocrystals, water, and NCPs are the most important structural elements. At this nanoscale level,
these components are organized into mineralized collagen fibrils (MCFs), and the individual MCF can be regarded as the building
block of bone.
Collagens and Collagen Fibrils

Collagen is a superfamily of chemically distinct but closely related proteins found in different quantities in the body. In total, > 25
different types of collagen molecules exist. The most abundant of these collagen types (type I, II, III, V and XI) assemble into
cylindrical-like structures, that is, the collagen fibril, with diameters varying from 20 to 500 nm and lengths up to 1 mm (Starborg
et al., 2013). Collagen type I is the most abundant of the collagen molecules and is the main constituent of collagen fibrils within
bone; small amounts of type III and V are also present. A characteristic of all collagen molecules is the close packing of three alpha
polypeptide chains (not to be confused with a-helices) into a right-handed twisted triple helix. The triple helix is mediated by the
high content of glycine amino acids (Gly). Glycines occupy every third position in the amino acid sequence of each a-chain and are
always positioned toward the core of the triple helix (Brodsky and Persikov, 2005). The location and high abundance of Gly allows
for the formation of a large number of hydrogen bonds (Launey et al., 2010; Ramachandran et al., 1973) as well as hydroxylation of
proline and lysine residues (Kivirikko et al., 1967) between the a-chains, both of which stabilize the triple helix into a collagen
molecule characterized by enhanced structural integrity.
Collagen Fibril Structure

Collagen fibrils are characterized by the 67 nm D-periodicity (Kadler et al., 1996), which results from the presence of overlap and
gap regions between self-assembled collagen molecules; this D-periodicity was first described by the Hodge–Petruska two-
dimensional model of collagen molecule packing. Orgel et al. showed that collagen fibrils are formed of quasihexagonally packed
collagen molecules, which are supertwisted into a right-handed structure across the longitudinal direction of the collagen fibril
(Orgel et al., 2006). Moreover, Orgel et al. proposed a microfibril model to describe the collagen fibril ultrastructure derived
from X-ray diffraction patterns. The microfibril model has a triclinic unit cell (a z 40.0 Å, b z 27.0 Å, c z 678 Å, a z 89.28,
b z 94.68 and g z 105.68) comprised of parts from five different collagen molecules (Fig. 10). As a result, a microfibril cannot
be considered as a functional structural unit but rather as a structural model. Currently, the Orgel-model of collagen fibril structure
is widely used in molecular dynamics simulations in the field of collagen biochemistry, structure, and mechanics (Orgel et al., 2006;
Perumal et al., 2008; Gautieri et al., 2011; Vesentini et al., 2013).
Fig. 10 Collagen structure. Panel (A) displays the front and side view of a collagen molecule showing the right-handed twist of the triple helix
(Jmol, collagen structure from Protein Data Bank: PDB 1CAG). Panel (B) shows the Hodge–Petruska model of the collagen molecules self-assembly
with overlap and gap regions. Panel (C) shows the a–b plane (top) of the Orgel microfibril model (bottom) with the quasihexagonal packing of
collagen molecules. The colors in the microfibril model represent different parts or portions of the collagen molecules and the c-axis is 67.8 nm.
Panel (D) shows an atomic force microscopy height topography image (top) of a collagen fibril and the corresponding height profile showing the
67 nm D-periodicity (bottom). Figure adapted from Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I
collagen in situ. Proceedings of the National Academy of Sciences of the United States of America 103, 9001–9005. https://doi.org/10.1073/pnas.
0502718103. Andriotis, O.G., Chang, S.W., Vanleene, M., Howarth, P.H., Davies, D.E., Shefelbine, S.J., Buehler, M.J., and Thurner, P.J. (2015).
Structure–mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model. Journal of The Royal Society Interface 12, http://rsi-
f.royalsocietypublishing.org/content/12/111/20150701.abstract with permissions. Copyright (2006) National Academy of Sciences, USA.
Collagen Fibril Hydration and Mechanics

Hydration water exists in two forms in collagen: bound water and unbound or freely moving water. Bound water is found in
close proximity to the collagen molecule, forms an organized structure surrounding the triple helix, and helps stabilize the
collagen molecule’s tight conformation (Bella et al., 1995). The unbound water is less organized and can freely move in the
intrafibrillar spaces. The intrafibrillar hydration level strongly influences the intermolecular distance. Early experiments using
X-ray diffraction to evaluate tendons from both murine and human donors showed that the intermolecular distance increases
with increasing hydration (Price et al., 1997). More recently, AFM experiments showed that native collagen fibrils swell upon
hydration (Heim et al., 2007), resulting in a three orders of magnitude decrease in the measured indentation modulus (Grant
et al., 2008). Collagen fibril hydration could also be influenced by the chemistry of the aqueous environment. Transverse elas-
ticity of individual collagen fibrils was shown to be tunable up to a sevenfold increase by merely changing the pH and ionic
strength (Grant et al., 2009). The importance of hydration in collagen fibril mechanics was further revealed in a recent study on
native collagen fibrils excised from the osteogenesis imperfecta mouse (OIM) model. OIM collagen fibrils were reported to
have reduced hydration levels compared to wild-type counterparts. This reduced hydration was accompanied by a fivefold
change in the transverse elasticity, measured using AFM cantilever-based nanoindentation experiments (Andriotis et al.,
2015). Although collagen fibril transverse elasticity is influenced by ionic strength, the tensile elastic modulus was shown to
be less sensitive to environmental salts in a separate study (Svensson et al., 2010). Svensson et al. proposed that this discrep-
ancy was due to the displacement of water molecules within the fibril upon exposure to environmental salts, which primarily
affect the radial properties, that is, swelling and elasticity, rather than the axial (Svensson et al., 2010). Molecular dynamic
simulations on collagen microfibril models show that a loss of water results in molecular unfolding of the collagen triple helix
(Gautieri et al., 2011); however, such unfolding only results in small changes in the longitudinal swelling of collagen (Masic
et al., 2015) compared to swelling in the radial direction. Although small changes occur in the length of collagen molecule
during dehydration, Masic et al. showed that osmotic pressure induced by partial dehydration generates tensile forces in
collagen (106 pN per collagen molecule) (Masic et al., 2015).
Mineralized Collagen Fibrils (MCFs)
At the nanoscale in bone, MCFs are the structural and functional elements that contain carbon-substituted hydroxyapatite crystals.
The crystals are plate-like structures with their c-axis oriented along the collagen longitudinal axis (Landis et al., 1996). MCFs are
formed through a multistage mineralization process, during which the collagen and noncollagenous proteins are believed to
play an active role, differing from the classical view of crystal formation (Nudelman et al., 2010). Generally, calcium-based minerals
in biological materials are believed to be formed through prenucleation clusters (Gebauer et al., 2008) before they transform into
a crystal (Pouget et al., 2009).
Mineralization of Collagen Fibrils

As noted, the mineralization of a collagen fibril differs from the classical view of crystal formation. Collagen fibrils have been shown
to mineralize in the presence of acidic proteins, for example, poly-l-aspartic acid, fetuin or NCPs (Nudelman et al., 2010;
Deshpande and Beniash, 2008; Price et al., 2009). Nudelman et al. proposed a mechanism for the mineralization of a collagen fibril
in the presence of polyAsp, a nucleation inhibitor, based on experiments combining cryogenic transmission electron microscopy
(cryoTEM) and low-dose selected-area electron diffraction (LDSAED) (Fig. 11) (Nudelman et al., 2010). In the first stage of this
mineralization process, calcium and phosphate ions form prenucleation sites with the polymer, that is, polyAsp. Then, the prenu-
cleation site stabilizes into a negatively charged polymer–amorphous calcium phosphate (ACP) complex. This polymer–ACP
complex then binds to positively charged sites in the collagen fibril located at the gap-overlap borders. Infiltration of minerals occurs
at these positively charged regions, resulting in a dense network of prenucleation clusters. Subsequently, these transform into ACP
clusters which finally form calcium phosphate crystals within the collagen fibril with the c-axis aligned with the collagen longitu-
dinal axis.
In addition to this intrafibrillar mineralization, collagen fibrils have extrafibrillar mineral, which is thought to be mediated via
NCPs (detailed in Noncollagenous proteins). Extrafibrillar mineral was first detected in neutron diffraction studies of mineralized
tendon and bone (Lees et al., 1984; Bonar et al., 1985); later, transmission electron micrographs of mineralized tendon, dentin, and
bone revealed differences in orientation and position of intra- and extrafibrillar mineral (Prostak and Lees, 1996; Lees and Prostak,
1988). As a result of AFM surface maps, Sasaki et al. estimated that up to 77% of the mineral in bovine bone is extrafibrillar (Sasaki
et al., 2002). Micromechanical models, designed to characterize the anisotropic behavior of heterogeneous materials such as bone,
are commonly employed to investigate the impact of individual constituents on the overall mechanical behavior of bone. Early
models of mineralized tissues supported the presence of extrafibrillar mineral and stressed their impact on the ultrastructural stiff-
ness of bone (Prostak and Lees, 1996; Hellmich and Ulm, 2002a, b). More recently, micromechanical models have been used to link
the apparent hardening effect in bone to energy dissipation in the extrafibrillar mineral and have been used to predict the strength
characteristics of bone from different species at various hierarchical levels (Fritsch et al., 2009; Morin et al., 2017). Foundational
work on quantitative validation of such models can be found in Hellmich and Ulm (2003) and Hellmich et al. (2004).
Mechanics of Mineralized Collagen Fibrils (MCFs)

In situ mechanical testing combined with X-ray diffraction reveals that tensile loading on micro-sized bone samples induces a coop-
erative deformation mechanism between mineral crystals and collagen fibrils (Jäger and Fratzl, 2000; Gupta et al., 2006b). This
results in load transfer between the inorganic (mineral crystals) and organic matrix (collagen fibril) (Jäger and Fratzl, 2000; Gupta
et al., 2006b). Gupta et al. reported differences in the load transfer mechanisms between wet and dry samples. Drying results in
a stiffer organic matrix (higher elastic modulus), promoting more effective load transfer from the organic to the mineral crystals.
Therefore, crystals bear a higher strain fraction leading to a stiffer overall sample.
Beyond elasticity, the presence of intrafibrillar mineral crystals affects yield and postyield behavior of bone at the nanoscale.
Molecular dynamics simulations have revealed that MCFs have a higher yield point and greater fracture toughness compared to
nonmineralized collagen fibrils (Buehler, 2007). Experimentally, the mechanical properties of native MCFs have been assessed
by in situ AFM with SEM imaging (Hang and Barber, 2011). In this work, exposed MCFs on the fractured surface of bone were first
imaged using SEM and then an epoxy droplet on the tip apex of an AFM cantilever was used to mechanically fix the exposed MCFs.
After fixation, the AFM cantilever was used to pull the fixed MCFs while measuring the applied force. The complex composition and
deformation mechanisms of MCFs resulted in bilinear stress–strain behavior, which was heavily dependent on the mineral density.
All MCFs were found to exhibit an initial linear response, which the authors attributed to loads transferring from the organic to the
inorganic matrix via uncoiling of the collagen molecules. Beyond the initial linear response, two distinct mechanical behaviors were
observed. High mineral density MCFs exhibited strain hardening whereas low mineral density MCFs plastically deformed. Strain
hardening was attributed to elevated stress transfer between collagen molecules while plastic deformation was attributed to inter-
molecular sliding. Both the stiffness of the initial linear-region and the behavior within the heterogeneous deformation zone were
dependent on the total mineral content. This observed nano-mechanical heterogeneity aids in energy dissipation, likely contrib-
uting to the fracture toughness of bone at larger length scales. Note that the samples in this study were exposed to vacuum, resulting
in considerable dehydration, thereby influencing the resulting deformation and mechanical behavior (Hang and Barber, 2011).
Herein, the structure and mechanics of native and mineralized collagen fibrils have been discussed. Regarding the process of
mineralization, there is evidence suggesting that NCPs could promote extrafibrillar mineralization by serving as points for
Fig. 11 Intrafibrillar collagen mineralization. (A) Prenucleation calcium clusters (in green) form into complexes with the polymer (orange), resulting
in more stable mineral droplets depicted as a red cluster (Left). Binding of the mineral droplets to a region on the collagen fibril (Right). (B) Depend-
ing on the size of the polymer, either the full droplet or only the calcium clusters enter the intrafibrillar space of the collagen fibril (Left). The intra-
fibrillar mineral droplets then diffuse within the fibril space (Right). (C) The mineral droplets gradually solidify into amorphous calcium phosphate
(ACP; black shown Top). The last step of intrafibrillar mineralization process includes the transformation of the ACP into apatite crystals (yellow)
oriented along the long fibril axis (Bottom). Figure adapted from Colfen, H. (2010). Biomineralization: A crystal-clear view. Nature Materials 9, 960–
961. https://doi.org/10.1038/nmat2911 with permissions. Copyright (2017) Nature Publishing Group. (D) A fibril mineralized for 48 h, where the
deformation caused by the presence of mineral can be observed (Top). Reconstructed cryo-electron tomography images reveal plate-shaped apatite
crystals (pink) embedded within the collagen matrix (Bottom). Adapted from Nudelman, F., Pieterse, K., George, A., Bomans, P.H.H., Friedrich, H.,
Brylka, L.J., Hilbers, P.A.J., de With, G., and Sommerdijk, N.A.J.M. (2010). The role of collagen in bone apatite formation in the presence of hydroxy-
apatite nucleation inhibitors. Nature Materials 9, 1004–1009. https://doi.org/10.1038/nmat2875. Copyright (2017) Nature Publishing Group.
prenucleation clusters to form. In addition to this role in the mineralization process, there is further evidence pointing to a nano-
mechanial function of the NCPs within bone.
Noncollagenous Proteins
In addition to collagen, carbon-substituted hydroxyapatite, and water, bone consists of a small fraction (< 10 wt%) of NCPs (many
of these are unstructured). These NCPs include osteopontin, osteocalcin, and bone sialoprotein, among others, and are mostly from
the small integrin-binding ligand N-linked glycoprotein (SIBLING) family (Fischer et al., 2001). Initially, the scientific focus on such
proteins was concentrated on their ability to steer and direct biomineralization. They offer attachment sites for collagen type I (Tye
et al., n.d.) and nucleation sites for carbon-substituted hydroxyapatite (Hunter and Goldberg, 1993) and cells (Fischer et al., 2001)
as well as being thought to control crystal size and shape (Qiu et al., 2004; Habelitz et al., 2004). In the early 2000s, high-resolution
imaging via AFM revealed unstructured material phases on fracture surfaces and between mineralized collagen fibrils; these phases
were attributed to the NCP fraction contained in bone and suggested a possible mechanical role of NCPs (Fantner et al., 2005). The
potential mechanical role of NCPs was further supported by in vivo experiments in which patches of purified proteins exhibited an
ability to repeatedly dissipate large amounts of energy during pull apart tests via sacrificial bonds and a so-called “hidden length
mechanism” (Fantner et al., 2007; Zappone et al., 2008; Adams et al., 2008). These sacrificial bonds are weak, reformable bonds
between neighboring mineralized collagen fibrils that break prior to the structural bonds of the mineralized collagen. The opening
of these sacrificial bonds reveals hidden length and the stretching of these bonds increases the total energy needed to fracture the
material, increasing overall material toughness. Studies involving animal models deficient of NCPs have also reported significant
reductions in bone strength as well as fracture toughness (Duvall et al., 2007; Thurner et al., 2010; Poundarik et al., 2012), neither
of which could be explained via structural or other compositional alterations (Thurner et al., 2010; Poundarik et al., 2012).
More recently, comparisons between interstitial and osteonal human bone showed changes in NCP content (Sroga et al., 2011).
However, whether NCP fractions change due to age and disease leading to similar significant changes in human bone material prop-
erties has not been investigated to date. This is despite reports that the protein matrix in bone does indeed change with age and
disease (Grynpas et al., 1994). It may well be that the reported shift from diffuse damage to microcrack accumulation associated
with aging bone is related to an increase in bone brittleness and fracture risk with age (Diab and Vashishth, 2007). The role of
NCPs in this phenomenon is not yet known. An additional finding of interest in this context is that diffuse damage in bone heals
without remodeling (Seref-Ferlengez et al., 2014). One may speculate that this is an effect mediated by NCPs; however, to this point
there exists no data to prove or disprove this hypothesis.
References
Abraham, A. C., Agarwalla, A., Yadavalli, A., McAndrew, C., Liu, J. Y., & Tang, S. Y. (2015). Multiscale predictors of femoral neck in situ strength in aging women: Contributions of
BMD, cortical porosity, reference point indentation, and nonenzymatic glycation. Journal of Bone and Mineral Research, 30, 2207–2214. https://doi.org/10.1002/jbmr.2568.
Acevedo, C., Bale, H., Gludovatz, B., Wat, A., Tang, S. Y., Wang, M., Busse, B., Zimmermann, E. A., Schaible, E., Allen, M. R., Burr, D. B., & Ritchie, R. O. (2015). Alendronate
treatment alters bone tissues at multiple structural levels in healthy canine cortical bone. Bone, 81, 352–363. https://doi.org/10.1016/j.bone.2015.08.002.
Adams, J., Fantner, G. E., Fisher, L. W., & Hansma, P. K. (2008). Molecular energy dissipation in nanoscale networks of dentin matrix protein 1 is strongly dependent on ion valence.
Nanotechnology, 19, 384008. https://doi.org/10.1088/0957-4484/19/38/384008.
Allen, M. R., McNerny, E. M. B., Organ, J. M., & Wallace, J. M. (2015). True gold or pyrite: A review of reference point indentation for assessing bone mechanical properties in vivo.
Journal of Bone and Mineral Research, 30, 1539–1550. https://doi.org/10.1002/jbmr.2603.
Andriotis, O. G., Chang, S. W., Vanleene, M., Howarth, P. H., Davies, D. E., Shefelbine, S. J., Buehler, M. J., & Thurner, P. J. (2015). Structure–mechanics relationships of collagen
fibrils in the osteogenesis imperfecta mouse model. Journal of The Royal Society Interface, 12. http://rsif.royalsocietypublishing.org/content/12/111/20150701.abstract.
Ascenzi, M.-G. (2012). The osteon: The micromechanical unit of compact bone. Frontiers in Bioscience, 17, 1551–1581.
Ascenzi, A., & Bonucci, E. (1967). The tensile properties of single osteons. The Anatomical Record, 158, 375–386. https://doi.org/10.1002/ar.1091580403.
Ascenzi, A., & Bonucci, E. (1968). The compressive properties of single osteons. The Anatomical Record, 161, 377–391.
Ascenzi, A., & Bonucci, E. (1972). The shearing properties of single osteons. The Anatomical Record, 172, 499–510. https://doi.org/10.1002/ar.1091720304.
Ascenzi, A., Baschieri, P., & Benvenuti, A. (1990). The bending properties of single osteons. Journal of Biomechanics, 23, 763–771. https://doi.org/10.1016/0021-9290(90)
90023-V.
Ascenzi, A., Baschieri, P., & Benvenuti, A. (1994). The torsional properties of single selected osteons. Journal of Biomechanics, 27, 875–884. https://doi.org/10.1016/0021-
9290(94)90260-7.
ASTM. (1997). E399: Standard test method for plane strain fracture toughness of metallic materials. West Conshohocken, PA: ASTM International.
ASTM. (2001). E1820: Standard test method for measurement of fracture toughness. West Conshohocken, PA: ASTM International.
Bella, J., Brodsky, B., & Berman, H. M. (1995). Hydration structure of a collagen peptide. Structure, 3, 893–906. https://doi.org/10.1016/S0969-2126(01)00224-6.
Bernhard, A., Milovanovic, P., Zimmermann, E. A., Hahn, M., Djonic, D., Krause, M., Breer, S., Püschel, K., Djuric, M., Amling, M., & Busse, B. (2013). Micro-morphological
properties of osteons reveal changes in cortical bone stability during aging, osteoporosis, and bisphosphonate treatment in women. Osteoporosis International, 24, 2671–
2680. https://doi.org/10.1007/s00198-013-2374-x.
Bini, F., Marinozzi, A., Marinozzi, F., & Patanè, F. (2002). Microtensile measurements of single trabeculae stiffness in human femur. Journal of Biomechanics. https://doi.org/
10.1016/S0021-9290(02)00182-3.
Black, J., Mattson, R., & Korostoff, E. (1974). Haversian osteons: Size, distribution, internal structure, and orientation. Journal of Biomedical Materials Research, 8, 299–319.
https://doi.org/10.1002/jbm.820080512.
Bonar, L. C., Lees, S., & Mook, H. A. (1985). Neutron diffraction studies of collagen in fully mineralized bone. Journal of Molecular Biology, 181, 265–270. https://doi.org/10.1016/
0022-2836(85)90090-7.
Boskey, A. L. (2013). Bone composition: relationship to bone fragility and antiosteoporotic drug effects. BoneKEy Reports, 2. https://doi.org/10.1038/bonekey.2013.181.
Braidotti, P., Branca, F. P., & Stagni, L. (1997). Scanning electron microscopy of human cortical bone failure surfaces. Journal of Biomechanics, 30, 155–162. https://doi.org/
10.1016/S0021-9290(96)00102-9.
Braidotti, P., Bemporad, E., D’Alessio, T., Sciuto, S. A., & Stagni, L. (2000). Tensile experiments and SEM fractography on bovine subchondral bone. Journal of Biomechanics, 33,
1153–1157. https://doi.org/10.1016/S0021-9290(00)00074-9.
Bridges, D., Randall, C., & Hansma, P. K. (2012). A new device for performing reference point indentation without a reference probe. The Review of Scientific Instruments, 83,
44301. https://doi.org/10.1063/1.3693085.
Brodsky, B., & Persikov, A. V. (2005). Molecular structure of the collagen triple helix. Advances in Protein Chemistry, 70, 301–339. https://doi.org/10.1016/S0065-3233(05)
70009-7.
Buehler, M. J. (2007). Molecular nanomechanics of nascent bone: Fibrillar toughening by mineralization. Nanotechnology, 18, 295102. http://stacks.iop.org/0957-4484/18/i¼29/
a¼295102.
Bumrerraj, S., & Katz, J. L. (2001). Scanning acoustic microscopy study of human cortical and trabecular bone. Annals of Biomedical Engineering, 29, 1034–1042. https://doi.org/
10.1114/1.1424908.
Burr, D. B., Schaffler, M. B., & Frederickson, R. G. (1988). Composition of the cement line and its possible mechanical role as a local interface in human compact bone. Journal of
Biomechanics, 21, 939–945.
Busse, B., Hahn, M., Soltau, M., Zustin, J., Püschel, K., Duda, G. N., & Amling, M. (2009). Increased calcium content and inhomogeneity of mineralization render bone toughness in
osteoporosis: Mineralization, morphology and biomechanics of human single trabeculae. Bone, 45, 1034–1043. https://doi.org/10.1016/j.bone.2009.08.002.
Carretta, R., Luisier, B., Bernoulli, D., Stüssi, E., Müller, R., & Lorenzetti, S. (2013a). Novel method to analyze post-yield mechanical properties at trabecular bone tissue level.
Journal of the Mechanical Behavior of Biomedical Materials, 20, 6–18. https://doi.org/10.1016/j.jmbbm.2012.12.003.
Carretta, R., Stüssi, E., Müller, R., & Lorenzetti, S. (2013b). Within subject heterogeneity in tissue-level post-yield mechanical and material properties in human trabecular bone.
Journal of the Mechanical Behavior of Biomedical Materials, 24, 64–73. https://doi.org/10.1016/j.jmbbm.2013.04.014.
Chan, Y. L., Ngan, A. H. W., & King, N. M. (2009). Use of focused ion beam milling for investigating the mechanical properties of biological tissues: A study of human primary
molars. Journal of the Mechanical Behavior of Biomedical Materials, 2, 375–383. https://doi.org/10.1016/j.jmbbm.2009.01.006.
Chang, W. C. W., Christensen, T. M., Pinilla, T. P., & Keaveny, T. M. (1999). Uniaxial yield strains for bovine trabecular bone are isotropic and asymmetric. Journal of Orthopaedic
Research, 17, 582–585. https://doi.org/10.1002/jor.1100170418.
Choi, K., & Goldstein, S. A. (1992). A comparison of the fatigue behavior of human trabecular and cortical bone tissue. Journal of Biomechanics. https://doi.org/10.1016/0021-
9290(92)90051-2.
Currey, J. D. (1975). The effects of strain rate, reconstruction and mineral content on some mechanical properties of bovine bone. Journal of Biomechanics, 8, 81–86. https://
doi.org/10.1016/0021-9290(75)90046-9.
Currey, J. D. (2002). Bones: structure and mechanics. Princeton: Princeton University Press.
Deshpande, A. S., & Beniash, E. (2008). Bioinspired synthesis of mineralized collagen fibrils. Crystal Growth & Design, 8, 3084–3090. https://doi.org/10.1021/cg800252f.
Diab, T., & Vashishth, D. (2005). Effects of damage morphology on cortical bone fragility. Bone, 37, 96–102. https://doi.org/10.1016/j.bone.2005.03.014.
Diab, T., & Vashishth, D. (2007). Morphology, localization and accumulation of in vivo microdamage in human cortical bone. Bone, 40, 612–618. https://doi.org/10.1016/
j.bone.2006.09.027.
Diez-Perez, A., Güerri, R., Nogues, X., Cáceres, E., Peña, M. J., Mellibovsky, L., Randall, C., Bridges, D., Weaver, J. C., Proctor, A., Brimer, D., Koester, K. J., Ritchie, R. O., &
Hansma, P. K. (2010). Microindentation for in vivo measurement of bone tissue mechanical properties in humans. Journal of Bone and Mineral Research, 25, 1877–1885.
https://doi.org/10.1002/jbmr.73.
Duvall, C. L., Taylor, W. R., Weiss, D., Wojtowicz, A. M., & Guldberg, R. E. (2007). Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of
osteopontin-deficient mice. Journal of Bone and Mineral Research, 22, 286–297. https://doi.org/10.1359/jbmr.061103.
Fantner, G. E., Hassenkam, T., Kindt, J. H., Weaver, J. C., Birkedal, H., Pechenik, L., Cutroni, J. A., Cidade, G. A. G., Stucky, G. D., Morse, D. E., & Hansma, P. K. (2005). Sacrificial
bonds and hidden length dissipate energy as mineralized fibrils separate during bone fracture. Nature Materials, 4, 612–616. https://doi.org/10.1038/nmat1428.
Fantner, G. E., Adams, J., Turner, P., Thurner, P. J., Fisher, L. W., & Hansma, P. K. (2007). Nanoscale ion mediated networks in bone: Osteopontin can repeatedly dissipate large
amounts of energy. Nano Letters, 7, 2491–2498. https://doi.org/10.1021/nl0712769.
Fazzalari, N. L., Forwood, M. R., Smith, K., Manthey, B. A., & Herreen, P. (1998). Assessment of cancellous bone quality in severe osteoarthrosis: Bone mineral density, mechanics,
and microdamage. Bone, 22, 381–388. https://doi.org/10.1016/S8756-3282(97)00298-6.
Ferreira, F., Vaz, M. A., & Simões, J. A. (2006). Mechanical properties of bovine cortical bone at high strain rate. Materials Characterization, 57, 71–79. https://doi.org/10.1016/
j.matchar.2005.11.023.
Fischer, L. W., Torchia, D. A., Fohr, B., Young, M. F., & Fedarko, N. S. (2001). Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochemical and
Biophysical Research Communications, 280, 460–465. https://doi.org/10.1006/BBRC.2000.4146.
Frank, M., Marx, D., Pahr, D. H., & Thurner, P. J. (2017). Mechanical properties of individual trabeculae in a physiological environment. In Proc. IASTED Int. Conf. Biomed. Eng.
(BioMed 2017). https://doi.org/10.2316/P.2017.852-023.
Fratzl, P. (2008). Bone fracture: When the cracks begin to show. Nature Materials, 7, 610–612. https://doi.org/10.1038/nmat2240.
Fritsch, A., Hellmich, C., & Dormieux, L. (2009). Ductile sliding between mineral crystals followed by rupture of collagen crosslinks: Experimentally supported micromechanical
explanation of bone strength. Journal of Theoretical Biology, 260, 230–252. https://doi.org/10.1016/j.jtbi.2009.05.021.
Gautieri, A., Vesentini, S., Redaelli, A., & Buehler, M. J. (2011). Hierarchical structure and nanomechanics of collagen microfibrils from the atomistic scale up. Nano Letters, 11,
757–766. https://doi.org/10.1021/nl103943u.
Gebauer, D., Volkel, A., & Colfen, H. (2008). Stable prenucleation calcium carbonate clusters. Science, 322, 1819–1822. https://doi.org/10.1126/science.1164271.
Giraud-Guille, M.-M., Besseau, L., & Martin, R. (2003). Liquid crystalline assemblies of collagen in bone and in vitro systems. Journal of Biomechanics, 36, 1571–1579.
Gong, J. K., Arnold, J. S., & Cohn, S. H. (1964). Composition of trabecular and cortical bone. The Anatomical Record, 149, 325–331.
Granke, M., Makowski, A. J., Uppuganti, S., & Nyman, J. S. (2016). Prevalent role of porosity and osteonal area over mineralization heterogeneity in the fracture toughness of human
cortical bone. Journal of Biomechanics, 49, 2748–2755. https://doi.org/10.1016/j.jbiomech.2016.06.009.
Grant, C. A., Brockwell, D. J., Radford, S. E., & Thomson, N. H. (2008). Effects of hydration on the mechanical response of individual collagen fibrils. Applied Physics Letters, 92,
233902. https://doi.org/10.1063/1.2937001.
Grant, C. A., Brockwell, D. J., Radford, S. E., & Thomson, N. H. (2009). Tuning the elastic modulus of hydrated collagen fibrils. Biophysical Journal, 97, 2985–2992. https://doi.org/
10.1016/j.bpj.2009.09.010.
Grynpas, M. D., Tupy, J. H., & Sodek, J. (1994). The distribution of soluble, mineral-bound, and matrix-bound proteins in osteoporotic and normal bones. Bone, 15, 505–513.
https://doi.org/10.1016/8756-3282(94)90274-7.
Güerri-Fernández, R. C., Nogués, X., Quesada Gómez, J. M., Torres del Pliego, E., Puig, L., García-Giralt, N., Yoskovitz, G., Mellibovsky, L., Hansma, P. K., & Díez-Pérez, A. (2013).
Microindentation for in vivo measurement of bone tissue material properties in atypical femoral fracture patients and controls. Journal of Bone and Mineral Research, 28, 162–
168. https://doi.org/10.1002/jbmr.1731.
Gupta, H. S., Schratter, S., Tesch, W., Roschger, P., Berzlanovich, A., Schoeberl, T., Klaushofer, K., & Fratzl, P. (2005). Two different correlations between nanoindentation modulus
and mineral content in the bone–cartilage interface. Journal of Structural Biology, 149, 138–148. https://doi.org/10.1016/j.jsb.2004.10.010.
Gupta, H. S., Stachewicz, U., Wagermaier, W., Roschger, P., Wagner, H. D., & Fratzl, P. (2006a). Mechanical modulation at the lamellar level in osteonal bone. Journal of Materials
Research, 21, 1913–1921. https://doi.org/10.1557/jmr.2006.0234.
Gupta, H. S., Seto, J., Wagermaier, W., Zaslansky, P., Boesecke, P., & Fratzl, P. (2006b). Cooperative deformation of mineral and collagen in bone at the nanoscale. Proceedings of
the National Academy of Sciences of the United States of America, 103, 17741–17746. https://doi.org/10.1073/pnas.0604237103.
Habelitz, S., Kullar, A., Marshall, S. J., DenBesten, P. K., Balooch, M., Marshall, G. W., & Li, W. (2004). Amelogenin-guided crystal growth on fluoroapatite glass-ceramics. Journal
of Dental Research, 83, 698–702. https://doi.org/10.1177/154405910408300908.
Hamed, E., Jasiuk, I., Yoo, A., Lee, Y., & Liszka, T. (2012). Multi-scale modelling of elastic moduli of trabecular bone. Journal of The Royal Society Interface. http://rsif.
royalsocietypublishing.org/content/early/2012/01/24/rsif.2011.0814.abstract.
Hang, F., & Barber, A. H. (2011). Nano-mechanical properties of individual mineralized collagen fibrils from bone tissue. Journal of The Royal Society Interface, 8, 500–505. https://
doi.org/10.1098/rsif.2010.0413.
Hansen, U., Zioupos, P., Simpson, R., Currey, J. D., & Hynd, D. (2008). The effect of strain rate on the mechanical properties of human cortical bone. Journal of Biomechanical
Engineering, 130, 11011-1–11011-8.
Hansma, P. K., Turner, P. J., & Fantner, G. E. (2006). Bone diagnostic instrument. The Review of Scientific Instruments, 77, 75105. https://doi.org/10.1063/1.2221506.
Hasegawa, K., Turner, C. H., Recker, R. R., Wu, E., & Burr, D. B. (1995). Elastic properties of osteoporotic bone measured by scanning acoustic microscopy. Bone, 16, 85–90.
Heim, A. J., Koob, T. J., & Matthews, W. G. (2007). Low strain nanomechanics of collagen fibrils. Biomacromolecules, 8, 3298–3301. https://doi.org/10.1021/bm061162b.
Hellmich, C., & Ulm, F. J. (2002a). Are mineralized tissues open crystal foams reinforced by crosslinked collagen?dSome energy arguments. Journal of Biomechanics, 35, 1199–
1212. https://doi.org/10.1016/S0021-9290(02)00080-5.
Hellmich, C., & Ulm, F.-J. (2002b). Micromechanical model for Ultrastructural stiffness of mineralized tissues. Journal of Engineering Mechanics, 128, 898–908. https://doi.org/
10.1061/(ASCE)0733-9399(2002)128:8(898).
Hellmich, C., & Ulm, F.-J. (2003). Average hydroxyapatite concentration is uniform in the extracollagenous ultrastructure of mineralized tissues: Evidence at the 1-10-mm scale.
Biomechanics and Modeling in Mechanobiology, 2, 21–36. https://doi.org/10.1007/s10237-002-0025-9.
Hellmich, C., Barthélémy, J. F., & Dormieux, L. (2004). Mineral-collagen interactions in elasticity of bone ultrastructuredA continuum micromechanics approach. European Journal
of Mechanics - A/Solids, 23, 783–810. https://doi.org/10.1016/j.euromechsol.2004.05.004.
Hengsberger, S., Boivin, G., & Zysset, P. K. (2002). Morphological and mechanical properties of bone structural units: A two-case study. JSME International Journal. Series C,
Mechanical Systems, Machine Elements and Manufacturing, 45, 936–943. https://doi.org/10.1299/jsmec.45.936.
Hengsberger, S., Enstroem, J., Peyrin, F., & Zysset, P. (2003). How is the indentation modulus of bone tissue related to its macroscopic elastic response? A validation study. Journal
of Biomechanics, 36, 1503–1509. https://doi.org/10.1016/S0021-9290(03)00131-3.
Hernandez, C. J., Tang, S. Y., Baumbach, B. M., Hwu, P. B., Sakkee, A. N., van der Ham, F., DeGroot, J., R.A. Bank, & Keaveny, T. M. (2005). Trabecular microfracture and the
influence of pyridinium and non-enzymatic glycation-mediated collagen cross-links. Bone, 37, 825–832. https://doi.org/10.1016/j.bone.2005.07.019.
Hodgskinson, R., Currey, J. D., & Evans, G. P. (1989). Hardness, an indicator of the mechanical competence of cancellous bone. Journal of Orthopaedic Research, 7, 754–758.
https://doi.org/10.1002/jor.1100070518.
Hu, S., Li, J., Liu, L., Dai, R., Sheng, Z., Wu, X., Feng, X., Yao, X., Liao, E., Keller, E., & Jiang, Y. (2015). Micro/nanostructures and mechanical properties of trabecular bone in
ovariectomized rats. International Journal of Endocrinology. https://doi.org/10.1155/2015/252503.
Hunter, G. K., & Goldberg, H. A. (1993). Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences of the United States of America, 90,
8562–8565. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC47397/.
Jäger, I., & Fratzl, P. (2000). Mineralized collagen fibrils: A mechanical model with a staggered arrangement of mineral particles. Biophysical Journal, 79, 1737–1746. https://
doi.org/10.1016/S0006-3495(00)76426-5.
Jee, W. S. S. (2001). Integrated bone tissue physiology: Anatomy and physiology. In S. C. Cowin (Ed.), Bone mechanics handbook (2nd edn, pp. 1–68). CRC Press. https://doi.org/
10.1201/b14263-3.
Jenkins, T., Coutts, L. V., D’Angelo, S., Dunlop, D. G., Oreffo, R. O. C., Cooper, C., Harvey, N. C., Thurner, P. J., & the O.S.E.O. (OStEO) Group. (2016). Site-dependent reference
point microindentation complements clinical measures for improved fracture risk assessment at the human femoral neck. Journal of Bone and Mineral Research, 31, 196–203.
https://doi.org/10.1002/jbmr.2605.
Jenkins, T., Katsamenis, O. L., Andriotis, O. G., Coutts, L. V., Carter, B., Dunlop, D. G., Oreffo, R. O. C., Cooper, C., Harvey, N. C., Thurner, P. J., & The OStEO Group. (2017). The
inferomedial femoral neck is compromised by age but not disease: Fracture toughness and the multifactorial mechanisms comprising reference point microindentation. Journal of
the Mechanical Behavior of Biomedical Materials, 75, 399–412. https://doi.org/10.1016/j.jmbbm.2017.06.036.
Jimenez-Palomar, I., Shipov, A., Shahar, R., & Barber, A. H. (2012). Influence of SEM vacuum on bone micromechanics using in situ AFM. Journal of the Mechanical Behavior of
Biomedical Materials, 5, 149–155. https://doi.org/10.1016/j.jmbbm.2011.08.018.
Jimenez-Palomar, I., Shipov, A., Shahar, R., & Barber, A. H. (2015a). Structural orientation dependent sub-lamellar bone mechanics. Journal of the Mechanical Behavior of
Jimenez-Palomar, I., Shipov, A., Shahar, R., & Barber, A. H. (2015b). Mechanical behavior of osteoporotic bone at sub-lamellar length scales. Frontiers in Materials, 2, 1–7.
Jirousek, O., Nemecek, J., Kytýr, D., Kunecký, J., Zlámal, P., & Doktor, T. (2011). Nanoindentation of trabecular bonedComparison with uniaxial testing of single trabecula.
Chemicke Listy, 105, 668–671.
Jungmann, R., Szabo, M. E., Schitter, G., Yue-Sing Tang, R., Vashishth, D., Hansma, P. K., & Thurner, P. J. (2011). Local strain and damage mapping in single trabeculae during
three-point bending tests. Journal of the Mechanical Behavior of Biomedical Materials, 4, 523–534. https://doi.org/10.1016/j.jmbbm.2010.12.009.
Kadler, K. E., Holmes, D. F., Trotter, J. A., & Chapman, J. A. (1996). Collagen fibril formation. The Biochemical Journal, 316(Pt 1), 1–11.
Katsamenis, O. L., Chong, H. M. H., Andriotis, O. G., & Thurner, P. J. (2013a). Load-bearing in cortical bone microstructure: Selective stiffening and heterogeneous strain
distribution at the lamellar level. Journal of the Mechanical Behavior of Biomedical Materials, 17, 152–165. https://doi.org/10.1016/j.jmbbm.2012.08.016.
Katsamenis, O. L., Jenkins, T., Quinci, F., Michopoulou, S., Sinclair, I., & Thurner, P. J. (2013b). A novel videography method for generating crack-extension resistance curves in
small bone samples. PLoS One, 8. e55641 https://doi.org/10.1371/journal.pone.0055641.
Katsamenis, O. L., Jenkins, T., & Thurner, P. J. (2015). Toughness and damage susceptibility in human cortical bone is proportional to mechanical inhomogeneity at the osteonal-
level. Bone, 76, 158–168. https://doi.org/10.1016/j.bone.2015.03.020.
Katz, J. L., Yoon, H. S., Lipson, S., Maharidge, R., Meunier, A., & Christel, P. (1984). The effects of remodeling on the elastic properties of bone. Calcified Tissue International, 36.
https://doi.org/10.1007/BF02406131.
Kindt, J. H., Thurner, P. J., Lauer, M. E., Bosma, B. L., Schitter, G., Fantner, G. E., Izumi, M., Weaver, J. C., Morse, D. E., & Hansma, P. K. (2007). In situ observation of fluoride-
ion-induced hydroxyapatite-collagen detachment on bone fracture surfaces by atomic force microscopy. Nanotechnology, 18, 1–8.
Kivirikko, K. I., Laitinen, O., & Prockop, D. J. (1967). Modifications of a specific assay for hydroxyproline in urine. Analytical Biochemistry, 19, 249–255. https://doi.org/10.1016/
0003-2697(67)90160-1.
Knapp, H. F., Reilly, G. C., Stemmer, A., Niederer, P., & Knothe Tate, M. L. (2002). Development of preparation methods for and insights obtained from atomic force microscopy of
fluid spaces in cortical bone. Scanning, 24, 25–33. https://doi.org/10.1002/sca.4950240104.
Koester, K. J., Ager, J., & Ritchie, R. O. (2008). The true toughness of human cortical bone measured with realistically short cracks. Nature Materials, 7, 672–677. https://doi.org/
10.1038/nmat2221.
Koester, K. J., Barth, H. D., & Ritchie, R. O. (2011). Effect of aging on the transverse toughness of human cortical bone: Evaluation by R-curves. Journal of the Mechanical Behavior
of Biomedical Materials, 4, 1504–1513. https://doi.org/10.1016/j.jmbbm.2011.05.020.
Kopperdahl, D. L., & Keaveny, T. M. (1998). Yield strain behavior of trabecular bone. Journal of Biomechanics, 31, 601–608. https://doi.org/10.1016/S0021-9290(98)00057-8.
Kuhn, J. L., Goldstein, S. A., Choi, R., London, M., Feldkamp, L. A., & Matthews, L. S. (1989). Comparison of the trabecular and cortical tissue moduli from human iliac crests.
Journal of Orthopaedic Research, 7, 876–884. https://doi.org/10.1002/jor.1100070614.
Lakes, R. (1993). Materials with structure hierarchy. Nature, 361, 511–515. https://doi.org/10.1038/361511a0.
Landis, W. J., Hodgens, K. J., Arena, J., Song, M. J., & McEwen, B. F. (1996). Structural relations between collagen and mineral in bone as determined by high voltage electron
microscopic tomography. Microscopy Research and Technique, 33, 192–202. https://doi.org/10.1002/(SICI)1097-0029(19960201)33:2<192::AID-JEMT9>3.0.CO;2-V.
Laugier, P., Saied, A., Granke, M., & Raum, K. (2013). Quantitative scanning acustic microscopy of bone. In , Vol. 1. Advances in acoustic microscopy and high re solution imaging:
from principles to applications (pp. 207–230). Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA. Chapter 9.
Launey, M. E., Buehler, M. J., & Ritchie, R. O. (2010). On the mechanistic origins of toughness in bone. Annual Review of Materials Research, 40, 25–53. https://doi.org/10.1146/
annurev-matsci-070909-104427.
Lees, S., & Prostak, K. (1988). The locus of mineral crystallites in bone. Connective Tissue Research, 18, 41–54. https://doi.org/10.3109/03008208809019071.
Lees, S., Bonar, L. C., & Mook, H. A. (1984). A study of dense mineralized tissues by neutron diffraction. International Journal of Biological Macromolecules, 6, 321–326.
Lewis, G., & Nyman, J. S. (2008). The use of nanoindentation for characterizing the properties of mineralized hard tissues: State-of-the art review. Journal of Biomedical Materials
Research Part B: Applied Biomaterials, 87B, 286–301. https://doi.org/10.1002/jbm.b.31092.
Lievers, W. B., Poljsak, A. S., Waldman, S. D., & Pilkey, A. K. (2010). Effects of dehydration-induced structural and material changes on the apparent modulus of cancellous bone.
Medical Engineering & Physics, 32, 921–925. https://doi.org/10.1016/j.medengphy.2010.06.001.
Lips, P., Courpron, P., & Meunier, P. J. (1978). Mean wall thickness of trabecular bone packets in the human iliac crest: Changes with age. Calcified Tissue Research, 26, 13–17.
https://doi.org/10.1007/BF02013227.
Litniewski, J. (2005). Determination of the elasticity coefficient for a single trabecula of a cancellous bone: Scanning acoustic microscopy approach. Ultrasound in Medicine &
Biology, 31, 1361–1366. https://doi.org/10.1016/j.ultrasmedbio.2005.06.009.
Lucchinetti, E., Thomann, D., & Danuser, G. (2000). Review micromechanical testing of bone trabeculaedPotentials and limitations. Journal of Materials Science, 35, 6057–6065.
https://doi.org/10.1023/A:1026748913553.
Luczynski, K. W., Steiger-Thirsfeld, A., Bernardi, J., Eberhardsteiner, J., & Hellmich, C. (2015). Extracellular bone matrix exhibits hardening elastoplasticity and more than double
cortical strength: Evidence from homogeneous compression of non-tapered single micron-sized pillars welded to a rigid substrate. Journal of the Mechanical Behavior of
Ma, S., Goh, E. L., Jin, A., Bhattacharya, R., Boughton, O. R., Patel, B., Karunaratne, A., Vo, N. T., Atwood, R., Cobb, J. P., Hansen, U., & Abel, R. L. (2017). Long-term effects of
bisphosphonate therapy: Perforations, microcracks and mechanical properties. Scientific Reports. https://doi.org/10.1038/srep43399.
Masic, A., Bertinetti, L., Schuetz, R., Chang, S.-W., Metzger, T. H., Buehler, M. J., & Fratzl, P. (2015). Osmotic pressure induced tensile forces in tendon collagen. Nature
Communications, 6, 5942. https://doi.org/10.1038/ncomms6942.
Mirzaali, M. J., Jakob Schwiedrzik, J., Thaiwichai, S., Best, J. P., Michler, J., Zysset, P. K., & Wolfram, U. (2015). Mechanical properties of cortical bone and their relationships with
age, gender, composition and microindentation properties in the elderly. Bone, 93, 196–211. https://doi.org/10.1016/j.bone.2015.11.018.
Mitchell, J., & van Heteren, A. H. (2016). A literature review of the spatial organization of lamellar bone. Comptes Rendus Palevol, 15, 23–31. https://doi.org/10.1016/
j.crpv.2015.04.007.
Mori, S., Harruff, R., Ambrosius, W., & Burr, D. B. (1997). Trabecular bone volume and microdamage accumulation in the femoral heads of women with and without femoral neck
fractures. Bone, 21, 521–526. https://doi.org/10.1016/S8756-3282(97)00200-7.
Morin, C., Vass, V., & Hellmich, C. (2017). Micromechanics of elastoplastic porous polycrystals: Theory, algorithm, and application to osteonal bone. International Journal of
Plasticity, 91, 238–267. https://doi.org/10.1016/j.ijplas.2017.01.009.
Mulder, L., Koolstra, J. H., den Toonder, J. M. J., & van Eijden, T. M. G. J. (2007). Intratrabecular distribution of tissue stiffness and mineralization in developing trabecular bone.
Bone, 41, 256–265. https://doi.org/10.1016/j.bone.2007.04.188.
Nalla, R. K., Kruzic, J. J., Kinney, J. H., & Ritchie, R. O. (2005a). Mechanistic aspects of fracture and R-curve behavior in human cortical bone. Biomaterials, 26, 217–231. https://
doi.org/10.1016/j.biomaterials.2004.02.017.
Nalla, R. K., Stolken, J. S., Kinney, J. H., & Ritchie, R. O. (2005b). Fracture in human cortical bone: Local fracture criteria and toughening mechanisms. Journal of Biomechanics,
38, 1517–1525. https://doi.org/10.1016/j.jbiomech.2004.07.010.
Nalla, R. K., Kruzic, J. J., Kinney, J. H., Balooch, M., Ager, J. W., & Ritchie, R. O. (2006). Role of microstructure in the aging-related deterioration of the toughness of human cortical
bone. Materials Science and Engineering: C, 26, 1251–1260. https://doi.org/10.1016/j.msec.2005.08.021.
Norman, T. L., Vashishth, D., & Burr, D. B. (1995). Fracture toughness of human bone under tension. Journal of Biomechanics, 28, 309–320. https://doi.org/10.1016/0021-
9290(94)00069-G.
Nudelman, F., Pieterse, K., George, A., Bomans, P. H. H., Friedrich, H., Brylka, L. J., Hilbers, P. A. J., de With, G., & Sommerdijk, N. A. J. M. (2010). The role of collagen in bone
apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nature Materials, 9, 1004–1009. https://doi.org/10.1038/nmat2875.
Oftadeh, R., Perez-Viloria, M., Villa-Camacho, J. C., Vaziri, A., & Nazarian, A. (2015). Biomechanics and Mechanobiology of trabecular bone: A review. Journal of Biomechanical
Engineering, 137, 108021–1080215. https://doi.org/10.1115/1.4029176.
Oliver, W. C., & Pharr, G. M. (1992). An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of
Materials Research, 7, 1564–1583. https://doi.org/10.1557/JMR.1992.1564.
Oliver, W. C., & Pharr, G. M. (2004). Measurement of hardness and elastic modulus by instrumented indentation: Advances in understanding and refinements to methodology.
Journal of Materials Research, 19, 3–20. https://doi.org/10.1557/jmr.2004.19.1.3.
Orgel, J. P. R. O., Irving, T. C., Miller, A., & Wess, T. J. (2006). Microfibrillar structure of type I collagen in situ. Proceedings of the National Academy of Sciences, 103, 9001–9005.
https://doi.org/10.1073/pnas.0502718103.
Perumal, S., Antipova, O., & Orgel, J. P. R. O. (2008). Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis. Proceedings of the
National Academy of Sciences of the United States of America, 105, 2824–2829. https://doi.org/10.1073/pnas.0710588105.
Peterlik, H., Roschger, P., Klaushofer, K., & Fratzl, P. (2006). From brittle to ductile fracture of bone. Nature Materials, 5, 52–55. https://doi.org/10.1038/nmat1545.
Pouget, E. M., Bomans, P. H. H., Goos, J. A. C. M., Frederik, P. M., de With, G., & Sommerdijk, N. A. J. M. (2009). The initial stages of template-controlled CaCO3 formation
revealed by cryo-TEM. Science, 323, 1455–1458. https://doi.org/10.1126/science.1169434.
Poundarik, A. A., Diab, T., Sroga, G. E., Ural, A., Boskey, A. L., Gundberg, C. M., & Vashishth, D. (2012). Dilatational band formation in bone. Proceedings of the National Academy
of Sciences of the United States of America, 109, 19178–19183. https://doi.org/10.1073/pnas.1201513109.
Poundarik, A. A., Wu, P.-C., Evis, Z., Sroga, G. E., Ural, A., Rubin, M., & Vashishth, D. (2015). A direct role of collagen glycation in bone fracture. Journal of the Mechanical Behavior
of Biomedical Materials, 52, 120–130. https://doi.org/10.1016/j.jmbbm.2015.08.012.
Price, R. I., Lees, S., & Kirschner, D. A. (1997). X-ray diffraction analysis of tendon collagen at ambient and cryogenic temperatures: Role of hydration. International Journal of
Biological Macromolecules, 20, 23–33. https://doi.org/10.1016/s0141-8130(97)01148-3.
Price, P. A., Toroian, D., & Lim, J. E. (2009). Mineralization by inhibitor exclusion: The calcification of collagen with fetuin. The Journal of Biological Chemistry, 284, 17092–17101.
https://doi.org/10.1074/jbc.M109.007013.
Prostak, K. S., & Lees, S. (1996). Visualization of crystal-matrix structure. In situ demineralization of mineralized turkey leg tendon and bone. Calcified Tissue International, 59, 474–
479. https://doi.org/10.1007/s002239900160.
Qiu, S. R., Wierzbicki, A., Orme, C. A., Cody, A. M., Hoyer, J. R., Nancollas, G. H., Zepeda, S., & De Yoreo, J. J. (2004). Molecular modulation of calcium oxalate crystallization by
osteopontin and citrate. Proceedings of the National Academy of Sciences of the United States of America, 101, 1811–1815. https://doi.org/10.1073/pnas.0307900100.
Rajagopal, K., & Srinivasa, A. (2009). On a class of non-dissipative materials that are not hyperelastic. Proceedings of the Royal Society A: Mathematical, Physical and Engineering
Science, 465, 493–500. https://doi.org/10.1098/rspa.2008.0319.
Ramachandran, G., Bansal, M., & Bhatnagar, R. S. (1973). A hypothesis on the role of hydroxyproline in stabilizing collagen structure. Biochimica et Biophysica Acta (BBA) - Protein
Structure, 322, 166–171.
Reilly, D. T., & Burstein, A. H. (1974). The mechanical properties of cortical bone. JBJS, 56. http://journals.lww.com/jbjsjournal/Fulltext/1974/56050/The_Mechanical_Properties_
of_Cortical_Bone.12.aspx.
Reilly, D. T., & Burstein, A. H. (1975). The elastic and ultimate properties of compact bone tissue. Journal of Biomechanics, 8, 393–405.
Reznikov, N., Shahar, R., & Weiner, S. (2014). Three-dimensional structure of human lamellar bone: The presence of two different materials and new insights into the hierarchical
organization. Bone, 59, 93–104.
Rho, J. Y., Ashman, R. B., & Turner, C. H. (1993). Young’s modulus of trabecular and cortical bone material: Ultrasonic and microtensile measurements. Journal of Biomechanics,
26, 111–119. https://doi.org/10.1016/0021-9290(93)90042-D.
Rho, J. Y., Kuhn-Spearing, L., & Zioupos, P. (1998). Mechanical properties and the hierarchical structure of bone. Medical Engineering & Physics, 20, 92–102.
Rho, J. Y., Roy, M. E., Tsui, T. Y., & Pharr, G. M. (1999). Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. Journal of
Biomedical Materials Research. https://doi.org/10.1002/(SICI)1097-4636(199904)45:1<48::AID-JBM7>3.0.CO;2-5.
Ridha, H., & Thurner, P. J. (2013). Finite element prediction with experimental validation of damage distribution in single trabeculae during three-point bending tests. Journal of the
Mechanical Behavior of Biomedical Materials, 27, 94–106. https://doi.org/10.1016/j.jmbbm.2013.07.005.
Ritchie, R. O., Kinney, J. H., Kruzic, J. J., & Nalla, R. K. (2005). A fracture mechanics and mechanistic approach to the failure of cortical bone. Fatigue and Fracture of Engineering
Materials and Structures, 28, 345–371. https://doi.org/10.1111/j.1460-2695.2005.00878.x.
Ritchie, R. O., Koester, K. J., Ionova, S., Yao, W., Lane, N. E., & Ager, J. W. (2008). Measurement of the toughness of bone: A tutorial with special reference to small animal studies.
Bone, 43, 798–812. https://doi.org/10.1016/j.bone.2008.04.027.
Roschger, P., Paschalis, E. P., Fratzl, P., & Klaushofer, K. (2008). Bone mineralization density distribution in health and disease. Bone, 42, 456–466. https://doi.org/10.1016/
j.bone.2007.10.021.
Rozental, T. D., Walley, K. C., Demissie, S., Caksa, S., Martinez-Betancourt, A., Parker, A. M., Tsai, J. N., Yu, E. W., & Bouxsein, M. L. (2017). Bone material strength index as
measured by impact microindentation in postmenopausal women with distal radius and hip fractures. Journal of Bone and Mineral Research. https://doi.org/10.1002/jbmr.3338.
Runkle, J. C., & Pugh, J. (1975). The micro-mechanics of cancellous bone. II. Determination of the elastic modulus of individual trabeculae by a buckling analysis. Bulletin of the
Hospital for Joint Diseases, 36, 2–10.
Ryan, S. D., & Williams, J. L. (1989). Tensile testing of rodlike trabeculae excised from bovine femoral bone. Journal of Biomechanics, 22, 351–355. https://doi.org/10.1016/0021-
9290(89)90049-3.
Sasaki, N., Tagami, A., Goto, T., Taniguchi, M., Nakata, M., & Hikichi, K. (2002). Atomic force microscopic studies on the structure of bovine femoral cortical bone at the collagen
fibril-mineral level. Journal of Materials Science. Materials in Medicine, 13, 333–337.
Schwiedrzik, J., Raghavan, R., Bürki, A., LeNader, V., Wolfram, U., Michler, J., & Zysset, P. (2014). In situ micropillar compression reveals superior strength and ductility but an
absence of damage in lamellar bone. Nature Materials, 13, 740–747. https://doi.org/10.1038/nmat3959.
Schwiedrzik, J., Taylor, A., Casari, D., Wolfram, U., Zysset, P., & Michler, J. (2017). Nanoscale deformation mechanisms and yield properties of hydrated bone extracellular matrix.
Acta Biomaterialia, 60, 302–314. https://doi.org/10.1016/j.actbio.2017.07.030.
Seref-Ferlengez, Z., Basta-Pljakic, J., Kennedy, O. D., Philemon, C. J., & Schaffler, M. B. (2014). Structural and mechanical repair of diffuse damage in cortical bone in vivo. Journal
of Bone and Mineral Research, 29, 2537–2544. https://doi.org/10.1002/jbmr.2309.
Seref-Ferlengez, Z., Kennedy, O. D., & Schaffler, M. B. (2015). Bone microdamage, remodeling and bone fragility: how much damage is too much damage[quest]. BoneKEy Reports,
4. https://doi.org/10.1038/bonekey.2015.11.
Silva, M. J., Brodt, M. D., Fan, Z., & Rho, J.-Y. (2004). Nanoindentation and whole-bone bending estimates of material properties in bones from the senescence accelerated mouse
SAMP6. Journal of Biomechanics, 37, 1639–1646. https://doi.org/10.1016/j.jbiomech.2004.02.018.
Skedros, J. G., Holmes, J. L., Vajda, E. G., & Bloebaum, R. D. (2005). Cement lines of secondary osteons in human bone are not mineral-deficient: New data in a historical
perspective. The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology, 286A, 781–803. https://doi.org/10.1002/ar.a.20214.
Sodek, J., Ganss, B., & McKee, M. D. (2000). Osteopontin. Critical Reviews in Oral Biology and Medicine, 11, 279–303. https://doi.org/10.1177/10454411000110030101.
Spiesz, E. M., Reisinger, A. G., Kaminsky, W., Roschger, P., Pahr, D. H., & Zysset, P. K. (2013). Computational and experimental methodology for site-matched investigations of the
influence of mineral mass fraction and collagen orientation on the axial indentation modulus of lamellar bone. Journal of the Mechanical Behavior of Biomedical Materials, 28,
195–205. https://doi.org/10.1016/j.jmbbm.2013.07.004.
Sroga, G. E., Karim, L., Colón, W., & Vashishth, D. (2011). Biochemical characterization of major bone-matrix proteins using nanoscale-size bone samples and proteomics
methodology. Molecular & Cellular Proteomics, 10, M110.006718. https://doi.org/10.1074/mcp.M110.006718.
Standring, S. (2016). Gray’s anatomy: The anatomical basis of clinical practice. Elsevier. https://books.google.at/books?id¼LjP9rQEACAAJ.
Starborg, T., Kalson, N. S., Lu, Y., Mironov, A., Cootes, T. F., Holmes, D. F., & Kadler, K. E. (2013). Using transmission electron microscopy and 3View to determine collagen fibril
size and three-dimensional organization. Nature Protocols, 8, 1433–1448. https://doi.org/10.1038/nprot.2013.086.
Sundh, D., Rudang, R., Zoulakis, M., Nilsson, A. G., Darelid, A., & Lorentzon, M. (2016). A high amount of local adipose tissue is associated with high cortical porosity and low bone
material strength in older women. Journal of Bone and Mineral Research, 31, 749–757. https://doi.org/10.1002/jbmr.2747.
Svensson, R. B., Hassenkam, T., Grant, C. A., & Magnusson, S. P. (2010). Tensile properties of human collagen fibrils and fascicles are insensitive to environmental salts.
Biophysical Journal, 99, 4020–4027. https://doi.org/10.1016/j.bpj.2010.11.018.
Swadener, J. G., Rho, J.-Y., & Pharr, G. M. (2001). Effects of anisotropy on elastic moduli measured by nanoindentation in human tibial cortical bone. Journal of Biomedical
Materials Research, 57, 108–112. https://doi.org/10.1002/1097-4636(200110)57:1<108::AID-JBM1148>3.0.CO;2-6.
Szabó, M. E., & Thurner, P. J. (2013). Anisotropy of bovine cortical bone tissue damage properties. Journal of Biomechanics, 46, 2–6. https://doi.org/10.1016/
j.jbiomech.2012.08.002.
Szabó, M. E., Taylor, M., & Thurner, P. J. (2011). Mechanical properties of single bovine trabeculae are unaffected by strain rate. Journal of Biomechanics, 44, 962–967. https://
doi.org/10.1016/j.jbiomech.2010.12.008.
Tang, S. Y., Zeenath, U., & Vashishth, D. (2007). Effects of non-enzymatic glycation on cancellous bone fragility. Bone, 40, 1144–1151. https://doi.org/10.1016/
j.bone.2006.12.056.
Thurner, P. J. (2009). Atomic force microscopy and indentation force measurement of bone. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology, 1, 624–649.
https://doi.org/10.1002/wnan.56.
Thurner, P. J., Chen, C. G., Ionova-Martin, S., Sun, L., Harman, A., Porter, A., Ager, J. W., 3rd, Ritchie, R. O., & Alliston, T. (2010). Osteopontin deficiency increases bone fragility
but preserves bone mass. Bone, 46, 1564–1573. https://doi.org/10.1016/j.bone.2010.02.014.
Townsend, P. R., Rose, R. M., & Radin, E. L. (2017). Buckling studies of single human trabeculae. Journal of Biomechanics, 8, IN5–IN6. https://doi.org/10.1016/0021-9290(75)
90025-1.
Turner, C. H. (1989). Yield behavior of bovine Cancellous bone. Journal of Biomechanical Engineering, 111, 256–260. https://doi.org/10.1115/1.3168375.
Turner, C. H., Rho, J., Takano, Y., Tsui, T. Y., & Pharr, G. M. (1999). The elastic properties of trabecular and cortical bone tissues are similar: Results from two microscopic
measurement techniques. Journal of Biomechanics. https://doi.org/10.1016/S0021-9290(98)00177-8.
Tye CE, Hunder GK, and Goldberg HA. Identification of the type I collagen-binding domain of bone Sialoprotein and characterization of the mechanism of interaction. The Journal of
Biological Chemistry 280(14): 13487–13492.
Uppuganti, S., Granke, M., Manhard, M. K., Does, M. D., Perrien, D. S., Lee, D. H., & Nyman, J. S. (2017). Differences in sensitivity to microstructure between cyclic- and impact-
based microindentation of human cortical bone. Journal of Orthopaedic Research, 35, 1442–1452. https://doi.org/10.1002/jor.23392.
Vashishth, D., Koontz, J., Qiu, S. J., Lundin-Cannon, D., Yeni, Y. N., Schaffler, M. B., & Fyhrie, D. P. (2000). In vivo diffuse damage in human vertebral trabecular bone. Bone, 26,
147–152. https://doi.org/10.1016/S8756-3282(99)00253-7.
Vesentini, S., Redaelli, A., & Gautieri, A. (2013). Nanomechanics of collagen microfibrils. Muscle, Ligaments and Tendons Journal, 3, 23–34. https://doi.org/10.11138/mltj/
2013.3.1.023.
Wagermaier, W., Gupta, H. S., Gourrier, A., Burghammer, M., Roschger, P., & Fratzl, P. (2006). Spiral twisting of fiber orientation inside bone lamellae. Biointerphases, 1, 1–5.
https://doi.org/10.1116/1.2178386.
Wang, X., Sudhaker Rao, D., Ajdelsztajn, L., Ciarelli, T. E., Lavernia, E. J., & Fyhrie, D. P. (2008). Human iliac crest cancellous bone elastic modulus and hardness differ with bone
formation rate per bone surface but not by existence of prevalent vertebral fracture. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 85B, 68–77. https://
doi.org/10.1002/jbm.b.30918.
Weaver, J. K. (1966). The microscopic hardness of bone. The Journal of Bone & Joint Surgery, 48, 273–288.
Weiner, S., & Wagner, H. D. (1998). THE MATERIAL BONE: Structure-mechanical function relations. Annual Review of Materials Science, 28, 271–298.
Yamada, S., Tadano, S., & Fukuda, S. (2014). Nanostructure and elastic modulus of single trabecula in bovine cancellous bone. Journal of Biomechanics, 47, 3482–3487. https://
doi.org/10.1016/j.jbiomech.2014.09.009.
Yeni, Y. N., & Norman, T. L. (2000a). Calculation of porosity and osteonal cement line effects on the effective fracture toughness of cortical bone in longitudinal crack growth.
Journal of Biomedical Materials Research, 51, 504–509. https://doi.org/10.1002/1097-4636(20000905)51:3<504::AID-JBM27>3.0.CO;2-I.
Yeni, Y. N., & Norman, T. L. (2000b). Fracture toughness of human femoral neck: Effect of microstructure, composition, and age. Bone, 26, 499–504. https://doi.org/10.1016/
S8756-3282(00)00258-1.
Zappone, B., Thurner, P. J., Adams, J., Fantner, G. E., & Hansma, P. K. (2008). Effect of Ca(2 þ) ions on the adhesion and mechanical properties of adsorbed layers of human
osteopontin. Biophysical Journal, 95, 2939–2950. https://doi.org/10.1529/biophysj.108.135889.
Zebaze, R. M. D., Jones, A., Welsh, F., Knackstedt, M., & Seeman, E. (2005). Femoral neck shape and the spatial distribution of its mineral mass varies with its size: Clinical and
biomechanical implications. Bone, 37, 243–252. https://doi.org/10.1016/j.bone.2005.03.019.
Zysset, P. K. (2009). Indentation of bone tissue: A short review. Osteoporosis International. https://doi.org/10.1007/s00198-009-0854-9.
Zysset, P. K., Edward Guo, X., Edward Hoffler, C., Moore, K. E., & Goldstein, S. A. (1999). Elastic modulus and hardness of cortical and trabecular bone lamellae measured by
nanoindentation in the human femur. Journal of Biomechanics, 32, 1005–1012. https://doi.org/10.1016/S0021-9290(99)00111-6.
Further Reading
Currey, J. D. (2002). Bones: Struture and mechanics. Princeton: Princeton University Press. ISBN: 9781400849505.
Fratzl, P. (2008b). Collagen: Structure and mechanics. New York: Springer. https://doi.org/10.1007/978-0-387-73906-9.
Reznikov, N., Shahar, R., & Weiner, S. (2014). Bone hierarchical structure in three dimensions. Acta Biomaterialia, 10, 3815–3826. https://doi.org/10.1016/j.actbio.2014.05.024.
Rosen, C. J., Bouillon, R., Compston, J. E., & Rosen, V. (2013). Primer on the metabolic bone diseases and disorders of mineral metabolism. Wiley. https://doi.org/10.1016/
S0021-9290(00)00074-9.
Cell Adhesion: Basic Principles and Computational Modeling
Diego A Vargas and Hans Van Oosterwyck, Biomechanics Section, KU Leuven, Heverlee, Belgium
Introduction 45
Basics of Cell Adhesion 46
Mechanotransduction 46
The Bell Model: Motivation Behind a First Model 47
Transcending Scales: The Subcellular and Multicellular Directions 48
The Integrin–Ligand Bond in More Detail 49
The Impact of Force Spectroscopy 51
Zooming In: Other Players at the Molecular Scale 52
Zooming Out: Cell Aggregates, Cell Sheets, Tissues 52
Refining Understanding Through Experiments 54
Imparting Forces at a Cellular Scale 54
Measuring Forces and Imaging at a Cellular Scale 56
Challenges and Conclusions 56
Further Reading 58
Glossary
Actomyosin contractility An actin network’s ability to contract due to pulling of myosin on actin filaments in the network
through a chemical-energy spending mechanism.
Agent-based model Phenomenological model that treats cells as individual units interacting according to a set of rules.
Apical domain Refers to cellular region opposite to the basal lamina in an epithelial layer. Physiologically it is exposed to
a lumen. It is rich in junctional proteins, making it distinct compositionally, structurally, and functionally from the lateral and
basal domains.
Cellularized materials Cell aggregate connected by intercellular junctions that interconnect internal cytoskeletons. Includes
cell sheets, cysts, amorphous aggregates, cancerous tumors, etc.
Continuum model Model in which matter is represented as a continuous homogeneous substance. Continuum models are
often deterministic in nature and defined by a set of analytic equations.
Force spectroscopy Category of techniques that measure forces in biological or molecular systems.
Glycocalyx Carbohydrate-rich layer connected to the cell membrane through proteoglycans and glycoproteins.
Ground-state configuration Configuration of a system characterized by the lowest possible energy value.
Mechanosensitivity Ability to sense a mechanical stimulus.
Mechanotransduction Process of converting a mechanical signal into a chemical signal.
Multiscale model Model that integrates processes that occur at different temporal or spatial scales.
Introduction
As a physical system, a cell’s interaction with its environment is dictated by electrical, chemical, and mechanical interactions of the
different components in the cell. Its response in turn affects interaction of the cell with the outside through feedback mechanisms.
The interface of the cell with its environment is its plasma membrane, rich in proteins that mediate this signal transduction. Those
involved in transduction of signals are transmembrane proteins that create a bridge between the extracellular and intracellular
worlds; however, these constitute only one piece of the puzzle. In the case of mechanical signals, these proteins will transmit forces
inside the cell to the cytoskeleton, the structure responsible for the shape and deformation of the cell. On the outside these proteins
bind the structural elements of the environment or other cells. This is not a straightforward process though. On the outside of the
cell, these proteins are modified changing how they interact with the environment. While inside, they do not bind the cytoskeleton
directly; multiple proteins make up this connection, many of them with their own complex and dynamic structures.
A study of cell adhesion must account for chemical as well as mechanical interactions of the cell and cellular components. Chem-
ical interactions have been studied for cellular components extensively using traditional binding kinetics assays. These experiments,
however, looked at proteins in isolation. Mechanical studies on the other hand are relatively recent and initially suffered from a para-
doxical limitation, they could only quantify the response of a cell as a whole. A theoretical approach through mathematical

46 Biomechanics j Cell Adhesion: Basic Principles and Computational Modeling
modeling was able to bridge this gap. Since the development of the first theoretical model, the different pieces that had been uncov-
ered by biochemists (e.g., diffusion rates along the plasma membrane and binding kinetics of protein pairs) were put together to
explain adhesion at the cell level. These initial theoretical studies were able to predict the existence of adhesion dynamics later
discovered, such as the catch bond in rolling leukocytes in our immune system.
Computational modeling has only expanded the use of theoretical models to study cell adhesion. The ability to use numerical
analysis as well as increased computational power has permitted the use of increasingly comprehensive models. This has allowed
computational modeling to keep up with the advancement of experimental techniques, playing the role of an equal partner in
development of model-driven hypotheses. Modeling techniques from different fields ranging from N-body simulations to
continuum mechanics have been used to mechanically model from a single adhesion receptor, through an entire cell, to cell
aggregates.
Basics of Cell Adhesion

In an organism, most cells are found in a scaffold of proteins and polysaccharides known as the extracellular matrix (ECM). The
exact composition of the ECM is tissue-dependent: it is heterogeneous in composition, and its function ranges from providing struc-
tural integrity and aiding in tissue organization to signaling, guiding cell survival, migration, proliferation, and differentiation, and
survival. The primary structural components of the ECM are fibrous proteins, which include collagen, elastin, fibronectin, and lam-
inin. Collagen, found in high abundance in many tissues, is a protein that self-assembles triple-helical molecules into thicker fibrils.
It is an adhesive component of the ECM, meaning cells can attach to it. The space in between the multiple proteins is occupied by
hydrophilic glycosaminoglycans and water. Based on its composition, mechanical properties of the ECM change, providing a series
of binding sites and a barrier to free diffusion of signaling molecules.
Integrins constitute the principal transmembrane adhesion molecule. An integrin receptor is active as a heterodimer, formed by
selective pairing of 1 of 18 known a-subunits and 1 of 8 known b-subunits, producing 24 distinct receptors. Named aptly, these
receptors integrate the intracellular and extracellular environments by linking the cytoskeleton to the ECM and provide a bidirec-
tional signaling path. Integrins bind specific amino acid sequences in some of the ECM fibrous proteins, notably the RGD motif
found in fibronectin, laminin, and, under some conditions, collagen. Integrins are the molecule modeled the most in computa-
tional studies. Nonetheless, it is not alone in forming an adhesion complex. The complete transmembrane macromolecular
complex is known as a focal adhesion (FA); and it is formed upon maturation of the nascent complex formed when integrins
bind a ligand in the ECM.
Similarly, intercellular adhesions consist of protein complexes with a transmembrane protein at their core. Based on their
composition, different types of intercellular adhesions exist in different tissues; these have differing degrees of strength and are
involved differently in tissue organization and signaling. These include gap junctions, tight junctions, adherens junctions (AJs),
and desmosomes. Gap junctions provide communication for chemical and osmotic regulation of the intracellular environment.
In a complementary role, tight junctions have a barrier function, preventing lateral diffusion of molecules in the extracellular space
between adhered cells, allowing for distinct chemical environments to be maintained. AJs and desmosomes anchor cells and
provide mechanical communication between cells. Further, desmosomes form patches that link intercellular junctions to interme-
diate filaments, while AJs exist along the cell–cell boundary and bind to the actin cytoskeleton to form adhesion belts. In both AJs
and desmosomes, members of the cadherin protein family bind to each other on opposing cells, forming homotypic bonds. Based
on the tissue type, different members of the cadherin family are found. This family includes N-cadherin, common in neural tissues,
P-cadherin, originally found in the placenta but increasingly in other tissues, VE-cadherin in endothelial cells, and E-cadherin in
epithelium.
Cadherins consist of a cytoplasmic domain, a transmembrane domain, and an extracellular region comprising multiple ectodo-
mains. Along with cadherin, AJs also include catenin molecules. Catenin molecules are the link between AJs with the cytoskeleton.
The individual or joint presence of the different catenin molecules in AJs indicates stability and maturity of junctions. b-catenin, g-
catenin, and p120-catenin can bind cadherin, with g-catenin substituting b-catenin as a junction matures. a-Catenin can bind either
b-catenin or g-catenin and the membrane–cytoskeletal protein vinculin: vinculin in turn binds actin, as it occurs in FAs, demon-
strating a similarity between mechanisms of cell–cell and cell–ECM adhesion.
Mechanotransduction
The structural commonalities between FAs and AJs are evident: they both share the structural components, bind the actin cytoskel-
eton, carry mechanical signals from the outside to the inside of the cell, and transform a mechanical stimulus into a chemical one.
The latter process is known as mechanotransduction. Thus the role of mechanical stimulation of cells has implications not only in
the shape and structural conformation of the cells, but also in biological processes. The targets of mechanotransduction include
regulatory proteins involved in determining cell growth, proliferation, differentiation, apoptosis, and organogenesis.
There is extensive evidence of the role of FAs and AJs in mechanotransduction. For example, FAs only mature once the cytoskel-
eton is bound, at which point integrins cluster increasing the size of the FA. Sufficient force is necessary for the FA to mature and
become stable; otherwise, the initial complex between integrins and the ECM will disintegrate.
Proteins with mechanosensitive functions include talin, vinculin, p130Cas, zyxin, and filamin A. The cytoskeleton plays the
biggest role. For example, tension on a FA could alter the conformation of scaffolding proteins; upon being stretched, talin reveals
Biomechanics j Cell Adhesion: Basic Principles and Computational Modeling 47
a binding site for vinculin which leads to the interaction of these proteins. In the case of p130Cas, tension increases its phosphor-
ylation status, which in turn activate multiple Rho GTPases (including Rac) involved in proliferation, differentiation, cell–cell adhe-
sion, and migration. Zyxin accumulates in areas of the actin fibers with large strains and recruits proteins involved in fiber repair.
Once a FA has reached maturity, it will activate additional proteins such as focal adhesion kinase (FAK), which is part of intracellular
signaling pathways involved in differentiation and migration. Tension on AJs also activates FAK, thus creating an interplay between
FAs and AJs.
These observations make it clear that a study of cell adhesion must incorporate chemical kinetics as well as mechanics. This was
observed in the first theoretical models and maintained through computational models.
The Bell Model: Motivation Behind a First Model
George Bell introduced in 1978 the first theoretical framework for the study of cell adhesion by considering spatial limitations (e.g.,
molecular orientation, membrane diffusion) in the reversible binding of a receptor and a ligand as well as the potential diversity of
molecular mechanisms that could exist. Although cells were known to have an electric charge, observations had made it evident that
nonspecific binding due to electrical forces are less relevant than specific-binding forces. The model is built from the assumption of
specific binding. The first steps in building a model were to define three aspects of the adhesion molecules:
1. Binding affinity: Described by rates of bond formation and breaking from elementary rate constants
2. Number of binding molecules: The number of receptors per unit surface area of the plasma membrane
3. Mobility along the membrane: Described by the rates of diffusion of the receptors along the membrane
Although Bell applied the same principles to cell–cell and cell–substrate adhesion, here the example of two adjacent cells having
complementary receptors is presented. The reaction is conceptually separated into two processes: The two adhesion molecules in
two neighboring cells encounter each other by coming into proximity (i.e., diffusion along the corresponding cell membranes)
and then create the adhesion complex. This initial encounter is modeled through conception of an intermediate “encounter”
complex, as in enzyme kinetics models (i.e., Michaelis–Menten). This reaction was written by Bell as:
dm
þ rþ
N1f þ N2f % N1 N2 % Nb (1)
dm
r
N1 and N2 are the total number of receptors per unit area (i.e., density) of membrane of cells 1 and 2, respectively. Nif is the
density of free receptors in cell i (i ¼ 1,2), N1N2 the encounter complex, and Nb the density of bound receptors. Given our current
knowledge of adhesion complexes, N1f and N2f can be taken to represent cadherins, and consequently, Nb represents an immature
AJ.
The rates d þ m and d m represent the rates of formation and dissolution of the encounter complex in a membrane, respectively,
and are thus dependent on the translational diffusion constants for free receptor motion in the membrane (Dm(Nif)) and an
encounter distance at which the molecules can form an encounter complex (R1,2):

dm
þ ¼ 2p Dm N1f þ Dm N2f (2)
2
¼ 2 Dm N1f þ Dm N2f R1;2
dm (3)
The rates rþ and r represent the forward and reverse rate constants for formation of the adhesion complex (Nb) from the
encounter complex (N1N2). Under assumption that the encounter complex is transient and exists in a very small concentration rela-
tive to the unbound concentrations of the receptors (N1f, N2f) and the adhesion complex (Nb), then by setting dN1N2/dt ¼ 0, Eq. (1)
can be simplified to:
kþ
N1f þ N2f % Nb (4)
k
The rate constants for this overall reaction of cell–cell adhesion formation (kþ, k) can be described in terms of the rate constants
of the intermediate steps:
dm
þ rþ
kþ ¼ (5)
þ rþ
dm
dm
r
k ¼ (6)
þ rþ
dm
Since the encounter complex is assumed to exist in small concentration, then Ni ¼ Nif þ Nb (i ¼ 1,2). The rate of adhesion
complex formation is shown to be:
dNb
¼ kþ ðN1 Nb ÞðN2 Nb Þ k Nb (7)
dt
This equation provided a theoretical description of the temporal evolution of adhesion between cells with a homogeneous distri-
bution and a constant number of adhesion molecules. It also provided an equilibrium concentration of cell–cell adhesions depend-
ing on binding dynamics. Even though up to that point the proposed model was novel and revealing, it was solely based on
diffusion and chemical kinetics. At the time Bell wrote this, there were already experiments that demonstrates the relevance of
mechanics on cell adhesion. Experiments would track how many cells (lymphocytes or fibroblasts) would remain attached to
lectin-coated fibers when shaken (with specified amplitude and frequency) in a fluid. Bell went on to consider adhesion force
and provided a way to couple forces and chemical kinetics.
Bell theorized that a force must be present to separate two cells: the probability of all adhesion complexes, which keep two cells
together, dissociating simultaneously is extremely small. To consider the effect of force on the rate of bond formation Bell postu-
lated that kinetic theory of the strength of solids could be applied to receptor–ligand bonds. In this theory, the lifetime of a bond
(between atoms in a solid) as a function of the force applied on the bond (f) is given by:
sð f Þ ¼ s0 exp½ ðgf E0 Þ=kT (8)
The lifetime of a bond (once formed) is dependent on a natural lifetime of the bond (s0) dependent on the frequency of oscil-
lation of atoms, the energy of a single bond (E0), and is scaled by the Boltzmann constant and temperature (kT). Additionally, g is
an empirical parameter (with units of length) accounting for structure of the solid. The negative exponent shows how increased
force reduces the lifetime of the bond (this behavior is used to classify this bond as a slip bond).
This equation can be integrated into Eq. (7) to be used in the context of cell adhesion by identifying that the inverse of the life-
time describes the rate of dissociation (i.e., s(f ¼ 0) ¼ (k) 1). Also, the force separating two cells (F) is taken to be distributed
equally between all adhesion complexes, therefore (f ¼ F/Nb). The resulting equation is:

dNb gF
¼ kþ ðN1 Nb ÞðN2 Nb Þ k Nb exp (9)
dt kT Nb
This is the first equation to take into account the effect of force applied on the cell in adhesion. Beyond this Bell mentioned other
more temporary factors that may affect binding, he termed these transients. In a display of premonitory abilities, Bell considered
transients such as the adsorption properties of ligand molecules for the case of cell–substrate adhesion, orientation of adhesion
molecules with respect to each other when they first encounter each other, and glycosylation of membrane proteins. All of these
transients would be explored in future studies by other researchers. In this way Bell’s work could be considered visionary. As
will be presented, today most models of adhesion are very specific to a cell type under specific conditions; nonetheless, there is
Bell to thank for the underlying theory.
Transcending Scales: The Subcellular and Multicellular Directions
In a fully formed organ, it is not difficult to conceive that the stroma and the parenchyma (structural and functional tissue, respec-
tively) would have different mechanical properties and that this fact plays a functional role. Given that many cells are sensitive to
force, do the mechanical properties of the distinct tissue determine the functionality of cells composing it, or alternatively, are
mechanical properties of the tissue a consequence of the identity or role of cells present? Given what we know about mechanotrans-
duction, both possibilities are true. Observations in developmental processes such as delamination of the neural crest or metastasis
in cancerous tumors, have given credit to the theory that differential adhesion of cells in tissue can be attributed the emergence of
supracellular organization (differential adhesion hypothesis or DAH). During these processes of cellular rearrangement, the same
cell can go from stiff and strongly attached to soft and detached. Further analysis of these processes revealed that there are distinct
molecular signatures of cells associated with the different cellular behaviors and ability to adhere.
Moving forward, it will become evident how after Bell’s seminal study theorizing about a single cell binding a surface or two cells
binding each other (referred to as cellular scale), researchers started thinking about the effects of mechanical properties at smaller and
larger spatial scales. At the subcellular scale, the mechanical properties of the plasma membrane or the response to force of single
molecules in the adhesion complex were explored and incorporated in cell adhesion models. The mechanics of subcellular elements
are considered all the way down to the atomic scale: although not considered here, steered molecular dynamics simulations look at
the effect of forces on single adhesion molecules using force fields to simulate the change in protein configuration with force
application.
In the opposite direction, there is the multicellular scale. Models of multiple cells, either forming a monolayer or a 3D mass, have
been developed explicitly incorporating cellular adhesion. These models can be used to study the mechanical (and functional)
properties of tissue.
The Integrin–Ligand Bond in More Detail

Dembo and colleagues set up a model to describe adhesion dynamics in a particular theoretical experiment, peeling a cell (or more
precisely a fragment of membrane) from a substrate, just as a strip of tape is peeled off a surface (Fig. 1). This “peel test” is an empir-
ical test used to assess the effectiveness of adhesives in industrial applications. Their purpose was to use the peel test as a means of
scientific exploration of biological adhesion and develop an analytical expression for steady-state peeling velocity for a biological
system (i.e., cell membrane). To do this, the equations for deformation of an elastic membrane are coupled with the equations for
the chemical kinetics of the adhesion molecules. This contrasts the Bell approach by including mechanical properties of the
membrane and describing the change in binding energy not just in terms of potential difference of bond breaking but mechanical
parameters of the adhesion molecule.
Fig. 1 shows the setup for the theoretical experiment. Every point in the membrane is tracked by using an arc length coordinate s:
the clamped end is designated by (s / -N) and the end being pulled by (s / þN). The point at which attachment begins is desig-
nated by (s ¼ 0), and the peeling velocity (Vpl) is defined in terms of the change in position of this point of detachment. As peeling is
a time-dependent process, the shape of the membrane at a time t is taken relative to the point at which the membrane detaches
(s ¼ 0). Thus the position of the membrane is described in Cartesian coordinates by x(s,t) and y(s,t); the x-axis is the surface of
attachment, and the y-axis describes the distance of the membrane to the surface. Tension applied on the free end of the membrane
is designated Tfx, and it is applied at an angle qfx with respect to the surface.
At each point along the length of the membrane, the density of adhesion molecules is considered: Nb(s,t) (in keeping with the
notation used to present the Bell model). Consequently, it can be stated that for the every point in the membrane: N1 ¼ N1f(s,t) þ
Nb(s,t). Because here the cell is attached to a surface, and not to a second cell, adhesion is assumed to be dependent solely on the
presence of the adhesion molecule on the membrane (i.e., no ligand molecule considered):
kþ ðyÞ
N1f ðs; t Þ % Nb ðs; t Þ (10)
k ðyÞ
Eq. (7) is modified accordingly:
vNb ðs; t Þ
¼ kþ ðyÞN1 ½kþ ðyÞ þ k ðyÞNb ðs; t Þ (11)
vt
In contrast to Eq. (7), Eq. (11) shows the rate constants for the overall reaction as a function of the distance between the
membrane and the surface (given by coordinate y(s,t)). Because adhesions are modeled as Hookean springs, Dembo and colleagues
Fig. 1 Schematic describing hypothetical setup modeled by Dembo et al. in which a fragment of cell membrane is peeled from a surface by
applying a tension Tfx on one end of the membrane while the opposite end is clamped. The membrane is attached to a surface via adhesion proteins
that have the mechanical characteristics of a Hookean spring and adhesion rates described by chemical kinetics (Bell model). The surface is taken to
be in the x-axis, while the y-axis describes the distance of the membrane from the surface. Tension is applied at a particular angle to this axis (qfx).
Reproduced from Dembo, M. et al. (1988). The reaction-limited kinetics of membrane-to-surface adhesion and detachment. Proceedings of the Royal
Society B: Biological Sciences 234, 55–83.
state that for thermodynamic consistency one must consider the change in a bond’s Gibbs free energy with the bond stress. The
relation between energy and stress is derived from the dependence on the bond’s free energy on bond strain:
Eh ¼ E0 þ 0:5kðy l0 Þ2 (12)
E0 represents the Gibbs free energy of an unstretched spring (same quantity as an Eq. 8), k is the Hookean spring constant, and l0
represents the resting length.
In determining the rate constants, Dembo and colleagues consider the existence of a transition state in bond formation. They
make two assumptions: First, the free energy of a molecule that is not attached is constant and independent of the distance to
its ligand, and second, the transition state is also described by a Hookean spring and thus has the same form of free energy (Eq.
12). In this way they apply the transition state theory and arrive at the following rate constants:

0:5kts ðy l0 Þ2
kþ ðyÞ ¼ kþ ðl0 Þexp (13)
kT

0:5ðk kts Þðy l0 Þ2
k ðyÞ ¼ k ðl0 Þexp (14)
kT
kts is the spring constant of the transition state, in contrast to that of the formed bond (k). Theoretically, this raised the possibility
of the existence of a bond for which the rate of disruption under tension is lower than when unstressed (this behavior is used to
classify this bond as a catch bond). This occurs when k < kts. These bonds would be directly observed much later in leukocytes under
various degrees of force in microfluidic chambers.
In fulfilling their objective of analytically describing peeling velocity, Eq. (11) is added a convection term describing the loss of
adhesion molecules due to peeling:
vNb ðs; t Þ vN ðs; t Þ
¼ Vpl b þ kþ ðyÞN1 ½kþ ðyÞ þ k ðyÞNb ðs; t Þ (15)
vt vs
The researchers go on to assume steady-state (v Nb(s,t)/vt ¼ 0), expanding Nb(s,t) as a power series in Vpl, and substitute with Eqs.
(13) and (14). This provides the following relation:

Nb ðs; t Þ ¼ ab0 þ ab1 Vpl þ ab1 Vpl 2 . ¼ ab0 þ O Vpl (16a)
h 2i
0Þ
N1 Keq exp 0:5kðyl
kT kþ ðl0 Þ
ab0 ¼ h i ; Keq ¼ (16b)
1 þ Keq exp
0:5kðyl0 Þ2 k ðl0 Þ
kT
Dembo and colleagues separate the tangential and normal components to stress for the Hookean springs along the membrane.
The tangential component, scaled by the density of adhesions at the specific location along the membrane, is given by:
vy
stan ¼ Nb ðs; t Þ k ðy l0 Þ (17)
vs
They then proceed to take advantage of another definition of tangential stress that uses the mechanical properties of the
membrane itself, specifically the modulus of bending (Mb):

v T þ 0:5Mb C2
stan ¼ (18)
vs
C represents the curvature of the membrane and T the tension. Finally, setting Eqs. (17) and (18) equal to each other and
substituting Nb(s,t) with the relation (16a and b), Dembo and colleagues arrive at the analytical description of the peeling velocity
in terms of both mechanical properties of the membrane and adhesion molecules:

vy v T þ 0:5Mb C2
O Vpl ¼ ab0 kðy l0 Þ (19)
vs vs
As a premise to their approach, the researchers reject the concept of using a continuous adhesive because of the relatively low
number of adhesion molecules found in a cell. They thus provide a way to spatially and temporally describe adhesion while
accounting for properties of the different components, in their case membrane and adhesion molecules. However, as discussed
previously, bonds between cells or between a cell and a substrate are not formed by a single pair of molecules, but they are
dynamic molecular complexes. Their composition may change spatially along the cellular surface, and in turn change the
mechanical properties of the membrane. They can also not only bind to similar complexes on other cells or surfaces (heterotypic
binding), but can bind to each other at different sites (homotypic binding) while still being able to form heterotypic bonds. The
diversity of binding mechanisms and complexity of force response became more evident when experimental techniques were
developed exclusively to study the binding forces between individual molecules. This category of techniques is known as force
spectroscopy.
The Impact of Force Spectroscopy

Force spectroscopy seeks to quantify the strength of individual bonds by looking at a spectrum of bond rupture force under tension.
The specifics of force spectroscopy will be explored in the next section (“Refining Understanding through Experiments” section),
where methods that measure response at the single cell level or even single molecule level (single molecule force spectroscopy
(SMFS)) will be presented. In the context of modeling, these techniques led for the first time to a less hypothetical consideration
of the effects of experimental factors on cell binding dynamics and the effect of individual bonds on one another in multiple bond
attachments.
Evans presented some of the pioneering work in SMFS beginning in 1997, with quantitative work on the bond lifetime under
tension. At that time it was possible to make measurements of single bonds. He developed a theoretical explanation for the force
response observed to increasing loading rates (force applied in time). Detachment force (or bond strength) had been shown to be
dependent on loading rate. What Evans noted was that the spectrum of bond rupture force as a function of loading rate can be
interpreted as a landscape displaying prominent energy barriers. This energy landscape reveals barriers that arise from molecular
interactions that are “difficult or impossible to detect in assays of near equilibrium dissociation,” in Evans’ words. These barriers
are what determine bond lifetime.
Experiments quickly showed that the response of a bond to tension was not linear; therefore, a Hookean spring is not an appro-
priate model. Taking the adhesion molecules to be highly flexible polymers, a better description of the dependence of force (f) on
adhesion molecule total length (x) is provided:
kT
f cb a (20)
1 Lxp
Lp and b are the contour length and persistence length of the adhesion molecule, respectively. Meanwhile, a and c are constants used
to describe the specific polymer and distinguish between different behaviors (e.g., a ¼ 1 and c ¼ 1 describe the polymer as a freely
jointed chain). Once again kT represents the Boltzmann constant multiplied by the temperature. In an experiment where the sepa-
ration speed (vs) is made to be constant, then the loading rate will be time dependent (df/dt ¼ rf(t)):

a kT
c Lp b vs
rf ðt Þ ¼ aþ1 (21)
1 vLspt
This is postulated for a single bond, and as such the transition and binding equilibrium is not presented in terms of adhesion
density but rather adhesion probability for a single bond. At each point in time, an adhesion molecules is said to have a probability
of being unbound Sf(t) or bound Sb(t): Sf(t) ¼ 1 Sb(t) (as there is no other possible state). The forward and backward transitions
between these states are described by the rate constants kþ(t) and k(t). Therefore, reminiscent of Eq. (10), binding and unbinding is
described by
kþ ðt Þ
Sf ðt Þ % Sb ðt Þ (22)
k ðt Þ
Since Bell postulated that the kinetic theory of the strength of solids could be applied to receptor–ligand bonds (Eq. 8), a relation
between force and unbinding rate had been proposed. In seeking to make a theoretical basis for bond behavior observed in force
spectroscopy experiments, in which the force applied is not constant, Evans was confronted with a system where force evolves in
time. This evolution is described by the loading rate, which implies that both the state probabilities and the rate constants can
be considered functions of force. Since the loading rate describes how time and force are related (df ¼ rf(t)dt), an equation
describing the change in binding probability with force can be postulated:
dSb ½k ð f Þ þ kþ ð f ÞSb ð f Þ kþ ð f Þ
¼ þ (23)
df rf ð f Þ rf ð f Þ
Analogous to Eqs. (9) (from Bell) and (11) (from Dembo and colleagues), this equation provides a quantitative relation
between binding dynamics and mechanical properties of the adhesion molecule. Evans’ equation is much more complete in
that it accounts for the different response to force by the molecule (i.e., rf(t)), the rate constants (i.e., kþ(f),k(f)), and the system
as a whole (i.e., Sb(f)).
Even when considering disruption of a single cellular bond, Evans believed that it is naïve to model the shortening of a bond’s
lifetime under external force as the lowering of a single activation (energy) barrier. There are in reality many widely distributed
atomic-scale interactions that create a complex energetic landscape. Evan provides the theoretical tools to model the response of
different proteins given their conformation, but also brings to our attention the large set of factors that come into play. There is
no general description for binding kinetics of proteins. Taking all of these factors into account, it is evident that binding kinetics
can only be known for the specific condition measured. Extrapolations to different applied forces or force-rates cannot be assumed
correct.
Zooming In: Other Players at the Molecular Scale

As much as Evans explores in detail the effect of adhesion molecule architecture on binding dynamics, he did not consider a bio-
logically active system where multiple molecules come into play. His work can be applied to an adhesion complex as a whole, yet he
did not address this explicitly as his work is very general to molecular adhesion in general. Subsequent models focusing precisely on
cell adhesion would address this; In one of the most refined models to date, Paszek and colleagues simulate an area of the cell
membrane explicitly considering individual integrin molecules, the glycocalyx (a carbohydrate-rich layer connected to the cell
membrane through proteoglycans and glycoproteins), and ligand molecules in the adjacent fraction of ECM. The model is set
up under the hypothesis that when an adhesive bond is formed, it induces a mechanical deformation of the membrane and
ECM, such that it modifies the distance between them. This change in distance can be cooperative to cell adhesion due to mechan-
ical coupling.
The glycocalyx is found in some bacterial cells as well as some mammalian tissues, including epithelial and endothelial. Despite
its function not being fully characterized, the large nature of carbohydrates has prompted theoretical studies on steric effects on
cellular systems and its role in disease. Rather than limiting their inquiry to the force felt at the receptor ligand complex, Paszek
and colleagues look at the source of the force itself by incorporating the compliance of the materials where ligand and receptor
molecules are embedded, cell membrane and ECM, respectively. The model considers the possibility that integrin clustering can
occur both from factors that control cell adhesion (e.g., ligand binding dynamics, matrix stiffness) and factors that mediate repul-
sion (e.g., glyxocalyx). It also seeks to determine how integrin clustering affects adhesion between the cell and ECM.
The model describes integrin–ligand bonds as Hookean springs with distance-dependent kinetic rates of bond association and
dissociation, just as Dembo and colleagues (Eqs. 13 and 14); however, it incorporates a lattice spring model (LSM) to represent both
the cell membrane and ECM. This consists in describing a body as a series of lattice points connected by harmonic springs; it is
a computationally efficient mesoscopic approach commonly used to study fracture mechanics. A schematic of the model used
by Paszek and colleagues is shown in Fig. 2.
A section, with an area of 1.4 1.4 mm2, of the interface between cell membrane and ECM is modeled as a single spring network.
Two separate LSM networks are used to represent the cell membrane (including the cortex) and the ECM, with a thicknesses of 40
and 400 nm, respectively. This corresponds to 70 70 3 lattice points for the former and 70 70 21 for the latter: The distance
between nodes (Dx) and the stiffness of the component springs (s) were selected such that the Young’s modulus (E) could be
adjusted according to the following equation:
5s
E¼ (24)
2 Dx
Integrin molecules, ECM ligand molecules (i.e., integrin binding sites), and the glycocalyx are located in between the two LSM
networks. Integrin molecules are bound to the top network (membrane and cortex); however, these can move off lattice when not
bound to the ECM. Integrin diffusion along the cellular membrane is modeled by a set of hop reactions in which the time evolution
of the system is given by a Gillespie algorithm: a dynamic Monte Carlo method allowing for discrete stochastic simulation where both
the time step duration and actual diffusion reactions taking place are variable at each step and calculated from random numbers.
The ligand molecules are fixed in certain nodes of the top of the spring network representing the ECM. Finally, the glycocalyx is also
represented as a series of Hookean springs always at node positions; these connect the top and bottom networks into a unified
mesh, for which forces are solved for each node based on minimization of the system’s energy.
With this model, mechanical properties of different cellular components (i.e., glycocalyx stiffness, glycocalyx thickness, and cell
cortex rigidity) are be modified. The effects of these changes are quantified in terms of cell adhesion, specifically the degree of integ-
rin clustering at the membrane and the cooperativity in binding. The authors found that cooperativity in integrin binding increased
with glycocalyx thickness, and that integrin clustering is primarily driven by bond formation. Similarly, an increased glycocalyx stiff-
ness caused enhanced cooperativity in integrin binding to the ECM and integrin clustering, as well as the formation of denser integ-
rin clusters. Finally, the study found that a minimum value of cortex rigidity is necessary to support cooperative binding of integrins.
Zooming Out: Cell Aggregates, Cell Sheets, Tissues

In the field of developmental biology, large homogeneous cell aggregates are studied for their composition, organization, and
mechanical properties. These systems can be grouped into a category of cellularized materials, which consist of a large number of cells
connected by intercellular junctions, linking neighboring cytoskeletons, providing an avenue for force transduction across multiple
cells. Forces applied on the ECM also become relevant to collective displacement, particularly in the case of epithelial sheets. To
capture their mechanical properties, these materials were initially studied as bulk materials. They were modeled through continuum
approaches, as was the case of tumor modeling. However, discretization of the bulk material is important when the material is
active, as is the case of living tissue. This led to the application of agent-based models for the study of cellular systems with tumor
growth models dominating the field.
In a popular modeling approach, individual cells are reduced to single points representing the center of mass. This is useful when
studying very large systems with hundreds or thousands of cells; however, mechanical interactions are simplified to pairwise inter-
actions described in the form of a force potential or Hertz contact mechanics (in which cellular interactions are modeled after that of
elastic spheres). These approaches have provided insight into phenomena such as collective cell dynamics and the emergence of
Fig. 2 Schematics showing different scales of cell adhesion models. Images A and B show a model at the subcellular level, representing a section
of membrane and ECM of surface area 1.4 1.4 mm2. Image C shows three isolated cells that form part of a larger hexagonal lattice used to repre-
sent an epithelial sheet. (A) Depiction of cell–ECM interface. Integrin molecules are on the bottom face of the membrane, while ligand molecules are
on the top face of the ECM. (B) The lattice spring model (LSM) representation of a 2D section of the cell–ECM section. Integrin molecules and the
glycocalyx are represented by springs as well. Integrin molecules are not spatially constrained to lattice nodes, while ligand molecules and the glyco-
calyx are. (C) Schematic showing apicolateral enrichment of cytoskeletal and junctional components in cells packed in epithelia (top). This packing is
modeled in 2D by using polygonal cells (numbered a) described by vertices (i,j) connected by cellular edges (lij) (bottom). Configuration of the polyg-
onal network is described by an energy function. (A and B) Reprinted from Paszek, M. J. et al. (2009). Integrin clustering is driven by mechanical
resistance from the glycocalyx and the substrate. PLoS Computational Biology 5(12), e1000604; (C) reprinted from Farhadifar, R., Röper, J., Aigouy,
B., Eaton, S. and Jülicher, F. (2007). The influence of cell mechanics, cell–cell interactions, and proliferation on epithelial packing, Current Biology
17(24), 2095–2104, Copyright (2007), with permission from Elsevier.
homeostatic stress distribution in tightly packed cellular systems. These approaches, nonetheless, fail to account for cell morphology
and are insufficient to describe cell adhesion in these contexts. To remedy this and look more carefully at the evolution of cellular
boundaries, vertex models and cellular Potts models (CPMs) were adopted from statistical mechanics.
Both types of models discretize the cell, and the cell geometry at each time step is determined through minimization of an energy
function calculated by summing up contributions of each discretized section. While CPMs discretize the cellular area in 2D (or
volume in 3D), vertex models discretize the perimeter in the form of vertices and line segments connecting them (Fig. 2C).
CPMs have proven very efficient for simulations looking into cell sorting of distinct cellular populations; they fail though to recreate
mechanical properties of the cellularized material as a whole. Meanwhile, vertex models focus entirely on cellular boundaries and
were developed with cell mechanics in the spotlight.
Farhadifar and colleagues developed a widely used vertex model to characterize the role of mechanical factors in the evolution of
epithelial tissue during wing formation in Drosophila melanogaster (fruit fly). The schematic presented in Fig. 2C shows three cells
represented by vertices and the line segments connecting them, representing the cellular boundaries. The epithelial sheet is repre-
sented by a 2D network under the assumptions that all cells are equal in height and that the epithelium is highly polarized (i.e.,
cortical proteins are only located in the apical domain of the cells): force application is restricted to the vertices.
The researchers seek to quantify the contributions to epithelial cellular configuration of cell–cell adhesion, actomyosin (cortical)
contractility, and cell elasticity. Rather than defining forces due to each factor explicitly, an energy-based approach is used to deter-
mine the configuration of the cellular system given vertex position. The energy function expresses the constraints related to the
ability of each cell to undergo a deformation due to these factors:
X X Ga X Ka 2
UðRi Þ ¼ Lij lij þ L2a þ Aa Aað0Þ (25)
<i;j> a 2 a 2
U is the energy function for a node (labeled i, with position Ri) in a cell population. The first summation term describes line
tensions (Lij) summed over cell boundaries between cells that contain vertex i. lij represents the length of the boundaries. Whether
Lij is positive or negative will determine if surface tension or cell–cell adhesion dominate the behavior of the system, respectively.
The second summation term describes the contribution of actomyosin contractility for each of cell containing vertex i in its perim-
eter (labeled a ¼ 1,.,Nc); the cells contract their cell perimeters (La) according to a coefficient (Ga). The third summation term
describes elasticity based on deviation of the cellular area (Aa) for each cell containing vertex i in its perimeter (labeled a ¼ 1,.,Nc),
from a preferred area (Aa(0)).
Given the energy function, the force acting on each vertex to reach a local minimum is obtained by calculating the derivative with
respect to the vertex coordinates:
vU
Fi ¼ (26)
vRi
By substituting Eq. (25) into (26) and computing the derivative, the equation of force can be rewritten for clarity:
X vlij X vLa X vAa
Fi ¼ Lij Ga Ka Aa Aað0Þ (27)
<i;j>
vRi a vRi a vRi
By varying the parameters describing linear tension (Lij) and cortex contractility (Ga) while keeping the target cellular area (Aa(0))
constant, Farhadifar and colleagues find the possible ground-states (lowest energy or most relaxed network configuration) of the
system for the different cellular properties.
The advantage of vertex models is that with them it is possible to look at large-scale cellular systems while still allowing param-
eters to have a physiological meaning that can be spatially defined and potentially measured experimentally. This is not usually the
case with continuum models. In the context of Drosophila wing development, Farhadifar and colleagues compared the simulated
system configurations with experimental observations. They counted the number of neighbors and how this value is distributed
in the population at the steady state. They also compared the evolution of the system upon removal of tension at specific cell–
cell boundaries through ablation of intercellular boundaries. The researchers were thus able to use the model to estimate the relative
magnitude of coefficients that describe line tension and cell contractility in epithelial tissue.
Refining Understanding Through Experiments
In the experiments cited by Bell, lymphocytes and fibroblasts were put in the same container as lectin-coated fibers, the containers
were shaken to apply shear forces to the cells, and then the number of cells that remained attached was quantified. This principle of
exposing cells to mechanical stimulation and quantifying adhesion has been maintained; however, the ways cells are stimulated and
visualized and measurements made have been refined.
The need to probe deeper comes from the fact that mechanosensitivity of cell adhesions is attributable to the complexity and
modular nature of the adhesion complexes. Mechanosensitive elements are found in every functional module of the adhesion
complex and in the ECM itself. Fibronectin, for example, stretches three to four times its rest length under cell imposed stress
and has been shown to have multiple hidden binding sites. Similar statements can be made for integrins in the membrane and
the actin polymerization machinery inside the cell. This fact is reflected in modeling in the trend to move to the subcellular scale,
as discussed earlier.
Both movements of experimental and modeling approaches to the subcellular scale are not coincidental, as these are comple-
mentary and insights gathered on each front spark new inquiries in the other. The refinement of experiments occurs due to advance-
ments in molecular probes, sensors, and imaging technology. These categories sometimes blend as ingenious scientists can modify
sensors to act as actuators, or imaging methods can be used to probe the cell or its environment. These advances in the field of cell
biology have changed our understanding of cell adhesion.
Imparting Forces at a Cellular Scale

Methods to probe a whole cell or its internal or external structures have been applied to study biological system since the late 1980s,
a few years after their development. Among the more popular ones are atomic force microscopy (AFM), laser optical tweezers (LOT),
magnetic tweezers, and biomembrane force probe (BFP) or micro-needle manipulation. The first two are widely available commer-
cially, making them more popular. In all methods, the probe can be functionalized, making them very versatile and thus used to
study a wide array of protein, molecular, and cellular interactions. Through this functionalization, it is possible to study response of
a single molecule; for this reason, these methods have been grouped under the umbrella term single molecule force spectroscopy
(SMFS). They can impart forces that range from 10 14 to 10 8 N and permit manipulation at a length scale with a range from 10 10
to 10 14 m.
Fig. 3 Experimental techniques used to impart forces at cell adhesions. (A) Schematic of atomic force microscopy (AFM). (B) Schematic of bio-
membrane force probe (BFP) or micro-needle manipulation. CHO cells expressing N-cadherin bind to RBC functionalized with mutant N-glycosylated
N-cadherin. Abbreviations: CHO, Chinese hamster ovary; ckN-Cad-Fc, Fc-tagged chicken N-cadherin; hN, wild-type human N-cadherin; RBC, red blood
cell. (A) Reproduced in part from Dufrêne Y.F. and Pelling A.E. (2013). Force nanoscopy of cell mechanics and cell adhesion. Nanoscale 5, 4094-
4104, with permission of The Royal Society of Chemistry. (B) Reproduced from Langer M., et al. (2012). N-glycosylation alters cadherin-mediated
intercellular binding kinetics. Journal of Cell Science 125(10), 2478–2485.
Atomic Force Microscopy (AFM): This technology consists of a small tip positioned at the end of a small cantilever which is then
used to probe the surface a sample. The deflection of the cantilever upon contact with the sample is measured from the deflection of
a laser beam reflected of it; this contact occurs over an area in the nanometer scale, allowing for single cell probing. Although
conceived as an imaging tool, it is now equally used to measure mechanical properties by functionalizing the tip and applying forces
at the molecular level (Fig. 3A). This system can measure forces at the scale of piconewtons. Studies targeting cell adhesion mole-
cules through AFM have uncovered the adhesion strength in living cells: Examples include measurements of cell–cell adhesion
through the glycoprotein contact site A (csA) in dictyostelium discoideum (slime mold) and measurements of cell–ECM adhesion
(i.e., binding between collagen-I and a2b1 integrins) in Chinese hamster ovary (CHO) cells.
Laser optical tweezer (LOT): Also known as optical traps, this method consists on trapping a bead in a focused laser beam due to
the attraction of the bead to dielectric particles in the vicinity of the focus. Under small movements (approximately 150 nm) from
the focus, the force experienced by the bead is linear with the displacement, thus providing a simple relationship that can be used for
probing the cellular systems. Optical tweezers can exert forces ranging from 0.1 to 1 nN. Magnetic tweezers are similar to LOT, but
a magnetic field is used instead of a laser to trap the bead. It has the advantage over the former method that the bead can be easily
rotated. For this reason, it is also known as magnetic twisting cytometry. Because cells are mostly unaffected by the magnetic field
(unlike in LOT where damage by bleaching or heating can occur), this option is particularly useful for probing studies inside the cell.
Biomembrane force probe (BFP): Also known as micro-needle manipulation. A cell is captured in a droplet using a micropipette.
Unlike the methods described earlier, the probe is not a rigid tip or sphere, but rather a cell itself. In many cases a red blood cell or
a lipid vesicle, which have no nucleus and whose mechanical properties have been characterized, is functionalized and used as
a probe by capturing it with a second micropipette as brought into proximity of the probed cell (Fig. 3B). Using a cell as the probe
provides the advantage that functionalization by chimeric proteins is easy, which in turn can be genetically modified upon synthesis
to see how specific amino acid residues affect the force response. This method has been used to study the mechanical response of E-
cadherin. Researchers have gone as far as using differently N-glycosylated (i.e., asparagine linked saccharide) E-cadherin molecules
in these experiments.
Of the methods presented earlier, AFM is arguably the most popular in the study of cell adhesion. This is the case, because cell
adhesion involves forces below the sensitivity of LOT or magnetic tweezers. And although it presents the disadvantage that it cannot
be used both outside and inside the cell, studies with LOT or magnetic tweezer that probe inside the cell remain scarce.
Measuring Forces and Imaging at a Cellular Scale

In addition to using probes, microfabrication techniques have been used to impart forces on cells. Controlled environments can be
created where cells can be exposed to specific forces, even at the site of FAs. For example, microfluidic channels can be made to
control the fluid flow and thus shear stress cells are exposed to. This presents a refinement over the original studies quantifying
the effect of shear stress on adhesion by shaking the container where cells resided. Also, rather than having cells sit on collagen
gels or synthetic gels entirely coated with ECM proteins (e.g., fibronectin, collagen), lithographic methods (chemical or photoacti-
vated) can be used to pattern the surface of a gel (usually polydimethylsiloxane (PDMS)) with protein islands of a desired geometry.
By using fluorescently tagged proteins, deformation of the gel by cellular tractions (force per unit area) can be quantified and the
spatial source of these tractions identified; this technique is known as traction force microscopy (TFM). TFM has been used exten-
sively due to its reproducibility and low cost. By using markers inside the substrate, whether fluorescent beads or fibrillar compo-
nents of the ECM, traction components in 3D can be calculated.
In a modality of TFM, cells are made to rest on PDMS pillars (or microposts) of a few micrometers in diameter, rather than on
a flat surface. The tractions can be easily known by taking into account mechanical and geometrical properties of the pillar.
However, the pillars can be used as more than sensors by introducing magnetic nanorods that can be controlled, providing
a way to exert force at the sites of cell adhesion by inducing torque in the pillars. This approach was developed by Sniadecki
and colleagues and was used to study the response of FAs in 3T3 fibroblasts on functionalized pillars with fibronectin. FA area
was monitored as a function of a force applied on the pillars containing the nanorods (Fig. 4A). A similar novel approach uses
optomechanical actuators (OMAs) to functionalize the substrate. The actuator consists of RGD-binding sites attached at the end
of a thermosensitive polymer with a nanorod core. Upon heating of the nanorod core with near-infrared light, the OMA contracts
exerting 13–50 pN forces on FAs. This method presents the advantage that it is up to two orders of magnitude faster than activation
of magnetic pillars. These methods work for cells on a 2D configuration (i.e., on a surface). For exploration of cellular adhesion in
physiological settings this presents a limitation, as most cells are in a 3D environment in vivo.
The trend in the field shows continuing specialization in specific molecules involved in mechanotransduction at the site of the
cell adhesions. In contrast to AFM and other SMFS techniques, which do not require imaging as the methods themselves provide
a reading of force, TFM does. This requires better imaging methods, the integration of novel microscopy techniques into the study of
cell adhesion. Laser scanning confocal microscopy (LSCM) has provided better resolution in 3D imaging by using a pinhole to
exclude fluorescent information from regions outside the plane of focus. AFM has been used simultaneously with LSCM to visualize
cell indentation simultaneously with measurements of elastic moduli of a cell. Similarly, cytoskeletal rearrangements have been
observed as the cell is mechanically stimulated. Imaging techniques with resolution beyond the diffraction limit, dubbed super-
resolution imaging (e.g., photo-activated localization microscopy (PALM)), have recently started to be implemented in the study
of cell adhesion. Though higher in resolution, most of these techniques are relatively slow, creating an image much slower than
molecular events occur at the adhesions.
A possible solution for this limitation is the use of sensors that produce a signal in response to force. This is the case of Förster
resonance energy transfer (FRET) sensors. These are sensors consisting of two fluorophores, a donor and an acceptor, whose prox-
imity determines whether energy transfer between donor and acceptor occurs and which is detected by a shift in emission wave-
length. By connecting the donor and acceptor by means of a calibrated molecular spring, FRET can act as a force sensor,
measuring the force acting on the molecular spring. A FRET tension sensor has been developed for vinculin, by replacing the flexible
linker connecting the head domain and tail domain of vinculin with an elastic domain flanked by fluorophores. This sensor is used
to measure tension at cell–cell and cell–ECM adhesions. These sensors have been used to correlate cellular traction with size and
duration of FAs (Fig. 4B); this revealed that FAs are responsive to mechanical simulation, undergoing remodeling upon increased
strain. Similarly, tension at AJs has also been quantified using VE-cadherin FRET sensors.
Challenges and Conclusions
The models presented in this work demonstrate two major trends in the study of cell adhesion mechanics: first, capturing the
mechanical properties of the cell–cell or cell–ECM bond itself, and second, accounting for the contribution of additional cellular
components beyond the adhesion molecules. Despite these common trends, the models clearly differ in the spatial scale of the
system they recreate. All the models, with the exception of the vertex model, start by focusing on the rates of protein–protein asso-
ciation (or dissociation) and attempt to describe how different parameters affect this rate. This provides a clue into what are some of
the major challenges in the study of cell adhesion.
Regarding the first trend, findings have demonstrated that a mechanical description of the adhesion molecule is not enough to
describe the evolution of cellular bonds. This is true, in part, due to the extensive number of molecules adhesion complexes
comprise, as well as the occurrence of extensive mechanical and chemical signaling. This is further complicated by mechanotrans-
duction, for which unlike in chemical signaling, there is no single ligand that elicits a response by a specific mechano-receptor.
Fig. 4 Novel experimental techniques used to measure forces at cell adhesions. (A) 3T3 fibroblast on microfabricated pillars in a magnetic field of
magnitude B ¼ 2T. Inset: Some of the pillars have a magnetic nanorod inside. This set up is used to measure and exert forces on cells at site of
adhesion (fluorescent labeling). (B) Schematic of action of vinculin FRET sensor in randomly migrating cells. Under high tension FRET efficiency
drops. Abbreviations: FN, fibronectin; FRET, Förster resonance energy transfer; mTFP, monomeric teal fluorescent protein; PM, plasma membrane; Vh,
vinculin head domain; Vt, vinculin tail domain. (A) Adapted from Sniadecki, N. J. et al. (2007). Magnetic microposts as an approach to apply forces
to living cells. PNAS 104(37), 14553-14558, Copyright (2007) National Academy of Sciences. (B) Reprinted from Hernández-Varas P. (2015). A
plastic relationship between vinculin-mediated tension and adhesions complex area defines adhesion size and lifetime. Nature Communications 6,
7524.
Continuing research in this direction entails further development of molecular sensors, such as FRET force sensors, and creative
experiments that can isolate roles of single molecules in the adhesion complexes. Application of SMFS techniques inside cells to
the level in which AFM or TFM is applied still needs to occur.
In addition to the difficulty in understanding the molecular mechanisms, it is important to recognize that there is also cell to cell
variability. The mechanisms do not work in the same manner in all cells and this should be acknowledged in modeling. This chal-
lenge is inherent to biological research. Even if this is accounted for by including stochasticity, cells are programmed differently (i.e.,
express different genes) in different tissues when they have different purposes. This means models are made specific to the cell
modeled, or more relevantly to the biological question probed.
The second trend can be seen as a tendency toward integrating models at different spatial scales (multiscale model): how many
cellular components are required to faithfully capture cellular behavior? Associated to this trend is the challenge common to
modeling, making the model complex enough to capture behavior of a complex system but simple enough that it can be efficiently
solved and studied. This tradeoff is most obvious in the models that try to capture large cellular systems or cellularized materials.
Tumor growth models were some of the first models of cellularized materials. Their evolution from continuum models to agent-
based models demonstrate the need to account for individual cell behavior to capture tissue behavior, and inherently a need to
model adhesion. These models have yet to integrate mechanics at the protein level. Yet a tumor growth model by Ramis-Conde
and colleagues is the first example of integrating cell mechanics and dynamics of adhesion in a flexible manner that could be applied
to many different cellular systems in 2D and 3D.
Increased computational power and development of numerous numerical methods have allowed for multiscaling. Additionally,
the use of modeling repositories and simulation environments exclusively for cell-based modeling (such as CompuCell3D devel-
oped at Indiana University, Chaste developed at the University of Oxford, etc.) have also provided a way to share models between
researchers in a way that these can be easily modified to overcome the challenge of the variability observed in biological systems.
The integration of mechanical description of cellular adhesions in these more widely used modeling platforms is not far down the
road.
Further Reading
Basic Principles
Bell, G. I. (1978). Models for the specific adhesion of cells to cells. Science, 200, 618.
Dembo, M., et al. (1988). The reaction-limited kinetics of membrane-to-surface adhesion and detachment. Proceedings of the Royal Society B, 234, 55–83.
Evans, E. (2001). Probing the relation between forcedLifetimedAnd chemistry in single molecular bonds. Annual Review of Biophysics and Biomolecular Structure, 30(1),
105–128.
Thomas Parsons, J., et al. (2010). Cell adhesion: Integrating cytoskeletal dynamics and cellular tension. Nature Reviews Molecular Cell Biology, 11, 633–643.
Computational Modeling
DiMilla, P. A., et al. (1991). Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophysical Journal, 60(1), 15–37.
Farhadifar, R., et al. (2007). The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing. Current Biology, 17(24), 2095–2104.
Kim, M.-C., et al. (2012). Integrating focal adhesion dynamics, cytoskeleton remodeling, and actin motor activity for predicting cell migration on 3D curved surfaces of the
extracellular matrix. Integrative Biology, 4, 1386–1397.
Paszek, M. J., et al. (2009). Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate. PLoS Computational Biology, 5(12).
Ramis-Conde, I., et al. (2008). Modeling the influence of the E-cadherin-b-catenin pathway in cancer cell invasion: A multiscale approach. Biophysical Journal, 95, 155–165.
Experimental Techniques
Grashoff, C., et al. (2010). Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature Letters, 466, 263–266.
Neuman, K. C., et al. (2008). Single-molecule force spectroscopy: Optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods, 5(6), 491–505.
Sniadecki, N. J., et al. (2007). Magnetic microposts as an approach to apply forces to living cells. PNAS, 104(37), 14553–14558.
Centrifugation and Hypergravity in the Bone
Carmelo Mastrandrea and Laurence Vico, Jean Monnet University, Saint-Étienne, France; and Lyon University, Lyon, France
Introduction 59
Disuse, Hypogravity, Microgravity, and Their Effects on the Bone 61
Bed-Rest Studies 62
Centrifugation Principles, Difficulties, and Side Effects 62
Hypergravity, Centrifugation, and Skeletal Changes in Humans 64
Centrifugation and Skeletal Changes in Animals 65
Centrifuge Acclimatization, Animal Size, and Animal Age as Research Considerations 66
Discussion and Conclusions 67
References 68
Further Reading 69
Glossary
Bone mineral content (BMC) A measurement of the amount of bone mineral within a specified area, usually measured in
grams (g).
Bone mineral density (BMD) An average measurement of the amount of bone mineral per unit area, usually measured in g/
cm2.
Dual emission X-ray absorption (DXA) A method for calculating the BMD of a tissue using X-ray imaging.
Hypergravity Conditions in which a body is exposed to an acceleration greater than that of its surrounding environment. At the
Earth’s surface this is an acceleration >9.81 m s2.
Hypogravity In this chapter hypogravity will be defined as an environment in which a body is exposed to acceleration lower
than that experienced at the Earth’s surface, or 9.81 m s2, but which is still >0 m s2.
ISS International Space Station.
Microgravity An environment in which a body experiences a negligible difference in acceleration relative to its environment,
such that the body is perceived to be weightless within this environment. Strictly speaking, this term is wrong since objects are
still in a strong gravitational field (the ISS altitude being 350 km, 92% of Earth’s gravitational force still acts on all objects
inside), but this term is commonly used and will therefore be applied in this article.
Introduction
Gravity, the force of attraction between two particles with mass, is the weakest of the four fundamental forces of the universe. With
a theoretically unlimited range, any particle in the universe, at any given time, experiences gravitational attraction to every other
particle in existence. However, as any two objects’ gravitational attraction diminishes exponentially as they move away from
each other, the majority of these forces produce a negligible, cumulative local effect. On Earth, the culmination of all the individual
gravitational forces in the universe produces a sea-level, groundward accelerative force of 9.81 m s 2 (1g), with the Earth producing
the majority of this; it is this continuous gravitational acceleration that has arguably provided the most significant evolutionary
stimulus for all nonaquatic life. Humans, as erect ambulatory creatures, have evolved complex physiological mechanisms to coun-
teract gravity and enable mobilization in our environment. For example, the cardiovascular system produces a powerful, continuous
pressure network through which blood travels to the whole body, supplying oxygen and nutrients to our tissues and organs; the
musculoskeletal system produces forces that overcome gravity and allow us to walk, run, and climb. Despite many important adap-
tations to the Earth’s gravitational field, it is the human skeleton and the associated gravitational effects on the bone that will be
discussed further in this article.
It may seem obvious that the human skeleton has evolved to provide a rigid framework for muscular attachment in order to
facilitate an individual’s movement. However, far from being an inert, passive framework, healthy adult bone is under continual
remodeling and repair. Three main cell types control this continuous process: osteoclasts, osteoblasts, and osteocytes. Osteoclast
cells, found throughout the skeleton, digest bone and release calcium and phosphate from mineralized mature bone into the blood.
This produces areas in which the bone has lost both its extracellular matrix and mineral content. Osteoblasts, cells that deposit new
extracellular bone matrix, subsequently replenish these areas with new naive bone that in time mineralizes to maturity. Following
the replenishment of an area of the bone, some osteoblasts transform into osteocytes, producing a connected cellular network

60 Biomechanics j Centrifugation and Hypergravity in the Bone
within the mineralized matrix of all bones. The remaining osteoblasts remain at the periosteum as lining cells. Osteocytes can stim-
ulate bone resorption in their immediate vicinity, possibly secondary to local bone damage or due to their own injury or death.
Osteocytes also act as mechanosensors. Sufficient mechanoactivation of the three distinct osteocyte mechanoreceptor classes –
ion channel, G-protein-coupled, and cytoskeletal-integrin complexes, either through direct cellular strain or due to changes in inter-
stitial fluid flow, leads to a signaling pathway that ultimately results in bone growth (Govey et al., 2015; Robling, 2012; Iwaniec and
Turner, 2016). This process of removal and replenishment, known as bone remodeling, is essential to the ongoing health of the
skeleton and creates a plasticity that is invaluable. Should a bone need to increase in strength, for example, due to heavy workload,
deposition of new bone exceeds removal, and the bone density increases or the bone hypertrophies. The opposite occurs in disuse,
where reabsorption of existing bone may exceed new bone deposition, resulting in an overall loss of bone mass.
In young animals including humans, new bone growth, known as bone modeling, occurs during childhood and adolescence.
Bone modeling produces the normal age-related growth in the size of the skeleton, and it is important to understand that bone
deposition during modeling is mainly under genetic control. It is this genetic control and mechanical stimulation in adolescence
that drives bone deposition at a significantly higher rate than would be typical of normal adult bone metabolism. Consequently,
research that aims to identify skeletal remodeling changes in young animals must consider the possibility of genetic factors driving
bone modeling that may otherwise confound results. In adults, bone modeling can occur at the periosteal margin, where load simu-
lation may cause a bone’s diameter to increase, but its length remains static. Unilateral increases in bone mineral content, bone
mineral density (BMD), and cortical thickness and an increase in bone diameter were found in the playing arm of tennis players
(Haapasalo et al., 1996, 2000). This offers an initial example of higher than normal use and load leading to anabolic bone changes
and bone growth in adults.
Bone loading or force directly imparted on the bone is now understood to be a major stimulus in its normal physiological main-
tenance and remodeling. However, it was not until the 1960s that Harold Frost, furthering theories on bone repair published by
Julius Wolff in 1892, incorporated strain and force as direct stimuli for bone deposition, maintenance, and loss (Frost, 1987). Frost
postulated that the growth and remodeling of any one particular area of the bone was a direct consequence of the forces experienced
in that particular area. Frost coined the term “mechanostat” to describe this phenomenon. His theory provides an initial framework
by which one can understand the effects of force and therefore gravity on bone homeostasis. As shown in Fig. 1, the magnitude of
regular forces exerted on the bone, measured in microstrains, directly corresponds to subsequent bone turnover; disuse at one
extreme causes bone loss, and overstress at the other risks fracture. The major sources of these regular forces are routine contact
with the ground created during activities such as walking, running, and jumping and direct muscle contraction that imparts forces
on the bone.
As the force needed to lift an object such as the leg is greater in hypergravity due to increased weight, one can comprehend that
higher g-forces result in greater muscle strain on the bone and that the forces of ground impacts when walking or running are also
increased. This in turn would cause increased bone deposition and in theory produce a higher bone mass in any one individual as
the gravitational force increased. The opposite would be true in a reduced gravitational environment. In the absence of gravity, the
resistance to movement is minimal, as weight is abolished and muscles no longer have to fight downward gravitational force. In
addition, weightlessness removes the compressive force of an individual’s body weight on weight-bearing bones within the skel-
eton, especially in the hips, legs, and feet. These two fundamental differences almost completely eliminate the bone’s anabolic
stimuli as according to Frost’s mechanostat theory. This understanding of the relationship between force, gravity, and bone mass
is of particular interest not only to the study and research of bone diseases such as osteoporosis, but also when considering the
detrimental effects of microgravity and hypogravity in astronauts, and the possible uses of hypergravity and centrifugation as a coun-
termeasure to microgravity-induced bone loss.
Fig. 1 Intepretation of Frost’s mechanostat relationship. The potential for bone mass change, represented by the thick curved line, varies for
differing magnitudes of forces imparted on bone mechanostat units (Frost, 1987).
Biomechanics j Centrifugation and Hypergravity in the Bone 61
The mechanostat theory therefore provides a theoretical understanding of how the bone will react to different magnitudes of
mechanical force. However, one must consider that the forces required to maintain bone mass in weight-bearing areas, such as
the hip, may not be the same as those required to maintain bone mass in the non-weight-bearing areas such as the arms or skull.
Subsequent research has therefore attempted to provide further classification for the stimuli and thresholds that lead to bone density
increases and, although still incomplete, provides a set of intriguing categories for consideration: (1) Different threshold strains
must exist in different types of bone; (2) peak magnitude, duration, and frequency of strains all contribute to the mechanostat
response; (3) the anabolic responses of the mechanostat diminish with age; (4) systemic interactions, such as circulating hormones,
cytokines, and nutrition, all alter responses to a given mechanical stimulus. (Skerry, 2006; Iwaniec and Turner, 2016). Skeletal
homeostasis therefore involves a complex set of interactions that results in an overall maintenance of bone mass, or an anabolic
or catabolic bone state, with routine muscle and ground impact forces seemingly playing an important role.
Disuse, Hypogravity, Microgravity, and Their Effects on the Bone
Spaceflight is often thought to take place in the absence of gravity; however, this is not true; even aboard the International Space
Station (ISS), astronauts still experience around 90% of the pull of the Earth’s gravitational field. As the space station and therefore
astronauts within it are orbiting the Earth at 27,600 km/h, their linear momentum prevents them from falling to the ground. The ISS
is orbiting in a state of equilibrium, with both crew and ship continually moving at the same speed and maintaining a constant
altitude. Therefore, astronauts experience microgravity, as they do not experience an accelerative vector in relation to their environ-
ment. Should the ISS accelerate, perhaps due to a rocket firing to elevate its altitude, those astronauts inside would experience an
accelerative force that may be a small percentage of the 1g experienced on Earth. For the duration of the rocket firing, astronauts
would experience hypogravity. Other times in which astronauts may experience hypogravity could be during extravehicular activity
on the moon or Mars, where the gravitational field is much weaker than that of Earth.
Bone loss in astronauts is common, and pre- and postflight measurements often show significant reductions in BMD. These
reductions differ between bones in the body, following a pattern of greater losses in those parts of the skeleton that bear more
weight. Fig. 2 shows collated, measured BMD changes in astronauts and in cosmonauts. This loss in bone mass causes a significant
increase in calcium liberation from the skeleton, with subsequent increases in calcium excretion in the urine and feces. Measured
calcium losses of 200–300 mg/day were typical during the Skylab missions (Rambaut and Johnston, 1979), and large negative
calcium balances have been recorded during numerous other missions (Whedon et al., 1976; Leach and Rambaut, 1977; Smith
et al., 1999). Not only is this vast volume of excreted calcium a worrying marker of bone loss, but also it increases the risk of
the development of urinary calculi. Should an astronaut develop such a complication, it could result in significant pain and risk
of serious infection, possibly requiring the termination of the mission.
In order to prevent such dramatic losses of bone mass, space agencies have employed microgravity countermeasures that aim to
increase the forces imparted on astronauts’ bodies and in turn reduce the loss of bone mass during flight. These typically include
exercise regimes involving simulated gravity and ground impact forces using, as an example, bungee cords to allow treadmill
running. Additionally resistance exercise is also possible using the advanced resistance exercise device aboard the ISS. Although
Fig. 2 Bone mineral density loss during spaceflight. (A) The percentage change in BMD following 6 month spaceflight at six different body loca-
tions. (B) Similar data collected from cosmonauts spending, on average, 188 days in space. As indicated, BMD loss appears to be greater in those
areas that typically bear more weight. (A) From Vico L. et al. (2010). Adaptation du squelette humain dans l’espace. 10.106/S0246-0521(10)52493-8.
(B) Data from LeBlanc A. et al. (2000). Bone mineral and lean tissue loss after long duration space flight. Musculoskeletal Neuronal Interaction 1(2),
157–160.
somewhat successful, these methods typically reproduce approximately 25% of an astronaut’s body weight intermittently during
exercise, therefore not replicating those forces experienced in the 1g environment on Earth, and bone loss still occurs. Alternatively,
continual compressive force through the use of a weight-loading g-suit may act to reduce the detrimental effects of unloading of the
spine, although the definitive effects of such garments are still being investigated. Disuse on Earth can cause similar, although less
dramatic, reductions in bone mass, with typical examples including immobilized patients, bed-rest studies, and water immersion.
Human research in space and on the ground can therefore provide invaluable information as to the processes that cause bone
changes in differing gravitational environments. New countermeasure research may achieve increases in the anabolic stimuli for
bone maintenance and repair in space, with one such possibility being the use of centrifugation, either continual or intermittent,
to provide an artificial gravity.
Bed-Rest Studies
Bed-rest studies aim to reproduce, as closely as possible, the effects of microgravity on the human body. They are performed in order
to provide a significantly cheaper and more accessible method for microgravity research, which would otherwise only be possible
during spaceflight. Six degrees of head-down tilt is now most commonly used as the model of microgravity in bed-rest studies, and
this figure was chosen following initial investigations concerning resultant physiological parameters and the subjects’ tolerance of
different degrees of head-down tilt. However, the physiological parameters recorded were cardiovascular, not skeletal, and the exact
angle and duration of head-down tilt should depend on the physiological system under investigation (Prisk et al., 2002; Hinghofer-
Szalkay et al., 2004). Typically, subjects in these studies are continually confined to their beds, unable to stand or sit in order to eat
and wash. Such research is therefore very difficult and time-consuming as a level of care akin to 24 h nursing is required for each
participant.
Centrifugation Principles, Difficulties, and Side Effects
Centrifugation as a spaceflight countermeasure has been historically proposed multiple times, with different sizes and speeds of
centrifugation suggested to achieve some degree of Earth-equivalent gravitational acceleration. However, despite the likely abolish-
ment of the majority of microgravity-induced deconditioning that would occur should this countermeasure be deployed, no perma-
nent centrifuge has ever been launched into or constructed in space. In the history of human spaceflight, centrifuges have only been
launched twice, aboard two Skylab missions, in which short-arm centrifuges were used for human neurovestibular study rather than
as a microgravity countermeasure.
In order to produce artificial gravitational acceleration, centrifuges spin a subject at high speed around a circular enclosure. The
size and rotational speed of a centrifuge are dependent on the desired artificial gravitational acceleration and in space can be calcu-
lated from the following equation:
Acceleration ðGravitational equivalentÞ ¼ Velocity2 =Radius
To give some examples, in order to achieve a gravitational acceleration equivalent to that at the Earth’s surface of 9.81 m s 2,
a theoretical space-based centrifuge would have the following approximate attributes:
A radius of 5 m and rotational speed of 13 rpm
On Earth or any other planet or the moon, one must also factor in the continuous surrounding gravitational field. As an additional
consideration, one must remain conscious of the Coriolis forces experienced by the subject. The Coriolis force defines the abnormal
motion of moving objects within a centrifuge from the viewpoint of an individual within that same centrifuge. As the rotational
speed of a centrifuge increases, it results in increasing disorientation, motion sickness, and difficulty performing tasks due to
constant rotation. This limits rotational speeds for long-duration centrifugation to approximately 3 rpm (Musgrave and Larsen,
2009). Therefore, in order to achieve a rotational speed no greater than 3 rpm and a continual gravitational acceleration of 1g,
the minimum required radius of a space-based centrifuge would have to be approximately 75 m or 150 m in diameter. Given
that the current modules that form the ISS are approximately 4.5 m in diameter and that the ISS is 108.5 m along its longest
axis, it becomes apparent that such a centrifuge would be impractical. It may be possible to rotate the spacecraft itself, much
like the space hotel in 2001: A Space Odyssey, thereby incorporating the centrifuge into the structure of the station. However,
such a station would come with immense cost and engineering difficulties.
With the aforementioned problems generating continual 1g acceleration, future proposed centrifuge countermeasures compro-
mise on certain parameters. Either the duration of centrifugation falls, thereby allowing high rotational speeds for short periods of
time, or the exposed gravitational acceleration falls, allowing for slower rotational speeds. Lower target gravitational accelerations
and intermittent rather than continual centrifugation also allow for smaller centrifuge sizes, an important consideration for space
countermeasure designs. Despite the theoretical benefits of hypogravity over microgravity (25%–50% of 1g rather than 0g), further
research must be performed in space to validate hypogravity as a countermeasure and to ensure adequate bone maintenance in
astronauts.
Terrestrial human and animal centrifugation takes place relatively regularly, either in the routine training of pilots who undergo
hypergravity stress training or in scientific research such as countermeasure trails in microgravity analogue bed-rest studies or hyper-
gravity animal studies. In human scientific research, centrifugation devices typically have a radius no larger than a few meters, and
centrifugation is intermittent; subjects do not perform complex tasks, and head movement can cause significant motion sickness
due to the Coriolis effect. It should be noted that the Russian Samara State Medical University (SSMU) has developed a human-
rated centrifuge for clinical use (SSMU; see Fig. 3). They state that it provides benefits for multiple physiological conditions, advo-
cating its use in bone fracture repair and secondary posttraumatic osteoporosis. We are not aware of any other hypergravity therapy
options currently available in other countries.
Animal research typically involves longer durations of centrifugation over many days to weeks, with continuous hypergravity
throughout the trial. Examples of human and animal centrifuges are shown in Fig. 3.
Larger centrifuges have been proposed in order to test the effects of continual hypergravity on human subjects, but none have ever
been constructed. Van Loon (2008) proposed the design and construction of a large-diameter centrifuge for continual hypergravity
experimentation. His design utilizes high-speed trains on a 942 m circular track, with carriages or modules to support long-duration
Fig. 3 Examples of human and animal rated centrifuges.

Fig. 4 Mean food consumption in centrifuged rats. Food consumption as a mean of six rats kept in continual 4.7g centrifugation. The marked
reduction in intake during the initial 2–3 weeks may lead to catabolic metabolism in these animals. Food consumption increased beyond that of non-
centrifuged rats if the hypergravity exposure continued for a sufficient period of time. From Oyama et al. (1965). Effects of prolonged centrifugation
on growth and organ development of rats. American Journal of Physiology 209(3), 611–615.
living. With rotational speeds under 3 rpm, human and animal subjects would experience long-duration, constant gravitational accel-
erations greater than 1g, and it offers a potentially exciting opportunity for both space and terrestrial medical research.
Animal centrifugation, both in space and on the Earth’s surface, has been performed multiple times in the past as a method to
study the effects of hypergravity. Given the smaller size of animals such as mice, smaller apparatus is required, and so cost and engi-
neering difficulty are reduced. Studies have been performed with gravitational accelerations in excess of 3g; however, one must be
careful when interpreting results from these experiments. The stress of animals in high-velocity centrifuge environments is known to
be elevated, with corticosterone levels rising during acclimatization to 2g and for the duration of a 21-day 3g centrifugation
(Guéguinou et al., 2012). Elevations in corticosterone also occur at 4g, as well as activation of the sympathetic nervous system
(Petrak et al., 2008). Additional evidence for animal stress in these environments includes the animal’s weight post centrifugation.
Wunder et al. (1966) investigated if the weight loss in centrifuged animals was due to nausea or motion sickness, a direct conse-
quence of the high rotational speeds. They found that in labyrinthectomized hamsters, in which motion could not be sensed by
the inner ear, a hypergravity-induced reduction in weight persisted, proving that reduced appetite as a direct consequence of nausea
is not the cause for weight loss in centrifugation studies. Wade et al. (2002) evaluated the metabolic rate of rats in hypergravity,
providing evidence that resting metabolism in these animals was elevated, a further mechanism that would lead to weight loss.
Whatever the reason, food consumption falls, and this plays a significant role in the bone formation ability of centrifuged animals
(see Fig. 4). Even at 1g centrifugation, rats spun in space at 53.3 rpm for 18.5 days showed significant suppression of weight gain
compared with control 1g animals kept in an identical, noncentrifuged environment (Spengler et al., 1983). As an elevation in
stress, circulating corticosterone and sympathetic activity and a reduction in food intake can all contribute to a reduction in
body weight and subsequent bone deposition; interpretation of results in experimental centrifugation research must be considered
carefully.
In summary, despite a clear understanding of the principles and design requirements for continual human hypergravity centri-
fugation either in space or on Earth, engineering and financial constraints prevent their use. Therefore, intermittent terrestrial centri-
fugation and continual animal centrifugation provide the majority of our understanding of the physiological changes to the skeletal
system in hypergravity.
Hypergravity, Centrifugation, and Skeletal Changes in Humans
Unfortunately, human centrifuge studies investigating skeletal changes in hypergravity are typically limited. The nature of such
experimentation and the need to provide exposure for long durations in order to achieve identifiable and reproducible results
prohibit their routine undertaking. In addition, it has historically been impossible to visualize microarchitectural bone changes,
as one could not dissect and section the femur of a living human subject. Recently, however, the development of high-
resolution peripheral quantitative computed tomography (HR pQCT) now allows for these investigations, although at this time
these experiments are yet to be performed.
Kos et al. (2013) and Rittweger et al. (2015) performed analyses of 1g centrifugation at the center of mass, 2.5G at the feet, as
a countermeasure to bed-rest-induced bone loss. Comparing short-duration, bed-rest-induced elevations in markers of bone resorp-
tion, they concluded that 30 min centrifugation per day, either as a single 30 min exposure or as six 5 min exposures, resulted in
a minimal reduction in markers of bone loss. This trial was short, lasting only 5 days for each condition. Smith et al. (2009) exam-
ined the effects of 1 h of centrifugation (1 g center of mass and 2.5 g at the feet) per day for 21 days, again in bed-rest subjects.
Measuring serum and urine bone resorption markers and performing dual-energy X-ray absorption (DXA) scans, they did not
find any differences in either markers of bone resorption, BMD, or bone mineral content (BMC) between control and experimental
groups. Finally, Iwase et al. (2004) identified a suppression of the bone resorption marker deoxypyridinoline in subjects under-
taking 14 or 20 days of bed rest, with 30 min per day 1.4g centrifugation with concurrent 60 W exercise. The full results of this
experiment were not, however, published, and the exercise intervention may have contributed to the protective effect seen in exper-
imental subjects.
The possible reason for a lack of statistical difference in these studies is due to the fact that bone mass changes take from many
weeks to months; therefore, identifiable changes are likely to be minimal after only a few days’ intervention. In addition, it would be
ethically unacceptable to allow experimental subjects in bed-rest groups to develop significant bone loss that may lead to compli-
cation in later life. Also, due to the huge cost and technical difficulties of caring for subjects who are confined to bed 24 h a day,
sample sizes are low, and this may impede the ability of studies to find statistically significant differences. Therefore, current human
trials are of limited value in this respect, although they do offer an interesting platform for the investigation of more rapid changes
such as cardiovascular fluid shifts and muscular degeneration that occur in microgravity.
One very interesting research paper focuses on the hypergravity-induced bone changes in fast-jet pilots. Naumann et al. (2001)
followed fighter pilot trainees over the course of a 1-year flight training course, in which they regularly experienced positive grav-
itational accelerations between 2g and 6g through their spines and pelvis. It was found that compared to age- and height-matched
control subjects, and following correction for changes in total body weight and fat mass, the trainee pilots showed an increased
BMD and BMC of 11.0% and 4.9% in the thoracic spine and pelvis, respectively. So far as we are aware, this is the only method
by which a comparison of bone parameters between 1g and accelerative forces greater than 1g has been performed in humans. These
results strongly suggest that regular hypergravity exposure has an anabolic effect on the bone; however, this exposure method is not
suitable for more standard research.
Centrifugation and Skeletal Changes in Animals
The centrifugation of many different animal species has been performed in the past, including chickens, dogs, rats, and mice. Both
on Earth and in space, animal centrifugation offers a method for the study of long-duration, continual hypergravity exposure on
bone parameters. Another advantage in using animals is that the effects of hypergravity during early life and adolescence can be
studied in order to identify any significant differences in primary bone growth. However, the number of studies that have investi-
gated these changes remains limited.
In 1977, Riggins et al. assessed chickens’ tibial strength following a gradual increase in gravitational acceleration to 3g over the
course of 18 weeks. They found that although the cortical bone thickness increased in the centrifuged animals, midshaft diameter
was significantly reduced, and bone density was unchanged. Despite little change in fracture patterns on stress testing, it appeared
that hypergravity induced some degree of bone growth and remodeling. Additional findings in hypergravity were also identified in
another study, where rats exposed to 2.8g centrifugation for 810 days showed an increase in femur density when compared with
similar control animals (Jaekel et al., 1977). Finally, Doden et al. (1978) exposed young beagle dogs to 3 months of continual
centrifugation at 2g, finding that the density of thinner bones was higher and thicker bones lower, in the centrifuged animals
when compared with controls.
More recent research has begun to identify the microscopic changes that occur in hypergravity. In 2015, Gnyubkin et al. pub-
lished their findings regarding the changes in bone deposition and resorption surfaces in the femur and vertebrae of C57BL/6
mice. Following 21 days of continual centrifugation at 2g, it was found that trabecular fraction and density increased in the femur
by 18% and 32% and in the vertebrae by 13% and 9%, respectively. Within the bone, osteoclast surfaces were reduced and miner-
alized surfaces increased, suggesting an overall reduction in bone resorption and an increase in bone formation. These differences
were accompanied by an increase in the number and volume of blood vessels in the distal femur metaphysis. As an additional
comparison, the same protocols and investigations were undertaken on another set of mice with one difference; in the second group
of animals, the gravitational acceleration was increased to 3g. In these animals, it was found that the bones showed cortical thinning,
osteoclast surface area increase, and reduction in the rate of bone formation. The author states that the changes in 3g may possibly
be due to increased stress rather than the true effects of hypergravity on the bone. Nonetheless, it was concluded that continual 2g
acceleration does cause an increase in bone deposition, with a possible detrimental effect of continual gravitational accelerations
around and above 3g in magnitude due to significant stress responses. In another study, Ikawa et al. (2010) identified a reduction
in the amount of bone resorption in ovariectomized rats over 28 days at 2.9g. Normally, estrogen, produced by the ovaries, inhibits
bone resorption and protects the female animal from osteoporosis; following ovariectomy, levels of estrogen decline, and bone
density falls. By comparing ovariectomized rats kept at 2.9g with those at 1g, it was found that hypergravity prevented much of
this bone loss, and the conclusion that 2.9g hypergravity inhibits bone resorption was therefore made. These findings correlate
with the decreased bone resorption surfaces in 2g mice found by Gnyubkin et al.; however, they directly conflict with the findings
of increased bone resorption surfaces in 3g mice. Finally, Kawao et al. (2016) investigated the effects of the vestibular system on
bone metabolism in hypergravity. They hypothesized that skeletal responses to gravitational accelerations are influenced by the
vestibular apparatus and that subsequent sympathetic signaling affects gravitational bone and muscle metabolism. Bilateral vestib-
ular lesions were created in young mice, leading to reductions in BMD and BMC. Beta-blockade in nonlesioned mice seemed to
inhibit the increase in body-weight-corrected BMC in hypergravity but did not achieve statistical significance. However, the authors
state that following vestibular surgery, mice did not eat properly for 2 weeks and immediately after this they were introduced into
a 3g environment, which is known to cause a reduction in food intake for another 2–3 weeks (see Fig. 4). Therefore, the mice may
not have eaten adequately for 4–5 weeks preceding sacrifice at 12 weeks of age, causing significant weight loss in lesioned mice,
a factor that can independently affect bone deposition. It should be noted that Wunder et al. (1966) induced vestibular lesions
Table 1 Animal and bone parameters following 18.5 days of spaceflight
Animal group Animal number Animal weight (g) Femur failure (torque, N cm) Femur density (g/cm3)
Ground control 5 289 9 32.2 9.6* 1.60 0.02

Ground simulated flight 5 294 16 26.1 6.9 1.60 0.01
0g flight 5 295 29 17.5 4.4 1.56 0.05
1g centrifugation flight 5 264 8 29.2 1.5* 1.61 0.01
*
Significantly different from the corresponding value for 0g flight group.
Adapted from Spengler, D., Morey, E., Carter, D., Turner, R., Baylink, D. (1983). Effects of spaceflight on structural and material strength of growing
bone. Proceedings of the Society for Experimental Biology and Medicine 174, 224–228.
in hamsters with no significant differences in weight at 1g and minimal differences in weight at 5g, findings that contradict Kawao
et al. Finally, by comparing bone parameters when corrected for body weight, values may be confounded by the significant reduc-
tion in body weight in hypergravity over such a short period of time. The actual skeletal density changes in differing gravitational
environments were relatively static, but due to a large loss of animal weight, the correction for body weight created a significant
difference. These three studies provide evidence for alterations to the skeleton in hypergravity, suggesting potential causes for these
changes. Animal age, size, duration of centrifugation, and nutrition were not consistent, and as discussed later, this may adversely
affect skeletal changes during centrifugation.
Centrifugation studies of animals in space have been previously achieved, aboard the Biocosmos 4 (Cosmos 936) satellite. Two
groups of male Wistar rats spent 18.5 days aboard the satellite before its return to Earth, with two additional control groups
spending 18.5 days in similar conditions on the ground. The pertinent results are shown in Table 1(Spengler et al., 1983).
Those rats in the simulated flight ground group experienced vibration and noise equivalent to those that flew aboard the Bio-
cosmos 4. From the data, one can see that induced femur failure occurred at much lower torque stresses in those animals having
spent 18 days in a 0g environment, and this is to be expected given the previous evidence presented in this article. Those rats that
were centrifuged in space (1g centrifugation flight group) had femur failure rates equivalent to those rats kept in similar environ-
ments on the ground. Despite the relatively short duration of the flight, the centrifuged animals were exposed to continual Earth-
equivalent gravitational acceleration, and this prevented the significant loss of femur strength and density seen in the 0g cohort.
However, mirroring the findings of ground-based centrifugation studies, there is a noticeable difference in the weight of these
animals, and this is likely to be secondary to the fast revolution speed of the centrifuge (53.3 3 rpm). Unfortunately, no video
telemetry of the rats exists, but it may be that they did not eat and as would have been expected. This would be a consequence
of both stress (as previously discussed) and due to the fast revolution speed and significant Coriolis forces causing disorientation
and nausea. Nonsacrificed rats did regain weight following landing (Spengler et al., 1983). As previously alluded to, stress due to
high centrifugation speeds must always be considered in experiments of this nature, with careful interpretation of subsequent
results. In addition, the acclimatization to centrifugation may play an extremely important role in the inconsistent results seen
in relatively short-duration, continual centrifugation research.
Centrifuge Acclimatization, Animal Size, and Animal Age as Research Considerations
Smith (1975) described in great detail the effect of alterations in gravitational acceleration on different organs in a variety of species.
He concluded that the tolerance of animals to both acute and chronic hypergravity was inversely proportional to the animal’s size
and weight. In addition, an organism’s skeleton must contribute to a larger percentage of overall body mass as the animal’s size
increases. This correlates with the animal findings in hypergravity, where the skeletal BMD increases as a proportion of total
body weight. Summarized by Clément and Slenzka (2006), in addition to the skeletal changes and the intrinsic ability of smaller
animals to resist hypergravity, posture and hydrostatic pressure greatly contribute to g-tolerance (see Fig. 5).
Therefore, one would expect smaller animals to better tolerate higher gravitational accelerations, with the subsequent produc-
tion of greater anabolic skeletal changes. However, using two studies discussed previously in this article (Gnyubkin et al., 2015;
Ikawa et al., 2010), it appears that mice had more detrimental skeletal changes, showing increased femur resorption areas, when
compared with the larger rats, which showed increased femur density, which is inconsistent with this theory. These discrepancies
may be due to centrifuge acclimatization, a direct consequence of the duration of centrifugation. Both centrifuged animal popula-
tions experienced weight loss compared with their control counterparts (12% and 20%, respectively), suggesting that the hypergrav-
ity cohort did not eat as much as the control cohort. This is a common finding in all centrifuge studies. What is different however is
the total duration of centrifugation. Gnyubkin et al. exposed mice to 21 days of centrifugation, whereas Ikawa et al. provided
28 days. Given the initial stress of centrifugation, 21 days may have not been enough time for the mice to adapt and subsequently
adequately respond to the hypergravity stimulus on the skeleton. Conversely, 28 days may have allowed the rats to resume normal
nourishment and for stress level to lower to a point at which the skeleton could anabolically respond to the hypergravity stimula-
tion. It would be expected, given the theories forwarded by Wolff, Frost, and Skerry, that the expected response of the skeleton
would be to increase in volume and density given the higher mechanical strain produced by this level of centrifugation. In
Fig. 5 The skeleton, hypergravity, and species size. (A) Relationship between nonaquatic organisms’ weights and their relative proportional skeletal
masses. (B) Peak survivable g-forces in relation to an animal’s size. Animal size is also inversely related to chronic hypergravity tolerance. From
Clément and Slenzka (2006). Gravitational biology. In: Fundamentals of space biology. Chapter 2. ISBN: 0-387-33113-1.
Table 2 Femur mass following long-duration hypergravity
Age matched 2.5g 3.5g 4.7g
Weight (g) 261 11 216 9 211 10 205 14

Femur mass (mg/100 g) 376 11 409 21 383 19 393 85
Femur mass (g) 0.98 0.88 0.81 0.81
All animals’ final weights are lower than their age-matched controls, due to the stresses inherent in centrifugation. Femur mass,
expressed as a proportion of overall animal mass, is increased in all animals; however, the absolute mass added on the bottom
row (g) is decreased, accounted for by the smaller animal size.
Adapted from Oyama, J., Platt, W. T. (1965). Effects of prolonged centrifugation on growth and organ development of rats.
American Journal of Physiology 209(3), 611–615.
longer-duration studies, hypergravity, even to levels of 4.7g, produces an anabolic effect on weight-bearing bones (Oyama and Platt,
1965); after 4.5 months of continual centrifugation, rats exposed to hypergravity developed increased femur masses, and when
expressed as a proportion of body weight, these findings show the relative anabolic effect of high gravitational acceleration (see
Table 2). These mice also experienced an initial weight loss due to decreased calorific intake (Fig. 4) (Oyama and Platt, 1965);
however, following acclimatization, the relative femur mass increased proportionally to the animal’s overall weight.
As an additional consideration, total activity and gait may be altered in hypergravity. In the developing mouse, continual hyper-
gravity is known to cause alterations to gait parameters including stride frequency and length, with walking causing extension of the
hind limbs beyond that normally shown in mice at 1g (Bojados et al., 2013). This may lead to differences in imparted strain on
joints and bones, in turn altering the skeleton’s response to load. Hypergravity may even cause a certain degree of immobility in
animals, with high accelerations leading to a reduction in walking and running distance. Therefore, further investigations into
voluntary movement, including running distances, must be made during hypergravity exposure in animals. This will ensure that
the skeleton is exposed to a similar frequency of ground impact forces and muscular contractions in both control and hypergravity
animals.
Consideration of animal age when investigating the consequences of the gravitational environment on skeletal parameters must
be addressed. Young animals and primary bone growth may be impacted negatively due to the lack of normal mechanical stimu-
lation during skeletal development. This is an important consideration for future physiological gravitational research. Martinez et al.
(1998) performed 14 days of continual centrifugation on rats at 2g. No significant differences in bone formation or cortical bone
composition were identified; however, it was noted that in these relatively young animals, hypergravity resulted in the stunting of
femur growth by approximately 3% and possible evidence for an earlier onset of bone maturation. Vico et al. (1999) compared the
effects of 4 days of continual 2g centrifugation with 1g controls in young Sprague Dawley rats. Within the primary spongiosa of the
tibia and femur, trabecular thickness and density were significantly elevated with a reduction in the overall bone length. These differ-
ences occurred despite a noted weight loss in the 2g animals. Therefore, the changes identified in these two papers indicate that
hypergravity may reduce the mature length of weight-bearing bones in juvenile animals but still indicate that overall bone density
is increased in a similar fashion to the mature skeleton.
Discussion and Conclusions
Currently, very little data exist with respect to the effects of hypergravity on the bone. In addition, the human research that has been
performed in this field typically involves a comparison between unloaded or microgravity analogue models and proposed, inter-
mittent countermeasures. Therefore, the majority of bone-related continual hypergravity experimentation has been performed on
animals, and even these are few in number. Despite this limitation, it can be concluded that in comparatively higher than normal
gravitational environments, the metabolic state of the bone is stable or anabolic relative to the mass of the organism. Given the
historical findings that show significant loss of appetite, growth retardation, hormonal alterations, and autonomic changes in
animal centrifuge studies, careful consideration must be employed in the interpretation of the results of these studies. True skeletal
alterations to hypergravity in research studies may only occur following the acclimatization of animals to centrifugation stresses, so
such experiments may require exposures of many months rather than a few weeks.
In humans, centrifugation remains as an untested microgravity countermeasure. It is the potential reduction in bone resorption
in hypergravity that is of greatest interest for space physiologists, as the loss of bone in microgravity causes considerable risk both
during and following flight, with current countermeasures that include resistance exercise and tethered treadmill running unable to
provide adequate long-duration skeletal protection. Therefore, the use of artificial gravity in space may provide a method to reduce
skeletal resorption with relatively physiologically normal bone stresses and without pharmacological intervention. However, one
must remain aware that with high continual gravitational acceleration, stresses on both the bone and organism as a whole may
be detrimental. Significant adverse physiological changes in humans who are exposed to continual or intermittent 1g centrifugation
in space may not occur, but given the findings in animal trials during continual gravitational acceleration in excess of 1g, future
ground-based human research is required. The increased bone densities in fighter pilots exposed to hypergravity, as previously dis-
cussed, suggests intermittent hyper gravity exposure, with adequate recovery for normal nourishment and a reduction in stress
responses, may provide the optimal circumstances for anabolic bone metabolism.
There is therefore a significant need, in both space and terrestrial medical research, for the future investigation of centrifugation
and the hypergravity consequences for the skeleton. Both as a countermeasure to microgravity-induced bone loss and as a possible
preventative measure for osteoporosis, hypergravity research offers exciting potential avenues for future therapeutic therapies.
However, given the significant costs involved in the construction of continual human hypergravity centrifuges, either in space or
on the ground, there is currently a significant fiscal barrier to this type of experimentation. Additionally, the duration of exposure
required to see significant beneficial changes in the bone density of humans during hypergravity centrifugation remains unknown.
Therefore, this field is open to both new physiological discoveries and development of possible future centrifugation treatments and
space countermeasures.
References
Bojados, M., Herbin, M., & Jamon, M. (2013). Kinematics of treadmill locomotion in mice raised in hypergravity. Behavioural Brain Research, 244, 48–57.
Clément G. Slenzka K. (2006) Gravitational biology. Fundamentals of space biology. Chapter 2 0-387-33113-1.
Doden, E., Oyama, J., & Amtmann, E. (1978). Effect of chronic centrifugation on bone density of the dog. Anatomy and Embryology, 153(3), 321–329.
Frost, H. M. (1987). Bone “mass” and the “mechanostat”: A proposal. The Anatomical Record, 219(1), 1–9.
Gnyubkin, V., Guignandon, A., Laroche, N., Vanden-Bossche, A., Normand, M., Lafage-Proust, M. H., & Vico, L. (2015). Effects of chronic hypergravity: From adaptive to deleterious
responses in growing mouse skeleton. Journal of Applied Physiology, 119(8), 908–917.
Govey, P., Kawasawa, Y., & Donahue, H. (2015). Mapping the osteocytic cell response to fluid flow using RNA-seq. Journal of Biomechanics, 48, 4327–4332.
Guéguinou, N., Bojados, M., Jamon, M., Derradji, H., Baatout, S., Tschirhart, E., Frippiat, J. P., & Legrand-Frossi, C. (2012). Stress response and humoral immune system
alterations related to chronic hypergravity in mice. Psychoneuroendocrinology, 37(1), 137–147.
Haapasalo, H., Sievanen, H., Kannus, P., Heinonen, A., Oja, P., & Vuori, I. (1996). Dimensions and estimated mechanical characteristics of the humerus after long-term tennis
loading. Journal of Bone and Mineral Research, 11(6), 864–872.
Haapasalo, H., Kontulainen, S., Sievanen, H., Kannus, P., Jarvinen, M., & Vuori, I. (2000). Exercise-induced bone gain is due to enlargement in bone size without a change in
volumetric bone density: A peripheral quantitative computed tomography study of the upper arms of male tennis players. Bone, 27(3), 351–357.
Hinghofer-Szalkay, H., Haditsch, B., Loder, I., Pilz, K., Rossler, A., & Jezova, D. (2004). Head down tilt at 6 degrees to 24 degrees can neutralize the cardiovascular effects of
LBNP at 15 or 35 mmHg. Aviation, Space and Environmental Medicine, 75(11), 947–951.
Ikawa, T., Kawaguchi, A., Okabe, T., Ninomiya, T., Nakamichi, Y., Nakamura, M., Uehara, S., Nakamura, H., Udagawa, N., Takahashi, N., Nakamura, H., & Wakitani, S. (2010).
Hypergravity suppresses bone resorption in ovariectomized rats. Advances in Space Research, 47(7), 1214–1224.
Iwaniec, U., & Turner, R. (2016). Influence of body weight on bone mass, architecture and turnover. Journal of Endocrinology, 230(3), R115–R130.
Iwase, S., Takada, H., Watanabe, Y., Ishida, K., Akima, H., Katayama, K., Iwase, M., Hirayanagi, K., Shiozawa, T., Hamaoka, T., Masuo, Y., & Custaud, M. A. (2004). Effect of
centrifuge-induced artificial gravity and ergometric exercise on cardiovascular deconditioning, myatrophy, and osteoporosis induced by a 6 degrees head-down bedrest.
Gravitational Physiology, 11(2), 243–244.
Jaekel, E., Amtmann, E., & Oyama, J. (1977). Effect of chronic centrifugation on bone density of the rat. Anatomy and Embryology, 151(2), 223–232.
Kawao, N., Morita, H., Obata, K., Tamura, Y., Okumoto, K., & Kaji, H. (2016). The vestibular system is critical for the changes in muscle and bone induced by hypergravity in mice.
Physiological Reports, 4(19), e12979.
Kos, O., Hughson, R., Hart, D., Clément, G., Frings-Meuthen, P., Linnarsson, D., Paloski, W., Rittweger, J., Wuyts, F., Zange, J., & Gorczynski, R. (2013). Elevated serum soluble
CD200 and CD200R as surrogate markers of bone loss under bed rest conditions. Bone, 60, 33–40.
Leach, C. S., & Rambaut, P. (1977). Biochemical responses of the Skylab crewmen: An overview. In R. S. Johnson, & L. F. Dietlein (Eds.), Biomedical results of Skylab (p. 204).
Washington, DC: NASA. SP-377.
Martinez, D. A., Orth, M. W., Carr, K. E., Vanderby, R., Vasques, M., Grindeland, R. E., & Vailas, A. C. (1998). Cortical bone responses to 2G hypergravity in growing rats. Aviation,
Space and Environmental Medicine, 69(6 Suppl), A17–A22.
Musgrave, G., & Larsen, A. (2009). Safety design for space systems, ISBN 978-0-7506-8580-1.
Naumann, F. L., Bennell, K. L., & Wark, J. D. (2001). The effects of þ Gz force on the bone mineral density of fighter pilots. Aviation Space and Environmental Medicine, 72(3),
177–181.
Oyama, J., & Platt, W. T. (1965). Effects of prolonged centrifugation on growth and organ development of rats. American Journal of Physiology, 209(3), 611–615.
Petrak, J., Mravec, B., Jurani, M., Baranov, M., Tillinger, A., Hapala, I., Frollo, I., & Kvetnansky, R. (2008). Hypergravity-induced increase in plasma catecholamine and corti-
costerone levels in telemetrically collected blood of rates during centrifugation. Annual of the New York Academy of Sciences, 1148, 201–208.
Prisk, G. K., Fine, J. M., Elliot, A. R., & West, J. B. (2002). Effect of 6 degrees head-down tilt of cardiopulmonary function: Comparison with microgravity. Aviation, Space and
Environmental Medicine, 73(1), 8–16.
Rambaut, P. C., & Johnston, R. S. (1979). Prolonged weightlessness and calcium loss in man. Acta Astronautica, 6(9), 1113–1122.
Rittweger, J., Bareille, M. P., Clément, G., Linnarsson, D., Paloski, W., Wuyts, F., Zange, J., & Angerer, O. (2015). Short-arm centrifugation as a partially effective musculoskeletal
countermeasure during 5-day head-down tilt bed rest-results from the BRAG1 study. Journal of Applied Physiology, 115(6), 1233–1244.
Robling, A. G. (2012). The interaction of biological factors with mechanical signals in bone adaptation: Recent developments. Current Osteoporosis Reports, 10(2), 126–131.
Skerry, T. M. (2006). One mechanostat or many? Modifications of the site-specific response of bone to mechanical loading by nature and nurture. Musculoskeletal and Neuronal
Interaction, 6(2), 122–127.
Smith, A. H. (1975). Principles of gravitational biology. In M. Calvin, & O. Gazenko (Eds.), Foundations of space biology and medicine (Vol II Book 1, pp. 129–162). Washington
DC: NASA.
Smith, S. M., Wastney, M. E., & Morukov, B. V. (1999). Calcium metabolism before, during, and after a 3-month space flight: Kinetic and biochemical changes. American Journal of
Physiology, 277, R1.
Smith, S. M., Zwart, S. R., Heer, M. A., Baecker, N., Evans, H. J., Feiveson, A. H., Shackelford, L. C., & LeBlanc, A. D. (2009). Effects of artificial gravity during bed rest on bone
metabolism in humans. Journal of Applied Physiology, 107(1), 47–53.
Spengler, D., Morey, E., Carter, D., Turner, R., & Baylink, D. (1983). Effects of spaceflight on structural and material strength of growing bone. Proceedings of the Society for
Experimental Biology and Medicine, 174, 224–228.
Van Loon, J. (2008). The human centrifuge. Microgravity Science Technology, 21, 203–207.
Vico, L., Barou, O., Laroche, N., Alexandre, C., & Lafage-Proust, M. H. (1999). Effects of centrifuging at 2G on rat long bone metaphyses. European Journal of Applied Physiology
and Occupational Physiology, 80(4), 360–366.
Wade, C., Moran, M., & Oyaya, J. (2002). Resting energy expenditure of rats acclimated to hypergravity. Aviation Space and Environmental Medicine, 73(9), 859–864.
Whedon, G. D., Lutwak, L., Rambaut, P. C., Whittle, M. W., Reid, J., Smith, M. C., Leach, C., Stadler, C. R., & Sanford, D. D. (1976). Mineral and nitrogen balance study
observations: The second manned Skylab mission. Aviation, Space and Environmental Medicine, 47(4), 391–396.
Wunder, C., Milojevic, B., & Eberly, L. (1966). Growth and Food consumption of labyrinthectomized hamsters during chronic centrifugation at 5G and 6G. Nature, 210(5032),
177–179.
Further Reading
LeBlanc, A., Schneider, V., Shackelford, L., West, S., Oganov, V., Bakulin, A., & Voronin, L. (2000). Bone mineral and lean tissue loss after long duration space flight.
Musculoskeletal Neuronal Interaction, 1(2), 157–160.
Riggins, R. S., & Chacko, K. A. (1977). The effect of increased gravitational stress on bone. Life Science and Space Research, 15, 263–265.
Samara Sate Medical University Website. (November 2016). Synergy project. Accessed on, 03. http://www.smuit.ru/projects/medicinskoe-priborostroenie/lechebnye-ehlektronnye-
pribory-medicinskogo-naznacheniya/gravitacionnyj-stend-sinergiya/.
Vico L, Pavy-Le Traon A (2010) Adaptation du squelette humain dans l’espace. https://doi.org/10.1016/S0246-0521(10)52493-8
Wolff, J. (1982). Das Gesetz der Transformation des Knochen. Berlin: Hirschwald.
Computational Modeling of Respiratory Biomechanics
Christian J Roth, Lena Yoshihara, and Wolfgang A Wall, Technical University of Munich, Munich, Germany
The Medical Reason 70

Structure of the Respiratory System 70
Reduced Models of the Conducting Zone 72
Continuum Models of the Conducting Zone 73
Reduced Models of the Respiratory Zone 74
Continuum Models of the Respiratory Zone 75
Reduced-Dimensional Coupled Lung Models 75
Single-Compartment Models 75
Multi-Compartment Models 76
Hybrid Representations of the Respiratory System 77
Hybrid Model (Conducting) 77
Hybrid Model (Respiratory) 78
Continuum Coupled Lung Models 79
Further Reading 80
Glossary
Acinus Composition of several air spaces (so-called alveolar ducts) distal to a terminal bronchiole. The acinus is the largest unit
of lung tissue in which all parts participate in gas exchange.
Compliance Measure for lung stiffness defined as quotient between volume change DV and associated pressure change DP.
Interdependence Interaction between neighboring air spaces competing for lung volume.
Parenchyma Lung tissue on a macroscopic point of view including air spaces, alveolar walls, small airways, and blood vessels.
Recruitment/Derecruitment Reopening (recruitment) and closure (derecruitment) of airways and/or lung tissue during the
respiratory process.
Resistance Measure for viscous and convective flow resistance in the conducting zone of the lung. The resistance is defined as
the quotient between pressure drop DP and associated flow DQ through an airway.
Surfactant Liquid lining of surface active agents on the inside of the alveoli. Surfactant reduces the surface tension on the gas
exchange interface and consequently facilitates breathing.
The Medical Reason
Respiratory biomechanics covers a variety of different complex and interacting phenomena including tissue mechanical, fluid
dynamical, gas and particle transport processes. Their underlying physics has been directly linked to observed immunological reac-
tions and to the current health state of a patient, leading to the fact that breathing is actually a mechanical problem. However, single
phenomena in respiration are difficult to measure in vivo due to both ethical and technical reasons. Therefore, advanced modeling
techniques have been developed to adequately represent important effects and to provide new insights into respiratory biome-
chanics in general as well as to promote suitable medical treatment in case of acute and chronic respiratory disease. Especially
approaches with the ability to represent patient-specific and regional information show the potential to deliver valuable informa-
tion on patient-tailored treatment in respiratory care. The current goal of developing predictive models based on the underlying
physics of the lung is driven by the awareness that it is more powerful to understand underlying processes in respiratory biome-
chanics than to only see their effects in medical imaging and clinical monitoring of a patient.
Structure of the Respiratory System
To allow for a better understanding of the governing physics in respiratory biomechanics, a deep knowledge about the anatomy and
physiology of the lung is indispensable. From the anatomical point of view (see Fig. 1), the lung as an organ is subdivided into the

Biomechanics j Computational Modeling of Respiratory Biomechanics 71
Fig. 1 Human anatomy showing the thoracic cage with all respiratory components highlighted. The conducting zone is visualized in blue and
comprises the first 16 generations of the airway tree. The respiratory zone is marked in pink and denoted as parenchyma on the macroscopic scale.
On the microscopic scale, lung tissue is composed of single alveoli arranged in a sponge-like structure. The alveolar walls contain the capillary
network and constitute the blood-gas barrier.
conducting zone (airway tree generations 1-16) and the respiratory zone (airway tree generations 17-23). The conducting zone is
characterized by a dichotomously branching tree of large to medium-size airway segments and its main task is to efficiently
distribute air towards the smaller structures of the lung. The respiratory zone starts with the terminal bronchioles leading to the
alveolar ducts which consist of single clustered air spaces, the so-called alveoli. The respiratory zone is ultimately responsible for
gas exchange with the blood. To allow for a maximum of exchange surface area, alveoli are embedded within a tissue scaffold
following the concept of a dense spherical packing (see Fig. 1). For many applications, it is not possible to resolve the complex
microstructure containing alveoli, alveolar walls and blood vessels in full detail. Therefore, the respiratory zone is often considered
from a macroscopic point of view. This macro-scale is referred to as lung parenchyma (see Fig. 1).
From the biomechanical point of view, the respiratory system can be seen as a complex multi-physics and multi-scale problem in
nature. Its full physical description is governed by airflow dynamics in the conducting airways, tissue mechanics of lung parenchyma
covered by a film of surfactant, gas transport and mixing processes as well as particle and aerosol transport on scales ranging from
a few centimeters to a few nanometres. Further, the single components visualized in Fig. 1 highly interact with each other. The defor-
mation of lung tissue introduced by contraction of the respiratory muscles and the diaphragm creates a negative pressure in the
pleural cavity and respiratory air spaces and thus induces flow in the airways. Airflow vice versa can lead to the movement of accu-
mulated liquid in occluded airways and triggers the regional inflation of lung tissue in healthy and diseased regions of the entire
organ.
72 Biomechanics j Computational Modeling of Respiratory Biomechanics
Fig. 2 Classification of respiratory biomechanics models. Approaches for the respiratory zone and for the conducting zone can be found in the red
and blue parts of the table, respectively, whereas coupled models combining both zones are given in the white part of the table. Reduced models are
highlighted in gray, continuum models in yellow and hybrid (i.e., combined continuum and reduced) models in green.
From this complexity it becomes clear that a simultaneous and full investigation of all aspects in respiratory biomechanics is
virtually impossible and in many cases not even reasonable. Different clinical questions are related to effects on different scales
that are governed by different physical fields. Only relevant phenomena have to be resolved in detail, while other effects can be
modeled appropriately. Finding suitable settings in mechanical ventilation of a patient for example, requires other effects and scales
to be resolved than particle and aerosol transport on the alveolar scale. These considerations confirm that there cannot be a one-size-
fits-all respiratory model but that each question in respiratory biomechanics requires a suitable approach for its investigation.
In this work, an overview of the current state of the art in modeling respiratory biomechanics is provided. In general, models for
the single zones as well as coupled models combining both zones are available. Each zone can be realized at different levels of reso-
lution, either as fully resolved three-dimensional continuum or as dimensionally reduced model. Dimensional reduction in this
context means any reduction of complexity either by elimination of spatial or governing physical details. Specific examples will
be presented in the following subsections. A classification with these two criteria (i) conducting zone/respiratory zone and (ii)
reduced/continuum based description is visualized in Fig. 2. In the following sections the visualized approaches will be discussed
in detail, with model complexity increasing successively.
Reduced Models of the Conducting Zone
Several decades ago, modeling approaches in respiratory biomechanics were restricted to reduced-dimensional observations of the
conducting zone only. These models were motivated by the fact that the anatomy of the branching airway tree has been well known
from lung casts which contained statistically relevant geometrical data up to the 16th generation of the airway tree. Specific effects
observed in lung physiology were attributed to this complex branching airway tree structure and the flow patterns within.
At that time, computational methods were not powerful enough to resolve the flow field in the entire conducting airway tree.
Therefore, simplified approaches computing a flow resistance for each airway segment based on its geometrical dimensions have
been used. These resistance models were mainly based on observations of laminar and turbulent flow in rigid pipes and the cor-
responding analytical solutions for example, Poiseuille’s resistance or modifications respecting turbulence.
The assumption required for such reduced-dimensional models of the conducting zone was the applicability of the derived
reduced-dimensional pressure-flow relationships in the airways under physiological conditions. Hence, all flow properties from
fully laminar to turbulent flow conditions have to be represented correctly. In particular, it is assumed that airflow within a single
airway segment is axisymmetric, fully developed, and that curvature of the single airway segments can be neglected.
These prerequisites allow a spatial reduction of the three-dimensional Navier–Stokes equations (potentially including fluid–
structure interaction effects) towards a one-dimensional (1D) formulation via integration within an airway cross section. It is further
assumed that the pulsatile character of this 1D formulation can be omitted in respiratory biomechanics as the pressure wave travels
throughout the entire airway tree in less than 0.1 ms and thus in a much smaller timescale than those relevant in respiration. Conse-
quently, an integration in axial direction can be performed leading to the zero-dimensional (0D, or lumped) description of fluid
flow in (compliant) airways. Another possible mathematical approach to build a formulation for airflow is the assumption that the
Womersley solution holds for each airway segment. Then, an equivalent impedance can be computed to account for the viscous
resistance, compliance, and inertance of airflow in each airway segment.
The resistances or impedances for single airway segments can then been combined to a network specified by the anatomy of the
airway tree and have been used for computations of airflow throughout the entire conducting zone.
This basic idea of dimensional reduction of the conducting zone has been an important first step towards building up the disci-
pline of airflow modeling in respiratory biomechanics. The applied resistance models were derived from basic physics of pipe flow
and perfectly valid in the physiological range of airflow in the lungs. One major limitation of these models has, however, been the
lack of respiratory tissue with all its important effects in lung physiology. Nevertheless, reduced-dimensional models of the conduct-
ing zone have successfully been used to investigate the resistance distribution across different generations of the airway tree. Further,
the effect of expiratory flow limitation has been investigated using the extension of flexible airway walls. Expiratory flow limitation
describes the maximum flow rate at which a subject is able to exhale. This flow rate cannot be increased with a higher forced positive
pressure generated by the respiratory muscles due to the involved compression of the flexible airways and the resulting increase in
flow resistance. Today, these models of the conducting zone have been extended by adequate representations of the respiratory zone
towards reduced-dimensional multi-compartment models presented later in this article (see “Multi-Compartment Models”
section).
Continuum Models of the Conducting Zone
If a detailed spatial resolution of the airflow field is crucial, the use of continuum mechanics based conducting zone models is
a prerequisite. In this case, airflow is modeled and simulated in two-dimensional or three-dimensional geometries of the conduct-
ing airways.
Depending on the specific application, different regions of interest are considered in existing models, starting with individual
airway models via single bifurcation models to multiple bifurcation models including different numbers of airway generations
between the upper respiratory tract and the terminal bronchioles.
Previously, idealized geometric representationsdthat is, individual tubes or a system of bifurcating tubesdhave been used
predominantly. Well-known examples are the Weibel and Horsfield lung models, which are based on general morphological
data about branching angles and generation-dependent airway diameters and lengths. Recently, the importance of patient-
specific geometric features for the development of airflow patterns has gained more attention. Consequently, many models are
meanwhile based on imaging-based geometries, that is, geometric reconstructions from, for example, bronchoscopic, X-ray, MR,
or CT data. However, due to the limited resolution of imaging techniques and the high computational cost related to patient-
specific (i.e., extremely complex) geometric models, it is not reasonable to resolve all airways in the conducting zone with such
a level of detail.
Airflow in the conducting zone is in general modeled by the incompressible Navier–Stokes equations. The assumption of incom-
pressibility is appropriate since typical Mach numbers under physiological conditions are below 0.2. It is known that a high-speed
jet is formed as air passes through the larynx. Resulting turbulence effects can affect flow patterns in the trachea and bronchi but are
believed to decay already after few generations of airways. Depending on the specific region of interest, some models (particularly of
the upper and central airways) consider the influence of potential turbulence effects either by resolving all relevant spatial and time
scales (i.e., direct numerical simulation) or by applying turbulence models such as large-eddy simulation (LES) or Reynolds-
averaged Navier–Stokes (RANS) models. For some particular investigations also multiphase or non-isothermal models can be used.
Most models of the larger airways consider the airway walls as being rigid. This assumption is often deemed suitable because the
walls of the trachea and the first airway generations mainly consist of cartilage and are consequently rather stiff. Hence, cross
sectional deformations in this region are usually small. However, in certain diseases (e.g., chronic obstructive pulmonary disease)
airway cross sections can change significantly even during normal breathing. Furthermore, the composition of the airway wall
changes over the generations and smaller airways are considerably more compliant. To account for the mutual interaction of airflow
and airway wall deformation in these cases, so-called fluid–structure interaction (FSI) models have been proposed as an alternative
to classical computational fluid dynamics (CFD) models. Apart from enabling the prediction of realistic airflow patterns in the
deforming airways for above-mentioned scenarios, FSI models also allow for the quantification of airway wall strains and stresses.
The latter are assumed to play an essential role, for example, for the alteration of cell shapes, biological signaling, and liquid secre-
tion. It has to be noted, though, that a detailed modeling of airway wall properties is indispensable for realistic FSI models. So far,
however, only few studies providing corresponding experimental data can be found in literature.
The airways are embedded in the surrounding lung tissue, which exerts stresses on the airway walls during breathing or mechan-
ical ventilation. Some models account for this so-called parenchymal tethering effect by introducing non-linear springs attached to
the outside of the airway walls or considering an additional, empirically derived tethering pressure to the airway model. This way,
the changes in flow characteristics as a consequence of disease-related alterations in the tethering forces (e.g., in asthma or fibrosis)
can be investigated theoretically.
Most existing continuum models of the conducting zone consider only a small part of the conducting zone, either since only
local effects are of interest or because the inherent complexity of the lung inhibits a detailed modeling of all airways down to
the respiratory zone. At the truncated airways in the models, suitable inflow and outflow boundary conditions have to be specified.
At the inlet of the modeled airway tree, usually a time-dependent parabolic or constant velocity profile or a (time-dependent)
pressure is prescribed. The definition of appropriate outflow conditions is more intricate. Boundary conditions at the outlets of the
modeled airway tree have to be chosen such that (i) flow separates realistically between the different branches; (ii) during expiration
(i.e., in case of a reversal of the flow direction), the problem remains well-posed and the volumes of previously inspired and expired
air at each outlet are equal. These requirements are often difficult to fulfill in practice since they actually necessitate a detailed
consideration of the unresolved downstream region. In most continuum models of the conducting zone, however, this influence
is either disregarded or taken into account in an often unphysiological way. At the truncated outflows, rather simple boundary
conditions are frequently prescribed. For instance, a priori defined outlet pressures, velocity profiles, or mass flow percentages
are commonly prescribed. Alternatively, a so-called individual path model can be used. In this case, some pathways are resolved
down to the peripheral airways, whereas most other pathways are truncated at low generations. The pressure or mass flow rate
at equivalent interior locations of the resolved pathways is then mapped randomly to the outlets of the truncated branches. Other
approaches have been based on subject specific boundary conditions to model the outflow from specific regions of the lung based
on imaging data. These imaging-based outflow rates prescribed at single outlets are only valid for the specific flow scenario under
image acquisition. However, they are of great value to validate a computed flow and radio marker distribution against available
clinical measurements (e.g., of hyperpolarised noble gas distribution) in this specific scenario.
The resulting numerical models can be applied for improving the general understanding of airflow in the conducting zone in
health and disease. For instance, the influence of the airway tree geometry on airflow dynamics has been studied extensively. Severe
differences with regard to airflow characteristics have been reported between patient-specific and idealized simplified geometries.
Airway curvature and complex shapes at junctions have been found to be responsible for more complex flow patterns (including
significantly more off-axis flow) in patient-specific geometries. Furthermore, the impact of pathological geometry changes (e.g.,
airway obstructions) on simulated airflow patterns has been surveyed. In case of airway constructions, for example, disturbed flows
(including flow recirculation zones) as well as an increase in both pressure drop and work of breathing have been shown. Besides,
the consequences of rapid inhalation (e.g., during sniffing) on airflow dynamics have been studied. While spontaneous fluctuations
associated with turbulence have been reported in the supraglottic region, these fluctuations have been found to decay rapidly. Apart
from the analysis of airflow patterns in the conducting zone, several studies have also been concerned with calculating airway wall
(shear) stresses, for example, during coughing. For instance, substantial shear stress levels have been detected at expiratory flow-rates
equivalent to cough, which can exacerbate damage to the epithelial layer of airway walls.
Another important application field of continuum models of the conducting zone is the investigation of airway stability and
reopening. Small airways are prone to fluid-elastic instabilities that can lead to their collapse and occlusion by a liquid bridge
formed by the airway liquid lining. Since a persistent occlusion of the airways can lead to a severe impairment of gas exchange,
medical treatment aims at a quick reopening of airways. At the same time, however, tissue forces resulting from the propagation
of the “air finger” into the liquid-filled airway have to be minimized to avoid cellular injury and inflammation. Continuum models
of the conducting zone can be used to determine (i) the propagation speed of the air finger as a function of the applied pressure and
(ii) the stresses in the airway wall.
Continuum models of the conducting zone are also the basis for the prediction of particle transport and deposition in the lung.
Example scenarios are targeted drug delivery and the inhalation of toxic pollutants from the environment. For this type of appli-
cation, the continuum models of the conducting zone have to be extended by a particle transport formulation and appropriate
absorption boundary conditions. Although in general a mutual interaction is possible, the effect of the particle transport on the
airflow is commonly deemed insignificant and, consequently, often neglected. In several studies, the influence of physical quantities
(e.g., flow rate and particle size) as well as model parameters (e.g., time-dependent vs. steady flow conditions, idealized vs. imaging-
based geometry) on particle deposition patterns and deposition efficiency have been investigated. It has been found that geometric
features of airways, particle distributions, and the history of airflow fields can affect particle deposition significantly.
Reduced Models of the Respiratory Zone
Similar to the conducting zone, the first models of the respiratory zone were restricted to reduced-dimensional observations. These
approaches were motivated by the fact that the respiratory zone is composed of a huge number of small clustered units (i.e., the
alveolar ducts) similar in their architecture. If the properties of a single alveolar duct can be expressed in a reduced-dimensional
sense, the entire respiratory zone can then be adequately described.
Models based on this idea have replicated alveolar tissue as a network of pin-jointed line elements (i.e., springs and dashpots)
representing bundles of collagen and elastin fibers. These line elements are arranged at the edges and across the surfaces of regular
polyhedra representing individual alveoli. Different quasi-static stress-strain behaviors are commonly attributed to the fiber bundles
(i.e., linear for elastin and highly nonlinear for collagen), whereas identical stress relaxation functions are assumed to define the
history of the stress response. Additionally, surface tension effects at the air-wall interface caused by a thin liquid lining on the inside
of the single alveoli, the so-called surfactant, can be respected using membrane elements across the surfaces of these polyhedra.
The main assumption that is required for a reduced-dimensional model of the respiratory zone is, that the chosen alveolar geom-
etry and arrangement as well as constitutive model for the line elements are representative for human lung tissue.
Inflation/deflation of such alveolar clusters then allows to compute quasi-static or dynamic relationships between pressure and
volume of an alveolar duct. These relationships provide a computationally very efficient representation of the respiratory zone based
on the underlying physics of collagen/elastin fibers and surfactant. They are used, for example, as terminal units in coupled lung
models to mimic the behaviour of the respiratory zone and to quantify regional inflation of lung tissue (see “Reduced-Dimensional
Coupled Lung Models” section). Further, reduced-dimensional models of the respiratory zone have also been used to study the
effect of different diseases on the behaviour of lung tissue. For instance, by eliminating single pin-jointed line elements within
a cluster, an emphysemic condition has been modeled. By this means, the failure of single alveolar walls on the microscopic scale
has been related to a disease-related tissue softening on the macroscopic scale. Finally, spring models of the respiratory zone have
been used to investigate the volume-competition between different lung regions during inflation in a simplified way and to quantify
interactions between neighboring air spaces in health and disease. Still, reduced models of the respiratory zone are only describing
one part of the lung, namely the respiratory zone. Therefore, they are limited to isolated observations of this region or have to be
coupled to an adequate model of the conducting zone, as presented in the “Reduced-Dimensional Coupled Lung Models” section.
Continuum Models of the Respiratory Zone
For a more detailed investigation of phenomena in the respiratory zone, continuum mechanics based models of single alveoli, alve-
olar ducts, or entire acini have been developed in the past. Many of these models are based on artificial geometric representations of
individual alveoli ranging from simple spherical or polyhedral shapes to more realistic irregular cells. Some approaches also use
imaging-based reconstructions, for example, obtained from synchrotron-based X-ray microscopic tomography. However, due to
the small size of the air spaces, in vivo imaging of alveolar structures remains difficult.
Continuum models of the respiratory zone have been used to study acinar flow phenomena in detail. Despite the low Reynolds
numbers, airflow in this region is still complex due to the rhythmic expansion and contraction of the tissue during respiration. For
instance, recirculating structures and radial flows induced by the septal wall movement can be found in the alveolar cavities. In addi-
tion to acinar flow patterns, the transport, mixing, and deposition of particles have been studied extensively. Applications include
the prediction of acinar aerosol deposition for therapeutic delivery and the assessment of health risks associated with inhaled
hazardous particles. Furthermore, the transport of oxygen molecules through the acinus and the exchange of oxygen at the
blood-gas barrier have been simulated. Finally, continuum mechanics based models of the respiratory zone have also been applied
to investigate alveolar stresses and strains. This way, local tissue strain “hotspots” have been identified which are at risk of overdis-
tension during mechanical ventilation. Since stresses and strains cannot be measured experimentally, continuum models of the
respiratory zone can make an important contribution to a better understanding of involved stress raising phenomena and associated
risks at tissue overdistension.
As with all isolated models of parts of the respiratory system, the formulation of physiologically reasonable boundary conditions
(i.e., flow boundary conditions, boundary loads, and deformations) is difficult. In reality, the modeled alveolar or acinar structure is
connected to the conducting zone and embedded in the surrounding respiratory zone. However, most existing models presented in
this section are not capable of considering these effects adequately. Therefore, continuum models of the respiratory zone are
primarily used for isolated, qualitative investigations.
Reduced-Dimensional Coupled Lung Models
In the majority of applications, both a representation of the conducting zone as well as the respiratory zone are necessary to cover all
aspects that are relevant for an accurate description of lung physiology. If, however, no detailed resolution of the flow field in the
conducting zone and no fully resolved continuum mechanical description of the respiratory zone is required, the lung can be rep-
resented using reduced-dimensional coupled approaches. These simplified reduced-dimensional representations of respiratory
biomechanics comprise approaches ranging from pure phenomenological single-compartment models to imaging-based, physio-
logically realistic approaches grounded on the physics of previously introduced airflow dynamics and tissue mechanics.
Single-Compartment Models
In analogy to the basic anatomy of the lung, single-compartment models consist of a single pipe representing the conducting
airways, which is connected to a single elastic compartment representing lung parenchyma. The motivation behind these models
is that the resistance of the entire conducting airway tree can be combined into a single equivalent (pipe) resistance. Further, the
compliance of all tissue components in the respiratory zone and the elastic properties of the chest wall are united into a single equiv-
alent (compartment) compliance. The mechanical behaviour of the lung can then be expressed via fitting these two parameters (i.e.,
resistance and compliance) to patient measurements using the method of least squares.
Since this approach is no physically based model in a strict sense, no modeling assumptions in a strict sense apply. The only two
necessary assumptions for single-compartment models are that the chosen model equation is able to replicate lung behaviour and
that a sufficient number of clinical measurements of pressure, flow and volume exists to fit the required parameters. A further
assumption that is implicit in the representation via a single compartment is that the fit mechanical behaviour is averaged over
the entire organ. This means that any regional differences in lung function are omitted in these models.
Depending on the complexity of the model, different realizations for resistance and compliance are conceivable. If both the resis-
tance and the compliance are constant the so-called linear single-compartment model is obtained. Several investigations have,
however, shown that both the conducting zone and the respiratory zone contain significant sources of non-linearity. To begin with
the respiratory zone, clinically measured transpulmonary pressures at different lung volumes recorded as quasi-static pressure–
volume (P–V) measurements have revealed that the lung shows a sigmoidal-like inflation/deflation behaviour. This behaviour
can be successfully described using exponential mathematical functions of transpulmonary pressure depending on lung volume.
Especially the lower and the upper sigmoidal part of such P–V curves are important in modeling respiratory biomechanics. The
lower part (i.e., at small pressures and lung volumes) reflects recruitment/derecruitment of air spaces from the initially degassed
state and the upper part (i.e., high pressures and lung volumes) characterizes tissue overdistension. These two phenomena cause
elevated stresses in the tissue and therefore require accurate quantification in clinically relevant modeling. Observing the conducting
zone, airflow through the larynx and in the larger airways can become turbulent especially at high flow rates or in cases of highly
pulsatile flows which leads to a flow-dependent non-linear resistance for example, described by Rohrer’s equation. Further, the pres-
sure difference between the inside of a conducting airway and its surrounding causes diameter changes of the compliant airway,
which in turn affect the airway’s resistance. This effect leads to resistance differences between inspiration and expiration due to
the varying pleural pressure that is propagated to the airway’s surrounding and ultimately defines expiratory flow limitation
explained previously.
In general, single-compartment models are a phenomenological description of lung mechanics based on mathematical equa-
tions. The required parameters, namely equivalent resistance and equivalent compliance, are fit from clinical pressure and flow
measurements allowing a quick and easy assessment of basic respiratory function. The required fitting algorithms for linear and
for non-linear models are easy to implement, they operate in real-time and deliver values that are easy to interpret in a clinical
setting. Still, these models do not have a strict physical background and rather have to be seen as a fitting technique. With more
effects included for example, non-linearity or a relationship for recruitment/derecruitment, single-compartment models become
more realistic and allow a better adaption to the physiology of the lung. On the other hand, each additional parameter requires
more reliable data for fitting and thus the model becomes less predictive, or is even not predictive at all. Finally, one major drawback
of the single compartment models is that no specific anatomy of the patient can be respected and no regional information on
mechanical overstraining or recruited/derecruited regions can be given. This means that if equivalent resistance and compliance
values lead to the conclusion that the lung of a specific patient is only working at 50%, no specification can be made whether
the entire lung is working at only 50% or if one part is working completely normal whereas the other one is not working at all.
Despite these limitations, single-compartment models are up to now the widest used modeling approach for respiratory biome-
chanics and still under development. They have successfully served as starting point for investigations of gas transport and transfer
into the blood and for general coupling with the cardiovascular system. Further, they have been used in diagnosis of respiratory
diseases as an easy to apply bedside tool in respiratory care. Recently, single-compartment models have been extended by a func-
tionality to quantify ventilator-associated lung injury resulting from recruitment/derecruitment and overstraining. Finally,
single-compartment models have successfully been applied in optimizing ventilatory settings in several randomized clinical trials.
Nevertheless as it has been proven that the extent of lung heterogeneity is directly linked to disease severity and mortality, it would
be desirable to use more precise regional models for this optimisation task.
Multi-Compartment Models
Multi-compartment models are the next step towards more realistic modeling of respiratory biomechanics. They comprise all
approaches that are characterized by multiple reduced-dimensional components for both the conducting and the respiratory
zone of the lung and mark the transition from pure phenomenological approaches towards physically motivated models in respi-
ratory biomechanics. In general, multi-compartment models are motivated by the idea that a reduced-dimensional description is
the most efficient way to describe respiratory biomechanics on the organ-level and the awareness that the lack of regional informa-
tion has to be overcome to allow precise conclusions in a clinical setting.
Pure phenomenological multi-compartment models are characterized by a parallel arrangement of single-compartment models
with distributed parameter values for equivalent resistance and compliance extended by models governing recruitment/derecruit-
ment dynamics. The same assumptions hold as for single-compartment models except for the assumption that the behaviour is
averaged over the entire organ (see “Single-Compartment Models” section). Required model parameters are still identified via
fitting to patient measurements.
Physically motivated multi-compartment models on the other hand are built upon the underlying physics. Specific assumptions
are made to enable the reduced-dimensional description of both the conducting and the respiratory zone. The one-dimensional,
zero-dimensional, or impedance-based representations of single airway segments of the conducting zone (see “Reduced Models
of the Conducting Zone” section) are then combined to a morphologically realistic tree structure using either data from lung casts
or tree-growing algorithms that generate a space-filling airway tree within a patient-specific imaging-based lung hull geometry. Addi-
tionally, each airway segment can be equipped with a representation of recruitment/derecruitment dynamics based on an additional
variable that describes the opening state and its progression. The respiratory zone at the terminal ends of the airway tree or in the
parallel arrangements of single-compartment models can also either be fit to pure phenomenological equations of lung tissue using
for example, the previously mentioned exponential compliance equations, or be derived from physically motivated descriptions of
lung tissue for example, based on alveolar duct models (see “Reduced Models of the Respiratory Zone” section). An important
recent extension related to the conducting zone in multi-compartment models is the consideration of the interplay between single
neighboring compartments, also known as lung interdependence, adding realistic stability to single inflating/deflating air spaces.
Essentially all multi-compartment models are a functional relationship between pressure and flow in the conducting and the
respiratory zone and allow for a spatial resolution of computed quantities in different regions of the lung. With the possibility
to respect spatially distributed material properties and regionally varying threshold reopening pressures as well as gravitational
effects, they allow a more realistic examination of lung function. Simple parallel arrangements of single-compartment models
are still phenomenological representations of lung mechanics that have to be fit to measurements and thus are easy to adapt to
a specific patient. For a satisfactory fit, the quality of the available measurements is decisive. The predictive character of these models
suffers from the fact that it is not understood what happens in scenarios beyond those where fitting data are available. Conclusions
on higher pressures than those measured are then only a more sophisticated mathematical extrapolation without deeper knowledge
about potential critical points in system behaviour and thus dangerous for prediction in a clinical application.
Physically based multi-compartment models allow a deeper insight into airflow throughout a network of compliant airway
segments and inflation of (visco-)elastic lung tissue. In these models, the descriptions of the conducting and the respiratory
zone are derived from physically sound airflow dynamics and tissue mechanics and extended by all capabilities that are necessary
to describe the behaviour of the lung. They can include interdependence as well as the dynamics of recruitment/derecruitment. Veri-
fication against continuum mechanical representations of the conducting zone show that results from the reduced-dimensional
models is in good agreement and even able to adequately take into account turbulence effects. Yet, the reduced-dimensional models
are fast in their computation and deliver pressure and flow data that are easy to interpret in a clinical setting. These models allow
a closer look into the black box of lung modeling and thus are more powerful than pure fitting approaches in terms of predicting
critical or extremely beneficial states of lung function. They require only few data for patient-specific calibration, which means that
they can deliver reliable data in the entire physiological pressure range in respiration. Furthermore, it is possible to integrate patient-
specific information from medical imaging in form of the lung contours that serve as a limitation of the artificially grown airway
tree.
So far, several questions in respiratory biomechanics have successfully been investigated using multi-compartment models. Most
importantly, the reopening dynamics of collapsed lung regions in acute respiratory distress syndrome have been assessed as a func-
tion of reopening pressure and time of the maneuver. In this context the optimal moments, pressures and duration of deep infla-
tions during mechanical ventilation could be determined. Further, it has been possible to predict flow limitations in a healthy
airway tree as well as the effect of heterogeneous bronchoconstriction and regional tissue heterogeneity on regional ventilation
in diseased lungs. Besides, the propagation of a liquid plug in a complex network of reduced-dimensional airways could be studied
and the associated frequency dependency of conducting airway and lung tissue behaviour could be determined. The aforemen-
tioned investigations address the basic concepts of cyclic closure/reopening and overstraining during mechanical ventilation of crit-
ically ill patients. The multi-compartment models have successfully enabled the identification of minimally injurious modes of
ventilation in this context.
Hybrid Representations of the Respiratory System
Hybrid models are the next level of detail in modeling respiratory biomechanics. They are used if only one zone requires specific
attention and has to be modeled as a continuum while the other one can be respected using a reduced-dimensional approach. By
coupling reduced and resolved representations, the interaction between the conducting and the respiratory zone of the lung is auto-
matically assured. In practice, there are two main possibilities how such hybrid models can be composed. First, a continuum
mechanical description of the conducting zone can be linked to a reduced-dimensional model of the respiratory zone. According
to the zone that is resolved as continuum, this variant is denoted “hybrid (conducting)” in the overview (see Fig. 2). Second,
a reduced-dimensional model of the conducting zone can be coupled to a continuum mechanical description of the respiratory
zone, denoted as “hybrid (respiratory).” The two variants will be discussed in detail in the following.
Hybrid Model (Conducting)

For some investigations, only the conducting zone is required to be resolved in full detail as far as possible from available imaging
data, while phenomena related to the terminal airways and the respiratory tissue are of no specific interest. Still, the influence of the
airways beyond full resolution and the respiratory zone cannot be neglected for a realistic description of airflow in the conducting
zone. Therefore, the effects of flow resistance in smaller non-resolved airways, and the inflation of lung tissue have to be modeled via
appropriate boundary conditions at the terminal ends of the fully resolved airway tree. The idea is to respect all downstream effects
that generate dynamic pressures during inflation/deflation of the lung in a reduced-dimensional fashion. Additionally, a storage
capacity for the air that leaves the fully resolved tree during inspiration, and can consistently re-enter the domain during expiration,
is provided. Hence, the model denoted as “hybrid (conducting)” is essentially a continuum description of the conducting zone
extended by appropriate methods to take into account all effects that occur downstream the fully resolved region.
One assumption that is required for this kind of hybrid models is that there are no recirculation zones present at the terminal
ends of the fully resolved conducting zone. Further, the elongation of airways in axial direction and the distortion of the airway tree
with regards to branching angles between different generations, introduced by the inflation of lung tissue, is usually not included in
this type of hybrid models. If elongation/distortion occurs, recirculation zones in the resolved tree could be influenced and inves-
tigations of, for example, particle transport and deposition as well as gas transport and mixing have to be carefully evaluated. For
such observations, and investigations in flow limitation or bronchoconstriction, fluid–structure interaction and parenchymal teth-
ering effects have to be considered (see “Continuum Models of the Conducting Zone” section).
The governing equations for airflow in the continuum part of the conducting zone of such hybrid models are the incompressible
Navier–Stokes equations. The non-resolved respiratory zone downstream of each resolved tree outlet is governed by a non-linear
relationship between pressure at the outlet cross section and flow into as well as volume of the region of the respiratory zone
attached. Different possibilities exist to derive such a relationship. It is for example, possible to couple the continuum conducting
zone to a reduced-dimensional multi-compartment tree-like model to account for the resistance and compliance of the smaller non-
resolved airways. Thereby, physically-based information can be included into the hybrid model such as the dimensions and connec-
tion of the non-resolved airways coming from a tree-growing algorithm. At the terminal ends of that tree, a reduced-dimensional
respiratory zone model is attached as known from physically based multi-compartment models. Alternatively, regional functional
diagnostics of a patient can be included to represent varying material properties of the different regions attached to the single
outlets. One popular approach in this context is to derive an equivalent regional resistance and compliance of the respiratory tissue
to be modeled and to prescribe a pressure-flow-volume relationship similar to the single-compartment equation at each outlet. In
this case, additional measurements are required to fit the involved parameters such as equivalent regional resistance and
compliance.
Both approaches then lead to a realistic pressure level over time at the outlets of the continuum mechanical conducting zone and
consequently to a correct pressure distribution in the entire domain. Neglecting the pressures resulting from non-resolved down-
stream passages can significantly corrupt flow patterns. Imagine for example, a case where airflow splits up between two almost
symmetrical daughter airways of a resolved domain. Without the representation of the downstream regions, airflow would
distribute almost equally between the two daughter airways. If however, one of these daughters supplies a stiffer lung region, in
reality airflow to this region would be much lower while the other region would be overdistended. Also, the transport of curative
aerosols to the stiffer region would be overrated and with it the potential for healing the increased stiffness.
This brief example already shows that it is extremely important to model the non-resolvable regions of the respiratory system,
especially in heterogeneous, that is, diseased lungs. A further important aspect is the storage possibility for air during inspiration. In
some modeling studies in respiratory biomechanics expiration is modeled via a prescribed negative inflow at the trachea inlet.
However, it is not clear from which of the single airway branches the outflowing air comes fromdnot to speak about the associated
dynamics. This may lead to the fact that at some outlets more air is expired than actually inspired before, a behaviour which is clearly
unphysical. In general, a storage capacity is an important aspect of the respiratory zone and has to be included in realistic models of
respiratory biomechanics. This is why an impedance modeling of the reduced-dimensional respiratory zone is in general not suit-
able, as the impedance does not include a storage capacity for air volume.
Important applications of the hybrid (conducting) models are in general all problems where a detailed flow field in resolvable
airways is required and where induced deformation and distortion of the bronchial geometry is small. These requirements usually
hold in large airways which are stiff enough and for small volume changes during respiration. Possible examples of such scenarios
have already been mentioned in the “Continuum Models of the Conducting Zone” section and virtually all of these investigations
would profit from a hybrid representation of the respiratory zone. One specific application in mechanical ventilation that is partic-
ularly suitable for this hybrid model is the ventilation with high frequencies and low tidal volumes known as high frequency oscil-
latory ventilation. In that case introduced deformation is low due to low tidal volumes and a resolved fluid field is required to fully
understand the complex mixing processes in the conducting system during this efficient technique of mechanical ventilation.
Hybrid Model (Respiratory)

A second approach of hybrid modeling is used if processes on the macroscopic tissue scale require specific attention (e.g., to
quantify tissue overdistension) whereas airflow in the conducting system is only of interest inasmuch as it is responsible for the
distribution of air in the respiratory zone. In this case, a suitable hybrid model can be realized by combining a physically based
reduced-dimensional model of the conducting zone with a continuum mechanical imaging-based description of the respiratory
zone. The geometry of the airway tree can be obtained using tree growing algorithms within the hull geometry of the lung
segmented from a CT or MRI scan.
The governing equations for the reduced-dimensional compliant airway tree in the conducting zone have already been intro-
duced along with multi-compartment models in the “Multi-Compartment Models” section. The respiratory zone can be modeled
both as a solid continuum and a porous medium. While the former model only accounts for solid mechanical phenomena, the
latter approach additionally considers airflow through the sponge-like parenchyma (see Fig. 1) in a volume averaged sense, that
is, without explicitly resolving the detailed microstructure. As regards the coupling of the conducting and respiratory zone models,
pressure and flow have to be in equilibrium at the ends of the reduced-dimensional airway tree and the associated tissue regions.
Hence, inflation of a tissue region can only happen if the conducting system supplies air to it and vice versa a prescribed deforma-
tion of a tissue region induces an airflow in the associated reduced-dimensional airway.
The derivation of this hybrid model with a reduced conducting and resolved respiratory zone requires some assumptions. For the
conducting zone, they are equal to those of multi-compartment models (see “Multi-Compartment Models” section). For the respi-
ratory zone, the assumptions are less restrictive and only require sufficiently resolved imaging data to extract the geometry of the
respiratory zone as well as a suitable continuum mechanically based constitutive law for lung tissue.
In brief, the “hybrid model (respiratory)” is a continuum model for inflation/deflation of the respiratory zone governed not only
by tissue/porous media mechanics but also by correct airflow dynamics to inflate/deflate a specific lung region. The benefit of the
hybrid approach comes from the fact that the conducting zone is represented in a dimensional reduced fashion. Consequently, pres-
sure and flow in the conducting airway network can be computed very efficiently. Further, more tree generations than obtainable
from imaging data can be integrated in the model using well-known tree-growing algorithms. In theory it is possible to create an
entire airway tree up to the 16th generation within the lung hull geometry segmented from medical imaging data. Then each
terminal end of the airway tree can be coupled to a continuum model of the associated respiratory region. In this context, equilib-
rium of pressure and flow through the airway tree and at the specific region to be modeled has to be ensured. Since the structure of
the lung parenchyma is modeled as a continuum, the interplay between neighboring air spaces during inflation/deflation is auto-
matically included and thus lung interdependence is inherently considered in the model. Further, due to the full representation of
the tissue, prescribing additional boundary traction forces resulting from kinematic constraints (e.g., from the thoracic cage or the
diaphragm) is straightforward. Moreover, not only traction forces but also deformation paths for example, obtained from a temporal
series of imaging data can be prescribed to the continuum representation of lung tissue.
Equipped with these properties, the “hybrid model (respiratory)” is applicable for detailed investigations on regional overstrain-
ing of lung tissue and resulting stresses. Therefore it is perfectly suited for investigations in ventilator-associated lung injury which
has been directly linked to structural stresses and strains. Further, the availability of a fully resolved structure is beneficial for the
identification of suspicious tissue regions in tumor tracking and associated radiation therapy applications from an inverse fit of
measured tissue movements. Recently, the effect of airway constriction and heterogeneous tissue elasticity on the distribution of
tissue stress and alveolar pressure have been evaluated based on this type of hybrid models. These are important information
for treatment planning in chronic obstructive pulmonary disease, emphysema, or fibrosis.
Further hybrid couplings are conceivable such as, for example, a continuum mechanical description of the large airways
combined with a reduced-dimensional description of the small airways in the conducting zone and a fully resolved representation
of the respiratory zone. The decision which part has to be resolved as continuum and which one is sufficiently modeled with
reduced approaches can be tailored to the current question to be investigated. In this way, very efficient and modular hybrid models
can be generated to exactly fulfill the specific requirements for the specific application.
Continuum Coupled Lung Models
Continuum mechanics based overall lung models offer the possibility to simulate coupled phenomena in two- or three-
dimensional representations of both the conducting and the respiratory zone at a high level of detail. For instance, a recently
proposed continuum overall lung model is based on the coupling of a three-dimensional model of the airway tree with a three-
dimensional model of lung parenchyma. Similar to the model denoted as “hybrid (respiratory),” the parenchyma model is divided
into subdomains associated with the outlets of airway tree now fully resolved as continuum. The volume of air passing through each
outlet is then constrained to equal the change in volume of the corresponding parenchyma subdomain. This mutual coupling of
airway and parenchyma models enables the simulation of reasonable deformation states and pressure levels since it links flow
quantities, for example, pressure and resolved velocity profiles in the conducting zone, to local stresses and strains in the respiratory
zone of the lung.
Concluding Remarks
In current literature a variety of approaches exist for modeling respiratory biomechanics and their sensible usage is largely depen-
dent on the problem to be investigateddsomething which is true for all meaningful modeling. For clinical monitoring of a patient
with plenty of measurement data available and a fast and only global classification required at bedside, phenomenological fitting
approaches are a quite reasonable choice. For more advanced investigations with single effects to be isolated or even to be predicted,
more realistic respiratory models grounded on the underlying physics of airflow dynamics and soft tissue mechanics are more
powerful and allow a deeper insight into physiological effects beyond classical black-box parameterisations of lung function.
In very specific cases, realistic predictions are possible based on models comprising the conducting or respiratory zone only. The
majority of phenomena in respiratory biomechanics, however, live from the interplay between the conducting and the respiratory
zone and how alterations in one zone ultimately affect the behaviour of the other. Therefore, many models consider both the con-
ducting and the respiratory zone as well as their interplay. Different levels of resolution for the two functional components have
been introduced, namely fully resolved continuum and reduced-dimensional representations.
Starting from phenomenological approaches that are purely based on parameter fitting of patient measurements, physically
based respiratory models have been defined with increasing complexity throughout this work. At first, single-compartment models
have been introduced which are often used in clinical monitoring to date. They have been extended towards physically based
reduced-dimensional multi-compartment approaches retaining the fast and efficient solution and offering reliable quality in the
prediction of regional pressures and ventilation. In case either the conducting or respiratory zone requires specific attention, hybrid
models can be defined where one zone can be realized by a continuum description while the remaining zone can be respected by
a reduced dimensional model. If a maximum level of detail is required either for extremely complex investigations or for validation
of reduced-dimensional models, both zones can be resolved as three-dimensional continuum.
Specific examples for potential applications of the single approaches have been presented in this article. They outline the fact that
there is no “one-size-fits-all” approach in respiratory biomechanics, but rather a toolbox of methods available to solve the current
problem at hand. An introduction towards this toolbox including model assumptions and a fair discussion of potentials and limi-
tations for each approach is provided in this work and guides the reader towards the best model for current and future investigations
in a clinical or scientific practice. We hope that this categorization sheds light on the current state of the art in modeling respiratory
biomechanics and further promotes the development of methods that ultimately improve diagnosis and advances in individual
patient treatment.
Further Reading
Amini, R., & Kaczka, D. W. (2013). Impact of ventilation frequency and parenchymal stiffness on flow and pressure distribution in a canine lung model. Annals of Biomedical
Engineering, 41, 2699–2711.
Bates, J. H. T. (2009). Lung mechanics: An inverse modeling approach (1st edn.). Cambridge: Cambridge University Press.
Bertram, C., & Gaver, D. P., III (2005). Biofluid mechanics of the pulmonary system. Annals of Biomedical Engineering, 33, 1681–1688.
Denny, E., & Schroter, R. C. (2000). Viscoelastic behavior of a lung alveolar duct model. Journal of Biomechanical Engineering, 122, 143–151.
Kleinstreuer, C., & Zhang, Z. (2010). Airflow and particle transport in the human respiratory system. Annual Review of Fluid Mechanics, 42, 301–334.
Ma, B., & Bates, J. H. T. (2010). Modeling the complex dynamics of derecruitment in the lung. Annals of Biomedical Engineering, 38, 3466–3477.
Maury, B. (2013). The respiratory system in equations. In A. Quateroni (Ed.), Modeling, simulation and applications (7th edn., pp. 1–278). Italia: Springer-Verlag.
Rausch, S. M., Haberthür, D., Stampanoni, M., Schitty, J. C., & Wall, W. A. (2011). Local strain distribution in three-dimensional alveolar geometries. Annals of Biomedical
Roth, C. J., Yoshihara, L., Ismail, M., & Wall, W. A. (2016). Computational modeling of the respiratory system: Discussion of coupled modeling approaches and two recent
extensions. Computer Methods in Applied Mechanics and Engineering, 314, 473–493.
Smith, B. J., & Bates, J. H. T. (2015). Variable ventilation as a diagnostic tool for the injured lung. IEEE Transactions on Biomedical Engineering, 62, 2106–2113.
Sznitman, J. (2013). Respiratory microflows in the pulmonary Acinus. Journal of Biomechanics, 46, 284–298.
Tawhai, M. H., & Bates, J. H. T. (2011). Multi-scale Lung Modeling. Journal of Applied Physiology, 110, 1466–1472.
West, J. B. (2012). Respiratory physiology–the essentials (9th edn.). Philadelphia, PA: Lippincott Williams & Wilkins.
Yoshihara, L., Roth, C. J., & Wall, W. A. (2016). Fluid–structure interaction including volumetric coupling with homogenized subdomains for modeling respiratory mechanics.
International Journal of Numerical Methods in Biomedical Engineering, 33, e2812.
Relevant Websites
Auckland, n.d.dhttp://www.abi.auckland.ac.nz/en/about/our-research/lungs-respiratory-system.html, Auckland Bioengineering InstitutedLungs and Respiratory System.

INRIA, n.d.dhttps://team.inria.fr/reo/respiration_modeling/, INRIA Paris-RocquencourtdRespiratory Modeling.
Technical University of Munich, n.d.dhttps://www.lnm.mw.tum.de/research/applications/biomedical-respiratory-system/, Insititute for Computational Mechanics, Technical
University of MunichdRespiratory Modeling.
Tulane University, n.d.dhttp://www.tulane.edu/dpg/index.html, Biofluid Mechanics Laboratory, Tulane University.
Constitutive Modeling of Soft Tissues
Michele Marino, Leibniz Universität Hannover, Hannover, Germany
Introduction 81
Structure–Mechanics Relationship 82
Basics of Continuum Mechanics 84
Hyperelastic Behavior 86
Material Objectivity 86
Stress–Strain Relationships 86
Material Symmetry 87
Invariant-Based Formulations 88
Material Stability and Mathematical Requirements 90
Incompressibility Condition 91
The penalty method 91
The augmented lagrangian method 93
Application and final remarks on incompressibility 93
State of the Art of Hyperelastic Constitutive Modeling 97
Multiscale structural approach 99
Comparison with experimental data 101
Inelastic Behavior 102
Viscoelasticity 105
Active Response 105
Plasticity 106
Damage 106
Growth and Remodeling 107
Open Problems and Future Challenges 109
Further Reading 110
Introduction
Constitutive modeling in mechanics is the art of describing the mechanical properties of materials through mathematical models,
that is by means of mathematical problems formulated in connection with physical concepts and experimental evidence. The effec-
tiveness of constitutive models plays a key role in the predictive capabilities of computational models of structures. In this frame-
work, computational models of biological structures have proved themselves to speed up the rate of scientific discovery in medicine
and to improve the effectiveness of clinical approaches. In silico analyses reduce indeed the need for expensive laboratory work and
clinical trials because they are able to reproduce different pathophysiological scenarios in a rapid and low-cost way. Accordingly, the
development of computational environments for biomedical research contributes to: (i) clarify the complex mechanobiological
equilibrium that maintain the physiological behavior; (ii) identify relationships between histological and biochemical alterations
with pathological responses; (iii) gain a better understanding of the etiology of diseases; (iv) support the tailoring of clinical treat-
ments to patient-specific features.
Soft tissues are biological tissues made up by cells, collagen, elastin, and ground matrix, not being mineralized. They provide the
essential link and support for organs and biological structures throughout the whole human body, typical examples being tendons,
ligaments, skin, muscles, blood vessels, heart, cornea, and intestine. Therefore, the mechanical response of soft tissues highly affects
the functionalities of many body systems in health and disease (e.g., musculoskeletal, cardiovascular, digestive). Hence, the consti-
tutive modeling of soft tissues is a frontier research challenge at the cutting edge of biomechanics.
As a general rule of thumb, constitutive models shall be able to reproduce the in vivo or in vitro mechanical behavior of tissues,
and in particular, the biomechanical phenomenon under investigation in the final numerical simulation. Moreover, the constitutive
description shall be based on model parameters whose value can be determined from in vitro or in vivo observations. Finally,
constitutive models shall respect mathematical requirements in order to be physically consistent and effective for conducting
numerical simulations.
The mechanical response of soft tissues is characterized by nonlinear stress–strain relationships associated with an incompress-
ible or quasi-incompressible behavior due to the high water content (i.e., characterized by negligible or small volume variations).
Moreover, tissue mechanics can be also affected by inelastic mechanisms, such as damage and viscous effects, as well as growth and
remodeling. This article opens with a brief overview of the structure–mechanics relationship in soft tissues and with the description
of basic ingredients in continuum mechanics needed for the development of constitutive models. The main part of this work

82 Biomechanics j Constitutive Modeling of Soft Tissues
addresses the constitutive modeling of soft tissues in terms of nonlinear hyperelasticity, anisotropic behavior, and incompressibility.
Although the description of techniques for facing computational issues on numerical simulations of biological structures is beyond
the scope of the present work, some hints on the treatment of the incompressibility constraint from a computational point of view
will be provided, especially considering the coupling with an anisotropic behavior. Afterward, the state of the art in terms of some
available constitutive approaches is presented with an insight on multiscale approaches. Finally, a general framework for the
modeling of possible inelastic effects occurring in soft tissues will be introduced, addressing viscosity, active response, plasticity,
damage, growth, and remodeling. Some remarks on open problems and future challenges conclude the article.
Structure–Mechanics Relationship
Collagen and elastin are the constituents mainly responsible for tissue elastic, stiffness, and strength properties. The high water
content in the ground substance also plays an important role from the mechanical point of view. Tissue constituents are organized
following a precise and hierarchical multiscale arrangement.
Elastin is assembled as a continuous network of fibers which are believed to have a key role in providing distensibility properties
and elastic recoil to tissues. Nevertheless, the stiffness of the elastin network is significantly lower than the one of collagen. Accord-
ingly, in elucidating the structure–mechanics relationship, the greatest attention is dedicated to collagen behavior.
Collagen can be found in form of fibers arranged in agreement with a regular (e.g., tendons) or an irregular (e.g., skin) pattern.
The arrangement of collagen fibers in regular tissues follows a predefined pattern and these can be conveniently classified in uni-
(e.g., tendons and ligaments) or multi- (e.g., arterial walls) directional. In unidirectional tissues, the main orientation of collagen
fibers is unique and fibers can be retained as parallel one to each other. A multidirectional tissue is intended to be made up of
a number of stacked thin layers, each of them with a regular unidirectional fiber arrangement.
The basic building blocks of collagen fibers are tropocollagen molecules. The latter are proteins that can be found in more than
27 forms, depending on the structure. Type I collagen is the most abundant in the human body, being the most important for main-
taining the structural integrity and a functional mechanics of soft tissues. Type I collagen molecules are made up by three polypep-
tide strands, each one being a left-handed helix (see Fig. 1). The three helices are twisted together into a triple helix quaternary
structure about 300 nm long and 1–2 nm in diameter, stabilized by interstrand hydrogen weak bonds.
Collagen molecules exhibit hydroxyproline-deficient sequences characterized by 60 residues, referred to as labile domains and
indicated as molecular kinks (see Fig. 1). The overall length of molecular kinks is about 20 nm and it is comparable with the value of
Fig. 1 Hierarchical multiscale arrangement of collagen constituents in soft tissues (top). Mechanical response of soft tissues (bottom): J-shaped
stress–strain relationship of a unidirectional tissue subjected to a uniaxial traction along the fiber direction, where the dominant mechanisms affecting
the toe, heel, and linear regions are highlighted.
Biomechanics j Constitutive Modeling of Soft Tissues 83
the persistence length for collagen (about 14 nm). The persistence length represents the minimum contour length over which
molecular segments fluctuate due to thermal energy. Therefore, molecular kinks are activated by thermal undulations, that is their
configuration follows a statistical distribution due to thermal excitation. Molecular kinks can be straightened by forces applied at
molecular ends that counteract thermal undulations, hence experiencing a transition from less ordered states (thermally activated
kinks) to more ordered ones (nearly straight molecule). In this regime, usually referred to as entropic elasticity, the mechanical
response of collagen molecules is mainly dominated by the flexural behavior of the polypeptide helices, rather than by the exten-
sibility of intramolecular covalent bonds. This contributes to collagen extensibility up to molecular contour length. Nevertheless,
collagen shows a significant level of molecular extensibility beyond its contour length. Accordingly, when molecular end-to-end
length approaches the contour length, covalent bonds within the polypeptide strands are stretched out, inducing the transition
from entropic elasticty to a different mechanism, which can be referred to as energetic elasticity.
In soft tissues, tropocollagen molecules self-assemble to form long and continuous cylinder-like structures, named fibrils, char-
acterized by a diameter between 50 and 500 nm (see Fig. 1). Collagen molecules are organized within fibrils by following a complex
three-dimensional crystallographic pattern. Nevertheless, simple arrangement models are effective in capturing the key mechanical
aspects of fibrils. For instance, according to the Hodge–Petruska scheme, fibrils can be successfully modeled as staggered arrays of
parallel macromolecules with an axial offset of about 67 nm and an equilibrium center-to-center distance of about 1.5 nm between
two transversally adjacent molecules. Within fibrils, molecules interact with each other by means of intermolecular covalent cross-
links (see Fig. 1). Accordingly, the elongation of collagen fibrils is affected by two mechanisms which can be retained as in series: the
stretching of collagen triple helices (depending both on entropic and energetic mechanisms) and molecular rearrangement mech-
anisms (mainly, intermolecular sliding). Since cross-links prevent intrafibrillar sliding, the load transmission within fibrils is highly
affected by the amount of cross-links. Indeed, the sliding-to-stretching ratio of the total fibril elongation highly depends on the
biochemistry of cross-links production and renewal.
Collagen fibrils are densely packed in bundles called fibers. Adjacent fibrils within fibers are stabilized by lateral fibril-to-fibril
proteoglycan filaments. A controversial matter is if proteoglycans play or not a significant role in loading transfer among adjacent
fibrils. Collagen fibers in soft tissues are characterized by a crimped microstructure (see Fig. 1). Fibers appear indeed as periodic-like
curvilinear structures with characteristic length period in the order of mm. The crimp amplitude of collagen fibers is highly variable
with tissue location and functional role, although generally in the order of one tenth of fiber period.
The mechanical response of soft tissues is highly nonlinear and characterized by J-shaped stress–strain curves. In the case of
a unidirectional tissue subjected to a uniaxial traction along the fiber direction, the stress–strain curve has been described as sub-
divided into three main regions where mechanisms occurring at very different length scales are dominant in each region (see Fig. 1):
1. Toe region (strain range z0%–2%): This is a low stiffness region associated with the straightening of the microscopic crimp in
collagen fibers.
2. Heel region (strain range z 2%–4%): This is a region associated with a significant stiffening response due to the straightening of
molecular kinks.
3. Linear region (strain range z4%–10%): This is a high stiffness region mainly related to fibril elongation (both collagen
stretching and sliding).
Accordingly, the structured and hierarchical arrangement of tissue constituents is the responsible for the peculiar mechanical
behavior of soft tissues. In many applications, soft tissue mechanics under relatively large deformations (up to about 10% nominal
strain) can be retained purely elastic. Approaches for the modeling of an elastic behavior will be introduced in what follows in the
framework of nonlinear hyperelasticity.
Nevertheless, the in vivo mechanical response of biological structures can be associated with the observation of mechanisms that
cannot be described by employing a hyperelastic framework. The mechanical response of soft tissues depends indeed on the loading
history. Addressing the cyclic loading–unloading testing of soft tissues, a hysteretic behavior (i.e., a stress-strain phase lag in the
cyclic process) is generally experienced, accompanied by relevant strain-rate effects (see Fig. 2). By repeated cycling, eventually
a steady state is reached at which no further change will occur. In this state, the tissue is said to be preconditioned.
Fig. 2 Inelastic behavior of soft tissues. Left: viscous-like response. Right: damage-related and plastic-like mechanisms.
Furthermore, as schematically depicted in Fig. 2, damage-related and plastic-like behaviors can be obtained when soft tissues
experience supraphysiological but subfailure loadings (e.g., during some surgical procedures such as balloon angioplasty in
arteries). In particular, significant residual strains arise upon loading removal when plastic-like mechanisms occur, while damage
is mainly associated with a degradation of tissue mechanical properties. In this framework, a behavior analogous to the Mullins
effect in rubber materials can be revealed. The Mullins effect consists in a degradation of material stiffness whenever the load
increases beyond its prior all-time maximum value. On the contrary, the final failure is associated with a drop in tissue stress.
Moreover, tissue mechanics can be altered by a change in tissue structure driven by the biological activity of cells in response to
both mechanical and biochemical stimuli. Finally, the presence of active elements in cells endows tissues with the ability to contract
and relax in response to biochemical signals, affecting the mechanical response of biological structures.
Some underlying nano- and microscale mechanisms responsible for the afore-introduced inelastic responses will be described
when the modeling of an inelastic behavior will be introduced in the framework of generalized standard materials.
Basics of Continuum Mechanics
Let E be a Euclidean space and V be the vector space associated with E. Moreover, denote by Lin the set of all linear transformations
of V into itself, namely the space of all second-order tensors. As notation rules, first- and second-order tensors are indicated, respec-
tively, in lowercase and uppercase boldface, while scalars are indicated as regular typeface. For a given (invertible) tensor A ˛ Lin, let
A 1, AT, Tr(A), and Det (A) denote the inverse, the transpose, the trace, and the determinant of A, with Adj (A) ¼ Det (A) A 1 being
the adjoint of A and Cof (A) ¼ (Adj (A))T the cofactor. Furthermore, given A, B ˛ Lin, the Frobenius inner product is denoted by A:
B ¼ Tr(ATB). Finally, Linþ collects transformations in Lin with positive determinant, R is the set of real numbers and Rþ collects
strictly positive real numbers. Introducing I ˛ Lin as the identity tensor, Q denotes the group of orthogonal tensors, i.e.,

Q ¼ Q ˛ Lin s:t: QT Q ¼ QQT ¼ I ; (1)
Qþ, the group of special orthogonal (rotation) tensors, that is

Qþ ¼ fQ ˛ Q s:t: Detð QÞ ¼ 1g; (2)
and U, the set of symmetric and positive definite tensors, i.e.,

U ¼ U ˛ Lin s:t: U ¼ UT and vT Uv > 0; cv ˛ V;vs0 : (3)
A biological medium is regarded as a continuum body occupying region Uo ˛ E in the reference configuration, parametrized in
xo ˛ V, and region U ˛ E in the current configuration, parametrized in x ˛ V (see Fig. 3). The deformation map 4 ˛ V maps point xo
(material coordinates) onto points x (spatial coordinates), resulting x ¼ 4(xo). In what follows, the subscript o denotes quantities in
Fig. 3 Theoretical framework for the mechanics of biological structures. Basic ingredients of continuum mechanics, material symmetry principles,
and representation of fiber reinforcements in soft tissues.
the reference configuration, while quantities in the current configuration have no subscript. At this standpoint, Grad (•) is intro-
duced as the gradient operator with respect to material coordinates and, introducing the time variable t, let x_ be the material time
derivative of quantity x.
The deformation map is locally described in terms of the deformation gradient tensor F, namely F ¼ Grad (4) ˛ Lin, which is
a two-point tensor transforming vectors from the reference to the current configuration. Local invertibility enforces the nonsingu-
larity of F, namely the Jacobian J ¼ Det (F) shall satisfy J > 0 or analogously F ˛ Linþ.
Introducing unit vectors vo, no, v, and n, the infinitesimal line vodxo, area vector nodAo, and volume dUo in the reference config-
uration transforms in the corresponding quantities vdx, ndA, and dU in the current configuration through:
vdx ¼ Fv o dxo ; ndA ¼ Cof ðFÞno dAo ; dU ¼ DetðFÞdUo : (4)
Moreover, the (unique) polar decompositions
F ¼ RU ¼ VR; R ˛ Qþ ; U; V ˛ U; (5)
highlight that each deformation gradient can be regarded as the superimposition of a rotation (associated with a rigid-body motion)
and a stretch, where U and V are named the right and left stretch tensors, respectively. Further common strain measures employed in
constitutive models are the right Cauchy–Green deformation tensor C ¼ FTF and the Green strain E ¼ (C I)/2.
Equilibrium is enforced by local balance equations, namely balance of mass, balance of linear and angular momentum, and laws
of thermodynamics. The balance of linear momentum and the second law of thermodynamics are here recalled, due to their rele-
vance for defining constitutive laws.
For a given kinematic quantity, chosen as reference measure for the development of the model, there exists a dual internal static
quantity which produces power for a motion associated with the time derivative of the chosen kinematic quantity. For instance, the
static quantity dual to the Green strain E is historically defined as the second Piola–Kirchhoff stress, denoted by tensor S.
In the case of conservative applied loads, the balance of linear momentum can be expressed in terms of a stationary condition for
the energy functional P ¼ P(4) ¼ !Uopint(4) dU þ Pext(4), namely
Z
4 ¼ argmin pint ðhÞdU þ Pext ðhÞ ; (6)
h ˛ C Uo
where C collects kinematically admissible transformations, pint(4) is the potential internal energy accumulated in the deformed
body per unit volume in the reference configuration, and Pext(4) is the potential energy of the external actions.
The potential energy in a thermodynamic system can be expressed by means of the mass specific free-energy functional jfe. For
reasons of material objectivity (detailed in the following), it is convenient to represent jfe as a function of the right Cauchy–Green
deformation tensor C, whose dependence on the deformation map 4, that is C ¼ C(4), is implicitly accounted for in what follows.
Moreover, in the framework of generalized standard materials, a set of internal variables V is introduced for describing possible
internal mechanisms. The potential internal energy pint of a deformed continuum body at constant temperature can be represented
in terms of the free-energy function jfe as:
pint ð4Þ ¼ Jfe ðC; VÞ ¼ ro jfe ðC; VÞ ; (7)
where Jfe is the free energy per unit volume and ro is the mass density in the reference configuration (here assumed to be constant in
time, if not differently specified). Addressing an isothermal and reversible thermodynamic process, !UoJfe dU represents the largest
quantity of work that can be gained from the deformed body.
In case of irreversible processes, a certain amount of work is lost and internal dissipation occurs. The second law of thermody-
namics enforces prescriptions on the internal dissipation Dint (per unit volume) which shall result nonnegative for any admissible
thermodynamic process. Neglecting thermal effects, the second law of thermodynamics is referred to as the Clausius–Duhem
inequality. In this framework, Dint represents the difference between the power produced by the internal static quantity and the
rate j_ fe of the free energy released by the deformed body. Accordingly, by choosing the Green strain E as reference kinematic quan-
tity (dual to the second Piola–Kirchhoff stress S), the Clausius–Duhem inequality prescribes:
C_
Dint ¼ S : E_ ro j_ fe ¼ S : ro j_ fe 0; (8)
2
for any admissible C_ and j_ fe . An admissible C_ corresponds to a motion which respects the constraints enforced by C, while
conditions on the admissibility of j_ fe derive from the choice of internal variables V and of their evolution equations.
Finally, other useful stress measures derived from the second Piola–Kirchhoff stress S are the first Piola–Kirchhoff stress P and the
Cauchy stress s:
1 1
P ¼ FS; s ¼ PFT ¼ FSFT : (9)
J J
The first Piola–Kirchhoff stress P is the static quantity dual to the deformation gradient F, while the Cauchy stress s produces
_ 1 . Balance of angular momentum prescribes that both S and s are symmetric tensors.
power for the spatial velocity gradient l ¼ FF
Finally, it is worth pointing out that the Cauchy stress s is a true measure of the force per unit area dA in the current (deformed)
configuration, the first Piola–Kirchhoff tensor P relates the force acting in the current configuration to the surface area element dAo in
the reference configuration (i.e., it is a two-point tensor), and the second Piola–Kirchhoff tensor S is the pull-back of P on the refer-
ence configuration (see Fig. 3).
Hyperelastic Behavior
A material is said to be hyperelastic or Green elastic when the energy stored upon deformation is fully released upon the removing of
the deforming cause. In other words, a hyperelastic material is conservative and a potential function exists, referred to as the strain
energy, that physically represents the potential mechanical energy stored in the body in the current configuration at constant
temperature. At this standpoint, function Jse is introduced as the strain energy density per unit volume in the reference configura-
tion. It is worth highlighting that Jse shall depend on a measure of deformation and, due to its physical meaning, it shall result in
a scalar-valued function taking positive values.
Constitutive models differentiate themselves in terms of different choices of the strain-energy function. The properties of the
strain-energy function directly follow physical and thermodynamical requirements, recalled in what follows.
First of all, it is worth pointing out that the strain-energy density Jse results to be function of material point xo for two
main reasons: (i) soft tissues are generally characterized by inhomogeneous mechanical properties within Uo; (ii) the defor-
mation is generally inhomogeneous within the body, leading to inhomogeneous values of Jse. Nevertheless, in order to
arrive at a more compact notation and if there is no danger of confusion, the dependence on xo of the individual functions
is generally omitted.
Material Objectivity
Material objectivity prescribes that the value of the strain-energy function is independent of superimposed rigid motions. Accord-
ingly, in order to rule out pure translations, the strain-energy density can be represented as a function JseF
of the deformation
gradient F, namely Jse ¼ Jse(F). Moreover, the value of Jse shall result independent from rotations superimposed to F. Therefore,
F F
it results:
Jse ¼ JFse ðFÞ ¼ JFse ðQFÞ; cQ ˛ Q: (10)
A strain-energy function satisfying Eq. (10) is said to be objective. Use of the polar decomposition in Eq. (5) and the choice
Q ¼ RT in Eq. (10) prescribe that the strain-energy function shall depend on the right stretch tensor U instead of F. In order to avoid
the polar decomposition and since C ¼ U2, the strain-energy function can be conveniently regarded as function of the right Cauchy–
Green deformation tensor C. Although choices based on different strain measures are also possible, material constitutive behavior
will be defined in what follows in terms of the strain-energy represented as:
Jse : Linþ 1Rþ ; Jse ¼ Jse ðCÞ: (11)
Stress–Strain Relationships
The relationship between stress and strain comes from energy definitions and thermodynamical requirements. For standard hypere-
lastic materials, the free energy Jfe is defined without considering any additional internal variable V, and equal to the strain energy
Jse(C), namely
Jfe ¼ Jfe ðCÞ ¼ Jse ðCÞ: (12)
By definition, the internal dissipation of a hyperelastic material shall be zero for any admissible deformation process. Accord-
ingly, considering Eqs. (7), (8), and (12), it results:
_
C_ _ se ¼ S 2 vJse : C ¼ 0 c admissible C: _
Dint ¼ S : J (13)
2 vC 2
The choice:
vJse
S¼2 ; (14)
vC
allows to a priori fulfill the requirement prescribed by Eq. (13) that holds for hyperelastic materials. The relationship S ¼ S(C)
obtained from Eq. (14) defines the constitutive law between tissue deformation and the resulting stress.
In order to account for the heterogeneous nature of soft tissues, where fibers are immersed in a soft ground matrix, a common
and general choice for the strain-energy density function in the homogenized body is to employ an additive decomposition for Jse
of the form:
vJM vJF
Jse ðCÞ ¼ VM JM ðCÞ þ VF JF ðCÞ; SðCÞ ¼ 2VM þ 2VF ; (15)
vC vC
where JM and JF, respectively, represent the matrix and the fibers contributions to the strain-energy density function, being,
respectively, averaged by means of the matrix VM and fiber VF volume fractions. If the reference configuration Uo is stress-free, then it
is referred to as a natural configuration and S(I) ¼ 0 holds.
Material Symmetry
Symmetries in the microstructural arrangement of constituents translate into symmetry properties of material behavior at the
continuum level. For instance, mechanical tests could be not able to distinguish the properties of a material in two distinct reference
configurations. Requiring the constitutive model to be consistent with material symmetries (if any), place further restrictions on the
strain-energy density function Jse(C), or analogously on stress function S(C).
Consider a change from Uo to a new reference configuration Uo* with material points identified by xo* and such that the trans-
o ¼ G belongs to the orthogonal group, namely G ˛ Q. Introducing F* as the nonsingular deformation
T
formation gradient Grad (x*)
gradient relative to Uo*, note that F ¼ F*GT, and F* ¼ FG, since G 1 ¼ GT (see Eq. 1). Moreover, the corresponding right Cauchy–
Green deformation tensor C* associated to F* results
C ¼ ðF ÞT ðF Þ ¼ GT CG; cG ˛ Q: (16)

Accounting for Eq. (16), the material symmetry group G with respect to the reference configuration Uo collects all the transfor-
mation gradients G such that the material response is independent from a change of the reference configuration, namely

G ¼ G ˛ Q s:t: Jse ðCÞ ¼ Jse GT CG ; cC ˛ Lin : (17)
Equivalently, in terms of stresses, material symmetry prescribes (see Fig. 3):

GT SðCÞG ¼ S GT CG ; cC ˛ Lin; cG ˛ G: (18)
The symmetry group G reflects the symmetry of the physical properties of the material. Therefore, it has to be specified case by
case, depending on the choice of the reference configuration and on microstructural properties.
If constituents are arranged with no apparent preferred orientation in the reference configuration, the material response obtained
from mechanical tests is identical for every rotation and reflection applied to Uo. In this case, the material is said to be isotropic in Uo
and it holds G h Q. The strain-energy density function can be represented in terms of isotropic scalar-valued tensor function Jiso
for which, by definition,

Jse ðCÞ ¼ Jiso ðCÞ ¼ Jiso GT CG ; cC ˛ Lin; cG ˛ Q : (19)
If constituents are arranged by following preferred orientations in the reference configuration, the material response obtained
from mechanical tests depends on the testing direction. In this case, the material is said to be anisotropic in Uo and it results G
3Q.
Soft tissues generally present anisotropic properties. For the sake of modeling, their anisotropic behavior is commonly described
by introducing a discrete collection of nF fiber families, each of them with direction described by the unit vector eo(a) ˛ V in the refer-
ence configuration with a ¼ 1, ., nF (see Fig. 3). In what follows, as a superscript rule, let a imply values in {1, ., nF}. Whenever
nF ¼ 1 fiber family is employed, superscript (a) is omitted.
In order to lose the dependence on the orientation of eo(a), that is a different response for e(a) o , the structural tensor
M ¼ eo(a) 5 eo(a) is employed in constitutive models. Geometrical symmetries in fiber arrangement are described by the invariance
(a)
set GM of structural tensors with respect to the reference configuration Uo, defined as

GM ¼ G ˛ Q s:t: GT MG ¼ M ; (20)
where M ¼ {M(1), ., M(nF)} and GTMG denotes{GTM(1)G, ., GTM(nF)G}.

From the Rychlewski’s theorem, the condition

Jse ðCÞ ¼ Jse GT CG ; cC ˛ Lin; cG ˛ G; (21)
is satisfied if and only if the strain-energy Jse can be represented by a function Jani whose list of arguments additionally includes the
structural tensors, namely
Jse ðCÞ ¼ Jani ðC; MÞ; (22)
which results to be an isotropic scalar-valued tensor function, namely

Jani ðC; MÞ ¼ Jani GT CG; GT MG ; cC ˛ Lin; cG ˛ Q: (23)
Indeed, it is immediate to verify that the isotropic tensor function Jani (i.e., for which Eq. 23 holds) respects:

Jani ðC; MÞ ¼ Jani GT CG; M ; cC ˛ Lin; cG ˛ GM 4Q; (24)
and thereby, the invariance set GM in Eq. (20) is a material symmetry group for anisotropic materials.
Invariant-Based Formulations
The construction of constitutive equations takes advantage from the classical invariant theory which allows to build isotropic func-
tions from a basis of invariants. The latter is made up by a minimal set of invariants from which all other invariants can be
generated.
By following the additive decomposition in Eq. (15), the matrix contribution JM is generally associated with an isotropic consti-
tutive response, while the fiber term JF collects the strain-energy contribution of the constituents which endow the tissue with an
anisotropic response. In turn, the latter is additively decomposed in order to account for the different fiber families. Accordingly, on
the basis of functions Jiso and Jani introduced in Eqs. (19) and (23), respectively, it results:
1 1 1 X nF
ðaÞ ðaÞ
JM ðCÞ ¼ Jiso ðCÞ; JF ðCÞ ¼ Jani ðC; MÞ ¼ v Jani ðC; MðaÞ Þ (25)
VM VF VF a¼1 F
where Jani
(a)
is the strain-energy contribution of the single fiber and vF(a) is the probability of finding a fiber with orientation e(a)
o such
P
that anF¼ 1vF(a) ¼ 1. It is worth pointing out that both Jiso(C) and Jani(C, M) are isotropic scalar-valued tensor functions. For the
anisotropic term, in particular, this is true thanks to the introduction of the notion of structural tensors. Polynomial invariants are
focused in the following, although other choices are possible.
A polynomial basis for the isotropic term Jiso(C) is made up by the set I iso¼ {I1, I2, I3}, where:
I1 ¼ Tr ðCÞ ¼ kFk2 (26a)
I2 ¼ TrðCof ðCÞÞ ¼ kCof ðFÞk2 ; (26b)
I3 ¼ DetðCÞ ¼ kDetðFÞk2 : (26c)

which geometrically represent, respectively, a measure of line, area, and volume change, as shown in Eq. (4). Introducing function
^ iso with scalar-valued arguments, it can be chosen:
J
^ iso ðI iso Þ;
Jiso ðCÞ ¼ J (27a)
and the corresponding second Piola–Kirchhoff stress tensor Siso results:
^ ^ iso vI2 vJ^ iso vI3 ^ ^ iso ^ iso ^ iso
vJiso vJiso vI1 vJ vJiso vJ vJ vJ
Siso ðCÞ ¼ 2 ¼2 þ þ ¼2 þ vI1 I Cþ Cof ðCÞ ; (27b)
vC vI1 vC vI2 vC vI3 vC vI1 vI2 vI2 vI3
where the following identities have been considered:
vI1 vI2 vI3
¼ I; ¼ I1 I C; ¼ Cof ðCÞ: (27c)
vC vC vC
A polynomial basis for the anisotropic term Jani(C, M) is made up by the set I ani¼ {I iso, I F(1), ., I F(nF), I M }, with I iso from
Eq. (26) and where I F(a) ¼ {I4(a), I5(a)} and I M ¼ {IM
(1)
,., IM (nF)}with:
ðaÞ ðaÞ ðaÞ
I4 ¼ TrðCMðaÞ Þ; I5 ¼ Tr C2 MðaÞ ; IM ¼ TrðMðaÞ Þ (28)
In particular, I M is generally ruled out since identically equal to the unitary set. Invariant I4(a) geometrically represents the square
stretch along the direction identified by eo(a), resulting I4(a) ¼ kFeo(a)k2.
Introducing I (a) ¼{I , I (a)} and function J ^ ðaÞ with scalar-valued arguments, it can be chosen:
ani iso F ani
ðaÞ ðaÞ
^ ðC; MðaÞ Þ ¼ J
J ^ ðaÞ
ani ani I ani ; (29a)
(a)
and the corresponding second Piola–Kirchhoff stress tensor Sani results:
ðaÞ ^ ðaÞ ðaÞ ^ ðaÞ vIðaÞ ^ ðaÞ ^ ðaÞ
ðaÞ vJani e vJani vI4 vJ e vJani ðaÞ vJ
Sani ðCÞ ¼ 2 ¼ Siso ðCÞ þ 2 þ ani 5
¼ S iso ðC Þ þ 2 M þ ani
ðCMðaÞ þ MðaÞ CÞ ; (29b)
vC ðaÞ vC
vI4
ðaÞ vC
vI5
ðaÞ
vI4
ðaÞ
vI5
where e
Siso is defined as:
^ ðaÞ ^ ðaÞ ^ ðaÞ ^ ðaÞ
e vJani vJ vJ vJ
Siso ðCÞ ¼ 2 þ I1 ani I ani
Cþ ani
Cof ðCÞ ; (29c)
vI1 vI2 vI2 vI3
and the following identities have been considered:
ðaÞ ðaÞ
vI4 vI5
¼ MðaÞ ; ¼ CMðaÞ þ MðaÞ C: (29d)
vC vC
It is worth highlighting that e
Sani ¼ 0 whenever I (ania) h I (Fa) .
In conclusion, the second Piola–Kirchhoff stress of soft tissues derived from the choice in Eq. (15) is
SðCÞ ¼ VM SM ðCÞ þ VF SF ðCÞ; (30a)
where, accounting for Eqs. (25), (27a), and (29a), it results

vJM 1 vJF 1 X nF
ðaÞ ðaÞ
SM ðCÞ ¼ 2 ¼ Siso ðCÞ; SF ðCÞ ¼ 2 ¼ v S ðCÞ; (30b)
vC VM vC VF a¼1 F ani
(a)
with Siso and Sani given in Eqs. (27b) and (29b).
Tendons and ligaments are made up by regular unidirectional tissues that can be modeled by introducing nF ¼ 1 fiber family. In
this case, tissue behavior results transversely isotropic since only one preferred orientation is present. As a matter of fact, the invari-
ance set in Eq. (20) is made up by all rotations about the eo-axis through an angle q, represented by tensor Rq (eo, q)˛ Q, as well as by
a reflection with respect to the plane with unit normal equal to eo, represented by tensor Qr (eo) ˛ Q. Accordingly, the symmetry
group Gti for a transversely isotropic tissue results
Gti ¼ f I; Qr ðeo Þ;Rq ðeo ; qÞ with 0 q < 2pg (31)
On the other hand, a large number of biological structures (e.g., arterial walls, heart valves) can be regarded as planar and made
up by regular multidirectional tissues that can be modeled by introducing nF ¼ 2 fiber families. The latter lie on tissue plane and
form an angle 2bo between each other, namely eo(1) , eo(2) ¼ cos (2bo). For the analysis of material symmetries, the superposition of
fibers may require a more sophisticated treatise than the analysis of the invariance set in Eq. (20), which is representative only for
symmetries in the geometric arrangement of fibers. Starting from the orientation eo(1) and eo(2) of fibers, the local orthonormal coor-
dinate basis (g1, g2, g3) can be introduced as:
ð1Þ ð2Þ ð2Þ ð1Þ
eo þ eo eo eo g1 g2
g1 ¼ ; g2 ¼ ; g3 ¼ ; (32a)
ð1Þ
eo þ
ð2Þ
eo
ð2Þ
eo eo
ð1Þ kg1 g2 k
such that:
ð1Þ ð2Þ
eo ¼ cosðbo Þg1 þ sinðbo Þg2 ; eo ¼ cosðbo Þg1 sinðbo Þg2 : (32b)
Recalling that Qr (a) represents a reflection with respect to the plane with a as unit normal and introducing Qr,• ¼ Qr (g•), it
results:
ðiÞ ð jÞ ðiÞ ð jÞ ðiÞ ðiÞ
Qr;1 eo ¼ eo ; Qr;2 eo ¼ eo ; Qr;3 eo ¼ eo ; (33)
with i, j ˛ {1, 2} and i s j. Hence, the following relationships hold:

ðiÞ ðiÞ T ð jÞ
Tr QTr;q CQr;q MðiÞ ¼ Tr CQr;q eo Qr;q eo ¼ TrðCMð jÞ Þ ¼ I4 ; (34a)
ðiÞ ðiÞ T ðiÞ

Tr QTr;3 CQr;3 MðiÞ ¼ Tr CQr;3 eo Qr;3 eo ¼ TrðCMðiÞ Þ ¼ I4 ; (34b)
where i, j, q ˛ {1, 2} and i s j. Without loss of generality, the anisotropic term is assumed to depend on the fourth invariant of
deformation only, namely J ^ ðaÞ IðaÞ . Based on the representation in Eq. (25) and accounting for relationships in Eq. (34), it
^ ðaÞ ¼ J
ani ani 4
holds (for q ˛ {1, 2}):
^ ð1Þ
ð2Þ ^ ð2Þ
Jani C; Mð1Þ ; Mð2Þ ¼ vFð1Þ J ð1Þ
ani I4 þ vF J
ð2Þ
ani I4 ; (35a)
ð1Þ ð2Þ
^
Jani QTr;q CQr;q ; Mð1Þ ; Mð2Þ ¼ vFð1Þ J ð2Þ ð2Þ ^
þ vF J
ð1Þ
ani I4 ani I4 ; (35b)
ð1Þ ð2Þ
^
Jani QTr;3 CQr;3 ; Mð1Þ ; Mð2Þ ¼ vFð1Þ J ð1Þ ð2Þ ^
þ vF J
ð2Þ
ani I4 ani I4 (35c)
ð1Þ ^ ð1Þ ð2Þ ^ ð2Þ

Accordingly, if it holds vF J ani ¼ vF Jani (i.e., roughly speaking, fiber families have the same mechanical response), it is imme-
diate to show that:

Jani C; Mð1Þ ; Mð2Þ ¼ Jani GT CG; Mð1Þ ; Mð2Þ ; (36)
for G ¼ I, Qr,1, Qr,2, Qr,3. Therefore, in this case, material behavior is orthotropic according to axes g1, g2, and g3 with
material symmetry group Gorth in the reference configuration Uo equal to:

Gorth ¼ I; Qr ðg1 Þ;Qr ðg2 Þ;Qr ðg3 Þ : (37)
Material Stability and Mathematical Requirements

The notion of material stability is related to the physical reasonability of the material response. A material is said to be stable if any
deformation increment from a given state needs a positive work to be produced.
In the framework of an isotropic behavior, material stability can be expressed by the Baker–Ericksen inequality which requires
that the maximum principal Cauchy stresses occur in direction of the maximum principal stretches. More generally, addressing both
isotropic and anisotropic responses, material is said to be stable if the Legendre–Hadamard inequality is respected at each material
point. The latter condition ensures the existence of traveling waves with real wave speeds in each direction. This requirement can be
verified by the analysis of the ellipticity of the acoustic tensor, obtained from the second derivative of the strain-energy function
JseF (F) with respect to the deformation gradient F. Nevertheless, the existence of minimizers of variational principles in finite elas-
ticity is not guaranteed for stable materials. Therefore, further restrictions shall be enforced on the strain-energy function for
ensuring the solution of boundary value problems associated with finite elasticity. Sufficient conditions for the existence of mini-
mizers are coercivity and sequential weakly lower semicontinuity (s.w.l.s.) of the strain-energy function.
The coercivity condition corresponds to enforce the growth condition that Jse F
(F) / þ N for extremely high strains. A possible
coerciveness formulation is to require that there exists a1 > 0, a2, p 2, q p/(p 1) and r > 1 such that:

JFse ðFÞ a1 kFkp þ kcof ðFÞkq þ ðDetðFÞÞr þ a2 ; cF ˛ Linþ : (38)
Moreover, a second growth condition is generally introduced in order to enforce that an infinite energy shall be required to anni-
hilate volume:
JFse ðFÞ/ þ N for DetðFÞ/0þ ; cF ˛ Linþ : (39)
By employing the additive decomposition in Eq. (15) with positive additive terms, the strain-energy density function is coercive
when, at least, one additive term respects the coercivity condition in Eq. (38). Accordingly, from a practical point of view, coercivity
is enforced on the matrix contribution JM of the strain-energy function and several expressions classically employed in the state of
the art for soft tissues respect this condition.
The sequential weakly lower semicontinuity of a strain-energy function is implied by its polyconvexity in the sense of Ball. Poly-
convexity is a stronger condition than s.w.l.s. but it results more feasible to handle from the mathematical point of view. The strain-
energy function Jse F
(F) is said to be polyconvex if there exists a convex function fj (in general, not unique),
fj ¼ fj ðA; B; cÞ; with A; B ˛ Lin; c ˛ R; (40)
such that
JFse ðFÞ ¼ fj ðF; AdjðFÞ;DetðFÞÞ: (41)
It is worth highlighting that all terms in Eq. (15) should satisfy the polyconvexity requirement. A construction principle for a pol-
yconvex strain-energy function is to introduce sets P iso (resp., P (ania)), collecting elements of I iso (resp., I (ania)) that result to be
convex functions in the list of arguments F, Adj (F) and Det (F). Then, accounting for Eqs. (27a) and (29a), an additive decompo-
sition of strain-energy terms J ^ ðaÞ is employed:
^ iso and J
ani
X
J^ iso ðI iso Þ ¼ ^ iso;j Pj jPj ˛ P iso ðI iso Þ;
J (42a)
j
ðaÞ X ðaÞ
^
J ðaÞ ^
J ðaÞ ðaÞ
ani I ani ¼ ani;j Pj jPj ˛ P ani I ani ; (42b)
j
^ ðaÞ shall result convex and monotonically increasing functions of arguments in sets P and
^ iso;j and J
where the single terms J ani;j iso
P ani .
( a)
Addressing isotropic contributions, some convex terms in P iso are:

I1 I2 pffiffiffiffi 1
I1 ; I1 ¼ 1=3 ; I2 ; 1=3
; I3 ; ln I3 ; ; (43)
I3 I3 I3
where terms with I3 at the denominator are useful for respecting the growth condition in Eq. (39). On the other hand, possible
convex anisotropic terms in P (a)
ani are:
ðaÞ
ðaÞ ðaÞ I4 ðaÞ ðaÞ ðaÞ
I4 ; I4 ¼ 1=3
; K1 ¼ I5 I1 I4 þ I2 ; (44a)
I3
ðaÞ ðaÞ ðaÞ ðaÞ ðaÞ

K2 ¼ I1 I4 ; K3 ¼ I1 I4 I5 : (44b)
Terms K1(a), K2(a), and K3(a) are introduced to account for invariant I5(a) which is not elliptic and hence nonpolyconvex. Their phys-
ical meaning can be elucidated by showing that:
ðaÞ ðaÞ 2
K1 ¼ Cof ðFÞeo ; (45a)
ðaÞ
K2 ¼ TrðCðI MðaÞ ÞÞ; (45b)
ðaÞ ðaÞ 2
K3 ¼ kCof ðFÞk2 Cof ðFÞeo : (45c)
(a) (a) (a)
Hence, K1 controls the deformation of the area element with unit normal eo , K2 the square stretch in the plane with unit
normal eo(a), and K3(a) the deformation of an area element with unit normal perpendicular to eo(a).
Incompressibility Condition
Soft tissues demonstrate a very slight volumetric compressibility (< 1%), and hence the constraint Det (F) z 1 shall be enforced.
This brings inherited computational issues that require the implementation of special numerical techniques. Among others, two
methods are here presented: the Penalty method and the Augmented Lagrangian method.
As preliminary material for both methods, it is useful to consider the multiplicative split of the deformation gradient F into the
volumetric part Fvol and the isochoric part Fdev such that:
F ¼ Fvol Fdev ¼ Fdev Fvol with Fdev ¼ J 1=3 F; Fvol ¼ J1=3 I; (46)
with Det (Fvol) ¼ J and Det (Fdev) ¼ 1. The volumetric part Fvol is associated with a motion that changes the volume but preserves the
shape. On the contrary, the isochoric part Fdev preserves the volume but changes the shape. For material objectivity requirements, let
Cdev ¼ Fdev
T
Fdev ¼ I3 1/3C be introduced as the isochoric part of the right Cauchy-Green deformation tensor.
For the sake of conciseness and without loss of generality, only nF ¼ 1 fiber family is employed in what follows when dealing
with the anisotropic contribution. Moreover, let the deviatoric operator Dev ((•)) be defined as Dev ((•)) ¼ (•) [(•): I]I/3.
The penalty method

The Penalty method consists to enforce the incompressibility constraint by an additive splitting of the isotropic strain-energy contri-
bution as
^ ðI3 Þ þ J
Jiso ðCÞ ¼ kJ ^ ðC Þ;
vol dev
(47)
iso iso dev
where k > 0 is a penalty parameter, ^ vol

J is a penalty function, and ^ dev
J is the isochoric strain-energy part. The term ^ vol
J shall be
iso iso iso
endowed by the following properties:
^ : Rþ 1Rþ Wf0g;
J
vol
^ is convex;
J
vol
^ ðI3 Þ ¼ 05I3 ¼ 1;
J
vol
(48a)
iso iso iso
such that it immediately follows that:
^
vJ
vol
iso
¼ 05I3 ¼ 1: (48b)
vI3
Addressing the isotropic matrix contribution of the Cauchy stress siso ¼ FSisoFT/J with Siso in Eq. (27b), the physical rationale
behind the constitutive assumptions in Eq. (47) is elucidated by considering the additive decomposition of siso in siso vol
and siso
dev
such that
Trðsiso Þ
siso ¼ svol
iso I þ siso ;
dev
svol
iso ¼ ; sdev
iso ¼ Devðsiso Þ: (49a)
3
Employing the constitutive choice in Eq. (47), the resulting stress can be split into a physically meaningful decomposition:
!
^ vol
vJ ^ vol
vJ 2 ^ dev
vJ
svol ¼ 2kJ iso
¼ k iso
; sdev
¼ Dev Fdev
iso T
F : (49b)
iso
vI3 vJ iso
J vCdev dev
Accordingly, addressing exclusively the isotropic part, the split in Eq. (47) ensures that change of volume and change in shape are
disjoint, being respectively related to a spherical or a deviatoric state of stress. The former corresponds to a stress state siso of the form
siso ¼ Tr(siso)I/3, while the latter is such that Tr(siso) ¼ 0. Indeed, any spherical state of stress will produce only a change of volume
and not a change of shape, since siso vol
s 0 (see Eq. 49a) and hence I3 s 1 from Eq. (48b). On the other hand, any deviatoric state of
stress will produce a change of shape but no change of volume, since it shall result siso vol
¼ 0 (see Eq. 49a) and hence I3 ¼ 1 from Eq.
(48b).
1=3
A convenient definition of the isochoric term is based on an invariant formulation where only the term I1 ¼ TrðCdev Þ ¼ I1 I3 is
employed:
Jdev ^ dev
iso ðCdev Þ ¼ Jiso ðI1 Þ (50)
Since I1 is polyconvex (see Eq. 43), Jdev (Cdev) is ensured to be polyconvex if function J ^
dev is convex and monotonically
2=3
increasing. On the other hand, the purely isochoric term I2 ¼ TrðCof ðCdev ÞÞ ¼ I2 I3 is not polyconvex.
Attention shall be paid when adopting the Penalty method in the presence of an anisotropic contribution due to the fiber term
JF. In agreement with the additive decomposition in Eq. (15) and positions in Eq. (25), the Cauchy stress contribution
sani ¼ FSaniFT/J, with Sani inEq. (29b), is added to the total Cauchy stress, which indeed results:
s ¼ siso þ sani ¼ svol vol dev dev

iso þ sani I þ siso þ sani : (51)
Here, the volumetric-isochoric split sani vol

¼ Tr(sani)/3 and sani
dev
¼ Dev(sani) is introduced, while siso
vol
and siso
dev
are given in Eq.
(49b).
The analysis is here restricted to the case where the anisotropic contribution is associated only with the fourth invariant of defor-
mation I4, namely when anisotropic stress contribution is related only to fiber elongation. As a consequence, stress sani is expected
pffiffiffiffi
to be one-dimensional and aligned with the unit vector u ¼ Feo = I4 .
Following the rationale employed in Eq. (47) for the isotropic part, a natural choice would be to introduce the dependence of the
strain-energy function on the deviatoric part Cdev of the right Cauchy–Green deformation tensor, namely
Jani ¼ Jdev ^ dev

ani ðCdev ; MÞ ¼ Jani ðI4 Þ; (52)
^ dev of a scalar argument is introduced. Therefore,
where I4 ¼ Tr(CdevM) is polyconvex (see Eq. 44) and the scalar-valued function J ani
fiber stress results
^ dev
2I4 vJ I

sani ¼ ani
u5u ; (53a)
J vI4 3
not contributing to the spherical part of the stress tensor, since
^ dev
2I4 vJ I

svol
ani ¼
ani
Tr u5u ¼ 0; (53b)
3J vI4 3
^ dev
2I4 vJ
sdev
ani ¼
ani
Devðu5uÞ: (53c)
J vI4
Therefore, a purely deviatoric state of stress does not change the volume since, from Eqs. (51) and (53b), it must result siso vol
¼0
and this holds for I3 ¼ 1 (see Eqs. 48b and 49b). On the other hand, a purely spherical state of stress induces a spherical state of
stress in the matrix (i.e., siso
vol
s 0), since the fiber contribution to the spherical stress state is a priori null (see Eq. 53b). Moreover, in
case of slight compressibility, the purely volumetric deformation of the matrix can be attained, resulting in a null deviatoric stress
state siso
dev
¼ 0. This leads to a null stress in the fibers because sani
dev
¼ siso
dev
¼ 0 (see Eqs. 49b, 51, and 53c). Therefore, in the formu-
lation of Eq. (52), a purely spherical state of stress can be accompanied by a null fiber stress and a null change of shape, in contrast
with the anisotropic properties of the material. Furthermore, as a further drawback, the resulting Cauchy stress sani in Eq. (53a) is by
no means one-dimensional, contradicting the physical sense.
To avoid these unrealistic physical responses, the dependence of the strain-energy function on the complete right Cauchy–Green
deformation tensor C could be adopted, namely
^ ani
2I4 vJ
^ ani ðI4 Þ;
Jani ðC; MÞ ¼ J sani ¼ u5u: (54)
J vI4
recovering the one-dimensionality of the resulting Cauchy stress. It is immediate to show that, in this case, fibers contribute to both
the spherical and the deviatoric parts of the stress tensor, namely
^ ani
2I4 vJ ^ ani
2I4 vJ
svol
ani ¼ ; sdev
ani ¼ Devðu5uÞ: (55)
3J vI4 J vI4
Accordingly, accounting for Eq. (51) and in contrast with the choice in Eq. (52), a deviatoric state of stress might be accompanied
by a volume change, because it results siso vol
¼ sani vol
, and hence I3 s 1 from Eqs. (48b) and (49b). On the other hand, a purely
spherical state of stress implies a stress state (and thereby, a deformation) in both matrix (siso vol
s 0) and fibers (sani
vol
s 0). Moreover,
a null deviatoric stress state implies siso ¼ sani , but not necessarily siso ¼ 0 and sani ¼ 0. Therefore, a spherical state of stress
dev dev dev dev
does not induce a spherical state of stress in the matrix and in the fibers, being accompanied by a change of shape which is in
full agreement with the anisotropic properties of the material. In other words, in the formulation of Eq. (54), change of volume
and change in shape are fully coupled and not related to a spherical or a deviatoric state of stress. Based on these considerations,
in the presence of an anisotropic behavior, a formulation based on the complete right Cauchy–Green deformation tensor C for the
anisotropic strain-energy term shall be adopted.
As a concluding remark, the effectiveness of the Penalty method for enforcing the incompressibility constraint in numerical appli-
cations depends on the value of the penalty parameter k since, only if k / þN, the volumetric isotropic stress contribution results
siso
vol
/ þ N for I3 s 1 (under the condition siso vol
¼ 0 for I3 ¼ 1, see Eqs. 48b and 49b). As a matter of fact, the fulfillment of equilib-
rium conditions in standard applications requires a finite norm of the total Cauchy stress, i.e. ksk þ N, and then a finite norm of
the single terms in Eq. (51). In particular, this has to occur for the volumetric isotropic term, i.e. siso
vol
þ N, which can be satisfied
only for I3 / 1 if k / þN. Accordingly, in numerical applications, high values of k shall be employed. It is worth highlighting that
the simultaneous fulfillment of equilibrium and incompressibility cannot be guaranteed in an exact way by the Penalty method.
The augmented lagrangian method

In the framework of Lagrangian duality and in agreement with Eq. (7), the Augmented Lagrangian method consists in introducing
an enriched potential energy pe int in Eq. (6) based on a free-energy Jfe generalized with respect to the one in Eq. (12). Considering
the strain-energy density function Jse, free-energy Jfe is enriched with a Lagrange multiplier ^p through the internal constraint func-
tion J^ , resulting
h
pint ð4Þ ¼ p ^ ð J Þ:
e int ð4; ^pÞ ¼ Jfe ðC; ^pÞ ¼ Jse ðCÞ þ ^p J (56)
h
^ is such that:
Here, function Jh
^ : Rþ 1R;
J ^ ð JÞ ¼ 05J ¼ 1:
J (57)
h h
Accordingly, the condition J^ ð J Þ ¼ 0 corresponds to enforce incompressibility. From Eq. (8), the internal dissipation density
h
Dint for incompressible materials with the potential internal energy density in Eq. (56) results:
^ vJ _
vJ vJse C
Dint ¼ S 2^p h 2 : ; (58)
vJ vC vC 2
and, in order to a priori enforce the nondissipative properties of hyperelastic materials (see Eq. 13), the constitutive choice:
^
vJ vJse
h 1
S ¼ ^pJ C þ2 ; (59)
vJ vC
replaces Eq. (14) for incompressible materials.
Employing the enriched internal energy functional density p e int in Eq. (56), the Lagrangian Sufficiency Theorem shows that there
exists a value p of the Lagrange multiplier ^p such that the elastic equilibrium is satisfied for J ^ ð JÞ ¼ 0, and hence J ¼ 1, fulfilling the
h
incompressibility constraint (see Eq. 57). Value p is referred to as the optimal Lagrange multiplier.
Since the optimal value p of the Lagrange multiplier is an unknown, this can be obtained either from an equilibrium condition
(generally determined from stationarity conditions of the total potential energy functional), or by means of an iterative procedure.
In the latter case, introducing constant kAL > 0, the iterative algorithm presented in Table 1 can be implemented. The following
section presents an applicative case where the Penalty method and the Augmented Lagrangian method are compared, highlighting
advantages and drawbacks of the two approaches.
Application and final remarks on incompressibility

Addressing an exemplary application, a tissue with nF ¼ 1 fiber family is addressed, subjected to the uniaxial traction along the fiber
direction. Due to material symmetry, the deformation gradient tensor F takes the form:
F ¼ lF ðu5eo Þ þ lt ðut1 5et1 þ ut2 5et2 Þ; (60a)
where lF and lt represent along-the-fiber and perpendicular-to-fiber stretches, respectively, (eo, et 1, et 2) forms an orthonormal
basis in the reference configuration, and (u, ut 1, ut 2) in the current configuration. Due to the simplicity of the case under
consideration, it results (u, ut 1, ut 2) h (eo, et 1, et 2).
As constitutive choices, the additive split in Eq. (15) of the strain-energy density function Jse and the positions in Eq. (25) are
employed. The isotropic contribution Jiso is made up by the sum of an isochoric term J ^ dev and the term kJ
^ vol , where k is the
iso iso
^ vol
penalty parameter and Jiso is a penalty function. The choices:
pffiffiffiffi
^ vol ðI3 Þ ¼ k
2
^ dev ðI1 Þ ¼ c1 ðI1 3Þ; kJ
J I3 1 (60b)
iso iso
2
Table 1 Pseudocode of an iterative algorithm for the solution of elastic equilibrium problems with
incompressible materials modeled via the Augmented Lagrangian method
1. Initialize p^1 ¼ 0 and J1 such that rJ ^ h ðJ Þr > TOL

1
2. LOOP n > 1 WHILE rJ ^ h ðJ Þr > TOL
n1
(a) Solve:
R
4n ¼ arg min e int ðh; pn1 Þd U þ Pext ðhÞ
p
h˛C Uo
(b) Compute Fn ¼ Grad (4n), Jn ¼ Det (Fn), Cn ¼ Fn TFn

^ h ðJn Þ
(c) Compute p^n ¼ p^n1 þ kAL J
END LOOP
3. Assign p ¼ p^n , 4 ¼ 4n and C ¼ Cn
are employed, with c1 > 0 being a stress-like parameter and where conditions in Eq. (48a) are satisfied. The anisotropic contribution
related to fibers is modeled either following a formulation based on Eq. (52),
^ ðI4 Þ ¼
dev k1
Jani ¼ J ani exp k2 ðI4 1Þ2 1 ; (60c)
2k2
or on Eq. (54)
^ ani ðI4 Þ ¼ k1
Jani ¼ J exp k2 ðI4 1Þ2 1 ; (60d)
2k2
where k1 > 0 is a stress-like parameter and k2 > 0 is a nondimensional constant.
The afore-introduced choices fully define the formulation employed for enforcing the incompressibility constraint via the
Penalty method. If imposing the incompressibility constraint via the Augmented Lagrangian method (see Eq. 56), the same consti-
tutive choices are employed for the strain energy density function Jse and, in agreement with conditions in Eq. (57), the constraint
function J^ is chosen as
h
^ ð JÞ ¼ J 1:
J (60e)
h
Employing the choices in Eq. (60), the Cauchy stress tensor s is characterized by the following relationships:
s : ðut1 5ut1 Þ ¼ s : ðut2 5ut2 Þ; (61a)
s : ðu5ut1 Þ ¼ s : ðu5ut2 Þ ¼ s : ðut1 5ut2 Þ ¼ 0 (61b)

Accordingly, two stress quantities are relevant for the present application: the along-the-fiber sF ¼ s: (u 5 u) and the
perpendicular-to-fiber st ¼ s: (ut 1 5 ut 1) Cauchy stresses. Hence, condition st ¼ 0 (i.e., null stress in the direction perpen-
dicular to fibers) ensures equilibrium together with Eq. (61b), the latter being a priori satisfied. In fact, the only nontrivial stress
component shall result sF because the tissue is subjected to a uniaxial traction along the fiber direction. The functional
dependencies

sF ðlF ; lt Þ Penalty method
sF ¼ s : ðu5uÞ ¼ (62)
sF ðlF ; lt ; ^pÞ Augmented Lagrangian method
and

st ðlF ; lt Þ Penalty method
st ¼ s : ðut1 5ut1 Þ ¼ (63)
st ðlF ; lt ; ^pÞ Augmented Lagrangian method
on stretches lF and lt, and eventually on Lagrange multiplier ^p, are highlighted. A displacement-based virtual test is here
considered, by choosing stretch lF as control variable.
Therefore, by adopting the Penalty method, tissue mechanical response is obtained by determining stretch lt, which can be
characterized by adopting:
strategy P1: Find lt such that st ðlF ; lt Þ ¼ 0
Clearly, by following strategy P1, the incompressibility constraint is not automatically fulfilled and should be a posteriori veri-
fied. Alternatively, since Det(F) ¼ lF(lt)2, stretch lt can be defined from
pffiffiffiffiffi
strategy P2: lt ¼ lF such that Det ðFÞ ¼ 1;
in order to a priori fullfill the incompressibility constraint. Nevertheless, it is not guaranteed that the equilibrium condition st ¼ 0
is satisfied and this should be a posteriori verified.
The obtained constitutive relationships between lF and sF and between lF and st are shown in Fig. 4, as well as the resulting
relationship between lF and lt, for both strategies P1 and P2 and for both constitutive choices in Eqs. (60c) and (60d).
It results that, by imposing st ¼ 0 (strategy P1) and adopting J ^ dev in Eq. (60c), the incompressibility constraint is violated since
ani
1/2
lt s lF , leading to a nonphysical behavior where the inverse proportionality between lt and lF is lost. On the other hand, if
the incompressibility constraint is a priori enforced (strategy P2), together with J ^ dev , then it results sts 0, violating equilibrium.
ani
These drawbacks are related to the non-one-dimensionality of the stress associated with fibers (see Eq. 53a) and are due to the
constitutive choice based on the formulation in Eq. (52).
On the contrary, by employing J ^ ani in Eq. (60d) as constitutive choice for the anisotropic term and thanks to the one-
dimensionality of the resulting stress, results obtained by employing strategies P1 and P2 are practically coincident such that the
incompressibility constraint and equilibrium can be retained as simultaneously respected, although not exactly.
By following the Augmented Lagrangian method, the values of the stretch lt and of the optimal Lagrange multiplier p can be
determined by applying:
strategy A1: Algorithm in Table 1 where step 2a reduces to :

Find lt;n such that st lF ; lt;n ; pn1 ¼ 0:
Fig. 4 The Penalty method: tissue with nF ¼ 1 fiber family subjected to uniaxial traction along the fiber direction (Eq. 60). Top: along-the-fiber
stretch lF versus along-the-fiber sF and perpendicular-to-fiber st Cauchy stresses. Bottom: lF versus perpendicular-to-fiber stretch lt. Results are
^ dev in Eq. (60c) or J
obtained by a priori enforcing equilibrium (i.e., strategy P1) or incompressibility (i.e., strategy P2), and by adopting either J ^
ani ani
in Eq. (60d) for the anisotropic term. Parameters: k ¼ 2 GPa, c1 ¼ 10 kPa, k1 ¼ 50 kPa, k2 ¼ 50.
The exact fulfillment of incompressibility and equilibrium is thereby paid in terms of a solution strategy that requires the imple-
mentation of an iterative procedure, on the contrary with respect to the Penalty method.
Alternatively, the optimal Lagrange multiplier can be determined from equilibrium conditions. Due to the simplicity of the
addressed application, an analytical solution strategy (here used as benchmark) can be pursued for a priori fulfilling incompressi-
bility and equilibrium with the Augmented Lagrangian method. Introducing Ct ¼ l t 2
, this is
pffiffiffi
strategy A2: lt ¼ lF such that Det ðFÞ ¼ 1 and
^ 1
vJse vJ h
p ¼ 2Ct such that st ¼ 0:
vCt vJ
The obtained constitutive relationships between lF and sF and between lF and st are shown in Fig. 5, as well as the resulting
relationship between lF and lt, for both strategies A1 and A2, compared with results obtained by means of the Penalty method
(solved through strategy P1), adopting the same value for the penalty parameter k. Fig. 5 shows also the evolution of the optimal
Lagrange multiplier p with lF, confirming the effectiveness of the iterative procedure in strategy A1 compared with the exact analyt-
ical solution from strategy A2.
Contrary to the Penalty method, the Augmented Lagrangian method allows to simultaneously and exactly satisfy both equilib-
rium and the incompressibility constraint employing low values of the penalty parameter k. This outcome allows to point out
a significant advantage of the Augmented Lagrangian method with respect to the Penalty method in terms of the (fourth-order)
tangent stiffness tensor C, that is
^
v2 J v2 Jse
h
C¼p þ (65)
vCvC vCvC
Fig. 5 The Augmented Lagrangian method: tissue with nF ¼ 1 fiber family subjected to uniaxial traction along the fiber direction (Eqs. 59, 60a, 60b,
60d, and 60e). Top: along-the-fiber stretch lF versus along-the-fiber sF and perpendicular-to-fiber st Cauchy stresses. Bottom: lF versus
perpendicular-to-fiber stretch lt (continuous lines) and optimal Lagrange multiplier p (discontinuous lines). Results are obtained by computing lt
and p via an iterative procedure (i.e., strategy A1) or via an analytical equilibrium relationship (i.e., strategy A2) and are compared with the Penalty
method by a priori enforcing equilibrium (i.e., strategy P1). Parameters: k ¼ kAL ¼ 10 kPa, c1 ¼ 10 kPa, k1 ¼ 50 kPa, k2 ¼ 50.
where p ¼ 0 for the Penalty method. Indeed, adopting the Penalty method, the tangent stiffness tensor C may result ill-
conditioned in the initial strain-range (i.e., close to the reference configuration) due to the high values of the penalty
parameter k requested to fulfill the incompressibility condition. This is not the case when employing the Augmented
Lagrangian method. In order to show this outcome, the condition number of the tangent stiffness tensor obtained from the
along-the-fiber uniaxial traction of tissues with nF ¼ 1 fiber family is reported in Fig. 6 as function of the applied stretch.
Addressing the initial strain range, the condition number results significantly higher for the Penalty method (strategy P1) than
for the Augmented Lagrangian one (strategy A1). In order to allow for the most fair comparison, the penalty parameter k
employed in the Penalty method has been reduced as far as possible, under the constraint that, in the addressed simple uniaxial
traction case, the incompressibility relationship is satisfied with the same level of accuracy than the one obtained from the
iterative strategy A1.
It is worth highlighting that, due to the anisotropic nature of soft tissues, the advantage on the condition number obtained
with the Augmented Lagrangian method is limited to the initial strain-range because, with increasing strain, fibers generate
large entries in the stiffness matrix, thus leading to a dramatic enhancement of the condition number. The negative implica-
tions for numeric connected to this outcome are open problems under investigation that have to be solved on the computa-
tional side.
A final remark is dedicated to numerical issues related to computational problems which involve spatial discretization
techniques (e.g., the finite element method) coupled with incompressibility conditions. As a matter of fact, employing the
Penalty method based on pure displacement formulations, the constraint condition J ¼ Det (F) ¼ 1 can only be fulfilled
with a considerable stiffening of the bending modes of individual elements, known as volume locking. In order to avoid
this drawback, mixed finite element formulations can be employed by introducing additional unknowns with respect to
displacements in the energy functional density. For instance, the expression in Eq. (56) for the Augmented Lagrangian
method represents a possible choice for a two-field mixed formulation, where the Lagrange multiplier ^p is introduced as inde-
pendent variable.
Fig. 6 Tissue with nF ¼ 1 fiber family subjected to uniaxial traction along the fiber direction (Eqs. 59, 60a, 60b, 60d, and 60e). Relationship
between lF and the condition number cond(C) of the tangent stiffness tensor (in log scale) obtained via the Penalty method (i.e., strategy P1 with
k ¼ 10 MPa) and the Augmented Lagrangian method (i.e., strategy A1 with k ¼ 10 kPa). Parameters: c1 ¼ 10 kPa, k1 ¼ 50 kPa, k2 ¼ 50.
Following a Hu–Washizu formulation, a more general and robust choice is based on a four-field functional which additionally
includes a volume change variable Q and a Lagrange multiplier pQ as additional unknowns to the enriched free energy in Eq. (56),
namely:
^ ðQÞ þ ^p J
Jfe ðC; ^p; Q; pQ Þ ¼ Jse ðCÞ þ kJ ^ ðQÞ þ p ðJ QÞ;
vol
(66)
iso h Q
where Jse(C) ¼ Jiso (Cdev) þ Jani(C, M) is the strain-energy density function (split in an isochoric isotropic term Jiso and an
dev dev
anisotropic term J ), J^ vol is a penalty function for the volumetric response (see Eq. 48a), and J
^ enforces incompressibility in
ani iso h
a Lagrangian framework (see Eq. 57). In this case, accounting for Eq. (13), the constitutive relationship results:
vJse ^ vol
vJ vJ^
S ¼ pQ JC1 þ 2 ; with pQ ¼ k iso
þ ^p h ; (67)
vC vQ vQ
where, in numerical applications, multiplier ^p can be updated following the rule in Table 1 (step 2c) or it is determined from
equilibrium relationships determined from stationarity conditions of the total potential energy functional.
State of the Art of Hyperelastic Constitutive Modeling

A large number of constitutive models for soft tissues available in the literature follows the additive decomposition in Eq. (15) of
the strain-energy Jse into a matrix JM and fiber JF term, respectively associated with an isotropic and an anisotropic response (see
Eq. 25). Collagen fibers surely contribute to material anisotropy, while the noncollagenous constituents (mainly, elastin) are gener-
ally associated with a purely isotropic response. Nevertheless, evidence supporting an anisotropic contribution of elastin is recently
available, being an open issue under investigation.
For the description of the isotropic behavior, R.W. Ogden has proposed an important class of polyconvex strain-energy density
functions that satisfy also the coercivity condition. In agreement with the formulation in Eq. (42a), the strain-energy density func-
tion for Ogden-type materials, in the form presented by P.G. Ciarlet with invariants from Eq. (43), is:
X
I
a =2
X
V
b =2
JM ðCÞ ¼ ai I1 i þ bv I2v þ Gð J Þ; (68)
i¼1 v¼1
where ai > 0, bv 0, ai, bv 1, and G:]0, þN[1 R is a convex function such that G(J) / þN as J / 0þ. A first example of Ogden-
type material is the compressible Mooney–Rivlin model, characterized by Eq. (68) with I ¼ V ¼ 1, a1 ¼ b1 ¼ 2 and G(J) ¼ g1J 2 g2
ln(J) where g1, g2 > 0. A second example is represented by the compressible Neo-Hookean model, characterized by Eq. (68) with
I ¼ V ¼ 1, a1 ¼ 2 and b1 ¼ 0.
In order to enforce incompressibility and employing a volumetric-deviatoric split in agreement with Eq. (47), the first two terms
of the strain-energy function in Eq. (68) represent the deviatoric term Jiso
dev
(replacing I1 and I2 with I1 and I2 , see Eq. 43), while the
^ vol
penalty term J is enriched by function G(J).
iso
Addressing the anisotropic behavior, important classes of strain-energy density functions in agreement with the formulation in
Eq. (42b) are based on the sets of invariants I 4 ¼ {I4(1), ., I4(nF)} and are based on an exponential JFe or a polynomial JFp
expression:
X ðaÞ
nF
ðaÞ k1 ðaÞ ðaÞ ðaÞ 2
JF ðC; MÞ ¼ JeF ðI 4 Þ ¼ vF ðaÞ
exp k2 I4 1 k3 1 ; (69a)
a¼1 2k2
X ðaÞ
ðaÞ k1 ðaÞ ðaÞ k2
nF ðaÞ
JF ðC; MÞ ¼ JpF ðI 4 Þ ¼ vF I
ðaÞ 4
1 k3 : (69b)
a¼1 k2
Here, k1(a) > 0 is a stress-like parameter associated with fiber stiffness; k2(a) > 0 dimensionless parameter governing fiber
nonlinearities; k3(a) 0 is a measure of fiber slackness. The Macaulay brackets hxi ¼ (x þ| x | )/2 (such that hxi ¼ 0 for x 0
and hxi ¼ x for x > 0) are employed for both physical and mathematical requirements. Firstly, since the anisotropic response
is generally associated with the presence of crimped collagen fibers, the fibrous constituents are assumed to bear load only in
traction mode (i.e., for I4(a) > 1 þ k3(a)). Moreover, the polyconvexity of the ansatz in Eqs. (69a) and (69b) can be obtained
thanks to the use of the Macaulay brackets, because the latter are instrumental for the increasing monotonicity of the
employed functions.
With reference to the existing literature, the well-established rationale, known as structural approach and proposed by G.A. Hol-
zapfel, T.C. Gasser and R.W. Ogden in 2000, is recovered by Eq. (69a) with k3(a) ¼ 0. In particular, the Holzapfel–Gasser–Ogden
ðaÞ
model has been originally formulated by using the invariant I4 based on Cdev, instead of I4(a), on the lines of the ansatz in Eq.
(52). As previously shown in the discussion of incompressibility, the latter choice leads to major drawbacks.
Since I4(a) is a polyconvex invariant, the polyconvexity of the strain-energy term JFe immediately follows. Moreover,
addressing the polynomial law JFp, the restriction k2(a) 1 ensures its polyconvexity, and k2(a) 2, a continuous fiber tangent
stiffness.
The stress obtained from Eqs. (69a) and (69b) satisfies a priori the stress free condition in the reference configuration, while
relationships between model parameters in Eq. (68) have to be introduced for the stress computed from the matrix term JM.
For instance, referring to a compressible Mooney–Rivlin formulation, the condition 2a1 þ 4b1 þ 2g1 g2 ¼ 0 is obtained. Further-
more, in order to fulfill the nonessential normalization condition J(I) ¼ 0, constants are generally introduced in the existing
literature.
Soft tissues with a multidirectional fiber arrangement (such as arterial tissues, heart valves, and myocardial laminae) might be
characterized by a high dispersion in fiber orientation which determines a complex multiaxial response with a high degree of anisot-
ropy. Accordingly, a high number of fibers families (i.e., nF [ 1) should be employed in order to reproduce a continuous angular
distribution. The latter shall be integrated during the analysis for obtaining the stress applied by fibers at each material point and by
employing the specific deformation at hand. This method, known as angular integration (AI) model, leads to issues in terms of
computational costs.
Alternatively, the generalized structural tensor (GST) approach has been introduced by T.C. Gasser, R.W. Ogden, and G.A. Hol-
zapfel in 2006. For each fiber family, the GST, denoted by M ^ ðaÞ , is defined as:

M^ ðaÞ ¼ kðaÞ I þ 1 3kðaÞ MðaÞ ; (69c)
F F
where M(a) ¼ eo(a) 5 eo(a) is the classical structural tensor associated with the mean fiber direction eo(a), and k(Fa)˛ [0, 1/3] is
a dispersion parameter, specific for each fiber family. In the limiting cases, k(Fa)¼ 0 corresponds to no dispersion (transverse
isotropy) and k(Fa)¼ 1/3 to a three-dimensional isotropic distribution of fiber orientation. In principle, the upper limit of k(Fa) is 1/2
(corresponding to a planar isotropic distribution of fibers with a preferred direction normal to that plane), but it has been shown,
for instance by G.A. Holzapfel and R.W. Ogden in 2010, that the range k(Fa)˛ (1/3, 1/2] can lead to unphysical results with non-
monotonic stress responses for increasing deformation.
ðaÞ
Within the GST approach, the generalized strain invariant Î4 ¼ TrðCM ^ ðaÞ Þ is used in constitutive relationships (for instance,
(a)
with the form of Eqs. 69a and 69b) instead of the classical one I4 , allowing to obtain a non-one-dimensional response for
each fiber family. Therefore, the multiaxial response associated with fiber dispersion can be captured maintaining a low value
for the fiber family number nF and avoiding AI techniques.
Nevertheless, attention shall be paid on how to account for the switch between fibers in tension and the ones in compression
when using the GST. Indeed, the Macaulay Brackets are not effective anymore, because all fibers are treated as a whole. Suitable
computational or theoretical approaches should be employed, as recently proposed, for example, by G.A. Holzapfel and R.W.
Ogden in 2015 or by M. Latorre and F.J. Montáns in 2016. Moreover, it is worth highlighting that, for a given strain-energy expres-
sion of an individual fiber used for AI, a different strain energy shall be employed within the GST approach for obtaining the same
results in terms of stress–strain relationships. For instance, even considering the same functional form, a different set of parameters
shall be employed for obtaining a correspondence between the AI and the GST approaches, especially for high fiber dispersions. In
other words, for a given (known) mechanical behavior of an individual fiber, an ad hoc calibration of model parameters shall be
performed for obtaining the corresponding behavior of the dispersed fiber pattern through the GST approach, like in a phenome-
nological framework.
Multiaxial effects can be also captured by employing strain invariants which control the deformation of area elements in the
strain-energy formulation (see Eq. 44). For instance, introducing the set K3 ¼ {K3(1), ., K3 (nF)} of invariants, a multiaxial stress
response (see Eq. 29b) can be obtained by employing the polynomial expression JFp2 defined as:
X ðaÞ
ðaÞ k4 ðaÞ ðaÞ k5
nF ðaÞ
JF ðC; MÞ ¼ Jp2
F ðI 4 ; K3 Þ ¼ JF ðI 4 Þ þ
p
vF
ðaÞ
K3 1 k6 ; (69d)
a¼1 k5
on the lines of the model by D. Balzani, P. Neff. J. Schröder and G.A. Holzapfel proposed in 2006.
A number of different soft tissues have been modeled by following expressions of the form in Eqs. (68) and (69). Without the
claim of being exhaustive, some examples are the models: of arteries by Gasser, Ogden, and Holzapfel proposed in 2006; of veins by
Alastrué, Peña, Martínez, and Doblaré in 2008; of the intestine by Ciarletta, Dario, Tendick in 2009; of the esophagus by Yang, Bach,
Zheng, et al. in 2006; of the cornea by Pandolfi and Manganiello in 2006; of the intervertebral disc by Eberlein, Holzapfel and
Schulze-Bauer in 2001; of ligaments by Holzapfel and Stadler in 2006; of cartilage by Pierce, Trobin, Trattnig et al. in 2009; and
of the myocardium by Holzpafel and Ogden in 2009.
The biomechanical properties of soft tissues are well reproduced by these models and, in general, by strain-energy func-
tions in Eqs. (68) and (69) or based on a similar ansatz. Nevertheless, in order to fit experimental data, the models shall be
suitably calibrated. Model calibration consists in the identification of the values of parameters that govern these laws (i.e., ai,
ai, ., for the isotropic part and k1(a), k2(a), ., for the anisotropic term). Collecting all model parameters in set § and
following a displacement-based approach, a suitable approach for model calibration consists in solving the optimization
problem:

§ ¼ argmin fobj ð§Þ^ ; (70a)
^
§˛A
where A is the set of admissible values of parameters and the objective function fobj can be defined as:
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u X n
^ 2
u1 ksexp ðCi Þ sðCi ; §Þk
^ ¼t
fobj ð§Þ : (70b)
n i¼1 max ksexp ðCi Þk2
i¼1;.;n
Here, Ci are the n values of the Cauchy–Green deformation tensor at which experimental data sexp are known, while sðCi ; ;
^ are model outcomes at C ¼ Ci, obtained by employing value §
; §Þ ^ of parameters. To fully characterize tissue anisotropic
properties, reference has to be made to triaxial mechanical tests, where planar information only (i.e., uni/bi-axial and, possibly,
shear tests) are suitable only in presence of special material symmetries. Clearly, other choices on the objective function are
possible.
It is worth highlighting that, although the fitting capabilities of constitutive laws in Eqs. (68) and (69) are generally good, the
values of the parameters do not have a straightforward relationship with structural features, such as collagen fiber radius and crimp
amplitude, intermolecular cross-link density, or molecular defects in the polypeptide sequence. Nevertheless, pathogenesis is asso-
ciated with an alteration in the structural properties of soft tissues. Computational models developed with the aim of analyzing the
response of pathological biological structures shall incorporate the structural differences of constitutive properties, as otherwise
simulations might not be good predictors for the actual in vivo stress and strain state.
To reach this aim, multiscale approaches for the constitutive modeling of soft tissues have been proposed. These approaches are
based on the development of models for mechanisms occurring down at the nanoscale (i.e., collagen triple helix elongation mech-
anisms), through the mesoscale (i.e., cross-linked molecular assemblies), up to the microscale (i.e., crimped fibers). Therefore, mul-
tiscale constitutive models allocate macroscopic stress to different micro- and nanostructural mechanisms with a special insight on
the structure–mechanics relationship.
Accordingly, multiscale constitutive models are powerful alternatives to the phenomenological expressions introduced in Eqs.
(68) and (69). Early ideas on microstructural relations for developing constitutive models have been proposed by Y. Lanir in the
1980s. Recent advancements in this framework have been obtained by explicitly considering nanoscale mechanisms in the model,
such as in the formulations developed by F. Maceri, M. Marino and G. Vairo in 2010 or by M.S. Sacks, W. Zhang and S. Wognum in
2016. In contrast to the structural approach introduced by G.A. Holzapfel and co-workers, these nano-micro-macro formulations
can be referred to as developed within a multiscale structural approach. The multiscale formulation based on the results proposed
by M. Marino, P. Wriggers and co-workers in 2017 is described in what follows.
Multiscale structural approach

The multiscale approach herein presented focuses on the fiber contribution JF to the strain-energy function, assumed to be repre-
sentative of the behavior of collagen fibers. The strain-energy term JF is defined as:
X
nF Z 1þhl4 1i
ðaÞ Z 1þðx1Þ
ðaÞ
JF ðC; MÞ ¼ Jm
F ðI 4 Þ ¼ vF EF ðhÞdh dx; (71)
a¼1 1 1
where l4(a) ¼ (I4(a))1/2 is fiber stretch and EF ¼ EF(l4(a)) is the equivalent tangent modulus of crimped collagen fibers. The latter is
function of the change of fiber along-the-chord length, measured via stretch l4(a), and it is obtained by means of a multiscale
Fig. 7 Multiscale constitutive approach: representation of stretch measures and structurally motivated parameters.
description of internal deformation mechanisms. For the sake of compactness, superscript (a), denoting different fiber families, is
omitted in what follows, although all the following introduced quantities generally vary family by family.
Collagen fibers are assumed to have a circular cross-section of radius rF and area measure AF ¼ prF2. As schematically depicted in
Fig. 7, the crimped structure of collagen fibers is taken into account by considering locally periodic fibers of along-the-chord period
length LF and amplitude HF in the current configuration (resp., LF,o and HF,o in the reference configuration).
Geometric sources of mechanical nonlinearities are accounted for by the functional dependence of fiber period and amplitude
on fiber stretch l4, namely LF ¼ LF (l4) and HF ¼ HF (l4). In particular, it is convenient to introduce fiber quarter-of-period ‘F
(l4) ¼ LF (l4)/4, where ‘F,o ¼ ‘F (1) represents the value in the reference configuration.
Material sources of mechanical nonlinearities are accounted for by means of fibril tangent modulus Ef that depends on fibril
stretch lf, in turn related to l4 by the interscale compatibility relationship Ff between microscale and mesoscale (here meso means
between micro and nano):
dlf l4 ‘2F;o þ HF dH F
Ff ðl4 ; HF Þ ¼ dl4
¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi; (72a)
dl4
l2F ‘2F;o þ H2F ‘2F;o þ H2F;o
formulated by considering lf as coinciding with the centerline stretch of a fiber with piecewise linear shape. Derivative dHF/dl4 will
be given in the following Eq. (75c).
Fibril stretch lf is associated with molecular stretch lm which, in turn, is function of entropy-related lms and energy-related lmh
molecular stretches (see Fig. 7). These functional dependences are taken into account via the interscale compatibility relationships
Ffm (from meso-to nano-scale), Fms, and Fmh (from nanoscale to atomistic scale). On the basis of simple equilibrium conditions
formulated assuming mechanisms as in series, the interscale compatibility relationships are obtained from the tangent modulus of
collagen fibrils Ef, of collagen molecules Em, and of entropy-related Ems and energy-related Emh mechanisms. Accordingly, it results

dl E ls ; lh
Ffm lsm ; lhm ¼ m ¼ f ms mh ; (72b)
dlf Em lm ; lm

dlsm Em lsm ; lhm
Fms lsm ; lhm ¼ ¼ s ; (72c)
dlm Esm lm

dlhm Em lsm ; lhm
Fmh lsm ; lhm ¼ ¼ h : (72d)
dlm Ehm lm
where

Em lsm ; lhm Lc kc ‘m;o
Ef lsm ; lhm ¼ ; (73a)
Lc kc ‘m;o þ Am Em lsm ; lhm

Esm lsm Ehm lhm
Em lsm ; lhm ¼ s ; (73b)
Esm lm þ Ehm lhm
and

kB T‘m;o ‘3c
Esm lsm ¼ þ1 ; (73c)
‘p ‘c Am 2 ‘c ‘m;o ls 3
m
( )
‘m;o ^
E
Ehm lhm ¼ ^
þ Eo ; (73d)
‘c 1 þ exp h ‘m;o lhm 1 ‘c εho
with kB being the Boltzmann constant and T being the absolute temperature. Moreover, referring to the entropic behavior of
collagen molecules (Eq. 73c), ‘p is the persistence length, ‘c is the contour length, ‘m,o is the end-to-end length in the reference
configuration (resulting ‘m,o ¼ ‘c ‘ks, with ‘ks being the length of molecular kinks), and Am is the cross-sectional area. Furthermore,
addressing the energetic regime (Eq. 73d), E ^ are, respectively, the low-strain and high-strain collagen tangent moduli, εoh is
ô and E
the uncoiling strain, and h is the uncoiling resistance. Finally, with reference to intermolecular sliding affecting fibril mechanics (Eq.
73a), Lc denotes the (mole fraction) density of intermolecular covalent cross-links, which are modeled with a linear elastic behavior
with stiffness kc.
Eqs. (72b) and (73a) (resp., Eq. 72c, 72d, and 73b) derive from the in series elasticity of molecular elongation and intermolecular
sliding (resp., entropic and energetic molecular elongation mechanisms). Eqs. (73c) and (73d) are, respectively, based on theoret-
ical results which recover the worm-like chain model for the description of entropic elasticity, and on atomistic computations that
elucidate energetic mechanisms.
The equivalent tangent modulus EF (l4) of crimped collagen fibers to be employed for the macroscale description of collagen
mechanics (i.e., in Eq. 71) is defined as EF (l4) ¼ CF (l4)/l4, with CF being the along-the-chord tangent modulus of an Euler–Ber-
noulli curvilinear beam whose geometry corresponds to the one of fibers (i.e., quarter-of-period ‘F and amplitude HF) and whose
material tangent modulus corresponds to the one of fibrils (i.e., Ef). Addressing a piecewise-linear beam shape, the incremental
application of the principle of Virtual Works gives (dependences omitted):

‘2F þ HF2 4H2 1
CF ¼ Ef qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi ‘F þ 2 F ‘2F þ HF2 ; (74)
‘2 þ H 2 3rF ‘F
F;o F;o
where it is worth recalling that Ef ¼ Ef(lh , lm ) (see Eq. 73), ‘F ¼ ‘F (l4) and HF ¼ HF(l4). The functional dependences lms ¼ lms(l4)
s h
and lmh ¼ lmh(l4) are obtained from Eq. (72) and the application of the chain rule, via the interscale compatibility relationships:
dlsm
¼ Fms lsm ; lhm Ffm lsm ; lhm Ff ðl4 ; HF Þ; (75a)
dl4
dlhm
¼ Fmh lsm ; lhm Ffm lsm ; lhm Ff ðl4 ; HF Þ; (75b)
dl4
with lms(1) ¼ lmh(1) ¼ 1. Moreover, function ‘F (l4) is chosen as ‘F (l4) ¼ l4‘F,o, while HF (l4) is obtained from the solution of the
geometry evolution equation:

dHF ‘F HF 4 ‘2F þ HF2 3rF2
¼ ; (75c)
dl4 l4 4HF2 ‘2F þ HF2 þ 3‘2F rF2
with HF (1) ¼ HF,o, which gives the evolution of crimp amplitude upon fiber deformation and it is derived from a second appli-
cation of the principle of Virtual Works on a curvilinear beam model. It is worth noting that the solution of Eq. (75) on the basis of
the family-specific fiber stretch l4(a) gives the family-dependent internal mechanisms, that is:
sðaÞ ðaÞ hðaÞ ðaÞ ðaÞ ðaÞ
lm ¼ lsm l4 ; lm ¼ lhm l4 ; HF ¼ HF l4 : (76)
Comparison with experimental data

The effectiveness of available constitutive modeling approaches in fitting experimental data is clearly highly depending case by case
(e.g., on the specific tissue under considerations, on the addressed mechanical tests, on the strain range of interest). Therefore,
general conclusive statements on fitting capabilities cannot be traced. On the other hand, an exhaustive analysis on specific case
studies is beyond the scope of present work which aims to be as general as possible.
Following these considerations, an exemplary comparison between modeling approaches and experimental data is presented in
one of the simplest available case study, that is one of the tendons. The latter are unidirectional tissues, with collagen fibers mainly
aligned along the loading direction. Therefore, in this case, a single collagen fiber family can be considered (i.e., nF ¼ 1) and data on
the uniaxial traction applied along-the-fiber direction are representative for the mechanics of these tissues. Tendons are here chosen
because their simple organization allows to obtain data on the evolution of structural features with strain, such as collagen fiber
crimp straightening upon tissue stretching. This allows for a full validation of the multiscale structural approach, although this
modeling rationale has been applied in the literature also to tissues with a multidirectional fiber arrangement (e.g., arterial tissues).
In the case under consideration, tissue deformation gradient tensor F results as in Eq. (60a). The strain-energy density function
Jse is additively decomposed, as in Eq. (15), into a matrix JM and fiber JF term, respectively, associated with an isotropic response
and an anisotropic response (see Eq. 25). Moreover, for dealing with the incompressibility constraint, an Augmented-Lagrangian
approach as in Eq. (56) is employed, considering J ^ as in Eq. (60e) together with an analytical solution strategy to a priori fulfill
h
2
both the kinematic condition lF ¼ l t and the equilibrium condition of null stress in the perpendicular-to-fiber direction (i.e.,
Table 2 Values of model parameters for fitting the data on the uniaxial traction of rat tail tendons in Fig. 8 for
e p
the exponential-based JF (see Eq. 69a) and polynomial-based JF (see Eq. 69b) terms
Model k1 (MPa) k2 k3
p
J F 215 2.16 0.045
e
J F 140 4.20 0.048
Table 3 Values of structural features in rat tail tendons introduced in the multiscale constitutive
m
description for the term JF in Eq. (71) and employed to compare model results with
experimental data in Fig. 8
Parameter Value Definition
rF 1.63 mm Collagen fiber radius

HF,o 13.04 mm Collagen fiber reference amplitude
LF,o 240 mm Collagen fiber reference period
Lc 1400 mmol mol 1 Inter-molecular cross-links density
kc 5 nN mm 1 Inter-molecular cross-links stiffness
‘ks 14 nm Collagen molecular kinks length
‘c 287 nm Collagen contour length
‘p 14.5 nm Collagen persistence length
Am 1.41 nm2 Collagen cross-sectional area
Eô 1 GPa Low-strain collagen tangent modulus
E^ 80 GPa High-strain collagen tangent modulus
h 22.5 Collagen uncoiling resistance
εo h 0.1 Collagen uncoiling strain
T 310 K Body temperature
Employed values belong to physiological ranges, as discussed in Marino M and Wriggers P (2017) Finite strain response of
crimped fibers under uniaxial traction: an analytical approach applied to collagen. J. Mech. Phys. Solids 98, pp. 429–453.
strategy A2). Addressing the matrix term JM, a compressible Mooney–Rivlin formulation, that is Eq. (68) with I ¼ V ¼ 1 and
a1 ¼ b1 ¼ 2, is chosen. Three possibilities are employed for the fiber strain-energy term JF: (i) the exponential-based law JFe in
Eq. (69a); (ii) the polynomial-based law JFp in Eq. (69b); (iii) the multiscale structural approach JFm in Eq. (71). Results are ob-
tained employing values of parameters in Tables 2 and 3 with nF ¼ 1, VM ¼ 0.43, VF ¼ 0.57, a1 ¼ b1 ¼ g1 ¼ g2/8 ¼ 10 kPa.
The obtained relationships between along-the-fiber nominal strain and stress are shown in Fig. 8, and compared with experi-
mental data obtained by K.A. Hansen, J.A. Weiss, and J.K. Barton in 2002. In addition, the multiscale approach gives also informa-
tion on the dependence of fiber crimp with stretch, that is function HF (l4) which is a measure of the average amplitude of collagen
fibers within the tissue. Function HF (l4) is compared with data on the amount of extinguished crimps at different strain levels,
obtained by means of nondestructive imaging analyses conducted in conjunction with mechanical tests. The effectiveness of the
employed constitutive approaches clearly arises, showing also that the multiscale approach is able to give a special insight on
the relationship between structure and mechanics.
Inelastic Behavior
The main inelastic effects that can be observed in soft tissues and that are specifically addressed in this work are:
• viscoelasticity, that is a time dependence of the stress–strain relationship;

• active behavior, that is the occurrence of a stress field originated by self-contracting elements, also in the absence of external
loads;
• plasticity, that is the occurrence of permanent strains, not associated with a degradation of the mechanical properties of tissue
constituents, at the removal of the deforming cause;
• damage, that is a degradation of tissue mechanical properties (e.g., stiffness, strength);
• growth and remodeling, that is an alteration of tissue mass and microstructure driven by mechanobiological mechanisms.
A general framework for the modeling of the afore-introduced inelastic effects is based on the choice of a multiplicative decompo-
sition of the deformation gradient F in an elastic Fe and an inelastic Fi term, holding (see Fig. 9):
F ¼ Fe Fi : (77)
Fig. 8 Comparison between constitutive models and experimental data on the uniaxial traction of rat tail tendons. Modeling rationale in agreement
with Eqs. (15) and (25): matrix term JM in Eq. (68) with I ¼ V ¼ 1 and a1 ¼ b1 ¼ 2; fiber term JF via the exponential-based law JF e in Eq. (69a),
the polynomial-based law JF p in Eq. (69b), and the multiscale structural approach JF m in Eq. (71). Incompressibility constraint treated via an
Augmented-Lagrangian approach (Eq. (56) with J ^ as in Eq. (60e)) and an analytical solution strategy. Results: along-the-fiber first Piola–Kirchhoff
h
stress P: (eo 5 eo) (top) and percentage amount of straight fibers, i.e., extinguished
pffiffiffiffi crimp function EC(l4) ¼ 1 HF (l4)/HF,o (only multiscale
approach, bottom), versus along-the-fiber nominal strain l4 1, with l4 ¼ I4 . Experimental data by Hansen KA, Weiss JA, Barton JK (2002)
Recruitment of tendon crimp with applied tensile strain. J. Biomech. Eng. 124, pp. 72–77. Parameters: nF ¼ 1, VM ¼ 0.43, VF ¼ 0.57,
a1 ¼ b1 ¼ g1 ¼ g2/8 ¼ 10 kPa (Tables 2 and 3).
The inelastic term Fi is associated with the onset and evolution of inelastic mechanisms and with an intermediate configuration
between the reference one Uo and the current one U. In principle, the intermediate configuration is incompatible from the kinematic
point of view and the elastic deformation Fe serves also to re-establish compatibility, together with describing the deformation due
to applied loads (see Fig. 9). In order to obey material objectivity requirements, the elastic part of the right Cauchy–Green defor-
mation tensor is introduced as Ce ¼ Fe TFe where the functional dependence Ce ¼ Ce(C, Fi) is conveniently highlighted since:

Ce C; Fi Þ ¼ FT 1
i CFi : (78)
In agreement with Eq. (7), inelastic mechanisms are described by means of a set V of internal variables, whose time dependence
determines the onset and evolution of inelastic mechanisms (see Fig. 9). The inelastic contribution Fi to the deformation tensor
clearly belongs to the set of internal variables. Moreover, for the sake of generality, tensor A and scalar a are here introduced to
enrich the set of internal variables for describing additional internal mechanisms associated with an inelastic response, resulting
V¼ {Fi, A, a}. Accordingly, free-energy Jfe can be defined as:
Jfe ¼ Jfe ðC; Fi ; A; aÞ: (79)
and the internal dissipation Dint in Eq. (8) results:

_
vjfe C vjfe vjfe vjfe
Dint ¼ S 2ro : ro : F_ i ro : A_ ro _
a: (80a)
vC 2 vFi vA va
Fig. 9 Modeling of the inelastic response of biological structures. Top: general modeling rationale represented by the multiplicative decomposition
of the deformation gradient F in elastic Fe and inelastic Fi terms, and the introduction of internal variables A and a. Bottom: schematic representation
of physical mechanisms originating inelastic effects in soft tissues.
On the basis of Eq. (80a), stress quantities Qi, Hi, and qi are introduced as dual static quantities to Fi, A, and a, respectively.
Introducing the constitutive relationships:
vjfe vjfe vjfe vjfe
S ¼ 2ro ; Qi ¼ ro ; Hi ¼ ro ; qi ¼ ro ; (80b)
vC vFi vA va
the Clausius–Duhem inequality reads as:
Dint ¼ Qi : F_ i þ Hi : A_ þ qi a_ 0; _ a:
c admissible F_ i ; A; _ (80c)
_ and a_ are given by evolution equations for the internal variables. The definition of these evolution equations
Admissible F_ i , A,
closes the constitutive formulation. If a potential function F of the generalized static quantities S ¼ {Qi, Hi, qi} exists, then evolu-
tion equations can be derived from the associate normality rule:
vF vF vF
F_ i ¼ z_ ; A_ ¼ z_ ; a_ ¼ z_ ; (81a)
vQi vHi vqi
where z_ is an indeterminate multiplier and F (S) is a convex function that may also depend on internal variables. The formulation is
completed by the classical Kuhn–Tucker conditions:
z_ 0; F 0; _ 0;
zF (81b)
ensuring that internal mechanisms evolve on the potential surface F ¼ 0 in the space of generalized static quantities S. It is
immediate to show that the Clausius–Duhem inequality in Eq. (80c) is a priori satisfied by the formulation in Eq. (81).
Ad hoc definitions, not motivated by thermodynamic considerations, of the evolution equations are also possible and have been
employed in the existing literature. For instance, in a strain-driven framework, the explicit definition of:
F_ i ¼ F_ i ðC; Fi ; A; aÞ; A_ ¼ A_ ðC; Fi ; A; aÞ; a_ ¼ a_ ðC; Fi ; A; aÞ; (82)
can be introduced, accompanied by the a posteriori verification of the dissipation inequality in Eq. (80c) in order to be thermo-
dynamically consistent.
Evidence and modeling approaches related to the afore-introduced inelastic mechanisms are presented in what follows. When-
ever necessary and referring to the ansatz in Eq. (25), only nF ¼ 1 fiber family is introduced for the sake of compactness, resulting
M¼ {M}, without loss of generality.
Viscoelasticity
The mechanical response of a viscoelastic material is characterized by a hysteretic and strain-rate behavior (see Fig. 2). Moreover,
viscoelastic properties are associated with stress relaxation (i.e., a time step-wise applied strain results in a decreasing stress) and
creep (i.e., a time step-wise applied stress results in an increasing strain).
A main source of viscoelasticity in soft tissues is related to the high water content and it is due to the flow of free water in the
ground matrix, driven by strain. This mechanism leads to a significant time-dependent behavior. A physically motivated approach
for modeling viscoelasticity due to water movement is given by the rationale of poromechanics. This approach couples equations
for the elastic response of the solid matrix with Navier–Stokes equations for the viscous fluid, as well as with Darcy’s law for the flow
of fluid through the porous matrix. A detailed description of poromechanics modeling is beyond the scope of present chapter, and
reference is made to the seminal papers and books by S. Cowin.
A second source of viscoelasticity in soft tissues can be associated with an intrinsic time dependence of the behavior of
tissue constituents. For instance, the deformation rate affects the unfolding pathways of the triple-helix structure of
tropocollagen molecules. The presence of water directly bound to collagen molecules contributes to the latter evidence
(see Fig. 9).
A general framework for the description of tissue viscoelasticity is to employ a generalized Maxwell model where the presence of
water is taken into account in a phenomenological way. The generalized Maxwell model is characterized by an additive decompo-
sition of the free energy in two contributions: Jv eq responsible for the thermodynamic equilibrium state at t / þN, and Jv eq
responsible for the thermodynamic nonequilibrium state of the material which may be seen as a “dissipative” potential (i.e., the
behavior of relaxation and/or creep).
The rheological description of a generalized Maxwell model consists in the parallel connection between a spring (associated with
term Jv eq and the entire kinematics, described by C) and a Maxwell element made up by the series of a dashpot (associated with the
inelastic part of the deformation tensor Fi) and a spring (associated with term Jv neq and the elastic part of the deformation tensor
Fe). In agreement with these observations, it results Jv eq ¼ Jv eq(C) and Jv neq ¼ Jv neq(Ce). Therefore, the generalized Maxwell
model motivates the ansatz:

Jfe ¼ Jfe C; Fi Þ ¼ Jeqv ðCÞ þ Jv ðCe ðC; Fi ÞÞ
neq
(83a)
resulting in (see Eqs. 7 and 80b):
vJneq
S ¼ Seq neq
v þ Sv ; Qi ¼ v ; Hi ¼ 0; qi ¼ 0; (83b)
vFi
where (see Eq. 78)
vJeq 1 vJneq
v ¼2
Seq v
; v ¼ 2Fi
Sneq v
FT : (83c)
vC vCe i
The constitutive framework is completed by the definition of Jv eq and Jv neq and of evolution equations for Fi, under the
constraint prescribed by the dissipation inequality in Eq. (80c). It is worth pointing out that, since the stress tensor S in Eq.
(83b) shall reach the thermodynamic equilibrium for t / þN, the constitutive model shall be defined in order to obtain
Sv neq |t / þ N ¼ 0.
Active Response
A specific class of cells (e.g., skeletal muscle cells, myocardial cells, smooth muscle cells) are able to contract and relax in absence of
applied loads (see Fig. 9). Cell contractile features are due to internal structures where calcium ions activate the interaction between
actin and myosin filaments. These mechanisms induce an inelastic strain field in the tissue, driven by biochemical and biophysical
actions, which significantly affect the distribution of stresses and strains in biological structures. Tissues characterized by such mech-
anisms are referred to as endowed with an active response.
Active response may be also associated with electroactive properties where mechanical forces and electric fields are
coupled. These properties endow tissues with the ability to spontaneously deform upon the application of an electric field.
The latter mechanism is of main relevance for the mechanical response, for instance, of the cardiac muscle and of the
intestine.
The mechanics of soft tissues with an active response can be described by considering the strain-energy function Jse, related to
the purely elastic deformation (i.e., Jse ¼ Jse(Ce)), and function Ja describing the contractile nature of the active part and depend-
ing on the overall deformation (i.e., Ja ¼ Ja(C)). Function Jse can be defined as in a hyperelastic framework and function Ja
accounts for biochemical and/or electrochemical fields.
Assuming an additive decomposition of the free energy, the ansatz of Eq. (83) can be employed, replacing Jv eq with Ja and
Jv neq with Jse. The formulation is completed by the definition of functions Jse and Ja, of evolution equations for Fi, under
the constraint prescribed by the dissipation inequality in Eq. (80c). If other fields are introduced, such as the electrical field for
cardiac or gastrointestinal tissues, further balance laws shall be accounted for.
Plasticity
The phenomenological response of a material undergoing plastic mechanisms is well elucidated by the analysis of the stress–strain
relationship obtained from a uniaxial traction test. Material response can be described as hyperelastic below a certain stress value,
denoted as elastic limit. Beyond the elastic limit, a plastic (equivalently, yielding) regime is attained. With reference to Fig. 2 and
addressing a positive stretching beyond the elastic limit, the slope of the stress–strain relationship significantly reduces, being equal
to zero in the ideal plastic case. Hence, a change in the shape of the specimen at (almost) constant stress is obtained. Moreover,
residual strains occur upon loading removal. In mechanics, plasticity is classically associated with the slip of dislocations in the crys-
talline structure of metals. Nevertheless, plastic-like mechanisms, leading to a nonreversible change of shape of biological structures,
occur also in soft tissues.
For instance, the alteration of the structure of the noncollagenous matrix may lead to the permanent slip of collagen fibers rela-
tive to each other, leading to a change of the reference state. Moreover, collagen fibers may experience nonelastic straightening upon
overstretching, not returning to the original unstrained wave pattern when residual strain appears in the tissue. This might be the
sign of a permanent alteration in the multiscale arrangement of collagenous structures within fibers. As schematically depicted in
Fig. 9, atomistic computations based on molecular dynamics simulations of collagenous assemblies show that two inelastic plastic-
like mechanisms may intervene: slip pulse and interstrand delamination. Slip pulse is a permanent intermolecular sliding where
weak bonds between adjacent molecules break and continuously reform (among different residues) during sliding. Interstrand
delamination is the loss of the structural integrity of collagen molecules due to the breaking of weak bonds between the polypeptide
strands within a single collagen triple helix. In this case, the permanent sliding of one polypeptide strand, with respect to the other
two, occurs.
Furthermore, the rearrangement and alterations of tissue constituents due to the evolution of internal plastic-like mechanisms
may lead to a stronger interaction between collagen molecules in fibers and between collagen fibers with the elastin network. This
phenomenon might be the source of the strengthening of tissues by plastic deformation, that is of hardening effects in soft
tissues.
A plastic behavior can be described by introducing the additive decomposition of the free energy in a strain-energy function Jse,
related to the purely elastic deformation (i.e., Jse ¼ Jse(Ce)), and the term Jp, depending on the hardening variable a and being
a measure of tissue strengthening with plasticity.
Accordingly, a possible ansatz is:

Jfe ¼ Jfe ðC; Fi ; aÞ ¼ Jse ðCe C; Fi ÞÞ þ Jp ðaÞ; (84a)

vJse T vJse vJp
S ¼ 2F1 F ; Qi ¼ ; Hi ¼ 0; qi ¼ : (84b)
i
vCe i vFi va
The constitutive framework is completed by the definition of material response in the elastic regime (i.e., function Jse), of hard-
ening mechanisms (i.e., function Jp) and of evolution equations for Fi and a, under the constraint prescribed by the dissipation
inequality in Eq. (80c). In this context, the evolution equation for Fi is generally referred to as the flow rule. If the formulation
of Eq. (81) is employed, the surface identified by F ¼ 0 in the space of generalized stresses is denoted as the yield surface. The
Kuhn–Tucker conditions ensure that the stress state never lies outside the yield surface. As a matter of fact, when plasticity evolves
(i.e., z_ > 0), the condition F ¼ 0 holds from Eq. (81b) or, in other words, the stress state remains on the yield surface. Nevertheless,
the shape and size of the surface may change as the plastic deformation evolves because F may depend on the internal variable a,
leading to the description of hardening effects.
Damage
Damage in materials initiates and evolves at strain/stress values beyond a certain limit which corresponds to the elastic limit. Below
the elastic limit, the material is within the elastic regime and it is generally described as hyperelastic. The onset and evolution of
damage is associated with a progressive reduction of material stiffness. In a strain-driven test, stress may also decrease, and this
phenomenon is known as softening. If damage evolution in the material is fast and the stress drops, failure is described as brittle
and tissue final failure is reached (see Fig. 2).
Damage can be associated with the process of initiation and growth of cracks occurring at a length scale comparable to the one of
the structure under consideration. This mechanism leads to the need of describing damage as the evolution of a discontinuity in the
integrity of the structure. This modeling approach is known as fracture mechanics. Different theoretical and computational
approaches exist for dealing with the discontinuous character of fracture mechanics (e.g., phase field approaches, the extended finite
element method, cohesive zone model) but the description of these is beyond the scope of present work.
On the other hand, the phenomenology of damage can be associated with an alteration of material features occurring at a length
scale significantly smaller than the one of the structure under consideration (e.g., like nucleation and growth of voids, nanoscale
and/or microscale defects of constituents). In this framework, stress measures obtained from constitutive models might represent
a homogenized quantity which implicitly accounts for the deterioration of the material. This modeling approach is known as
continuum damage mechanics.
In this context, possible sources of damage in soft tissues are the rupture of covalent bonds within polypeptide strands of
collagen molecules or the degradation of covalent cross-links between collagen molecules (see Fig. 9). For instance, the amount
of intermolecular cross-links may decrease due to biochemical reactions mediated by enzymes. Collagen-related damage mecha-
nisms determine a degradation of the mechanical properties of collagen fibrils, in turn associated with degraded mechanical prop-
erties at tissue level.
In the framework of continuum damage mechanics, the mechanical response of soft tissues undergoing damage can be modeled
by means of the strain-energy Jse, which describes the hyperelastic material response in the elastic regime, modulated by the contin-
uous internal variable a which describes damage. Variable a is valued in [0, 1], being equal to 0 if the soft tissue is sound (i.e., non-
damaged and behaving as purely elastic) and equal to 1 if it is fully damaged (i.e., not able to carry load). Values of a ˛ (0, 1)
describe a partially damaged situation. It is worth highlighting that the inelastic part of deformation Fi, and hence the multiplicative
decomposition in Eq. (77), is not needed for modeling damage effects associated with softening. Damage is described by intro-
ducing the damage function g, valued in [0, 1], and such that:
vg
gð0Þ ¼ 1; gð1Þ ¼ 0; g0ðaÞ ¼ 0: (85)
va
An ansatz for the free energy for describing damage in soft tissues is:
Jfe ¼ Jfe ðC; aÞ ¼ gðaÞJse ðCÞ; (86a)

vJse
S ¼ 2g ðaÞ ; Qi ¼ 0; Hi ¼ 0; qi ¼ Jse ðCÞ: (86b)
vC
From Eqs. (85) and (86b), it is immediate to show that stress S corresponds to the one obtained in a hyperelastic framework for
a ¼ 0 (i.e., when damage is not activated). Since function g(a) is monotonically decreasing (see Eq. 85), the onset and evolution of
damage (i.e., when a increases) is associated to a stress decrease, up to the limit case S|a / 1 ¼ 0 (see Eq. 85), recovering a softening
response.
In order to move toward a structurally motivated framework, damage can be associated to a single constituent and introduced in
the definition of the strain invariants. For instance, addressing collagen damage, let eI4 ¼ eI4 ðC; aÞ be a generalized fourth invariant of
deformation, defined as:
eI4 ðC; aÞ ¼ g ðaÞ½I4 ðCÞ 1 þ 1 ¼ g ðaÞ½TrðCMÞ 1 þ 1; (87a)
where g(a) respects the conditions in Eq. (85).
In this framework, a structurally motivated formulation of the free energy of soft tissues undergoing damage is:
Jfe ¼ Jfe ðC; aÞ ¼ VM JM ðCÞ þ VF JF ðeI4 ðC; aÞÞ; (87b)
S ¼ VM SM þ VF e
SF ðeI4 ; aÞM; qi ¼ VF g’ðaÞ½I4 ðCÞ 1 SF ðeI4 Þ; (87c)
with Qi ¼ 0, Hi ¼ 0 and
vJM e vJF
SM ¼ 2 ; SF ðeI4 ; aÞ ¼ g ðaÞSF ðeI4 Þ; SF ðeI4 Þ ¼ : (87d)
vC veI4
Model response when damage is not activated (i.e., a ¼ 0) is purely elastic. In fact, it results eI4 ðC; 0Þ ¼ I4 ðCÞ (see Eqs. 85 and
87a) and the fiber stress tensor corresponds to the one that would be obtained in a hyperelastic framework, that is equal to VFSF
(I4)M. Moreover, accounting for Eq. (85), fiber stress eSF in Eq. (87c) results inversely proportional to damage, up to the limit
case e
SF ja/1 ¼ 0 (see Eq. 85 and 87d) where only the matrix contribution SM contributes to the overall second Piola–Kirchhoff
stress S.
The definitions of the strain-energy function Jse (as in a hyperelastic framework), of damage function g(a), and of evolution
equations for a complete the damage formulation, under the constraint enforced by the dissipation inequality in Eq. (80c).
Growth and Remodeling

Living tissues continually undergo a turnover of constituents via a delicate balance between the production of new material and the
removal of the old one (see Fig. 9). A change in the rates of turnover of constituents can lead to atrophy (i.e., decrease in the overall
mass of material, also known as resorption) or hypertrophy (i.e., increase in the overall mass of material, also known as growth). In
the literature, growth and resorption are collected under the term growth. A distinction is made between growth on the surface (i.e.,
appositional growth) and the one within the volume (i.e., interstitial growth) of tissues. Present work addresses volumetric growth,
since the most relevant for soft tissues. Clearly, mass growth and volume variations are related through the mass density.
On the other hand, remodeling describes the changes in the histology and biochemistry of tissues associated with an alter-
ation of material mechanical properties (e.g., stiffness, strength, and anisotropy). It may be related to coarse changes in tissue
composition (e.g., excess of fibrous material known as fibrosis), as well as to fine alterations of tissue microstructure (e.g., realign-
ment, thickening, and crimp alterations of collagen fibers) and of biochemical properties (e.g., in density of intermolecular cross-
links).
Remodeling is often used jointly with growth, although being two independent mechanisms. However, remodeling can be
a manifestation of growth at spatial scales lower than the tissue one (e.g., in fibrosis). Moreover, deposed/resorbed mass
associated with interstitial growth may be initially incompatible from a kinematic point of view with the preexisting material,
and remodeling could smooth this incompatibility. Accordingly, an intimate connection between growth and remodeling
arises.
Since cells respond to mechanical stimuli, applied loads have a strong influence on growth and remodeling. For instance, cells
align as a consequence of the applied stresses and/or strains, hence controlling the direction of the secreted collagen fibers which
realign in agreement with the current stress and/or strain fields.
Growth and remodeling in soft tissues can be modeled by following two different rationales: a constrained mixture model and
a kinematic approach. The theory of constrained mixtures is based on the assumption that tissue constituents exhibit distinct refer-
ence configurations and rates of turnover. At this standpoint, full mixture equations for mass balance are used in combination with
classical equations for linear momentum balance written in terms of rule-of-mixture relations for the stress response. The evolution
of the reference configurations and rates of turnover of tissue constituents are defined in order to maintain a preferred (homeostatic)
biomechanical state, incorporating biologically driven mass density productions and survival functions within constitutive rela-
tions. A detailed description of the constrained mixture approach is beyond the scope of present work. To this aim, reference is
made to the seminal works by J.D. Humphrey and C. Cyron.
On the contrary, the kinematic approach identifies a unique reference configuration. The overall deformation tensor is decom-
posed into elastic and growth components along the lines of Eq. (77). In the framework of open system thermodynamics, the kine-
matic approach focuses on volumetric growth, where the intermediate configuration reached by tensor Fi physically represents the
(possibly incompatible) grown configuration without any elastic deformation.
In order to describe growth, density ro in the reference configuration at the grown state (i.e., at current time) is introduced as an
internal variable. Clearly, the balance of mass for open systems shall be taken into account, considering the rate of change of density
r_ o in the reference configuration, a possible influx/outflux of matter R (e.g., cell migration) and a volumetric mass source R (e.g., cell
proliferation, apoptosis). Therefore, denoting by Div (•) the divergence operator, it holds:
Z t
ro ¼ ro ðt Þ ¼ ro þ r_ o dt; with r_ o ¼ DivðRÞ þ R: (88)
o
where ro is the density in the reference configuration at the initial state (i.e., at initial time t ¼ 0). It is worth pointing out that growth
in soft tissues can be described by assuming that the density of the newly grown tissue has the same density as the initial tissue.
Accordingly, a mass change is accompanied by a volume change. Hence, it results ri ¼ ro ¼ const, where ri ¼ ro/Det (Fi) is the
density in the intermediate configuration.
In this case, the source term R can be directly related to the deformation tensor Fi,
resulting R ¼ ro DetðFi Þ Tr F_ i F1
i . Clearly, a number of different choices are also possible, based on ad hoc hypotheses on tissue
growth.
In order to describe remodeling, the rearrangement of fibers is herein considered as an illustrative example. To reach this goal,
the structure tensor M is introduced as an internal variable, obtaining an alteration of anisotropic material properties with
remodeling.
In analogy with the ansatz of Eq. (79) with A ¼ M and a ¼ ro, the free-energy function jfe per unit mass is introduced as:
jfe ¼ jfe ðC; Fi ; M; ro Þ (89a)
Moreover, the Clausius–Duhem inequality shall be formulated considering open thermodynamical systems, allowing for the
exchange of mass with the exterior, in order to account for the living nature of soft tissues. Accordingly, the dissipation inequality
considers a mass specific entropy term s0 that takes into account external entropy flux and/or source. The Clausius–Duhem
inequality in Eq. (8) for open systems reads as:
C_
Dint ¼ S : ro j_ fe ðC; Fi ; M; ro Þ ro Ts0 0; (89b)
2
for any admissible thermodynamical state.
Accounting for the constitutive relationships in Eq. (80b), together with mass balance in Eq. (88), the Clausius–Duhem
inequality in Eq. (89b) for growth and remodeling reads:
Dint ¼ Qi : F_ i þ Hi : M
_ þ Dm ðr_ o ; s0 Þ 0; c admissible F_ i ; M;
_ r_ o ; (89c)
with the term Dm, associated with the exchange of mass, being:
Dm ðr_ o ; s0 Þ ¼ qi r_ o ro Ts0 ¼ qi ½Div ðRÞ þ R ro Ts0 : (89d)
A possible formulation for the free energy in growth and remodeling accounts for the elastic deformation from the intermediate
to the current configuration by means of the (volume specific) strain-energy function Jse, depending on the elastic part of defor-
mation Ce and weighted by the relative density (ro/ro )n, with n being a model parameter. Due to the remodeling of anisotropic
directions, the explicit dependence of the strain energy on the structure tensor M is highlighted, that is Jse ¼ Jse(C, M). Therefore,
the (mass specific) free-energy function jfe reads:

1 ro n
jfe ðC; Fi ; M; ro Þ ¼ Jse ðCe ðC; Fi Þ; MÞ ; (90a)
ro ro
resulting in (see Eq. 80b):
n n
ro vJse T r vJse
S¼2 F1 F ; Qi ¼ o ; (90b)
ro i
vCe i ro vFi
n n
r vJse r
Hi ¼ o ; qi ¼ ð1 nÞ o Jse ðCe ; MÞ: (90c)
ro vM ro
Within the present ansatz, a possible definition of the extra entropy term s0 follows from requiring a null dissipation associated
with the mass term Dm in Eq. (89d), obtaining:

1 n ro n
s0 ¼ Jse ðCe ; MÞ½DivðRÞ þ R : (90d)
T ro r0
The constitutive framework is completed by the definition of material response in the elastic regime (i.e., function Jse as in
a hyperelastic framework), of evolution equations for Fi and M, as well as of mass flux R and source R, under the constraint
prescribed by the dissipation inequality in Eq. (89c). Remarkably, a homogenized constrained mixture model, which gathers the
mechanistic description of constituents-dependent growth and remodeling with the simplicity deriving from a gross description
of the main kinematic effects, has been recently developed by C. Cyron and co-workers.
Open Problems and Future Challenges
In silico analyses support nowadays the biomedical research in improving the effectiveness of the clinical outcome and in clarifying
unknown and unexplored pathways that affect the functional behavior of biological structures. The understanding of the physical
mechanisms behind the pathophysiological behavior of soft tissues, from the cellular to the organ scale, would open the door to
ground-breaking results both in research and in clinics. Indeed, the ultimate goal is to turn heuristic knowledge into predictive capa-
bilities, through the quantitative modeling of the fundamental interactions between mechanics and biology.
The development of reliable constitutive models of soft tissues stands out as one of the major challenges in biomechanics. In this
framework, both computational and theoretical issues arise. From the computational standpoint, the coupling of anisotropic and
incompressible behavior in soft tissues inherits numerical stability problems. Moreover, especially when inelastic mechanisms are
addressed, the implementation of constitutive models in numerical frameworks can be challenging. Finally, fast (ideally, real-time)
simulations of large and realistic biological structures are needed for applying in silico approaches in clinics. Accordingly, special
care shall be paid to verify the convergence rate of numerical algorithms, with constitutive models playing a major role on this issue.
Issues on the theoretical formulation of novel constitutive models are associated with the need of capturing more and more
experimental evidence that arise in tissue pathophysiology. Model generalizations shall not be paid in terms of loss of material
stability mathematical requirements. Moreover, the description of a wide range of mechanical responses is generally accompanied
by the increase of the number of parameters and these, generally, have a phenomenological meaning. Therefore, issues on model
calibration appear, especially in a patient-specific framework.
A common practice is to employ population-based mean data for material properties, even though this can introduce some inac-
curacies when referred to specific scenarios. Furthermore, even assuming an accurate calibration, the evolution of the value of
phenomenological parameters with the onset and evolution of pathologies is an open issue. In addition, possible constitutive inho-
mogeneities associated with pathological localized tissue defects are generally neglected. Typical disorders (e.g., aneurisms, kerato-
conus, arthrofibrosis) are indeed associated with structural defects at different length scales: in content of tissue constituents, in
shape of collagen fibers, in collagen genetic pattern, in density of intermolecular cross-links. Microstructural and biochemical alter-
ations reflect in a nonphysiological mechanical response of soft tissues, such as hyperextensibility or weakness.
Multiscale approaches allow to incorporate structural information in a straightforward way, since model parameters have a clear
physical meaning. Therefore, these models can be fed by patient-specific histological and biochemical information, accounting also
for possible pathological alterations. The analytical rationale of available multiscale approaches, well-established in a hyperelastic
framework, allows to maintain a low computational cost, despite the gained insight on the structure–mechanics relationship.
Nevertheless, pathologies are often related to an inelastic tissue behavior but the application of a multiscale rationale to inelas-
ticity is still an open issue. In this field, some results are available at the scale of collagen fibrils, starting from the modeling of
damage- and plastic-related mechanisms affecting collagen mechanics (e.g., slip pulse, interstrand delamination), proposed by
M. Marino and G. Vairo in 2014 and afterwards generalized by M. Marino in 2016. Advances on this issue, up to the tissue scale,
would help to clarify the dominant viscous/damage/plastic mechanisms in pathological conditions across the length scales, contrib-
uting in developing targeted therapeutic approaches.
Moreover, addressing growth and remodeling, multiscale approaches would allow to explicitly account for the strict relationship
between tissue macroscopic response and remodeling mechanisms. Indeed, a number of classical approaches in growth and remod-
eling employ phenomenological expressions of fiber strain-energy functions, with fiber orientation being the only structural infor-
mation. Therefore, apart from fiber angle, the remodeling-induced evolution of parameters governing strain energies is generally
neglected. At most, only a phenomenological rationale, tough to be validated and calibrated, can be employed. On the other
hand, multiscale approaches are promising alternatives, opening the door to the development of a new class of models for growth
and remodeling. A multiscale rationale allows indeed to define remodeling laws directly from biological evidence and clinical cases,
since model parameters are measurable properties describing tissue histology and biochemistry (e.g., the radius and crimp ampli-
tude of collagen fibers, the density of intermolecular cross-links).
Furthermore, one of the major challenges is related to the living nature of soft tissues, shedding the light on the role of biochem-
istry in tissue active response, as well as in growth and remodeling. Tissue mechanical response is indeed the result of biochemical
processes regulated by cell–cell signaling pathways. Cells collect, produce, and release a wide range of ions and molecules (e.g.,
nutrients, hormones, enzymes, growth factors, drugs). The transport and diffusion of ions and molecules govern a cascade of
biochemical reactions that may activate intracellular contractile elements and/or alter the composition, the properties and the
arrangement of tissue constituents. Cell–cell signaling pathways are continuously active in soft tissues, governing the natural turn-
over of constituents. Pathologies develop when an imbalance of mechanobiological processes occurs, inducing nonphysiological
histology and biochemistry.
In turn, biochemical pathways are strongly affected by mechanics. For instance, the osmotic pressure in the interstitial fluid of
soft tissues surely plays a role in the diffusion of ions and molecules. In addition, since biological cells respond to mechanical
stimuli by altering the biochemical environment (possibly involving the nervous system), mechanical quantities also affect trans-
port phenomena in terms of molecular sources and sinks. Accordingly, mechanics and biochemistry shall be taken into account
within a unique framework, represented by a closed-loop feedback system. Furthermore, electrochemical mechanisms can also
affect molecular pathways in specific tissues (e.g., myocardium, gastrointestinal tissue), adding the need of accounting for the elec-
trical field in the formulation of the constitutive model. Therefore, the development of constitutive models of soft tissues within
multiphysics frameworks is highly needed. In this context, chemo-mechano-biological modeling strategies for the coupling of arte-
rial growth and remodeling with biochemical reactions have been traced, for instance, by P. Aparício, M.S. Thompson, and P. Wat-
ton in 2016. Along the same lines, an insight on the effects of the remodeling of fine tissue structural properties (i.e., the coupling
with a multiscale constitutive approach), as well as on molecular transport mechanisms affecting cell–cell signaling pathways, has
been provided by M. Marino, G. Pontrelli, G. Vairo, and P. Wriggers in 2017.
Advances on the afore-introduced challenges arise as urgent priorities of the biomechanical research community. These would
allow indeed to gain a novel insight on the living and adaptive properties of biological tissues, properties which distinguish the
constitutive modeling in biomechanics from the one in the framework of standard “dead” materials.
Further Reading
Ambrosi, D., Athesian, G. A., Arruda, E. M., Cowin, S. C., Dumais, J., et al. (2011). Perspectives on biological growth and remodeling. Journal of the Mechanics and Physics of
Solids, 59, 863–883.
Balzani, D., Brinkhues, S., & Holzapfel, G. A. (2012). Constitutive framework for the modeling of damage in collagenous soft tissues with application to arterial walls. Computer
Methods in Applied Mechanics and Engineering, 213–216, 139–151.
Cowin, S., & Doty, S. B. (2007). Tissue mechanics. New-York: Springer ScienceþBusiness Media, LLC.
Cyron, C., & Humphrey, J. (2017). Growth and remodeling of load-bearing biological soft tissues. Meccanica, 52, 645–664.
Fratzl, P. (2008). Collagen: Structure and mechanics. New York: Springer.
Gasser, T. C., & Holzapfel, G. A. (2002). A rate-independent elastoplastic constitutive model for biological fiber-reinforced composites at finite strains: Continuum basis, algorithmic
formulation and finite element implementation. Computational Mechanics, 29, 340–360.
Holzapfel, G. A., & Ogden, R. W. (2010). Constitutive modelling of arteries. Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 466,
1551–1597.
Maceri, F., Marino, M., & Vairo, G. (2010). A unified multiscale mechanical model for soft collagenous tissues with regular fiber arrangement. Journal of Biomechanics, 43,
355–363.
Marino, M. (2016). Molecular and intermolecular effects in collagen fibril mechanics: A multiscale analytical model compared with atomistic and experimental studies. Biomechanics
and Modeling in Mechanobiology, 15, 133–154.
Marino, M., & Wriggers, P. (2017). Finite strain response of crimped fibers under uniaxial traction: An analytical approach applied to collagen. Journal of the Mechanics and Physics
of Solids, 98, 429–453.
Marino, M., Pontrelli, G., Vairo, G., & Wriggers, P. (2017). A chemo-mechano-biological formulation for the effects of biochemical alterations on arterial mechanics: The role of
molecular transport and multiscale tissue remodelling. Journal of the Royal Society Interface, 14, 20170615.
Menzel, A., & Kuhl, E. (2012). Frontiers in growth and remodeling. Mechanics Research Communications, 42, 1–14.
Sacks, M. S., & Wei, S. (2003). Multiaxial mechanical behavior of biological materials. Annual Review of Biomedical Engineering, 5, 251–284.
Sacks, M. S., Zhang, W., & Wognum, S. (2016). A novel fibre-ensemble level constitutive model for exogenous cross-linked collagenous tissues. Interface Focus, 6, 20150090.
Schröder, J., & Neff, P. (2003). Invariant formulation of hyperelastic transverse isotropy based on polyconvex free energy functions. International Journal of Solids and Structures,
40, 401–445.
Continuum Mechanics and Its Practical Applications at the Level of Scaling
Laws
Ko Okumura, Department of Physics and Soft Matter Center, Ochanomizu University, Tokyo, Japan
Introduction 111
Examples From Classic Problems 111
Stokes’ Law of Drag Friction 111
Hertz’s Law of Elastic Deformation 112
Bending of a Plate 112
Capillary Rise 113
Practical Applications 114
Microfluidic Devices for Liquid Mixing 114
Kirigami Approach to Control Elastic Modulus 115
Conclusion 117
Acknowledgments 118
References 118
Introduction
In this article, we try to explain the usefulness of arguments at the level of scaling laws by examples in the context of continuum
mechanics and its application relevant to biomedical sciences. Scaling laws are relations between physical quantities in which
all the physical quantities appear in terms of powers, whereby a power of x is expressed in the form xa where a is a real number.
For example, the relation U ¼ mV2/2, which gives the kinetic energy U of a particle of mass m moving at a velocity V, is expressed as
U x mV2 at the level of scaling laws. In general, a scaling relation is often expressed by using the symbol “x.” For example, a scaling
law “A x B” is a substitute of a more formal expression “A ¼ kB” with a dimensionless numerical coefficient k, the order of magni-
tude of which is not significantly different from one. In other words, the expression “A x B” means that the quantities A and B are
dimensionally equal.
This article is prepared for students and researchers who once studied the basics of continuum mechanics (theory of linear elas-
ticity and hydrodynamics). However, this article does not require rich experiences in mathematical handling of equations in the
textbooks. The basic knowledge of surface tension and capillary phenomena will help the readers to deepen their understanding.
But this is not compulsory, and a brief introduction will be given. For readers who are tempted to study (again) continuum
mechanics in a conventional way by following equations, we recommend textbooks such as Landau and Lifshitz (1987) (for
Sections “Stokes’ Law of Drag Friction” and “Capillary Rise”) and Landau and Lifshitz (1975) (for Sections “Hertz’s Law of Elastic
Deformation” and “Bending of a Plate”).
This article is organized as follows. First, we derive some scaling laws for classic problems to show examples of scaling argu-
ments. Second, we explain examples from recent studies, in which scaling laws are theoretically derived and experimentally
confirmed. Finally, we summarize advantages of the arguments at the level of scaling laws.
Examples From Classic Problems

Stokes’ Law of Drag Friction
Let us consider a sphere with radius R, which is moving with velocity V in a fluid with viscosity h, as is shown in Fig. 1. In the limit in
which the viscous effect is larger than the inertial effect, the drag force acting on the sphere from the liquid can be estimated at the
R
V
Fig. 1 Stokes’ friction: a sphere with radius R is moving at a velocity V in a liquid with viscosity h.

112 Biomechanics j Continuum Mechanics and Its Practical Applications at the Level of Scaling Laws
level of scaling laws in the following manner. From Newton’s law of viscous force, the stress (s) acting on a surface is given by
viscosity times velocity gradient at the surface:
sxhVV
in which the expression VV symbolically stands for the velocity gradient. In the present case, the velocity is of the order of V near the
surface of the sphere, but it decreases as we go away from the sphere, and the characteristic decay length is R: dimensionally, the
velocity gradient scales as V/R. The stress at the surface is thus given by hV/R. The drag force is given by the total force given by the stress
times the area of the surface. In the present case, since the area of the sphere scales as R2, the drag force F scales as h(V/R)R2:
FxhVR (1)
More precise calculation gives a numerical coefficient 6p at the cost of lengthy calculation: F ¼ 6phVR.
Hertz’s Law of Elastic Deformation

Let us consider a sphere with radius R in contact with a plate, where the distance between the center of the sphere and the plate is
smaller than R, by the length d as in Fig. 2. Within the limit where R is very much larger than d, the elastic energy stored in the sphere
can be estimated at the level of scaling laws in the following manner. Let us remember that the elastic energy u stored per unit
volume in an elastic body with elastic modulus E subject to the strain ε scales as Eε2:
uxEε2 (2)
In the present problem, the strain decays as we go away from the contact area and the characteristic decay length is the length of
AC in the figure, which is called “a”: elastic deformation is localized in a volume xa3. Note that, since R [ d, the length a is much
smaller than R, and this is the only length scale that characterizes the deformation of the sphere. This point would be better under-
stood if one realizes that the boundary condition for the elastic deformation should be described by a (not d). Thus, the deforma-
tion is localized in a region near the contact area whose volume scales as a3. From this scaling view, the strain scales as d/a, and thus,
the elastic energy per unit volume E(d/a)2 is stored in a region with a volume xa3: the total elastic energy stored in the sphere U is
given by E(d/a)2a3. In fact, the length a is determined once R and d are given: for the right triangle OAC, Pythagorean theorem gives
the relation R2 ¼ a2 þ (R d)2, from which we obtain
dxa2 =R (3)
With this relation, U is recast into the following form:
pffiffiffi
UxE Rd5=2 (4)
Since the force F is given by the derivative dU/dd we obtain a nonlinear expression for F:
pffiffiffi
FxE Rd3=2 (5)
Note that at the level of scaling laws, the derivative with respect to d is equivalent to “division by d,” that is, d 5/2
/d ¼ d 3/2
.
Bending of a Plate
Let us consider a plate with thickness h, whose length and width are a and b, respectively. When this plate is bent as in Fig. 3, we
regard the plate as a collection of curved thin sheets of thickness dx whose surface S(x) is a part of the surface of a cylinder with radius
R þ x ( h/2 < x < h/2), that is, S(x) ¼ bl(x) where
lðxÞ ¼ ðR þ xÞq with a ¼ Rq (6)
Assume that the strain of the sheet in the middle whose section is represented by the dashed curve in Fig. 3 (i.e. the sheet whose
area is S(0)) is zero (if we set the strain of the surface S(0) to be ε ¼ ε0, the integral in Eq. (8) below has the minimum at ε0 ¼ 0):
εðxÞ ¼ ðlðxÞ aÞ=a ¼ x=R (7)
A a
C
δ
Fig. 2 Hertz’s contact: a sphere with radius R is pushed against the plate by the length d.
Biomechanics j Continuum Mechanics and Its Practical Applications at the Level of Scaling Laws 113
h
δ
O
Fig. 3 Bending of a plate. The length of the curved dashed line (a part of the arc of a circle with radius R) is a ¼ Rq, and the width of the plate (i.e.
the length of the plate in the direction perpendicular to the sheet) is b.
The strain ε(x) of the surface S(x) is positive (negative) for x > 0 (x < 0). Since the energy per unit volume scales as Eε2 with
Young’s modulus E, the total energy stored in the plate is calculated as
Z h=2
Ux dxEεðxÞ2 SðxÞxabEh3 =R2 (8)
h=2
in the small ε limit. Note that, at the level of scaling laws, the integration by x is equivalent to “multiplication by x.” By virtue of
Pythagorean theorem, the deflection at the center d (see Fig. 3) satisfies the relation R2 ¼ (R d)2 þ (a/2)2 in the small ε limit, which
gives the relation Rd x a2. Thus, the energy is written as a function of d as
U ðdÞxEbh3 d2 =a3 (9)
The force in the direction of d in this case is again given by “division by d,” which is linear in d:
FxEbh3 d=a3 (10)
Capillary Rise
Some basic knowledge of capillary phenomena will be, though not compulsory, helpful to understand this section. The key words
are as follows: surface (interfacial) tension, surface (interfacial) energy, contact angle, capillary length, and capillary rise. These key
concepts are briefly explained below.
A liquid surface has a positive energy proportional to the area, and thus, the area always wants to shrink. Therefore, any line on
the surface is pulled from both sides by the surface in the direction perpendicular to the line as in Fig. 4A (the force vectors are on the
surface; since the two forces are equal in magnitude and directed in the opposite directions with each other, the total force on the
line is zero). The force per unit length is called surface tension and denoted as g. Likewise, any solid surface or solid–liquid interface
(usually) has a positive energy; because of the energy, any line on the surface is pulled from both sides by the surface in the perpen-
dicular directions. The corresponding forces per unit length for the solid surface and the solid–liquid interface are denoted as gS and
gSL, respectively.
When a drop of a liquid with surface tension g is placed on a solid substrate with surface tension gS, the drop makes an angle q at
the edge on the substrate as in Fig. 4B, and this angle is called the contact angle. As is clear from the force balance in the horizontal
direction, we obtain Young’s relation: gS ¼ g cos q þ gSL.
In general, capillary force acts strongly at small scales (compared with gravitational force), and gravitational
pffiffiffiffiffiffiffiffiffiffi force acts strongly at
large scales; the two forces become comparable at a length scale called the capillary length k1 ¼ g=rg with the liquid density r
and the gravitational acceleration g. This scale can be estimated by balancing the surface energy and the gravitational energy of
a spherical drop, at the level of scaling laws: gR2 x rR3gR (the latter scales as the gravitational potential energy of a drop at height
xR). Solving this relation in terms of R, we obtain the size of liquid drop for which capillarity and gravity compete with each other,
and this size is identified with the capillary length k 1.
When a vertical tube of inner radius R is in contact with a bath of a liquid whose viscosity is h, the liquid starts to rise into the
tube and then reaches the final height zf, if R k 1 and q < p/2 (see Fig. 4C). This is understood in terms of force by considering
interfacial forces acting on the liquid cylinder of length z. At the top, the column is pulled upward by the force 2pR(g cos q þ gSL); at
(A) (C) γ γ
G
γSL γSL
γ θ
γ
S
(B) z
L
G
γ
L 2R
θ G
γS γSL S
L
γSL γSL
Fig. 4 Capillary phenomena. (A) Surface tension. The dashed line of unit length on the surface is pulled from both sides by the forces with magni-
tude g. The two arrows representing the two forces g are perpendicular to the dashed line, and the force vectors are on the surface. (B) Force balance
at the edge of a liquid drop placed on a solid substrate. Three forces are acting on the cylinder of unit length whose section is given by the dashed
circle. L, S, and G stand for the liquid, solid, and gas phases, respectively. (C) Capillary rise. Interfacial forces acting on the liquid column between
the two dashed straight lines.
the bottom it is pulled downward by the force 2pRgSL: in total, the column is pulled by the net interfacial force 2pRg cos q, which
balances with the gravity acting on the column, at the equilibrium. The final height z ¼ zf is determined by the force balance
2pRg cos q ¼ rpR2zfg. When z is much smaller than zf, there is a regime in which the interfacial force and viscous friction become
the two important players (in fact, when z is very small, we have another regime in which the interfacial force competes with inertia,
de Gennes et al., 2013). In the so-called viscous regime, the force balance can be expressed as Rg cos q x h(V/R)Rz at the level of
scaling laws. This is because the viscous friction per unit area on the surface of the liquid column scales as hV/R (at the center of
the column, the liquid velocity scales as V; at the surface, which is away from the center by the distance R, the velocity is zero:
the velocity changes by V over the length R, which means the velocity gradient scales as V/R) and the surface area of the cylinder
scales as Rz. The balance Rg cos q x hVz can be rewritten as g cos q x h(z/t)z because at the level of scaling laws V x dz/dt x z/t (if
z is given by some powers of t, the derivative with respect to t is equivalent to “division by t”). From this, we obtain
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
zx tgRcosq=h (11)
Practical Applications
Microfluidic Devices for Liquid Mixing
Recently, in many biomedical applications, mixing of small amounts of liquids becomes an important issue, and microfluidics is
promising in this context (Beebe et al., 2002; Squires and Quake, 2005). However, conventional microfluidic devices require pumps
to transport liquids inside channels, and such a pumping system makes the dimension of the whole device considerably large. Here,
we present examples of microfluidic devices that do not require pumps; thus, these devices can be easily used outside of
laboratories.
The key point of the device is to use open capillaries whose sections are rectangular (width w and depth d). Such a rectangular
groove at submillimeter scale (x 0.1 mm) is now easy to make outside the clean room, by using a micromilling system.
To use such rectangular open capillary channels for microfluidic devices, it is important to know the dynamics of capillary rise for
the rectangular open capillary. By virtue of argument at the level of scaling laws, quite similar to the one we obtain in Eq. (11), we
can show the following formula for the height z of the column after the time t in the deep channel limit (d [ w) (see Tani et al.,
2015 for the details):
gwt=3hÞ1=2 for t s
zxðe (12)
with

gh= r2 g 2 w3
s ¼ 12e (13)
Here, h is the viscosity of the liquid and ge ¼ gðcosq þ ðcosq 1Þw=2dÞ with g the surface tension of the liquid and q the contact
angle the liquid makes on the surface of channel walls.
The scaling law is shown to be robust through agreement between theory and experiment. Although the formula is theoret-
ically valid in the limits d [ w and t s, the formula is shown to be practically correct as long as d > w and t < s, as demonstrated
later.
(B)
(A) 103
d w ν
20 0.2 0.225 500
2
10 0.4 0.230 100
0.4 0.222 500
15 1.0 0.220 50
d w ν 101
zLC [mm]
1.0 0.220 100
zLC / zf
0.2 0.225 500 1.0 0.221 500
10 0.4 0.230 100 Theory
0.4 0.222 500 100
1.0 0.220 50
5 1.0 0.220 100
1.0 0.221 500 10−1
0
0 5 10 15 20 25
t [100s] 10−2 −3
10 10−2 10−1 100 101 102
t/τ
Fig. 5 Data collapse showing the agreement between theory and experiment. (A) z vs. t. (B) z/zf vs. t/s. The parameters d and w are given in mm,
and the kinematic viscosity n ¼ h/r is given in cS. From Tani, M., Kawano, R., Kamiya, K. and Okumura, K. (2015). Towards combinatorial mixing
devices without any pumps by open-capillary channels: Fundamentals and applications. Scientific Reports 5, 10263.
The agreement was shown clearly via “data collapse.” Eq. (12) can be recast into the following dimensionless expression:
z=zf xðt=sÞ1=2 (14)
with
2eg
zf ¼ (15)
rgw
The original relation given in Eq. (12) shows that, when z is plotted as a function of t, plot curves obtained for different param-
eters should look different. However, according to Eq. (14), when y h z/zf is plotted as a function of x h t/s by calculating the
renormalization factors zf and s by using corresponding parameters, all the plot should look the same and represented by
y ¼ kx1/2. This is clearly demonstrated in Fig. 5.
The data collapse shown in Fig. 5 gives the value of numerical coefficient k, the only quantity that we do not derive theoretically.
In addition, as mentioned earlier, the collapse shows that Eq. (12) is valid in a robust way: the data collapse well, as long as d > w
and t < s while, theoretically, Eq. (12) becomes exact in the limit d [ w and t s.
Practically, the scaling law can be used as a guiding principle for designing complex open capillary devices. The robustness is
practically important because deep channels are difficult to make (because micromilling tips are easily broken): availability of
a simple law for easy-to-fabricate nondeep channels should be a great advantage.
Potential weakness of open capillary system for contamination and evaporation effects was examined in Tani et al. (2015). It was
shown that open capillary devices are practically useful, by means of two examples.
The first example is shown in Fig. 6. This device has four mixing spots, in which we can put four different liquids. When we put,
in the circular spot in the left, liquid M to be reacted with the four liquids, by virtue of capillary force, liquid M is transported and
arrived at the four spots simultaneously, which is quantified by the plot in Fig. 6B.
The second example is shown in Fig. 7. This device mixes two liquids placed at two spots that are transported to the central spot
(shown in Fig. 7A) in order to initiate the mixing. As indicated in Fig. 7B, this device can estimate the reaction time without the
influence of evaporation and contamination.
Kirigami Approach to Control Elastic Modulus

The principle of kirigami is making many cuts on a sheet and stretching it to have a three-dimensional structure. The Japanese word
“kiri” stands for cut, and “gami” stands for paper. Recently, many engineering applications of kirigami have been demonstrated,
such as foldable actuators (Hawkes et al., 2010), self-folding shape-memory composites (Felton et al., 2014), stretchable
lithium-ion batteries (Song et al., 2015), stretchable electrodes (Shyu et al., 2015), stretchable graphenes (Blees et al., 2015; Qi
et al., 2014), and solar tracking batteries (Lamoureux et al., 2015). One of the features of kirigami is the high stretchability.
Here, we discuss the physics of the high stretchability of kirigami at the level of scaling laws (Isobe and Okumura, 2016).
We consider a simple example of kirigami illustrated in Fig. 8A, in which the parameters N , d , w, and h are defined. The force is
plotted as a function of extension in Fig. 8B, which demonstrates clearly a first linear elastic regime terminated by a sharp transition.
As illustrated in the photographs shown below the plot (Fig. 8B), this sharp transition corresponds to the transition from the in-
plane deformation to the out-of-plane deformation. We can estimate the corresponding energies as a function of the extension D at
(A) (B)
1.1
10.00s
1
Normalized Mean Brightness

0.9
0.8
0.7
0.6 S1
0.5 S2
S3
0.4 S4
5 mm
0.3
0 2 4 6 8 10 12
t [s]
Fig. 6 Mixing of liquids with four different acidities with BTB solution. (A) Result of the reaction. (B) Brightness analysis. S1–S4 stands for the four
rectangular spots of the device shown in (A). From Tani, M., Kawano, R., Kamiya, K. and Okumura, K. (2015). Towards combinatorial mixing devices
without any pumps by open-capillary channels: Fundamentals and applications. Scientific Reports 5, 10263.
(A) (B)
30
Mean Brightness
20
40min
10
1
2
3
0
0 10 20 30 40 50 60
t [min]
Fig. 7 Expression of GFP by an open capillary device. (A) Result of the expression. (B) Brightness analysis. From Tani, M., Kawano, R., Kamiya, K.
and Okumura, K. (2015). Towards combinatorial mixing devices without any pumps by open-capillary channels: Fundamentals and applications.
Scientific Reports 5, 10263.
the level of scaling laws in the limit w [ d by arguments similar to the one we employed for the derivation of Eq. (9) (see Isobe and
Okumura, 2016 for the details). As a result, we obtain the stiffness in the first in-plane regime K1:
2NK1 xEd3 h=w3 (16)
with Young’s modulus E and the critical extension at the transition point Dc:
Dc =ð2NÞxh2 =d (17)
These scaling laws are shown to be correct via “data collapse.” For example, Eq. (16) can be recast into the following dimension-
less expression
K1 =ðEhÞxðd=wÞ3 (18)
for a fixed N. The original relation given in Eq. (16) shows that, when K1 is plotted as a function of h, plot curves obtained for
different parameters should look different. However, according to Eq. (18), when y h K1/(Eh) is plotted as a function of x h d/w, all
the plot curves should look the same and represented by y¼ kx3, with k given by the agreement between theory and experiment. This
is clearly demonstrated in Fig. 9. Similar data collapse is obtained for Eq. (17).
The scaling laws given in Eqs. (16) and (17) clarify the physical mechanism of the high stretchability of kirigami. In addi-
tion, the laws established with numerical coefficients are useful for tuning the elastic modulus of sheet materials in the in-plane
regime by adjusting the parameters: we can change the modulus of various sheet materials in a broad range by the kirigami
structure without losing flatness (planeness). One interesting application would be the application of the kirigami structure
for cell sheets.
(A) w + 2d (B) 3
1
w 2.5 0.8 Initial
Regime
d 0.6
F [N]
2 0.4
0.2 Transition
F [N]
1.5 0
0 0.5 1 1.5 2 2.5 3
Δ [mm]
1
2Nd 0.5
0
0 20 40 60 80 100 120 140
d
Δ [mm]
Initial Regime Second Regime Final Regime
2 mm
Fig. 8 (A) Simple model kirigami structure. (B) Force vs. extension. From Isobe, M. and Okumura, K. (2016). Initial rigid response and softening
transition of highly stretchable kirigami sheet materials. Scientific Reports 6.
(A) (B)
2
(d,w) (d,w)
(1,15) 0.1 (1,15)
(1.5,15) (1.5,15)
1.5 (2,15) (2,15)
(3,15) (3,15)
K1 [N/mm]
(3.5,15) 0.01 (3.5,15)

K1/Eh
1 (2,10) (2,10)
(2,20) (2,20)
(2,30) (2,30)
slope 3
0.5 0.001
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.01 0.1 1
h [mm] d/w
Fig. 9 Data collapse showing the agreement between theory and experiment for N ¼ 10. (A) K1 versus h. (B) K1/(Eh) versus d/w. The parameters
d and w are given in mm. From Isobe, M. and Okumura, K. (2016). Initial rigid response and softening transition of highly stretchable kirigami sheet
materials. Scientific Reports 6.
Conclusion
As seen in the classic examples, we see that some well-known expressions can be derived by a simple dimensional analysis if we give
up deriving numerical coefficients. Note that, when developing arguments at the level of scaling laws, differentiation and integration
can be replaced by division and multiplication, respectively. This approach at the level of scaling laws is useful in practical problems.
Although such an approach is theoretically valid only in certain limits, scaling laws can be clearly checked by data collapse, and in
some cases, they are robust, that is, valid for a wide range of parameters (Yokota and Okumura, 2011; Okumura, 2015); from the
data collapse, we can experimentally derive numerical coefficients, which we do not obtain from theoretical analysis. It is stressed
that finding scaling laws is not necessarily easy in general because scaling laws are valid in certain limits and we do not know a priori
within which limits the scaling law in question becomes correct and we need to do experiments changing experimental parameters
in wide ranges to find the answer. However, once a scaling law is established, the physics is clarified, and the law is useful as a guiding
principle for practical applications because, without trials and errors, we can tune parameters for desired purposes. This approach is
very popular especially in the field of soft matter physics, and monographs based on this approach are available for polymer physics
(de Gennes, 1979) and capillary phenomena (de Gennes et al., 2005) (see also minireview articles on strength and toughness of
biological composites (Okumura, 2015) and some other phenomena (Okumura, 2016, 2017)).
Acknowledgments
This work was partly supported by Grants-in-Aid for Scientific Research (A) (No. 2014-PM01-02-01) of JSPS, Japan, and by ImPACT Program of
Council for Science, Technology and Innovation (Cabinet Office, Government of Japan).
References
Beebe, D. J., Mensing, G. A., & Walker, G. M. (2002). Physics and applications of microfluidics in biology. Annual Review of Biomedical Engineering, 4, 261–286.
Blees, M. K., et al. (2015). Graphene kirigami. Nature, 524, 204–207.
de Gennes, P. G. (1979). Scaling concepts in polymer physics. Ithaca, NY: Cornell University Press.
de Gennes, P.-G., Brochard-Wyart, F., & Quéré, D. (2013). Capillarity and wetting phenomena: Drops, bubbles, pearls, waves. New York: Springer Science & Business Media.
de Gennes, P.-G., Brochard-Wyart, F., & Quéré, D. (2005). Gouttes, Bulles, Perles et Ondes (2nd edn.). Paris: Belin.
Felton, S., Tolley, M., Demaine, E., Rus, D., & Wood, R. (2014). A method for building self-folding machines. Science, 345, 644–646.
Hawkes, E., et al. (2010). Programmable matter by folding. Proceedings of the National Academy of Sciences of the United States of America, 107(12441–12445).
Isobe, M., & Okumura, K. (2016). Initial rigid response and softening transition of highly stretchable kirigami sheet materials. Scientific Reports, 6, 24758. https://doi.org/10.1038/
srep24758.
Lamoureux, A., Lee, K., Shlian, M., Forrest, S. R., & Shtein, M. (2015). Dynamic kirigami structures for integrated solar tracking. Nature Communications, 6, 8092. https://doi.org/
10.1038/ncomms9092.
Landau, L., & Lifshitz, E. (1975). Elasticity theory. New York: Pergamon Press.
Landau, L., & Lifshitz, E. (1987). Fluid mechanics (2nd edn.). Oxford: Pergamon.
Okumura, K. (2015). Strength and toughness of biocomposites consisting of soft and hard elements: A few fundamental models. MRS Bulletin, 40, 333–339.
Okumura, K. (2016). Simple views on different problems in physics: From drag friction to tough biological materials. Philosophical Magazine, 96, 828–841.
Okumura, K. (2017). Viscous dynamics of drops and bubbles in Hele-Shaw cells: Drainage, drag friction, coalescence, and bursting. Advances in Colloid and Interface Science, 255,
64–75. https://doi.org/10.1016/j.cis.2017.07.021.
Qi, Z., Campbell, D. K., & Park, H. S. (2014). Atomistic simulations of tension-induced large deformation and stretchability in graphene kirigami. Physical Review B, 90, 245437.
Shyu, T. C., et al. (2015). A kirigami approach to engineering elasticity in nanocomposites through patterned defects. Nature Materials, 14, 785–789.
Song, Z., et al. (2015). Kirigami-based stretchable lithium-ion batteries. Scientific Reports, 5, 10988.
Squires, T. M., & Quake, S. R. (2005). Microfluidics: Fluid physics at the nanoliter scale. Reviews of Modern Physics, 77, 977.
Tani, M., Kawano, R., Kamiya, K., & Okumura, K. (2015). Towards combinatorial mixing devices without any pumps by open-capillary channels: Fundamentals and applications.
Scientific Reports, 5, 10263.
Yokota, M., & Okumura, K. (2011). Dimensional crossover in the coalescence dynamics of viscous drops confined in between two plates. Proceedings of the National Academy of
Sciences of the United States of America, 108, 6395–6398.
CT-Based Bone and Muscle Assessment in Normal and Pathological Conditions
Paolo Gargiulo, Magnus K Gislason, Kyle J Edmunds, and Jonathan Pitocchi, Reykjavik University, Reykjavik, Iceland
Ugo Carraro, IRCCS Fondazione Ospedale San Camillo Venezia-Lido, Venezia, Italy
Luca Esposito, Massimiliano Fraldi, Paolo Bifulco, and Mario Cesarelli, University of Naples Federico II, Naples, Italy
Halldór Jónsson, University of Iceland, Reykjavík, Iceland; and Landspitali University Hospital, Reykjavík, Iceland
Introduction 119
Muscle Research and Medical Imaging: A Focus on Muscle Changes and Degeneration 119
Modeling Material Properties of the Bone 120
Quantitative Muscle Assessment 121
Nonlinear Trimodal Regression Analysis 121
Applications 123
Healthy, elderly, and pathological HU distribution parameters 123
Muscle assessment in patients undergoing total hip arthroplasty 125
Three-Dimensional approach: Virtual histology 126
Quantitative Bone Assessment in THA 127
Bone Profiling and Gain and Loss for 2-D-Based Assessment 127
Applications 127
Bone gain and loss for 3-D-based assessment 127
Limitations 130
CT-Based Finite-Element Modeling 131
Loads and Constrains 131
Aseptic Mobilization: Stress Shielding 132
Topology Optimization 132
Application: Preoperative Assessment 132
Conclusion 132
References 133
Further Reading 133
Introduction
Muscle Research and Medical Imaging: A Focus on Muscle Changes and Degeneration
Muscle degeneration, characterized by the progressive loss of function, strength, and mass of the muscle, coupled with substitution
of healthy muscle fibers with increased content of fibrous collagen and fat, has been consistently implicated as an independent
mortality risk factor in aging individuals and those who suffer from neuromuscular primary pathologies and injuries (Goodpaster
et al., 2006). When associated with aging, muscle changes are defined as sarcopenia, and its prevalence has been observed to incur
significant declines in overall physical activity and quality of life. However, despite its growing interest in clinical aging research,
a precise, sensitive, and quantitative method for defining etiology, diagnosis, and managements, in particular those related to
prevention and rehabilitation, remains debated.
Despite the absence of a universal definition, extant literature commonly associates sarcopenia with the loss of both skeletal
muscle function and structure, and many mechanisms that govern these changes have been implicated (Fielding et al., 2011).
The most prevalent of these mechanisms identifies the loss of muscle mass with infiltration of noncontractile tissues, which in
turn confers an increased risk for frailty, disability, reduced mobility, hospitalization, and finally mortality. In an increasingly aging
world, identifying a normative clinical definition for sarcopenia is of considerable importance.
Many of the mechanisms that elicit muscle degeneration in sarcopenia have been analogously identified within the context of
neuromuscular injury or pathologydmost notably with regard to spinal cord injuries (SCI). The dramatic deleterious changes in
muscle function and composition exhibited in SCI patients have been suggested as accelerated analogues to the changes evident
in sarcopenia, as paralysis from lower motor neuron denervation drastically and immediately reduces skeletal muscle function
and then mass, whose extent is worsened by the associated increases of muscle adiposity and fibrosis. Severe skeletal muscle degen-
eration due to other metabolic and oncological illnesses, known as cachexia, has been likewise associated with muscle fiber loss and
an increase in relative muscle adiposity and fibrosis, which are likewise been correlated to increased rates of cachectic patient
morbidity and mortality. In general, the increasing prevalence of sarcopenic and cachectic muscle degeneration necessitates
a gold standard for a quantitative assessment of muscle quality (Carraro et al., 2016).

120 Biomechanics j CT-Based Bone and Muscle Assessment in Normal and Pathological Conditions
Fig. 1 Coronal view from a lower limb section underlining. Fat tissues 200 and 10 HU (A). Water, muscle, and connective tissues 9 and 200
HU (B). Bone cancellous 201 and 500 HU (C). Cortical bone 501 and 2000 HU (D). Hounsfield distributions associated to soft tissues (E) and the
bone (F).
This article will focus on quantified analysis of X-ray computed tomography (CT) imagesda modality whose image matrices
consist of linear attenuation values that are calculated from the specific X-ray absorption characteristics of present tissue. These
linear attenuation coefficients may then be linearly transformed into a scale known as the Hounsfield unit (HU) scale.
Fig. 1 shows how different tissues displayed with CT images are associated to their corresponding HU distribution. In detail, we
have Fig. 1A–D showing a lower limb section, coronal view, where, respectively, fat tissues, muscle and connective tissues, trabec-
ular bone, and cortical bone have been underlined with different colors. Fig. 1E and F shows their correspondent HU distributions.
The number of pixels within the volume of interest is higher for muscle tissues compared to fat (Fig. 1E) and for trabecular bone
compared to cortical bone (Fig. 1F).
Therefore, when considering CT images of soft tissue, HU distributions typically range from negative values around 200 HU
for fat, up to 200 HU including muscle tissues and connective tissue. Further sections of this article will illustrate a method for quan-
tifying these HU distributions.
Modeling Material Properties of the Bone

In order to derive the mechanical properties of the bone from the CT scan data, CT numbers or HU is first converted into bone
densities, and then, the bone material propertiesdin terms of Young’s modulidare estimated from these data. The relationship
between bone density and CT numbers has been assessed to be linear by scanning a phantom, with known material densities,
together with the patient bones. The relationship between bone density and bone elasticity has been deeply discussed. In a recent
Biomechanics j CT-Based Bone and Muscle Assessment in Normal and Pathological Conditions 121
literature review, Helgason et al. (2008) selected a total of 18 studies and 22 different density–stiffness relationships that have been
derived from these experimental tests. Results appear largely dispersed, and the considerable differences between the investigated
studies are related to the complexity of comparison of the empirical data.
Different normalization criteriadin terms of apparent density and strain ratedwere used to decrease interstudy differences, but
the testing, the measurement procedures and instrumentation, the specimen geometry, the holders, the boundary conditions, and
the anatomical site of the samples still play an important role. Morgan et al. (2003) investigated the bone site specificity. To this
purpose, they tested 142 specimens from 61 cadavers measuring on-axis elastic moduli and apparent densities from different
anatomical sites with an experimental protocol that minimized the end artifacts and specimen misalignments. The observed site
specificity is attributed to intersite variations in trabecular architecture, and when the site-specific structure was taken into account,
there were no differences in terms of tissue moduli.
Also, the relationship between bone density and strength of trabecular bone has been examined. Volume fraction and bone
architecturedwhich in turn hugely depend on anatomical site, age, sex, and various pathologiesdaffect the strength of the trabec-
ular bone causing a remarkable discrepancy in the value of the failure stresses. In fact, probing the effects of aging, McCalden et al.
(1997), testing 255 specimens of cancellous bone from 44 human femora, highlighted the decrement in bone volume fraction of
8.5% per decade and the consequences on the mechanical competence of the bone due to quantitative changes rather than qual-
itative changes; moreover, the ultimate stress is reduced by almost 7% per decade for the human proximal femur and by almost 11%
per decade for vertebral bone. Zysset et al. (1991) in a pioneer study reviewed the several efforts oriented to relate measures in the
bone with density and strength also using a power-law function, even if in a static regime. Moreover, it is not currently known if this
relationship depends on anatomical site.
The relationship between bone density and CT numbers can be described by the following equation:
rapp ðPÞ ¼ 0:000412HU þ 1:018668
where P denotes the generic point inside the femur. Moreover, the value of ash density, rash(P), is related to apparent density,
rapp(P), by means of the following equation:

0:522 rapp ðPÞ þ 0:007 if rapp < 1
rash ðPÞ ¼
0:779 rapp ðPÞ 0:025 if rapp 1
As a consequence, nonhomogeneous and isotropic behavior of bone femur models can be assumed, and at each point P, the
following correlation between Young’s Modulus, E, and the local density, rash ¼ rash(P), can be chosen:
8
< 33900rash ðPÞ2:2 if rash 0:27
EðPÞ ¼ 5307rash ðPÞ þ 469 if 0:27 < rash < 0:6
:
10200rash ðPÞ2:01 if rash 0:6
where E represents the actual local stiffness (MPa) at the generic point P.
The choice of a very fine mesh in the FE model ensures that structural gradients over the representative volume element result in
very small, avoiding conflicts in terms of the relation between structural gradients and material elastic symmetries.
Postelastic behavior or bone tissue can be taken into account. Since values of the ultimate tensile stress of trabecular bone equate
about 0.79 of the compressive yield stress and values of the ultimate tensile stress of cortical bone are about 0.76 of compressive
yield stress, symmetrical bilinear isotropic hardening material models can be adopted, neglecting the tiny difference. The yield stress
defined as that corresponding to the mean value of yield strain can be set to 0.7, while the tangent modulus can be set to five
hundredths of the corresponding elastic modulus, depending on local density. Poisson’s ratio of the bone is typically set to 0.4.
Quantitative Muscle Assessment
As previously mentioned, this article highlights the use of entire HU distributions to assess muscle quantity and quality. In this
section, we will analyze soft tissue (fat, water and loose, and connective and healthy muscle) considering the information within
the HU interval from 200 to 200.
We will begin here by highlighting a method that allows quantifying Hounsfield distribution based on nonlinear trimodal
regression analysis. Follows three examples from individuals representing whole spectrum of different soft tissue distributions:
healthy control, elderly subject, and subject with degenerative muscle pathology. Finally, leading from the profiling, a full-
dimensional approach is introduced to underline the utility of virtual histology.
Nonlinear Trimodal Regression Analysis

Based on what we can see in Fig. 1E, we may define any distribution of soft tissue as based on standard Gaussian distributions: that
is to say that the distribution of HU values correlating to that tissue varies according to a normal probability density function
described by the following standard equation:
N ðxmÞ2
4ðx; m; s; aÞ ¼ pffiffiffiffiffiffi e 2s2 (1)
s 2p
where 4 represents the probability density function of the tissue type, N is the Gaussian distribution’s relative amplitude, m is the
location of the distribution’s mean, and s is the width of the distributiondall of which may be evaluated as a function of each pixel
or voxel’s computed HU value (variable x).
Next, we may observe the aforementioned notion that segmented soft tissue in CT images generates HU distributions with three
distinct tissue domains: fat, water-equivalent and loose connective tissue, and muscle and fibrous connective tissue. These three
tissue types have linear attenuation coefficients that occupy distinct HU domains, namely, from 200 to 10 HU, from 9 to
40 HU, and from 41 to 200 HU, respectively (Edmunds et al., 2016). Indeed, if we take a CT image cross section and isolate all
pixel values from 200 to 200 HU (the following linear transformation from CT bin number), the following characteristic curve
is generated:
It is clear from the shape of the earlier distribution that optimum regression analysis will require the definition of a generalized
soft tissue distribution function that contains each of the three aforementioned tissue types (Fig. 2). Writing this in simple summa-
tion notation, we now have the following:
X3 Ni
2
ðxmi Þ
4ðx; mi ; si ; ai Þ ¼ pffiffiffiffiffiffi e 2si 2 (2)
i¼1 si 2p
Finally, one may additionally observe the inwardly sloping asymmetries within the fat and muscle peaks. To optimize our
regression analysis, it is crucial to account for this asymmetrydto do this, we employ the use of the Gaussian skewness
parameter:
X3 ðxm Þ2
Ni 2s i2 ai ðx mi Þ
4ðx; mi ; si ; ai Þ ¼ pffiffiffiffiffiffi e i erfc pffiffiffi (3)
i¼1 si 2p si 2
where erfc is the error function and a is the distribution skewness. Note that, for the purposes of our definition, the skewness for the
central peak (water-equivalent and loose connective tissue) may be assumed to be zero, thereby effectively removing its error
function component.
Utilizing this definition, we can now generate a theoretical curve via precise regression analysis. To do this, we begin by employ-
ing an iterative generalized reduced gradient algorithm via the minimization of the sum of standard errors at each CT bin value, x.
After running hundreds of iterations, the standard error at each point will be gradually and progressively reduced. Finally, the sum of
all standard errors may be utilized to compute the overall R2 value for the theoretical curve’s fit to the image datada procedure that
utilizes the following set of equations:
Fig. 2 Diagram depicting the three components of the trimodal radio-densitometry distribution utilized in this study. This figure illustrates the loca-
tion and skewness of each probability density function, with tissue types as previously defined.
RSS 2 RSS
R2 ¼ 1 R ¼1 (4)
SST SST
Xn
SST ¼ i¼1
ðyi yÞ2 (5)
Xn
RSS ¼ i¼1
ðyi f ðxi ÞÞ2 (6)
In summary a CT-scanned muscle volume generates 11-parameters that identify the muscle in unique way N, m, s, and a for fat,
water-equivalent connective tissue, and muscle tissue. These distribution parameters may then be exported for further analyses and
comparison.
Applications
Healthy, elderly, and pathological HU distribution parameters
To ascertain potential differences in muscle degeneration pathways, as evidenced by subtle changes in HU distributions, the afore-
mentioned regression analysis method has been utilized on the CT scans of the three previously mentioned subjects: healthy
control, elderly, and pathological. Results from the HU distribution profiling for each of these subjects are depicted in Fig. 3.
As is evident from the results displayed in Fig. 3, there are significant qualitative differences between the shapes of the HU distri-
butions of the healthy, elderly, and pathological subjects. The curve of the healthy subject exhibits a definitively high-amplitude
muscle peak and a comparatively blunted fat peak, whereas the fat and muscle components in the elderly subject’s curve are decid-
edly the opposite in appearance. Contrastingly, the pathological subject elicited a distribution with heavily skewed fat and muscle
peaks that were likewise closer together and shifted toward negative HU values.
When compared according to the typical metric of average HU value, it is evident that the healthy subject’s average HU value was
significantly shifted toward the muscle peak in the distribution. However, the average HU values of the elderly and pathological
subjects were nearly indistinguishable from one another. To better explore the clearly obtuse differences in their distributions,
each of the 11 regression analysis parameters was compiled and compared. The qualitatively distinguishable differences between
HU distributions are further exemplified by these results in Fig. 4.
Fig. 3 Radio-densitometry distributions showing their respective nonlinear regression curves and average HU values. (A) The control subject’s
curve showed a large muscle peak at around 55 HU, which directly contrasted with (B) the elderly subject’s distribution. (C) However, the patholog-
ical subject’s distribution was much lower in total pixel count (due to lower overall mass within the leg volume) and elicits fat and muscle peaks that
are similar in amplitude with a large connective tissue regime between them. Likewise, the control subject’s average HU value was remarkably higher
than the other subjects, whose values were similarly negative. All regression analyses yielded R2 values above 0.99 (Edmunds et al. 2016).
Fig. 4 Results from the nonlinear trimodal regression analyses of each HU distribution. (A) The amplitude parameter, N; (B) the location parameter,
m; (C) the width parameter, s; and (D) the skewness parameter, a. Note that the skewness for the connective tissue peak was assumed to be zero in
accordance with our hypothesis of its being a normal distribution (Edmunds et al., 2016).
As is evident in Fig. 4, each distribution parameter confers its own distinct differences and relationships between the three
subjects. For example, the elderly subject’s fat amplitude is at least fourfold larger than those of the other subjects but for the control
subject’s muscle amplitude is largest by at least twofold. However, the pathological subject has the highest connective tissue
amplitudeda parameter that, in general, increases nearly linearly between subjects, from the lowest value in the control subject.
A potential physiological explanation for this variation in tissue amplitudes could reside with their precise definitionsdmost
importantly, for the connective tissue distribution. This central tissue regime accounts for any water-equivalent and loose fibrous
tissues, which, in addition to being generally present within healthy leg volumes, are increasingly evidenced in degraded, unhealthy
muscle with significant fatty infiltration.
An analogous linearity is present when observing fat tissue skewness, which is likewise lowest in the control subject and highest
in the pathological subject. These data suggest that the fat tissue variance in positive distribution asymmetry may likewise be due to
the progressive fatty infiltration into the much higher HU value muscle tissue. However, since this relationship is not commensurate
in the muscle peak’s negative skewness, it is clear that skewness alone may not adequately describe present physiology.
Moving to the location parameter, it is evident that fat location values are almost identical between subjects but muscle peak
locations are singularly high in the elderly subject; these data associated to low amplitude in the muscle suggest muscle degener-
ation that have as results that the only remaining tissue within the muscle volume is a fibrous connective tissue. Similarly, the
control subject has a singularly high connective tissue location value, although the difference is less extreme. While less significant,
perhaps, it remains evident that the central connective tissue distribution shifts toward lower, “fatter” HU values in these elderly and
pathological subjects, which is again evidence of muscle degeneration.
Finally, the width parameter exhibits noticeable differences between subjects. The control subject has the widest fat distribution,
but their muscle width is at least half of the other subjects’ values. Furthermore, while the elderly and pathological subjects exhibit
similar fat and muscle widths, the elderly subject exhibits a much wider connective tissue distribution. A precise physiological inter-
pretation of width as a parameter is difficult to obtain, but it is clear that sharper muscle and fat peaks confer to a more homogenous
distribution of tissue HU values, suggesting that a reduction in the width parameter would confer to a commensurate reduction in
muscle degeneration.
Muscle assessment in patients undergoing total hip arthroplasty

The potential utility of the regression analysis method previously detailed may be further tested with a cohort of total hip arthro-
plasty (THA) patients to assess changes in their upper leg muscle following hip replacement surgery. To do this, one may observe
HU distributions from these patients from both presurgical and 1-year postoperative CT scans, followed by the assembly of distri-
bution parameters for both their healthy and operative legs. Results from these analyses are depicted in Fig. 5.
As is evident in Fig. 5, the results from the THA cohort analyses further support many of the aforementioned relationships
between distribution parameters and the degree of muscle degeneration. To clarify this interpretation, it is useful to operate under
the physiological assumption that these patients’ operative legs would be naturally underutilized compared with their healthy legs.
Firstly, in general, all patients’ fat amplitudes are shown to decrease, while muscle amplitudes increase 1 year following surgery.
Additionally, operative leg muscle amplitudes elicit a significant increase after this timespan. Furthermore, regarding the location
parameter, there are minimal shifts evident in fat and muscle peaks, but there are notable increases in connective tissue location
values in both the healthy and operative legs 1 year after surgery. This shift toward healthy muscle suggests the overall efficacy
of corrected ambulation and 1 year of normative use. Indeed, once again, this is most evident and singularly significant in the oper-
ative leg.
Fig. 5 Results from the nonlinear trimodal regression analyses of the n ¼ 15 THA cohort. (A) The amplitude parameter, N; (B) the location param-
eter, m; (C) the width parameter, s; and (D) the skewness parameter, a. Note that $ and * denote P < 0.05 and the results are presented for before
(b) and 1-year after (a) surgery (Edmunds et al., 2016).
The width parameter elicits no significant or meaningful changes in either leg, but in accordance with our previous observation,
the connective tissue peak widths are significantly larger than muscle or fat, which are both nearly identical in both legs. While not
immediately useful in a physiological sense, this is further evidence that each distribution parameter seems to have its own sensi-
tivity with respect to the entire population.
Finally, Fig. 5 shows that the skewness parameter decreases in magnitude in both the healthy and operative fat peaks but remains
relatively diminished and constant in the muscle peak, with one important exception: the preoperative muscle peak has significantly
higher skewness than each of the others. We previously saw that the pathological example subject had a much more negative fat
skewness than the other two example subjects. This suggested the presence of fatty infiltration and/or buildup of intramuscular
fat in their degenerating muscle. This notion is similarly evident here, as the operative legs of the THA cohort elicit significantly
more extreme negative skewness’s compared with other fat peak values.
Altogether, the results from utilizing the reported regression analysis method indicate significant improvement in muscle quality
in both legs following THA surgery. These data further support the notion that each HU distribution parameter may have particular
specificities regarding muscle assessment, which further supports the method’s utility as a robust and straightforward indicator for
muscle degeneration.
Three-Dimensional approach: Virtual histology

Carrying out such analysis on volumetric data can yield a three-dimensional representation of the overall state of an anatomically
defined full muscle (Gargiulo et al., 2010; Carraro et al., 2015). Fig. 7 shows the three-dimensional modeling of the tibialis anterior
muscle for three different subjects: a healthy control subject, a healthy elderly subject, and an SCI patient with different degrees of
muscle changes in the legs. Accordingly, the figures show that the relative content of the three main components of a human muscle
varies to a great extent. Indeed, the red color represents healthy muscle tissue, the blue color represents loose connective tissue (with
tissue density very near to water), and the yellow color represents fat. Interpretation of the images has to take into account that the
longitudinal three-dimensional reconstruction of the full tibialis anterior in the three subjects is based on the cortical layer of the
muscle in which the healthy muscle fibers of the full muscle are separated by loose connective tissue from the adjacent muscles.
Indeed, even in a normal healthy person, the muscle shows large amounts of nonmuscle tissues. The true discriminative analysis
is the one in which the percentage content in the 3-D reconstruction of the muscle are presented. In the case of a normal healthy
person (left panels), the healthy muscle is of about 80%, the loose connective is around 20%, and fat is around 1%. In the elderly
subject (central panels), the three values are near 50%, 45%, and 5%. Interestingly, the values in the SCI person (pathological
subject) identify the unilateral lesion present in that case. It is worth stressing that similar percentage changes were observed in
the content of different tissues when analyzed by morphometry of muscle biopsies harvested from the SCI patient (Fig. 6)
(Kern et al., 2010).
Fig. 6 Segmented soft tissues and compositions within the tibialis anterior are from each subject’s 3-D upper leg volumes. Three tissue types of
distinct radiodensitometric domains were utilized for the purposes of this study as follows: from 200 to 10 HU, from 9 to 40 HU, and from 41
to 200 HU representing, respectively, fat (yellow), loose connective tissue and atrophic muscle (cyan), and normal muscle (red). (A) The control
subject’s muscle composition is composed primarily of healthy muscle tissue, whereas (B) the elderly subject exhibited markedly more fat and
connective tissue, to the detriment of healthy muscle. (C) However, the pathological subject, whose left leg was affected by the sciatic nerve denerva-
tion, exhibited an almost identical healthy leg composition compared with the elderly subject but almost entirely fat and connective tissue in the path-
ological leg. It should be noted that, for the purposes of comparing pathological muscle degeneration with sarcopenic degeneration, only the
radiodensitometric distributions from subjects’ left legs were utilized in this study (Edmunds et al., 2016).
Quantitative Bone Assessment in THA
The bone quality and the bone’s mechanical strength play a pivotal role in the success of THA. Many studies have looked at how the
bone is likely to respond to external forces before and after THA, based on mechanical testing or computational studies. Clinical
assessment of bone material distribution is an important factor in determining the treatment for patients or monitoring the changes
in bone quality over a period of time. For THA, it is important to have information about the mechanical strength of the bone, prior
to surgery and after surgery to monitor the possible aseptic loosening of the implant. Once a metal implant has been placed into the
femoral canal or the acetabular cup, the stress state of the surrounding tissue will change as mechanical forces are proportionally
distributed to the stiffness of the material. Since the stiffness of the prosthesis is in general an order of magnitude higher than for the
bone tissue, the prosthesis becomes the main structural element, therefore reducing the load onto the surrounding bone tissue. The
phenomenon is called stress shielding. This results in that bone tissue around the prosthesis becoming less dense through the
remodeling process governed by Wolff’s law. The loss of density of the bone can have serious implications with regard to the fixation
of the implant that can become loose. The loosening of the prosthesis is a common reason for revision surgery. It is inevitable that
the bone will undergo changes in morphology and strength following a THA procedure. It is however important to be able to
monitor where the changes occur to determine where the bone density decreases (loss) and where it increases (gain). Using CT scans
that are taken 24 h and 1 year postoperatively, to create a three-dimensional model of the femur, can determine where the bone loss
and gain have been occurring, respectively.
Bone Profiling and Gain and Loss for 2-D-Based Assessment

Using the same methodology as described earlier and in Eqs. (1) and (2), it is possible to quantify the bone material distribution
either at given cross sections (2-D) or over the whole bone (3-D). Like muscles, the bone will show one or two peaks at the density to
which most pixels belong. For slices around the epiphyses of the long bones such as at the femur and the tibia, a cancellous and
a cortical peak can be seen, but for slices around, the diaphysis will just show a single cortical peak since no cancellous bone is present
at that location. Fig. 7 shows the different cross sections analyzed and the resulting bone density profiles from each cross section.
From Fig. 7, it can be seen how the different profiles correspond to different cross sections around the femur. Slices A and B show
a peak toward the low-density spectrum representing the cancellous bone at those locations, whereas slices C–E show the peak at the
higher-density regions representing the cortical bone. The information obtained from the analysis of the peak will give information
about the material characteristics such as the volume of the cancellous or the cortical bone, the stiffness, and the mass.
Analysis of individual slices using image registration and processing could be beneficial for the clinical environment, both in
terms of reducing radiation doses to the patients and the possibility of automatizing the bone segmentation procedure. The two
scans from the 24 h and 1 year are superimposed using bony landmarks of the femur. The two slices from the same cross section
are then compared, and if the difference in pixel values exceeds a given threshold (111 HU), it is determined that there has become
a change in the bone material. If the difference is positive, there is a gain, and if it is negative, there is a loss. Fig. 8 shows how the
formation and erosions of bone can be seen in a single slice around the lesser trochanter. Such analysis can give clinicians infor-
mation both about the mechanical strength based on the bone mineral density distribution (Pétursson et al., 2015) of the bone
at selected regions and information about the spatial progression of the bone remodeling process over a given time period to be
able to assess how the bone is adapting to the prosthesis.
Applications
Analysis was carried out on three patients undergoing THA, looking both at the bone profiles and using the Gaussian fit model and
the image registration method. The results from the parameter fitting can be seen in Fig. 9.
Looking at the fitted variable coefficients, it is possible to look at the material characteristics of the bone in each slice. The
percentage difference between the value of the parameters representing the amplitude (changes in the amount of material) and
the location (changes in the density) over a period of 1 year can be seen in Table 1.
From Table 1, it can be seen that there is a tendency in the bone to shift toward the lower-density regions for the cancellous bone
around the greater (subjects 2 and 3) and the lesser trochanter (subject 1). This can also be seen around the cortical region at the
distal aspect of the bone but to a much lesser content.
The results from the parametric fitting can then be compared with the finding using the 2-D image registration to identify the
amount of gain and loss in each slice. The full results of the two-dimensional image analyses can be seen in Table 2. (See Table 3.)
From Table 2, it can be seen that the area around the lesser and the greater trochanter is more susceptible to bone loss over
a period of 1 year, whereas the distal aspect is more likely to show higher levels of bone gain.
Bone gain and loss for 3-D-based assessment

For the 3-D analysis, image registration needs to be carried out to align the two scans so that the femur is in identical position
between the scans. The image registration and realigning were carried out using Mimics (Materialize). The image registration
was carried out to identify the following anatomical landmarks:
• The most distal tip of the stem

• Protuberance under the greater trochanter
Fig. 7 Selected slices for the two-dimensional analysis of the bone profiles.
Fig. 8 Slices at the lesser trochanter at 24 h postoperatively (left), 1 year postoperatively (middle), and the difference between the gain (green) and
loss (red) of bone in the slice over a 1-year period (right).
Fig. 9 Pixel distribution at the cross section and the fitted Gaussian curves through the dataset.
• Superior aspect of the greater trochanter

• Lesser trochanter
• Gluteal tuberosity in the axial view
• Protuberance of the pectineal line in the axial view
The two scans were aligned using a common line defined using the longitudinal axis of the stem of the prosthesis. The two scans
were then resliced according to the new orientation ranging from the proximal end of the femur to 2 cm distally to the distal tip of
the prosthesis. Segmentations of both femurs were carried out incorporating pixel values between 255 and 3070 HU. Using Boolean
operators, the prosthesis is deducted from the model, and finally, adjustments are made in order to remove image artifacts that arise
Table 1 Percentage difference in the values of the fitted parameters describing the amplitude and density values of the bone material within each
slice
Region Subject 1 (%) Subject 2 (%) Subject 3 (%)
Amp Loc Amp Loc Amp Loc

Greater trochanters 38.8 10.3 8.7 32.5 15.0 26.3
Lesser trochanter 11.4 23.7 2.0 8.3 17.1 14.4
5 cm proximal to the distal part of the stem 9.8 5.2 12.0 22.7 5.1 20.9
Distal part of the stem 2.8 1.0 2.6 2.2 43.2 5.4
2 cm distal to the distal part of the stem 5.7 1.2 10.5 0.5 10.1 2.9
A positive value indicates an increase over a period of 1 year, and a negative value indicates a decrease.
Table 2 Two-dimensional analysis of the bone gain and loss of slices at various regions of the femur over a period of 1 year
Subject 1 (%) Subject 2 (%) Subject 3 (%)
Region Loss Gain Loss Gain Loss Gain

Greater trochanters 23.1 2.3 5.6 2.9 9.8 11.2
5 cm proximal to the distal part of the stem 7.0 0.6 12.4 0.1 0.6 14.7
Table 3 Results from the bone gain and loss from three different subjects
Subject 1 (%) Subject 2 (%) Subject 3 (%)
Region Loss Gain Loss Gain Loss Gain

Greater trochanter 1.8 2.7 1.1 1.0 1.6 4.0
5 cm proximal to the distal part of stem 5.2 3.0 8.0 0.7 5.5 1.7
from the presence of the metal implant during the scanning protocol. With the scans now aligned, resampled according to pixel size,
a direct comparison is carried out on a voxel basis. To estimate the gain, the 24 h scan is subtracted from the 1-year scan where
positive pixel values indicate bone gain. The opposite was done to work out the bone loss where the 1-year scan was subtracted
from the 24 h and positive pixel values indicate the extent of the loss. A cohort of patients has been analyzed, and the results
show that the medial proximal area of the femur around the lesser trochanter is most susceptible for bone loss. Some bone gain
can be seen at the various regions of the femur, mostly at the distal part. The results from the gain and loss can be summarized
in Table 1. The table represents the percentage of voxels that have decreased in density (loss) and increased density (gain).
Using three-dimensional representation of the bone gain and loss will give the clinician detailed information about the location
and the extent of the bone loss. Fig. 10 shows the extent of the bone gain and loss in 3-D for three subjects.
From the image, it can be seen that the three-dimensional changes in the bone density vary greatly between the subjects. All show
loss around the lesser trochanter area and some gain toward the distal aspect of the prosthesis. Three-dimensional analysis will give
a detailed representation of the bone gain and loss, but the process is time-consuming, and therefore, two-dimensional analysis is
more appropriate in the clinical practice.
Limitations
For the analysis, there are several limitations with regard to the quantification of the gain and loss. The greatest limitation is that the
scans between 24 h and 1-year postoperative scan can vary in terms of resolution, slice thickness, distance between slices, body
posture of the patient, etc. Additionally, it is possible that the prosthesis has shifted, thus compromising the integrity of the image
registrations based on the position of the prosthesis. The comparison between the voxels is based on reslicing and resampling of the
1-year postoperative scan that can introduce errors to the analysis. Additionally, partial volume effect can play an important role
during the bone segmentation. As can be seen from the results of both the two-dimensional analysis and the three-dimensional
analysis, the results between individuals vary to a great extent. A larger cohort and more vigorous subdivision of groups (fixation,
gender, age, and prosthesis type) is needed to be able to get a more detailed view of the material and morphological changes in the
bone over a period of 1 year.
Fig. 10 Location of the bone gain and loss over a period of 1 year postoperatively for three subjects. The yellow color indicates no changes, red
color indicates bone loss, and green color indicates bone gain.
CT-Based Finite-Element Modeling

CT is a methodology used to acquire volumetric densities of tissues. CT-based finite-element (FE) models have been exploited for
analyzing the micro- and macroscopic mechanical behavior of bone sites and for studying growth, remodeling, and morphogenesis
phenomena. Many research groups have focused their activities on the development of rapid and automated FE preprocessing
(Breusch et al., 2001).
In principle, there are two basic concepts of generating FE models from CT scans: geometry-based and voxel-based. The voxel-
based meshing technique is achieved by matching each CT voxel to a single FE, and the main advantage of this strategy is that it is
a simple and automated technique that can deal with any shape of arbitrary complexity. However, curved surfaces cannot be prop-
erly represented by brick elements. Hence, where voxel meshes can provide accurate internal stresses and strains, the jagged-edged
surface causes peak stresses and strains, which is a disadvantage when accurate mechanical data are needed at surfaces. Moreover,
unstable elements (i.e., elements insufficiently anchored to the whole model and thus potentially involved in partial rigid-body
motion) can be generated, which is a crucial problem in obtaining consistent FE models, hindering mechanical analyses (Lotz
et al., 1990). In the following figure, two FE models are shown: on the left side geometry-based model and on the left side
voxel-based model of prosthesized femur (Fig. 11).
Loads and Constrains
Preclinical endurance tests on hip implants require defining realistic in vivo loads from younger and more active patients. These
loads require simplifications to be applicable for simulator tests and numerical analyses. Bergmann et al. (2001) measured the
contact forces in the joint with instrumented hip implants in 10 subjects during nine of the most physically demanding and
Fig. 11 Geometry-based (left) and voxel-based model of prosthesized femur.

frequent activities of daily living. Typical levels and directions of average and high joint loads were extracted from the intra- and
interindividually widely varying individual data. Because the magnitudes and orientations of peak forces substantially vary among
the activities, load scenarios that reflect a collection of time-dependent high forces should be applied rather than using unidirec-
tional forces.
Aseptic Mobilization: Stress Shielding
Bad implant position or imprecise indexing of the prosthesis can determine aseptic mobilization phenomena that could result in
collateral effects in the long period. After hip replacement, a frequent complication may also occur, represented by a mechanical
loosening of the implant. This is revealed by implant movement and remodeling of the bone around the prosthesis, bone remod-
eling being the physiological dynamic response of the bone to the environmental stress level. From the mechanical point of view,
the factors influencing the primary stability of the stem depend on biomechanical interaction between femur and prosthesis. The
difference in stress before and after THA can be calculated and divided by the stress occurring pre-THA to determine the stress shield-
ing increase (SSI) for that region.
The before and after ratios are then volume-averaged over a specific region to calculate SSI for that region. Since the von Mises
stress is strictly nonnegative, positive stress difference values indicate a decrease of the stress level in post-THA situation, therefore
stress shielding. The explicit expression for SSI is the following: vanishing SSI means vanishing stress shielding and indicates an
optimal condition. On the contrary, negative values of the SSI indicate an increase of stress when the prosthesis is present, and
thus, they can be interpreted as a measure of stress concentrations, especially, if the actual stress in the bone exceeds yield strength
or physiologically based thresholds.
Topology Optimization
With particular interest on THA, optimization of orthopedic prostheses can be employed to minimize the probability of implant
failure or maximize prosthesis reliability. This goal is often identified with the reduction of stress concentrations at the interface
between bone and these devices. However, aseptic loosening of the implant is mainly influenced by bone resorption phenomena
revealed in some regions of the femur when prosthesis is introduced. As a consequence, bone resorption appears due to stress
shielding, that is to say the decrease of the stress level in the implanted femur caused by the significant load carrying of the prosthesis
due to its higher stiffness. A maximum stiffness topological optimization (TO)-based strategy can be utilized for nonlinear static FE
analyses of the femur–implant assembly, with the goal of reducing stress shielding in the femur and furnishing guidelines for rede-
signing hip prostheses.
Application: Preoperative Assessment

Using the FE method as a clinical tool for surgical planning can give the clinician important information about the possible effects
that the procedure is likely to have on the bone. During the press fitting, the prosthesis is hammered into the femoral canal, thus
causing an elevated strain level onto the cortical shell. Should excess force be applied, there is a risk of causing periprosthetic fracture
of the femur. The interference fit between the bone and the prosthesis was modeled using the FE method, by applying radially
directed displacement boundary conditions on the surface of the femoral canal. By analyzing the strain field at the exterior surface
of the bone, it is possible to determine which patients would be eligible for the press-fitting procedure and which should undergo
cemented fixation. Fig. 12 shows the strain distribution of three individuals that show various mechanical response of the femur.
From the figure, it can be seen that two of the three subjects show levels of strain that exceeds the theoretical fracture limit of the
cortical bone. One of those two patients did suffer a periprosthetic fracture during the press-fitting surgery.
The FE method used in conjunction with CT data can give important information to the clinician about the overall state of the
bone. Such surgical planning tool not only can increase safety during the operation but also can help to identify the optimum treat-
ment for the patient resulting in lower revision rates.
Conclusion
Evaluating the material composition of both the muscle and bone in healthy subjects, it is possible to further understand the mech-
anisms that lead to the degradation of the tissues in pathological conditions and carry out the appropriate treatment. However, it
must be taken into account the variability of the HU values that depend on different factors such as CT scan device, scanning
protocol, region of interest, and artifacts. The methods presented here use thresholds and intervals based on the experimental
data and calibration measurements performed with the CT devices employed in these studies. Finally, this article demonstrates
how various different techniques can be used to assess the bone and muscle, both qualitatively and quantitatively.
Fig. 12 Three-dimensional strain distribution showing the anterior surface of the femur for three patients.
References
Bergmann, G., Deuretzbacher, G., Heller, M., Graichen, F., Rohlmann, A., Strauss, J., & Duda, G. N. (2001). Hip contact forces and gait patterns from routine activities. Journal of
Biomechanics, 34(7), 859–871.
Breusch, S. J., Lukoschek, M., Kreutzer, J., Brocai, D., & Gruen, T. A. (2001). Dependency of cement mantle thickness on femoral stem design and centralizer. The Journal of
Arthroplasty, 16(5), 648–657.
Carraro, U., Edmunds, K. J., & Gargiulo, P. (2015). 3D false color computed tomography for diagnosis and follow-up of permanent denervated human muscles submitted to home-
based functional electrical stimulation. European Journal of Translational Myology, 25(2), 129–135.
Carraro, U., Kern, H., Gava, P., et al. (2017). Recovery from muscle weakness by exercise and FES: Lessons from masters, active or sedentary seniors and SCI patients. Aging
Clinical and Experimental Research, 29, 579–590.
Edmunds, K. J., Árnadóttir, Í., Gíslason, M. K., Carraro, U., & Gargiulo, P. (2016). Nonlinear Trimodal regression analysis of Radiodensitometric distributions to quantify Sarcopenic
and sequelae muscle degeneration. Computational and Mathematical Methods in Medicine, 10. pp. Article ID 8932950.
Fielding, R. A., Vellas, B., Evans, W. J., et al. (2011). Sarcopenia: An undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences.
International working group on sarcopenia. Journal of the American Medical Directors Association, 12(4), 249–256.
Gargiulo, P., Kern, H., Carraro, U., et al. (2010). Quantitative color three dimensional computer tomography imaging of human long term denervated muscle. Neurological Research,
32(1), 13–19.
Goodpaster, B. H., Park, S. W., Harris, T. B., et al. (2006). The loss of skeletal muscle strength, mass, and quality in older adults: The health, aging and body composition study. The
Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 61(10), 1059–1064.
Helgason, B., Perilli, E., Schileo, E., Taddei, F., Brynjolfsson, S., & Viceconti, M. (2008). Mathematical relationships between bone density and mechanical properties: A literature
review. Clinical Biomechanics, 23, 135–146.
Kern, H., Carraro, U., Adami, N., et al. (2010). Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower
motor neuron lesion. Neurorehabilitation and Neural Repair, 24, 709–721. https://doi.org/10.1177/1545968310366129.
Lotz, J. C., Gerhart, T. N., & Hayes, W. C. (1990). Mechanical properties of trabecular bone from the proximal femur: A quantitative CT study. Journal of Computer Assisted
Tomography, 14, 107–114.
McCalden, R. W., McGeough, J. A., & Court-Brown, C. M. (1997). Age-related changes in the compressive strength of cancellous bone. The relative importance of changes in
density and trabecular architecture. The Journal of Bone and Joint Surgery. American Volume, 79, 421–427.
Morgan, E. F., Bayraktar, H. H., & Keaveny, T. M. (2003). Trabecular bone modulus–density relationships depend on anatomic site. Journal of Biomechanics, 36, 897–904.
Pétursson, T. H., Edmunds, K. J., Gíslason, M. K., et al. (2015). Bone mineral density and fracture risk assessment to optimize prosthesis selection in total hip replacement.
Computational and Mathematical Methods in Medicine, 2015, 162481, 7 pages.
Zysset, P. K., Hayes, W. C., & Piazza, S. J. (1991). Biomechanics of fracture risk prediction of the hip and spine by quantitative computed tomography. Radiologic Clinics of North
America, 29, 1–18.
Further Reading
Carter, D. R., & Hayes, W. C. (1977). The compressive behavior of bone as a two-phase porous structure. Journal of Bone and Joint Surgery, 59, 954–962.
Fischer, D., Kley, R., Strach, K., Meyer, C., Sommer, T., Eger, K., et al. (2008). Distinct muscle imaging patterns in myofibrillar myopathies. Neurology, 71(10), 758–765.
Hansson, T. H., Keller, T. S., & Panjabi, M. M. (1986). A study of the compressive properties of lumbar vertebral trabeculae: Effects of tissue characteristics. Spine, 11, 56–62.
Keaveny, T. M. (2001). Strength of trabecular bone. In S. C. Cowin (Ed.), Bone mechanics handbook. New York: CRC Press.
Kopperdahl, D. L., & Keaveny, T. M. (1998). Yield strain behavior of trabecular bone. Journal of Biomechanics, 31, 601–608.
Kornblum, C., Lutterbey, G., Bogdanow, M., Kesper, K., Schild, H., Schröder, R., et al. (2006). Distinct neuromuscular phenotypes in myotonic dystrophy types 1 and 2. Journal of
Neurology, 253(6), 753–761.
Mosekilde, L., & Mosekilde, L. (1986). Normal vertebral body size and compressive strength: Relations to age and to vertebral and iliac trabecular bone compressive strength. Bone,
7, 207–212.
Lang, T., Cauley, J. A., Tylavsky, F., Bauer, D., Cummings, S., & Harris, T. B. (2010). Computed tomographic measurements of thigh muscle cross-sectional area and attenuation
coefficient predict hip fracture: The health, aging, and body composition study. Journal of Bone and Mineral Research, 25(3), 513–519.
Linde, F., Hvid, I., & Pongsoipetch, B. (1989). Energy absorptive properties of human trabecular bone specimens during axial compression. Journal of Orthopedic Research, 7,
432–439.
Liu, M., Chino, N., & Ishihara, T. (1993). Muscle damage progression in Duchenne muscular dystrophy evaluated by a new quantitative computed tomography method. Archives of
Physical Medicine and Rehabilitation, 74(5), 507–514.
Lotz, J. C., Gerhart, T. N., & Hayes, W. C. (1990). Mechanical properties of trabecular bone from the proximal femur: A quantitative CT study. Journal of Computer Assisted
Tomography, 14, 107–114.
McBroom, R. J., Hayes, W. C., Edwards, W. T., Goldberg, R. P., & White, A. A., III (1985). Prediction of vertebral body compressive fracture using quantitative computed
tomography. The Journal of Bone and Joint Surgery. American Volume, 67, 1206–1214.
Rohlmann, A., Zilch, H., Bergmann, G., & Kolbel, R. (1980). Material properties of femoral cancellous bone in axial loading. Part I: Time independent properties. Archives of
Orthopaedic and Traumatic Surgery, 97, 95–102.
Swash, M., Brown, M. M., & Thakkar, C. (1995). CT muscle imaging and the clinical assessment of neuromuscular disease. Muscle and Nerve, 18(7), 708–714.
Zysset, P. K., Hayes, W. C., & Piazza, S. J. (1991). Biomechanics of fracture risk prediction of the hip and spine by quantitative computed tomography. Radiologic Clinics of North
America, 29, 1–18.
Knowledge Extraction From Medical Imaging for Advanced Patient-Specific
Musculoskeletal Models
Marie-Christine Ho Ba Tho and Tien Tuan Dao, Sorbonne Universités, Paris, France; and Université de Technologie de Compiègne,
Compiègne, France
Introduction 135
Geometrical Properties Extraction From Computed Tomography and MRI Modalities 136
Mechanical Properties Extraction From Computed Tomography Modality 136
Biochemical Properties Extraction From Advanced MRI 137
Loading and Boundary Conditions Extraction From Advanced MRI 138
Muscle Force Extraction From Magnetic Resonance Elastography 139
Reliability and Uncertainty Data 140
Conclusions and Perspectives 141
Acknowledgments 141
References 141
Glossary
Advanced medical imaging Relates to new imaging sequences and protocols, which are developed commonly for research
purposes. However, these sequences are sometimes used in routine practice for preclinical tests.
Advanced patient-specific modeling Is an engineering process to develop mathematical representation of a physical system
with properties extracted directly from patient data. Patient-specific data may include geometry, mechanical properties,
biochemical properties, loading, and boundary conditions.
Finite-element modeling Is a continuum modeling approach to solve a physical problem by approximating the solutions on
a set of finite elements. This approach is commonly used for studying the stress–strain relationships of the biomaterials.
Knowledge extraction Is the information processing aiming to extract and establish useful knowledge from data.
Material-driven meshing Is the meshing process to discretize the model geometry into finite elements based on the knowledge
of involved materials.
Rigid body modeling Is a mechanistic modeling approach to simulate the kinematics, kinetics, and muscle force behaviors of
the musculoskeletal system.
Introduction
The article will address the methodology we have developed in order to model bone and joints with appropriate geometric and
mechanical properties derived from medical imaging. Medical imaging system such as MRI (magnetic resonance imaging) CT
(computed tomography) is commonly used to evaluate musculoskeletal disease. Numerical methods are used for solving physical
and mechanical engineering problems. These numerical methods are appropriate for modeling such complex system as human
bone and joints and musculoskeletal systems. Literature review demonstrated the extensive use of finite-element modeling in
biomechanics. During the last decade, the virtual physiological human a framework supported by the European Commission
has allowed research based on personalized, predictive, and integrative medicine with in silico modeling at different scales of
the body. Patient-specific computer modeling has been developed since the last decade, but still the specificity is not fully described
or is limited to patient geometry. Concerning bone and joints modeling it is possible to obtain geometry and mechanical properties
derived from CT, but using medical images one should be paid attention on the reliability of the images quality and the control of
the acquisition parameters (Ho Ba Tho, 2003; Hellmich and Kober, 2006). Besides these extensive numerical models, most of
models are derived from CT data and few from MRI. Few consider appropriate material properties derived from tissue characteriza-
tion obtained from medical images, as they mostly are issued from the literature (data or relationships).
The methodology we have developed is based on a semiautomatic generation of a three-dimensional geometric model of bone
and joints, muscles anatomy derived from medical imaging CT, or MRI data (Ho Ba Tho, 1993). Predictive relationships obtained
from the previous work demonstrated significant correlation between the material properties and quantitative measurements
derived from imaging techniques (Rho et al., 1995). Then, from the same source of medical imaging data, numerical models
with individualized geometric and mechanical properties were developed (Couteau et al., 1998). Concerning soft tissue same
methods are applied (correlation between in vivo tissue characterization and in vitro) or direct assessment could also be performed

136 Biomechanics j Knowledge Extraction From Medical Imaging for Advanced Patient-Specific Musculoskeletal Models
using MRE (magnetic resonance elastrography) (Bensamoun et al., 2008). Concerning the in vivo forces, dynamic MRI (Dao et al.,
2013) and MRE (Bensamoun et al., 2013) are able to provide data which could be exploited via a musculoskeletal model in order to
predict in vivo loads by dynamic inverse analysis.
This article aimed to present state-of-the-art methodologies for extracting knowledge from medical imaging for advanced
patient-specific modeling of the musculoskeletal system.
Geometrical Properties Extraction From Computed Tomography and MRI Modalities
Scientific and technological progresses of the medical imaging have advanced the knowledge of structure–function relationships
inside the human body in a more quantitative and precise manner (Abramson et al., 2015; Ren et al., 2016). Computed tomog-
raphy (CT) and magnetic resonance imaging (MRI) are two most important imaging modalities, which allows detailed 3D struc-
tures of the biological tissues and organs to be commonly acquired and used for numerical modeling (Ho Ba Tho, 2003). CT
modality has been commonly used for hard tissue (e.g., bone) characterization, while MRI modality has been applied for soft tissue
(e.g., muscle, tendon) characterization (Frangi et al., 2016). These conventional imaging modalities allow rigid body and finite-
element models of the human musculoskeletal system to be generated in a subject- or patient-specific manner (Ho Ba Tho,
2003; Blemker and Delp, 2005; Dao et al., 2012) (Fig. 1). Based on the raw images, image processing may be applied to extract
geometrical properties of the musculoskeletal system like muscle volume, physiological cross-sectional area, or cartilage thickness
(Dao et al., 2011).
Mechanical Properties Extraction From Computed Tomography Modality
Mechanical properties of bone have been studied for over three decades in order to understand the mechanical behavior of bone in
the process of fracture risk, repair, and bone-related disease. Besides, few data were available or insufficient for human bone
modeling. Human bone is highly heterogeneous and anisotropic material. It can be compared to composite materials; it is
made of two different tissue spongious bone (high porosity) and cortical bone (compact bone) depending on the anatomical loca-
tion (Ho Ba Tho et al., 1991).
In order to associate geometric and mechanical properties, we assume that measurements derived from medical imaging could
predict material properties. We have investigated the relationships between CT number derived from CT imaging technique and
mechanical properties of bone. The CT number characterize a linear coefficient of attenuation of X-ray within the tissue. For the
CT scan, the pixel values are represented by an empirical number called CT number expressed in Houndsfield Units (HU). Predictive
relationships between elastic properties and density and CT numbers for different human bone have been provided (Rho et al.,
1995). Finally from CT images, one could model geometric and mechanical properties of the subject (Couteau et al., 1998).
Recently, we developed material-driven mesh techniques allowing to drive the mesh with the knowledge of the material prop-
erties (Nguyen et al., 2016) (Fig. 2).
(A) (B)
SAC
Distance (mm)
1.50 ASIS
Condyles femoraux 1.39
1.29
1.18
Medial THIG
Lateral 1.07
.964
.857
.750 KNE
Posterieur .643
.536
.429 TIB
Plateau tibial
.321
.214
.107
ANK
0. HEE
TOE
Anterieur
Fig. 1 Patient specific: geomtrical and FEM model (A) and rigid body model (B) derived from medical image (Dao et al., 2012).
Biomechanics j Knowledge Extraction From Medical Imaging for Advanced Patient-Specific Musculoskeletal Models 137
(A) (B) (C)

Max: 28653 Max: 28653
200 160 25000
1500
Young’s modulus (Mpa)

25000
Young’s modulus (Mpa)

140
Hounsfield unit (HU)

20000 20000
150
1000 120
15000 15000
100 100
10000 10000
500 80
50
60 5000 5000
0 Min: 824 Min: 824

0 50 100 150 200 20 40 60 80 100 120 140 160 180
Pixel Pixel
Fig. 2 Material distributions on segmented CT slices (A), material-driven meshes (B), and cross sections with Young’s modulus mapping (C).
Biochemical Properties Extraction From Advanced MRI
To provide patient-specific data at the tissue scale, advanced MRI sequences such as T2 mapping, diffusion weighted, and diffusion
tensor imaging have been a potential solution (Dao et al., 2013). These techniques were commonly used to provide microstructural
properties of the tissues of interest (e.g., cartilage, intervertebral disk (IVD)). In fact, T1r time reflects the loss of macromolecules.
The T2 relaxation time estimated reflects the change of material (water and proteoglycan) properties. Apparent diffusion coefficient
estimated from diffusion-based MRI reflects the molecule mobility of these material properties of the IVD. Diffusion-weighted
image deals with the amount of water diffusion occurring within a voxel. It is well known that the diffusion in disk tissue is aniso-
tropic. Consequently, this shows the direction-dependent character of disk structures. On the other side, diffusion tensor imaging
quantifies the direction and magnitude of water diffusion in three dimensions in each voxel. In fact, diffusion-based imaging can be
used as a potential microstructural marker for revealing early degeneration states of the IVD. Furthermore, these properties can be
used to develop the accurate numerical IVD model at the tissue level. Among the available studies in the literature, the investigation
of T1r-weighted MRI sequence on the IVD is complete. This MRI sequence has been used for assessing the in vivo and in vitro IVD
tissue. In an in vitro study, Johannessen et al. (2006) reported a fair correlation (R coefficient ranges from 0.58 to 0.70) between T1r
time and the biochemical properties of the IVD. In another study of the same research group (Nguyen et al., 2008), they showed
a fair correlation (R ¼ 0.59) between T1r time and the mechanical properties of the IVD. Moreover, T1r time has been reported for
the degenerated IVDs (Auerbach et al., 2006). This property has been considered as a novel biomarker for the assessment of disk
degeneration and low back pain (Borthakur et al., 2011). However, despite its great potential capacity, this MRI sequence could not
be used in any MRI machine without additional technical implementation (Taheria and Sood, 2006). For this purpose, T2 mapping
and diffusion-based MRI sequences could be used as alternative solutions thank to their available implementation in classical
medical imaging machines (Dao et al., 2013, 2014) (Fig. 3). Moreover, based on the correlation outcomes between in vitro
image-derived properties and biomechanical property measurements, finite-element models may be developed with patient-
specific biochemical property information (Fig. 4) (Nguyen, 2016).
Fig. 3 The T2 relaxation time estimated from the T2-mapping MRI images: (A and D) raw MRI image; (B and E) T2 maps of in vivo and in vitro
IVD; (C and F) ADC maps of in vivo and in vitro IVD; (G) a dissected view of a cadaveric IVD. The T2 color codes mean that the outer AF is in blue
color, the inner AF is in green color, and the NP is in red color. The ADC color code means that the outer AF is in blue color, the inner AF is in green
color, and the NP is in yellow color.
(A) (B) (C)

200
T2 relaxation time (ms)

150
100
50
Fig. 4 T2 image (A), meshed IVD model (B), and water content distribution on the meshed model (C) (Nguyen, 2016).
Model derived from CT
Model derived from MRI
Fig. 5 T2 image (A), meshed IVD model (B) and water content distribution on the meshed model (C) (Nguyen, 2016).
From data obtained for CT and MRI, the patient-specific numerical model can be derived from extracted material properties of
CT (vertebrae) and MRI (disk). According to our knowledge it is a first numerical model of patient-specific lumbar spine including
patient material properties of the vertebrae and the IVD (Fig. 5). The next step would be to integrate the patient-specific forces for
specific movement.
Loading and Boundary Conditions Extraction From Advanced MRI
Finite-element simulation of the musculoskeletal system during dynamic movements requires complex loading and boundary
conditions. The coupling between rigid body and finite-element models has been commonly performed to achieve this challenge
(Halloran et al., 2012). Muscle forces and joint loading, estimated from rigid body modeling, are usually prescribed as loading and
boundary conditions in finite-element models. These quantities may be computed in a straightforward manner for the upper and
lower limbs. 3D motion capture systems like VICON has been commonly used for acquiring simulation kinematics (Dao et al.,
2012). However, the kinematics of the lumbar spine system is still very hard to be acquired due to the complexity of the lumbar
spine geometries. Lumbar spine ranges of motion are commonly acquired using medical imaging (e.g., 2D radiography Frobin et al.,
1996 or biplanar radiography Rillardon et al., 2005) or dual fluoroscopic imaging (Xia et al., 2010) or Upright MRI (Alyas et al.,
2008) or motion capture techniques (e.g., electromagnetic tracking system Wong and Lee, 2004) or computerized dynamic motion
analysis devices (Mannion and Troke, 1999) or 3D motion tracking system with implanted bone pins (Rozumalski et al., 2008).
Medical imaging techniques provide internal accurate lumbar spine ranges of motion, while motion capture provides external
ranges of motion. However, imaging techniques provide only quasi-static motions rather than real dynamic motion (Powers
et al., 2003). Moreover, due to limited range of motion and spatial/temporal image resolution, medical imaging approach needs
further developments and investigations to provide accurate dynamic motion data. Furthermore, invasive character of some tech-
niques limits their use in vivo (Frobin et al., 1996; Rillardon et al., 2005; Rozumalski et al., 2008) even they provide accurate motion
data. Recently, we developed dynamic MRI for acquiring lumbar spine kinematics (Dao et al., 2015) (Fig. 6). Then, these data have
been used for estimation of the lumbar spine muscle forces as well as for reassessment of kinematic of model outcomes (Fig. 7). In
fact, noninvasive conventional dynamic MRI technique opens new perspectives to provide in vivo spinal kinematic and muscle
force data reflecting the real lumbar spine motions. Then, these data may be used as loading and boundary conditions for the
finite-element model in a patient-specific manner (Toumanidou et al., 2016).
Fig. 6 Dynamic MRI images during hyperlordosis motion.
Fig. 7 Patient-specific lumbar spine model: frontal view (A) and lateral view (B).
Muscle Force Extraction From Magnetic Resonance Elastography
At the present time, the optimization technique has been accepted as a unique solution to estimate healthy muscle forces (Erde-
mir et al., 2007) but abnormal behavior of the musculoskeletal system, due to muscle diseases (Dao et al., 2012), cannot be
modeled and simulated. Generic-parameterized musculoskeletal models are mainly composed of the Hill-based model, which
is the most used rheological model to assess the muscle tensile forces. Some authors investigated the sensitivity study of abnormal
behavior of muscle due to aging-affect (Thelen, 2003) or paralyzed effect (Law and Shields, 2005) by adjusting the contractile
properties of the Hill-based model. However, there is no way to validate such parameters adjustment strategies. In fact, the quan-
tification of these forces requires more intrinsic properties of muscles which can be determined in vivo with the determination of
the sarcomere contractile dynamics properties (Llewellyn et al., 2008) or the characterization of the muscles mechanical proper-
ties (Ringleb et al., 2007; Bensamoun et al., 2007). Recently, we proposed a new direction to estimate lower limb muscle forces by
introducing in vivo muscle elastic properties, leading to future simulation of abnormal muscles (Bensamoun et al., 2013)
(Table 1, Fig. 8). In fact, the MR-elastography technique could provide a full database of active and passive elastic properties
of different types of muscle allowing for the measurement of in vivo muscle forces, corresponding to each muscle, in order to
better understand abnormal muscle behavior or to characterize the effect of age and growing processes on the musculoskeletal
system.
Table 1 Tensile forces (mean SD in Newton) of vastus medialis muscle at different phases during gait
Tensile VM force during the stance phase (0%–60%) Tensile VM force during the swing phase (60%–100%)
Heel strike Midstance Toe-off Initial swing Midswing Terminal swing
Model (0%–2%) (10%–30%) (50%–60%) (60%–73%) (73%–87%) (87%–100%)
Voigt 791 2 220 25 170 24 511 73 294 61 93 31

Springpot 793 1 157 22 108 21 272 106 104 81 14 24
Fig. 8 The musculoskeletal model of the lower limbs.
Reliability and Uncertainty Data
The accuracy, validation of predictive numerical models needed to be investigated, quantified as the clinical diagnostic will depend
on. In fact, data are extracted from medical images so one should pay attention on the reliability of the images quality and the
control of the acquisition parameters (Ho Ba Tho, 2003). The reliability of assessment of the knowledge (geometry, mechanical
properties, loads, and boundary conditions) derived from medical imaging (or literature data) are to be addressed and see their
impact on the numerical developed and its propagation until the predictive results. Data are extracted from medical images,
such as relationships with biochemical, physics, mechanical properties, their certification should be required as illustrated in Fig. 9.
As an example, the effect of geometrical uncertainties due to segmentation error was performed on the quantification of joint
loading and muscle forces of the lower limb system (Dao et al., 2012). In fact, the use of medical images for the patient-specific
model leads to new error, which should be characterized and quantified.
Concerning mechanical properties of the biological material investigated, it is quite difficult or impossible to say that it is the
best description of the mechanical behavior or the law. There is obviously a need to validate or to establish statistical, uncertainty
modeling. Random and epistemic uncertainties are two types of uncertainties of biomechanical data. Random uncertainty relates to
the variability of repeatable acquisitions artifacts, hardware/software errors, or human errors (e.g., intersubject, intrasubject, inter-
operator, intraoperator) on the measurements. Epistemic uncertainty exists due to the lack of information/knowledge (e.g.,
modeling hypothesis or limited experiments). To model these uncertainties, precise and imprecise probabilities should be used.
Recently, probability-box structure has been used for modeling heterogeneous data uncertainty such as medical imaging, mechan-
ical properties, physiological and clinical data leading to quantify their propagation effect on the simulation outcomes (Dao and Ho
Ba Tho, 2015, 2017) (Fig. 10). Furthermore, expert judgment may be used to assess the reliability of the biomechanical data from
the literature. Advanced data modeling theories like belief theory may be applied for this purpose (Hoang et al., 2016).
Fig. 9 Same section location with different acquisition “hardware” parameters (A) and “software” parameters (B).
Fig. 10 Example of P-Box on thigh mass properties (A) and its impact on estimation of muscles forces expressed in range of values (B).
Conclusions and Perspectives
Geometrical, material, forces knowledge derived from advanced medical imaging are of interest to provide full patient specificity.
Besides, one should note that the accuracy, reliability of such models have to be investigated in order to provide an objective tool for
aided decision to clinicians. In that context we have developed a methodology to model uncertainty related to heterogeneous data
obtained experimentally and/or from the literature such as medical imaging, mechanical properties, forces, physiological and clin-
ical data, and its propagation in the model. Precise and imprecise probabilistic approach have been applied to achieve this chal-
lenging issue. This new approach allowed to interpret predictive results with a level of confidence. In fact, a future generation of
musculoskeletal models will be developed to provide reliable biomarkers for clinical decision support system of the musculoskel-
etal disorders in the framework of in silico and personalized medicine.
Acknowledgments
The authors acknowledge the financial support of the Collegium-UTC CNRS INSIS and the Labex MS2T through the program Investments for the
future managed by the National Agency for Research (Reference ANR-11-IDEX-0004-02).
We are grateful and acknowledge the MYSPINE European project (FP7/2007–2013, no. 269909) for providing data for this present work.
References
Abramson, R. G., Burton, K. R., Yu, J. P. J., Scalzetti, E. M., Yankeelov, T. E., Rosenkrantz, A. B., Mendiratta-Lala, M., Bartholmai, B. J., Ganeshan, D., Lenchik, L., &
Subramaniam, R. M. (2015). Methods and challenges in quantitative imaging biomarker development. Academic Radiology, 22(1), 25–32.
Alyas, F., Connell, D., & Saifuddin, A. (2008). Upright positional MRI of the lumbar spine. Clinical Radiology, 63(9), 1035–1048.
Auerbach, J. D., Johannessen, W., Borthakur, A., Wheaton, A. J., Dolinskas, C. A., Balderston, R. A., Reddy, R., & Elliott, D. M. (2006). In vivo quantification of human lumbar disc
degeneration using T1q-weighted magnetic resonance imaging. European Spine Journal, 15(3), S338–S344.
Bensamoun, S. F., Ringleb, S. I., Chen, Q., Ehman, R. L., An, K. N., & Brennan, M. (2007). Thigh muscle stiffness assessed with magnetic resonance elastography in hyperthyroid
patients before and after medical treatment. Journal of Magnetic Resonance Imaging, 26, 708–713.
Bensamoun, S., Glaser, K. J., Ringleb, S. I., Chen, Q., Ehman, R. L., & An, K. N. (2008). Rapid magnetic resonance elastography of skeletal muscle using one dimensional
projection. Journal of Magnetic Resonance Imaging, 27, 1083–1108.
Bensamoun, S. F., Dao, T. T., Charleux, F., & Ho Ba Tho, M. C. (2013). Estimation of muscle force derived from in vivo MR elastography tests: A preliminary study. Journal of
Musculoskeletal Research, 16(3) (2013), 1350015.
Blemker, S. S., & Delp, S. L. (2005). Three-dimensional representation of complex muscle architectures and geometries. Annals of Biomedical Engineering, 33, 661–673.
Borthakur, A., Maurer, P. M., Fenty, M., Wang, C., Berger, R., Yoder, J., Balderston, R. A., & Elliott, D. M. (2011). T1r magnetic resonance imaging and discography pressure as
novel biomarkers for disc degeneration and low back pain. Spine, 36(25), 2190–2196.
Couteau, B., Ho Ba Tho, M. C., Darmana, R., Brignola, J. C., & Arlaud, J. Y. (1998). Finite element modelling of the vibrational behaviour of the human femur using CT-based
individualized geometrical and material properties. Journal of Biomechanics, 31(4), 383–386.
Dao, T. T., & Ho Ba Tho, M. C. (2015). Assessment of parameter uncertainty in rigid musculoskeletal simulation using a probabilistic approach. Journal of Musculoskeletal Research,
18(3) (2015) 1550013.
Dao, T. T., & Ho Ba Tho, M. C. (2017). A consistent data fusion approach for uncertainty quantification in rigid musculoskeletal simulation. Journal of Mechanics in Medicine and
Biology, 17(3), 1750062.
Dao, T. T., Pouletaut, P., Goebel, J. C., Pinzano, A., Gillet, P., & Ho Ba Tho, M. C. (2011). In vivo characterization of morphological properties and contact areas of the rat cartilage
derived from high resolution MRI. Biomedical Engineering and Research (IRBM), 32(3), 204–213.
Dao, T. T., Marin, F., Pouletaut, P., Aufaure, P., Charleux, F., & Ho Ba Tho, M. C. (2012). Estimation of accuracy of patient specific musculoskeletal modeling: case study on a post
polio residual paralysis subject. Computer Method in Biomechanics and Biomedical Engineering, 15(7), 745–751.
Dao, T. T., Pouletaut, P., Robert, L., Aufaure, P., Charleux, F., & Ho Ba Tho, M. C. (2013). Quantitative analysis of annulus fibrosus and nucleus pulposus derived from T2 mapping,
diffusion-weighted and diffusion tensor MR imaging. Computer Methods in Biomechanics and Biomedical Engineering: Imaging & Visualization, 1(3), 138–146.
Dao, T. T., van Rijsbergen, M., Pouletaut, P., Charleux, F., Ito, K., & Ho Ba Tho, M. C. (2014). In In vitro assessment of micro-structural properties of intervertebral disc using 1.5T
magnetic resonance T2 and ADC mappings7th World Congress of Biomechanics, July 6–11, 2014, MITdBoston, Massachusetts, USA.
Dao, T. T., Pouletaut, P., Charleux, F., Lazáry, Á., Eltes, P., Varga, P. P., & Ho Ba Tho, M. C. (2015). Multimodal medical imaging (CT and dynamic MRI) data and computer-graphics
multi-physical model for the estimation of patient specific lumbar spine muscle forces. Data and Knowledge Engineering, 96-97, 3–18.
Erdemir, A., McLean, S., Herzog, W., & van den Bogert, A. J. (2007). Model-based estimation of muscle forces exerted during movements. Clinical Biomechanics, 22(2), 131–154.
Frangi, A. F., Taylor, Z. A., & Gooya, A. (2016). Precision imaging: More descriptive, predictive and integrative imaging. Medical Image Analysis, 33, 27–32.
Frobin, W., Brinckmann, P., Leivseth, G., Biggemann, M., & Reikeras, O. (1996). Precision measurement of segmental motion from flexiondExtension radiographs of the lumbar
spine. Clinical Biomechanics, 11(8), 457–465.
Halloran, J. P., Sibole, S., van Donkelaar, C. C., van Turnhout, M. C., Oomens, C. W., Weiss, J. A., Guilak, F., & Erdemir, A. (2012). Multiscale mechanics of articular cartilage:
potentials and challenges of coupling musculoskeletal, joint, and microscale computational models. Annals of Biomedical Engineering, 40(11), 2456–2474.
Hellmich, C., & Kober, C. (2006). Micromechanics-supported conversion of computer tomographic (CT) images into anisotropic and inhomogeneous finite element models of organs:
the case of a human mandible. PAMMdProceedings in Applied Mathematics and Mechanics, 71–74.
Ho Ba Tho, M. C. (1993). Logiciel de pre´traitement d’images me´dicales SIP 305 ÓInserm 1993.
Ho Ba Tho, M. C. (2003). Bone and joints modelling with individualised geometric and mechanical properties derived from medical images. Computer Mechanics and Engineering
Sciences, 4(3&4), 489–496.
Ho Ba Tho, M. C., Rho, J. Y., & Ashman, R. B. (1991). Atlas of mechanical properties of human cortical and cancellous bone. In G. Van der Perre, & A. Lowet (Eds.), In vivo
assessment of bone quality by vibration and wave propagation techniques, part II (pp. 1–38). Leuven: ACCO press.
Hoang, T. N., Dao, T. T., & Ho Ba Tho, M. C. (2016). Clustering of children with cerebral palsy with prior biomechanical knowledge fused from multiple data sources. Lecture Notes
in Computer Science (Springer), 9978, 359–370.
Johannessen, W., Auerbach, J. D., Wheaton, A. J., Kurji, A., Borthakur, A., Reddy, R., & Elliott, D. M. (2006). Assessment of human disc degeneration and proteoglycan content
using T1r-weighted magnetic resonance imaging. Spine, 31(11), 1253–1257.
Law, L. A., & Shields, R. K. (2005). Mathematical models use varying parameter strategies to represent paralyzed muscle force properties: a sensitivity analysis. Journal of
Neuroengineering and Rehabilitation, 2, 2–12.
Llewellyn, M. E., Barretto, R. P. J., Delp, S. L., & Schnitzer, M. J. (2008). Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature,
454, 784–788.
Mannion, A., & Troke, M. (1999). A comparison of two motion analysis devices used in the measurement of lumbar spinal mobility. Clinical Biomechanics, 14(9), 612–619.
Nguyen, H. Q. (2016). Material-driven mesh derived from medical images for biomechanical system. In Application on modeling of the lumbar spine. Université de Technologie de
Compiègne. PhD thesis.
Nguyen, A. M., Johannessen, W., Yoder, J. H., Wheaton, A. J., Vresilovic, E. J., Borthakur, A., & Elliott, D. M. (2008). Noninvasive quantification of human nucleus pulposus
pressure with use of T1rho-weighted magnetic resonance imaging. The Journal of Bone and Joint Surgery. American Volume, 90(4), 796–802.
Nguyen, H. Q., Dao, T. T., Rassineux, A., & Ho Ba Tho, M. C. (2016). Material-driven mesh of the lumbar spine derived from CT data. Computer Methods in Biomechanics and
Biomedical Engineering: Imaging & Visualization, 1–9. https://doi.org/10.1080/21681163.2016.1188729.
Powers, C. M., Kulig, K., Harrison, J., & Bergman, G. (2003). Segmental mobility of the lumbar spine during a posterior to anterior mobilization: assessment using dynamic MRI.
Clinical Biomechanics, 18(1), 80–83.
Ren, Y., Chen, G. Z., Liu, Z., Cai, Y., Lu, G. M., & Li, Z. Y. (2016). Reproducibility of image-based computational models of intracranial aneurysm: A comparison between 3D
rotational angiography, CT angiography and MR angiography. Biomedical Engineering Online, 15(1), 50.
Rho, J. Y., Ho Ba Tho, M. C., & Ashman, R. B. (1995). Relations of mechanical properties to density and CT numbers in human bone. Medical Engineering & Physics, 17(5),
347–355.
Rillardon, L., Campana, S., Mitton, D., Skalli, W., & Feydy, A. (2005). Evaluation of the intervertebral disc spaces with a low dose radiographic system. Journal de Radiologie, 86(3),
311–319.
Ringleb, S. I., Bensamoun, S., Chen, Q., Manduca, A., Ehman, R. L., & An, K. N. (2007). Applications of magnetic resonance elastography to healthy and pathologic skeletal muscle.
Journal of Magnetic Resonance Imaging. Invited Review., 25(2), 301–309.
Rozumalski, A., Schwartz, M. H., Wervey, R., Swanson, A., Dykes, D. C., & Novacheck, T. (2008). The in vivo three-dimensional motion of the human lumbar spine during gait. Gait
& Posture, 28(3), 378–384.
Taheria, S., & Sood, R. (2006). Spin-lock MRI with amplitude- and phase-modulated adiabatic waveforms: An MR simulation study. Magnetic Resonance Imaging, 24, 51–59.
Thelen, D. G. (2003). Adjustment of muscle mechanics model parameters to simulate dynamic contractions in older adults. Journal of Biomechanical Engineering, 125, 70–77.
Toumanidou, T., Dao, T. T., Ho Ba Tho, M. C., & Noailly, J. (2016). In Exploring the interactions between muscle function and intervertebral disc multiphysics in the healthy and the
degenerated lumbar spine12th World Congress on Computational Mechanics (WCCM), 24–26 July 2016, Seoul, Korea.
Wong, T. K. T., & Lee, R. Y. W. (2004). Effects of low back pain on the relationship between the movements of the lumbar spine and hip. Human Movement Science, 23(1), 21–34.
Xia, Q., Wang, S., Kozanek, M., Passias, P., Wood, K., & Li, G. (2010). In-vivo motion characteristics of lumbar vertebrae in sagittal and transverse planes. Journal of Biomechanics,
43, 1905–1909.
Mathematical Quantification of the Impact of Microstructure on the Various
Effective Properties of Bones
Miao-Jung Y Ou, University of Delaware, Newark, DE, United States
Annalisa De Paolis and Luis Cardoso, The Graduate School of The City University of New York, New York, NY, United States
Introduction 143
Quantification of the Influence of Material Microstructure on Its Effective Properties 145
Integral Representation Formulas (IRFs) of Effective Properties 145
Permeability and Tortuosity of Anisotropic Materials 147
Fabric tensor 147
Permeability tensor as a function of fabric 147
Generalized IRF for anisotropic permeability 147
Computation of Fabric Tensors and Static Permeability of Cancellous Bone Samples 148
Sample Preparation 148
Micro-CT Image Acquisition 148
Image Processing 148
Global Measurements of Microarchitecture 149
Directional Measurements of Microarchitecture 149
Realignment of Images to Principal Axes of Symmetry 149
Pore Diameter Measurements 149
Computational Fluid Dynamic Simulations 149
Numerical Results 150
Discussion 152
Acknowledgments 153
References 153
Introduction
Poroelastic composites are two-phase composite materials consisted of elastic solid frames with fluid saturated pore space. The
study of poroelasticity plays an important role in biomechanics, seismology and geophysics due to the nature of objects of research
in these fields, for example, fluid saturated rocks, sea ice and cancellous bone.
As the starting point of a systematically study of the microstructure influence on all the effective properties, we assume that
constituent materials, that is, the solid matrix and the pore fluid are isotropic and that the anisotropy of the effective properties
of the poroelastic composites is determined by the microstructure. For cancellous bones, this assumption was experimentally vali-
dated in Odgaard et al. (1997). We would like to remark that in the trabeculae of cancellous bone, there exist pores much smaller
than the thickness of trabeculae (see e.g., Cowin, 1999; Gailani and Cowin, 2011; Morin and Hellmich, 2014; Scheiner et al., 2016)
and the references therein. The two-phase model considered in this paper does not take into account the effects caused by the exis-
tence of these pores or any other structure smaller than these pores. For discussion of possible anisotropy of the trabeculae and the
cortical bones (see Fritsch et al., 2009; Hellmich and Ulm, 2005; Ascenzi et al., 2008).
The physical properties of these composites depend not only on the constituent materials but also on the microstructure of pore
space and how the viscous pore fluid interacts with the solid frame. The bulk properties are described by various effective parameters.
They play the role of coefficients in the integral-differential equations governing wave propagation through poroelastic materials
when the wavelength is much bigger than the scale of microstructure. To be more precise, let f be the volume fraction of the fluid,
u and U the displacement of the solid part and the fluid part, respectively. Define v d vtu (solid velocity), w d f(U u) (fluid
displacement relative to the solid), q d vtw, z d V $ w. Let (x1, x2, x3) be the coordinates that coincide with the principal direc-
tions of the static permeability tensor K0, which is symmetric and positive definite (Biot, 1962). Biot (1956a, b) considered sepa-
rately the low frequency case and high frequency case, which are defined by a cut-off frequency above which the Poiseuille flow
assumption for the pore fluid breaks down. It is known that the drag force exerted on the viscous pore fluid is dominated by
the inertia term for low frequency and by the viscous term for high frequency (Johnson et al., 1987).
In Johnson et al. (1987), a description of the inertia/viscous drag effects valid over the entire frequency range was derived by
a causality argument and hence it unifies Biot’s theory for low frequency and high frequency poroelastic wave equation. We refer
to this set of poroelastic wave equations as the Biot-JKD equations.
The Biot-JKD equations in three-dimensional space consist of the stress–strain relations and the six equations of motion. Let

3 ij d
1 vui þ vuj , then the stress–strain relation is given by Biot (1962).
2 vxj vxi

144 Biomechanics j Impact of Microstructure on the Various Effective Properties of Bones
0 1 0 10 1
s11 cu11 cu12 cu13 cu14 cu15 cu16 Ma1 311
B C B CB C
B s22 C B cu12 cu22 cu23 cu24 cu25 cu26 Ma2 C B C
B C B CB 322 C
B C B CB C
B s33 C B cu13 cu23 cu33 cu34 cu35 cu36 Ma3 CB 333 C
B C B CB C
B s23 C ¼ B cu cu24 cu34 cu44 cu45 cu46 Ma4 C B C (1)
B C B 14 CB 2323 C
B C B CB C
B s13 C B cu15 cu25 cu35 cu45 cu55 cu56 Ma5 CB 2313 C
B C B CB C
B s12 C B cu cu26 cu36 cu46 cu56 cu66 Ma6 C B C
@ A @ 16 A@ 2312 A
p Ma1 Ma2 Ma3 Ma4 Ma5 Ma6 M z
where p is the pore pressure, ciju are the elastic constants of the undrained frame, which are related to the elastic constants cij of the
drained frame by ciju ¼ cij þ Maiaj, i, j ¼ 1, /, 6. In terms of the material bulk moduli ks and kf of the solid and the fluid, respectively,
the fluid–solid coupling constants ai and M are given by
8
> 1 X3
>
> 1 c for j ¼ 1; 2; 3
>
< 3ks k¼1 jk
aj d
>
> 1 X3
>
>
: c for j ¼ 4; 5; 6
3ks k¼1 kj
ks
Md
1 k=ks f 1 ks kf
c11 þ c22 þ c33 þ 2c12 þ 2c13 þ 2c23
kd
9
RN
Define the Laplace transform of a function f(x, t) to be bf ðx; sÞd f ðx; t Þ est dt and let g denote the inverse Laplace transform of
0
g(x, s). The equations of motion in the Biot-JKD equations are (see e.g., p. 265 of Carcione, 2001)
X3
vsjk vvj vqj
¼r þ rf ; j ¼ 1; 2; 3 (2)
k¼1
vxk vt vt

vp vvj rf vqj
¼ rf þ j ; j ¼ 1; 2; 3
a (3)
vxj vt f vt
pffiffiffiffiffiffiffi
where a j ðt Þ is the inverse Laplace transform of the dynamic tortuosity function aj(u) with sd 1u, rf and rs the density of the pore
fluid and of the solid, respectively, and r d rs (1 f) þ frf.
The convolution term denoted by * in (3) signifies that the dynamical interaction between solid and fluid are frequency depen-
dent (Biot, 1956b; Johnson et al., 1987). The low frequency Biot’s equation (Biot, 1956a) corresponds to a j ðt Þ ¼ Tj dðt Þ þ khf
j rf
Hðt Þ,
where h is the dynamic viscosity of the pore fluid, kj the static permeability in the xj direction, Tj the infinite-frequency tortuosity in
the xj direction, d(t) the delta function and H(t) is the Heaviside function. The dynamic tortuosity functions aj are the quantification
of the fluid–solid inertia coupling and the viscous energy dissipation in the poroelastic material.
Osteoporosis is characterized by a decrease in bulk strength of the cancellous bone matrix mainly due to the deterioration of the
microstructure, Fig. 1. Currently, bone mineral density (BMD) is the gold standard for in vivo assessment of the fracture risk of
bones and is measured using X-ray absorptiometric techniques (Chaffai et al., 2000). However, only 70%–80% of the variance
of bone strength is accounted for by bone density. As the brittleness of bone depends on more factors than bone density, biologists
believe that quantitative ultrasound techniques (QUT) could provide an important new diagnostic tool (Langton and Njeh, 2003;
Fry et al., 1978; Fellah et al., 2004). Moreover, in contrast to X-ray densitometry, ultrasound does not ionize the tissue, and its imple-
mentation is relatively inexpensive. Since the loss of bone density and the destruction of the bone microstructure are most evident in
osteoporosis cancellous bone, it is natural to consider the possibility of developing accurate ultrasound models for the isonification
of cancellous bone, which is a poroelastic composite of elastic solid trabecula with pore space filled with fluid, Fig. 1. Biot’s theory for
poroelastic waves (Biot, 1956a, b) predicts a fast- and a slow compressional wave and a shear wave in a poroelastic material. The
slow compressional wave does not exist for elastic materials, so the detection of two different compressional waves signifies the
poroelastic property of a specimen. Hosokawa and Otani (1997) (see also McKelvie and Palmer, 1991) identified fast and slow
compressional waves in cancellous bone. The experiment suggested therein leads to a multiparameter inverse problem for the effective
parameters in the Biot equations. It would be of enormous clinical advantage if an accurate method could be developed using ultra-
sound interrogation to determine whether one had osteoporosis. However, even in the low-frequency range, solving the multipa-
rameter inverse problem is a daunting task due to the number of effective parameters involved and the ill-posed nature of the
problem. Also, some of the parameters such as the tortuosity T and the diffusion correlation length L are difficult to measure.
On the other hand, since all the effective parameters of a poroelastic sample are tied to the same microstructure, it is natural to study
how the microstructure affect each of the effective parameters and use it as an underlying common feature in the aforementioned
inverse problems.
In this paper, we will focus on the dynamic permeability tensor K(u) and the dynamic tortuosity tensor a(u). For poroelastic
materials, K(u) and a are defined in the frequency domain as
Biomechanics j Impact of Microstructure on the Various Effective Properties of Bones 145
Fig. 1 (A) A 12 12 12 mm3 cubic VOI was obtained at the center of the calcaneum. The fabric tensor, denoted by F, was measured and
images were rotated in DataViewer until the principal directions were coincident with the faces of the cubic sample. The VOI was digitally cropped to
7 7 7 mm3 at the center of each rotated cubic sample to perform micro-computational fluid dynamic (mCFD) analyses. (B–D) A 7 7 7 mm
fluid volume was added before and after the fluid mask on each of the three directions analyzed for all trabecular bone samples.

b u K ðuÞ
iuf U b ¼ Vb b
p þ rf u2 u (4)
h

aðuÞrf ð iuÞ2 Ub u
b ¼ Vb b ;
p þ rf u2 u (5)
These relations hold if the fluid in the pore space is Newtonian, however, they are in general considered valid for Reynolds
numbers, Re < 10. Eqs. (4), (5) imply the following relation between K and a
ihf 1
aðuÞ ¼ K ðuÞ for us0; (6)
urf
pffiffiffiffiffiffiffi
where id 1. The infinite-frequency tortuosity T d limu / N a(u) and the static permeability K0 d K(0). It was shown in
Johnson et al. (1987) and Auriault et al. (1985) that K and a stay the same if the elastic solid in its frame is replaced with a rigid
solid.
Quantification of the Influence of Material Microstructure on Its Effective Properties

Integral Representation Formulas (IRFs) of Effective Properties
The IRF for the dielectric property of a composite material with two isotropic constituents was first derived in Golden and Papani-
colaou (1983). Naturally, the dielectric property of a composite material should depend on the dielectric properties of the constit-
uent materials and on the microstructure. The thrust of this IRF is that it quantifies the influence of the former through their ratio
appearing in the integrand of the IRF while encoding all the microstructure influence in the positive measure in the IRF. The nth
moment of the measure was shown to be related to the (n þ 1)-point correlation function of the microstructure, n ¼ 0, 1, 2,/.
The IRF was derived by applying Herglotz functions theory. This result has been applied to study bone structures (Kenneth
et al., 2011). The generalization of IRFs from dielectric composites to elastic composites has been discussed in Milton (2002)
(see the references therein) and in Ou (2012). In this section, we will focus on the IRF for dynamic permeability derived in Avella-
neda and Torquato (1991) by summarizing the key ingredients in its derivation and discuss some of its implications.
The IRF of the dynamic permeability of an isotropic porous media of rigid solid matrix saturated with fluid with kinematic
viscosity n is
Z
v Q1 QdGðQÞ
K ðuÞ ¼ (7)
F 0 1 iuQ
where G(Q) ¼ 0 for all Q 0 and G(Q) ¼ 1 for Q Q1 for some positive constant Q1, whose meaning will be made precise later.
Similar to the features of the IRFs mentioned above, (7) provides a representation of K that quantifies its dependence on
frequency through the integrand while encoding all its microstructure dependence through the measure dG F . Here F is the
formation factor.
One key ingredient in the derivation of (7) is the following spectral properties of the Stokes equation. Let Jn and 3 n be the eigen
functions and eigen values of the Stokes equation, V 1 the region occupied by the pore fluid and vV the fluid–solid interface
DJn þ VQn ¼ 3 n Jn and V,Jn ¼ 0 in V 1 ; Jn ¼ 0 on vV; Qn dðv3 n Þ1 ; (8)

0 < 3 1 3 2 / and / N as n / N. The eigenfunctions are orthonormal in the sense
3n
Z
1
Jm ðxÞ,Jn ðxÞdx ¼ dmn ; ðKronecker deltaÞ (9)
jV 1 j V 1
RN
In terms of these eigenfunctions, the Laplace transform b v ðx; sÞd 0 vðx; t Þest dt of the solution to the linearized Navier-Stokes
equation with a forcing term in an arbitrary direction e with magnitude v0,
!
vv p
¼ V þ nDv þ v0 edðt Þin V 1 ; V,v ¼ 0 in V 1 ; v ¼ 0 on vV; (10)
vt rf
can be represented as
X
N
1
b
v ðx; sÞ ¼ v0 bn Jn ðxÞ ; (11)
n¼1
1=Qn þ s
Another key ingredient in the derivation is the functional representation of K(u), derived from homogenization theory, as the
spatial average of b
v
n XN
b2n
K ðsÞ ¼ b
v ðx; sÞ,e ¼ nf : (12)
v0 n¼1
1=Q nþs
where the second equality results from substituting (11) in to the functional expression. This leads to the IRF in (7) and the explicit
P
P b2n
expressions for the formation factor F d (f nN¼ 1 bn2) 1 and GðQÞd PQNn Q 2 .
b
n¼1 n
It was proved in Miao-Jung Yvonne (2014) that the well-known JKD dynamic permeability
,0sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1
4iT 2 K 20 rf u iTK0 rf u
K ðuÞdK0 @ 1
D
A (13)
hL2 f2 hf
can indeed be represented as the IRF in (12) with Q1 ¼ xp and a probability measure dGðuÞ ¼ cI ðuÞ jðuuÞ du þ xrp dxp , where
pffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
C uðC uÞ C þ C2 þ4C22 2C2 xp ðxp C1 Þ
jðuÞdp C2 2 þuðC1 uÞ ; C2 dFkn 0 ; C1 d4CL2 Fk0
; xp d 1 2 1 ; rd ; cI the characteristic function of the interval [0, C1], du
½ 2 1 2 2xp C1
the Lebesque measure and d is the Dirac measure. This implies that the microstructure information influences L, the electrically
weighted average of volume-to-surface ratio of the dynamically connected pore space, through the first moment and the second
moment of measure dG as follows
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u
u 2K0 T
L¼t h i
m2 ðdGÞ
f 2 1
ðm1 ðdGÞÞ
qffiffiffiffiffiffiffiffi
It is interesting to note that the empirical formula for L suggested by JKD (Pride, 1992) is Lz 2TK f=4 . Another implication of
0
(12) is that the dynamic tortuosity function a(u) also assumes an IRF which contains a simple pole at u and a Herglotz function.
Consequently, given infinite-tortuosity T and data of K(u) at M distinct frequencies, a(u) can be approximate to very high accuracy
as (Miao-Jung Yvonne, 2014).
ihf XM
rj 1
aðuÞz þTþ ; rj > 0; < pj < 0 (14)
rf K0 u j¼1
iu pj Q1
This result provides an efficient numerical scheme for handling the memory term in (3).
Permeability and Tortuosity of Anisotropic Materials

The engineering community has a long history of trying to incorporate the influence of the microstructure, for example, anisotropy,
of composite materials into the effective properties. Among them, the permeability tensor and the effective elasticity tensor are of
special interest in the poroelasticity community. A number of experimental, analytical and numerical studies have investigated the
relationship between the permeability tensor and measures of the pore architecture. Such studies have shown that the permeability
tensor depends on the size, shape and directionality of pores. A simple relationship between the permeability tensor and a tensorial
descriptor of the spatial directionality of pores, the fabric tensor, A, was previously proposed by our group, and it is presented below.
We will also present in this section new results based on generalizing the IRF in (7) to arbitrary anisotropic cases.
Fabric tensor
The fabric tensor serves as a measure of the degree of structural anisotropy of the porous medium (Cowin, 1985, 2004; Cowin and
Cardoso, 2011, 2014). Use of the fabric tensor is restricted to materials with orthotropic or higher symmetry. The eigenvectors of F
are the principal axes of material symmetry of the porous solid medium and the eigenvalues of F provide a measure of the distri-
bution of porous volume fraction in the direction of the principal axes of material symmetry. As with any symmetric positive defi-
nite second order tensor in 3D, the fabric tensor may be represented as an ellipsoid. The ellipsoid is one with three unequal axes for
an orthotropic fabric, one unique axis for a transversely isotropic fabric (forming an ellipsoid of revolution about the unique axis)
and the ellipsoid becomes a sphere in the case of an isotropic fabric. The fabric tensor is a good measure of the pore structure anisot-
ropy in cancellous bone tissue, as well as the mechanical and fabric main directions that coincide in cancellous bone. The second
rank fabric tensor A is dimensionless, symmetric, and its invariants I, II and III are related to the traces of A, A2 and A3 by
1 1
I ¼ tr ðAÞ; II ¼ tr ðAÞ2 tr A2 ; III ¼ tr ðAÞ 3tr A2 þ 2tr A3
2 6
The fabric tensor is normalized by setting tr(A) ¼ 1.
Permeability tensor as a function of fabric

The relationship between the second-rank intrinsic permeability tensor K, and the fabric tensor A is obtained by assuming that K is
an isotropic function of A. The relationship between two second-rank symmetric tensors in which one is an isotropic function of the
other then produces the relationship (Cowin and Cardoso, 2011).
0 1
X
3
Kij ð0Þ ¼ k0 @K1 dij þ K2 Aij þ K3 Aiq Aqj A (15)
q¼1
with Ki, i ¼ 1, 2, 3 being functions of f, II and III and k0 dp2 ð2K1 þ K2 Tr ðAÞ þ K3 TrðA,At ÞÞ represents the value of the permeability
2
tensor when it is averaged over all possible directions at a point.

The expression in (15) takes into account dissipation phenomena due to viscous losses, however, it is adequate only for low
frequencies of fluid motion and needs to be corrected to take into account the change in fluid flow regime occurring between
low and high frequencies of wave propagation (Cardoso and Cowin, 2011; Cowin and Cardoso, 2011). This correction results
in the following expression for the dynamic permeability
0 1
J1 ð fdÞ @ X3
Kij ðuÞ ¼ k0 1 2 K1 dij þ K2 Aij þ K3 Aiq Aqj A; (16)
fd J0 ð fdÞ q¼1
qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
f d iurf =h is the viscous skin depth, d the squared average diameter or pores, and J0, J1 are the zerothorder and first-order
Bessel functions of first kind, respectively. This correction was originally introduced by Johnson et al. (1987) describing a dynamic
permeability in a porous medium system characterized by cylindrical tubes.
The local fabric tensor A can be obtained from processing the digitized images of samples for parameters by fitting the Mean
Interception Length (MIL) in all directions by ellipsoids (Harrigan and Mann, 1984; Tabor, 2009; Tabor and Rokita, 2007).
An interesting feature of this empirical approach is that other effective properties such as the drained elasticity tensor can also be
regarded as certain combinations of the fabric tensor with coefficients dependent on f, II and III (Cardoso and Cowin, 2011; Cowin,
1985, 1986). Despite its empirical nature, it is shown in Tabor (2009) that fabric tensor is able to explain at least 90% of the vari-
ation of the apparent elastic constants.
Generalized IRF for anisotropic permeability

To generalize the IRF for isotropic (or sometimes referred to as scalar-valued) K in (Avellaneda and Torquato, 1991) to the perme-
ability function for anisotropic materials, we note the direction-dependent version of the Laplace transformed (10) is
.
i
sb p r þ nDb
v i ¼ V b v i þ v0 ei ; V,b
v i ¼ 0 in V 1 b
v ¼ 0 on vV (17)
v i has the
where ei is the unit vector in the ith direction. Similar to the expression in (11), the direction-dependent solution b
following expression
X
N
1
v i ðx; sÞ ¼ v0
b bin Jn ðxÞ ; (18)
n¼1
1=Qn þ s
with bni defined as

Z
1
bin ¼ ei , Jn ðxÞdx eei ,hJn i (19)
jV 1 j V1
Finally, using the functional representation of permeability by L. Tartar in Sánchez-Palencia (1980), we have the expression for
the (i, j) component of K,
nD i E XN j
bin bn
kij ðsÞ ¼ b
v ðx; sÞ,ej ¼ nf : (20)
v0 n¼1
1=Qn þ s
In matrix form, it is expressed in terms of the outer products hJni 5 hJni
nD i E XN
hJn i5hJn i
K¼ b
v ðx; sÞ,ej ¼ nf : (21)
v0 n¼1
1=Qn þ s
P
Define F d (f nN¼ 1hJni 5 hJni) 1, which is termed the formation tensor in the physics literature, and note that hJni 5 hJni
is a real-valued, symmetric, nonnegative matrix for each n. This means K is a matrix-valued function holomorphic in Cy(N, 3 1]
on the complex s-plane and can be represented as an IRF with real, positive semidefinite matrix-valued distribution G(Q) d
P
fF( Qn < QhJni 5 hJni)
Z Q1
QdGðQÞ
K ðsÞ ¼ nF 1 (22)
0 1 þ sQ
Note that G(Q) ¼ 0 for Q 0 and G(Q) ¼ I for Q > Q1. This IRF provides a precise characterization of microstructure’s influence
on K through the matrix-valued positive semidefinite measure dl(Q): ¼ F 1QdG(Q), which is clearly independent of u. Recall that
s d iu. Most importantly, this IRF separates K(s)’s dependence on the frequency u from its dependence on the microstructure.
Hence it provides the foundation of dehomogenization, which is to obtain information on dl(Q) from values of K at distinct
frequencies.
We would like to emphasize that the complexification, which is necessary in deriving the IRF, is applied to the frequency u, not
the space variable x and hence it does not restrict the applicability of our approach to problems in R2. Even though this approach
works in Rn for any n 2, we are mainly interested in problems in R3. As was in the isotropic case (Miao-Jung Yvonne, 2014), with
a change of variable x d 1/s and R(x) d s(nF)K(s), we see that R(x) is a matrix-valued Stieltjes function (Fritzsche et al., 2015).
Z Q1
QdGðQÞ
RðxÞ ¼ (23)
0 xQ
Computation of Fabric Tensors and Static Permeability of Cancellous Bone Samples

Sample Preparation
Three human calcanei were obtained from the National Disease Research Interchange (NDIR; Philadelphia, PA) resource center.
Bone donors were female who died of cardio/pulmonary failure, drug/alcohol abuse, cancer or natural causes. Calcanei were har-
vested within 24 h postmortem, and immediately fixed in 10% formaldehyde. Upon arrival at our laboratory, calcanei were care-
fully cleaned free of soft tissues and immersed in formaldehyde at 20 C for one more day and stored at 4 C.
Micro-CT Image Acquisition

Calcanei were thawed to room temperature prior to scanning with an 1172 SkyScan high-resolution mCT system (SkyScan, Bel-
gium). X-ray projections were acquired at a nominal isotropic voxel size resolution of 13.47 mm using a 0.5 mm aluminum filter
to eliminate beam-hardening artifacts. X-ray projections were generated every 0.2 of rotation, obtaining 900 consecutive projec-
tions. To produce high-contrast, low-noise images the projections were averaged four times, and a median filter was used to prevent
speckle noise formation. Due to the large size of specimens, four consecutive vertical connected scans were needed to scan each
bone; each of these scans consumed approximately 11 h. Hydroxyapatite rods (HA, 2 mm diameter, 0.25 and 0.75 gHA/cm3)
were scanned using the same protocol to calibrate images for tissue mineral density, TMD (Palacio-Mancheno et al., 2014).
Image Processing
Approximately 4500 images per sample were reconstructed from X-ray projections using the back-projection reconstruction algo-
rithm in NRecon software (Skyscan, v.1.6.1.1, SkyScan). Hounsfield unit (HU) and tissue mineral density calibration procedures
were performed in CTAn software (CT Analyzer, v.1.6.1, SkyScan, Belgium). Four volumes of interest (VOIs) were selected from
regions that contained water, air, 0.25 or 0.75 gHA/cm3. The mean grayscale index value from water and air was used to calibrate
images in HU, and the mean grayscale index values from the 0.25 and 0.75 gHA/cm3 mineral rods were used to generate a calibration
curve between the grayscale color in each pixel and the corresponding mineral density in gHA/cm3. After image density calibration,
the separation (image segmentation) between mineralized and soft tissues in each scan was performed using a mean global
threshold value. The threshold value was determined by analyzing the images using an edge detection algorithm (ImageJ v 1.37,
National Institutes of Health). The tissue mineral density threshold obtained through this edge detection procedure was
0.40 gHA/cm3 (Souzanchi et al., 2012). More precise calibration methods have been proposed in bone biomechanics; these
more precise calibration methods also account for the attenuation behavior of organics and water, rather than neglecting the latter
two (see Blanchard et al., 2016 and references therein).
Global Measurements of Microarchitecture

For each VOI, global architectural parameters were measured, including the porosity (f), trabecular number (Tb.N), trabecular
thickness (Tb.Th), and trabecular separation (Tb.Sp). Since images were calibrated for mineral density, volumetric bone mineral
density (vBMD) and TMD were obtained using built-in algorithms in CTAn software employing the guidelines for assessment of
bone microarchitecture using mCT.
Directional Measurements of Microarchitecture

The directional variation of pore orientation was calculated via the measurement of the mean intercept length (MIL) tensor (Har-
rigan and Mann, 1984) M using CTAn software. Then, values of the fabric tensor A eigenvalues F1, F2 and F3, were obtained by taking
the inverse square root of the eigenvalues of MIL tensor M. Fabric components were normalized by dividing each one by the sum
F1 þ F2 þ F3. The three eigenvectors of A represent the principal axes of material symmetry, which also correspond to the principal
orientations of trabeculae.
Realignment of Images to Principal Axes of Symmetry

A 12 12 12 mm3 cubic VOI was obtained at the center of the coronal plane (medial–lateral–inferior–superior directions in the
calcaneum). The fabric tensor was measured on each VOI to determine the relative alignment between the principal directions of the
fabric and the three orthogonal planes formed by the faces of the cubic sample. Images were rotated in DataViewer and the fabric
was re-measured until the principal directions were coincident with the faces of the cubic sample. The VOI was digitally cropped to
7 7 7 mm3 at the center of each rotated cubic sample to perform micro-computational fluid dynamic (mCFD) analyses aligned
to the principal directions of bone microarchitecture, see Fig. 1A. A 7 7 7 mm fluid volume was added using IrfanView (v.4.44)
before and after the VOI on each of the three directions analyzed for all trabecular bone samples, see Fig. 1B–D, (Palacio-Mancheno
et al., 2014).
Pore Diameter Measurements

Because of the anisotropy of the porous media, it is unlikely that the cross section area and the equivalent pore diameter will be
similar along the three interrogated directions in each sample. Therefore, we measured the average pore diameter in all the images
(xy-, xz- and yz-planes) normal to the direction of the permeability test. The average pore diameter for each direction was obtained
from 2D measurements of trabecular separation (Tb.Sp.) in CTAn.
Computational Fluid Dynamic Simulations

The stack of images representing the VOI and the added fluid domains was imported in Mimics Research (version 19, Materialize,
Leuven, Belgium), and the regions corresponding to bone and fluid were segmented apart using an automated thresholding algo-
rithm (Fig. 1B–D). The 3D mask of the fluid comprehensive of the fluid domains and the fluid inside the trabecular bone was trans-
ferred from Mimics into 3Matic Research (version 11, Materialize, Leuven, Belgium) for the generation of a 3D adaptive volumetric
mesh. The finite element mesh was then exported from 3Matic into Abaqus/CFD (v. 6.14 Simulia, Providence, RI), where the CFD
problem was solved. The transient and steady state fluid dynamic behavior of the fluid inside the trabecular bone pores was inves-
tigated using an explicit time-marching integration approach. The fluid was assumed to be Newtonian, incompressible and viscous.
The fluid flow behavior is governed by the Navier-Stokes equations for an incompressible, viscous fluid where g is the acceleration of
gravity and v and p are respectively the flow velocity and the pressure,

vv
rf þ v,Vv ¼ Vp þ nDv þ rf g; V,v ¼ 0 (24)
vt
The CFD simulations were conducted using a static pressure difference of 0.1 Pa across the trabecular bone sample. The outer
boundary of the fluid pore space was considered to be perfectly rigid, with no slip, and no penetration boundary wall conditions.
Since the fluid saturating the cancellous bone structure in our experiments is water, the fluid mass density rf ¼ 1000 Kg/m3, bulk
modulus Kf ¼ 2.25 GPa and viscosity n ¼ 10 3 Pa s. The time step increment was selected as 0.01 s, and the time integration
parameter in Abaqus was set by default to q ¼ 0.5, producing a second order accurate semiimplicit method suitable for time accu-
rate transient analysis. The total simulation time was 5 s in order to capture both transient and steady state response in all simu-
lations. The CFD was performed using tetrahedral fluid elements (FC3D4). An adaptive algorithm (3Matic) was used to create the
fluid mesh (De Paolis et al., 2017). CFD simulations were performed on a high-end workstation with 24 core Xeon CPUs 2.7GHz,
graphic accelerator with 2700 GPU cores, and 512 GB of RAM. The CPU time for each simulation varied between 1–3 h per
simulation.
Numerical Results
Global and directional parameters of microarchitecture were measured on the trabecular bone samples analyzed in this study.
The porosity of the three samples analyzed in this study was 76%, 84% and 94%, spanning a range of values that are char-
acteristic of normal and osteoporotic bone. Trabecular thickness and separation are global measurements of the porous
medium, and were found as Tb.Th. ¼ 182, 133, 125 mm, and Tb.Sp. ¼ 615, 398, 806 mm, respectively for the three bone
samples.
Directional measurements of permeability were obtained from mCFD numerical simulations on each of the trabecular bone
samples tested along the three principal directions of microarchitecture. Plots of the pressure, the fluid velocity, and the fluid stream-
lines in the principal directions of these samples are shown in Fig. 2. The three principal components of the static permeability
tensor were determined from averaged fluid velocity, fluid viscosity and pressure gradient applied on each test. Directional measure-
ments of the pore architecture (i.e., pore diameter and fabric), fluid velocity and static permeability are reported as a function of the
x, y and z direction in Table 1. In Fig. 3, the data for the fabric components, permeability.
and pore size is reported for each direction on the three bone samples. It can be observed that the preferential alignment of pores,
along direction z, has the largest magnitude for the fabric, permeability and equivalent diameter pore size, see Fig. 3A–C. Also, there
is a strong linear trend between permeability and fabric, see Fig. 3D; however, we can observe that such linear correlation is
Fig. 2 (A–C) pressure, (D–F) fluid velocity, and (G–I) stream lines in the principal directions of three bones with 76%, 84% and 94% porosity.
Table 1 Fabric, static permeability and pore size as functions of directions x, y and z
Direction Pore diameter (mm) Fabric Fluid velocity (mm/s) Static permeability (m2)
Bone 1 f ¼ 76% x 387 0.288 39.38 2.10E09

y 478 0.274 23.75 1.26E09
z 468 0.438 132.02 7.02E09
Bone 2 f ¼ 84% x 441 0.291 91.34 5.37E09
y 640 0.333 126.28 7.43E09
z 740 0.377 183.45 10.79E09
Bone 3 f ¼ 94% x 814 0.257 274.52 18.07E09
y 785 0.62 353.31 23.25E09
z 931 0.382 395.08 26.00E09
Fig. 3 (A) Fabric, (B) permeability and (C) pore size reported as a function of directions x, y and z, and porosity. (D) Trend between permeability
and fabric eigenvalues, and (E) trend between permeability and pore size for each bone sample.
dependent on the porosity of the sample, since the linear relationship for each sample seems shifted to the left for low porosities and
towards the right for high porosities. Similarly, there is a clear inverse correlation between permeability and pore size, which is also
modulated by the porosity in each sample, see Fig. 3E. The computed static permeability, the fabric tensor, the pore size, the
porosity and k0 for each bone sample are then used to compute the corresponding values of k0K1, k0K2 and k0K3 in (15). With
the computed pore size, the graphs of the dynamic permeability defined in (16) and the corresponding dynamic tortuosity are eval-
uated and shown in Fig. 4. In Fig. 5, the relationship between the average micro-velocities measured at the pore level in the mCFD
simulations is compared with the apparent level fluid flux, corresponding to the product of the porosity times the apparent level
macro-velocity (Darcys velocity).
Fig. 4 Dynamic permeability defined in (16) as a function of frequency for the 76%, 84% and 94% porosity bone from left to right in the top row
panels, and the corresponding tortuosity is also shown as a function of frequency in the three bottom panels for the same bone samples. From
Johnson, D. and Myklebust, H. (1967). Learning disabilities: Educational principles and practices, New York, 1967, Grune and Stratton, Inc, p. 37.
Fig. 5 Relationship between pore micro-velocities and the product of the porosity and the apparent level macro-velocity (Darcys velocity).
Discussion
The numerical results presented in this work suggest a strong trend between the fabric tensor and the static permeability tensor,
which is different for each bone, possibly because their different porosity level. This numerical result supports the notion that
anisotropy of the permeability tensor in trabecular bone is a consequence of the porosity and mainly the fabric tensor describing
the directionality of the pores. Also, an inversely proportional trend was observed between pore size and permeability, suggesting
that the pore shape is also affecting importantly the permeability of the porous media. The static permeability presented in Table 1
exhibits a variability (10 9–10 8 m2) that falls within the data in studies reporting experimental measurements of the permeability
(10 12–10 8 m2) on cancellous bone (Abdalrahman et al., 2015; Accadbled et al., 2008; Baroud et al., 2004; Benalla et al., 2014;
Birmingham et al., 2013; Bleiler et al., 2015; Cardoso et al., 2013; Coelho et al., 2011; Coughlin and Niebur, 2012; Cowin and
Cardoso, 2014; Grimm and Williams, 1997; Kameo et al., 2016; Kohles et al., 2001; Kohles and Roberts, 2002; Kreipke and Niebur,
2017; Li et al., 1987; Metzger et al., 2015; Nauman et al., 1999; Pakula et al., 2008; Sandino et al., 2014; Souzanchi et al., 2013;
Syahrom et al., 2015; Tae-Hong and Hong, 2000; Teo and Teoh, 2012). The variability of the intrinsic permeability in porous media
is due to the dependence of the permeability on the porosity (Nauman et al., 1999; Grimm and Williams, 1997; Benalla et al., 2013)
and the microstructure of the sample (Nauman et al., 1999; Kohles et al., 2001; Baroud et al., 2004; Kohles and Roberts, 2002).
Further studies comprising a larger number of samples are needed to extend and generalize the results outlined by the examples
treated here.
Based on our experience with IRFs, the n-point correlation functions form a natural hierarchy for quantifying the difference
between microstructures. The volume fraction (i.e., porosity) is related to only the 1-point correlation functions, which are low
in the hierarchy in the sense that among the microstructures that share the same porosity, we can use their two-point correlation
functions to further divide them into subgroups; the microstructures within each subgroup are more similar to each other than those
with those outside the subgroup. This process can be continued by looking into higher order correlation functions. With the IRF
approach, we expect the information of n-point correlation functions to be coded in the moments of the positive measure in the
IRF. Based on the definition of permeability tensor from homogenization, we see that these moments depend not only on the
microstructure but also on the spectral properties of the Stokes’ operator acting in the pore space. Similarly, the measure in
the IRF for effective elasticity tensor is influenced by the microstructure and the linear elasticity operator. In other words, an effective
property’s dependence on the microstructure is a consequence of the interaction between the underlying operator (e.g., Stokes oper-
ator for permeability) and the microstructure. To be more specific, for example, in the IRF of dielectric properties of two-phase
composite materials, the nth moments of the measure is the n þ 1-fold convolution of the fundamental solution of the Laplacian
operator and the characteristic function that defines the microstructure. We are currently working on generalizing the dielectric
result to static permeability. Another future work from here is to extend the dehomgenization scheme in Miao-Jung Yvonne
(2014) to anisotropic case described in (22) and compare the measure F 1qdG with the fabric tensor. Another future direction
is to apply mCFD to compute the dynamic permeability from its definition (i.e., Stokes equation with time-harmonic pressure
gradient), instead of the from (22). Using these data, we can then compute using the algorithm presented in Miao-Jung Yvonne
(2014) to compute the rj and pj in (14) for each principal direction and use them to compute the moments of the measures in
the IRFs; these moments will serve as characterization of the viscodynamic properties of the bone samples in addition to the
infinite-frequency tortuosities.
Acknowledgments
The work of Miao-jung Yvonne Ou was partially sponsored by the US National Science Foundation grant NSF-DMS-1413039. This work of Luis
Cardoso and Annalisa De Paolis was supported by NSF (CMMI-1333560, MRI-0723027, and MRI-1229449), and National Institute of Health grant
NIHDK103362.
References
Abdalrahman, T., Scheiner, S., & Hellmich, C. (2015). Is trabecular bone permeability governed by molecular ordering-induced fluid viscosity gain? Arguments from re-evaluation of
experimental data in the framework of homogenization theory. Journal of Theoretical Biology, 365, 433–444.
Accadbled, F., Ambard, D., De Gauzy, J. S., & Swider, P. (2008). A measurement technique to evaluate the macroscopic permeability of the vertebral end-plate. Medical
Engineering & Physics, 30(1), 116–122.
Ascenzi, M.-G., Gill, J., & Lomovtsev, A. (2008). Collagen orientation patterns around osteocyte lacunae in human secondary osteons by confocal microscopy. Journal of
Biomechanics, 41(16), 3428–3437.
Auriault, J. L., Borne, L., & Chambon, R. (1985). Dynamics of porous saturated media, checking of the generalized law of Darcy. The Journal of the Acoustical Society of America,
77, 1641.
Avellaneda, M., & Torquato, S. (1991). Rigorous link between fluid permeability, electrical conductivity, and relaxation times for transport in porous media. Physics of Fluids A: Fluid
Dynamics, 3, 2529.
Baroud, G., Falk, R., Crookshank, M., Sponagel, S., & Steffen, T. (2004). Experimental and theoretical investigation of directional permeability of human vertebral cancellous bone for
cement infiltration. Journal of Biomechanics, 37(2), 189–196.
Benalla, M., Palacio-Mancheno, P. E., Fritton, S. P., Cardoso, L., & Cowin, S. C. (2013). Dynamic permeability of the lacunar–canalicular system in human cortical bone.
Biomechanics and Modeling in Mechanobiology, 1–12.
Benalla, M., Palacio-Mancheno, P. E., Fritton, S. P., Cardoso, L., & Cowin, S. C. (2014). Dynamic permeability of the lacunar–canalicular system in human cortical bone.
Biomechanics and Modeling in Mechanobiology, 13(4), 801–812.
Biot, M. A. (1956a). Theory of propagation of elastic waves in a fluid-saturated porous solid. I. Low-frequency range. The Journal of the Acoustical Society of America, 28, 168.
Biot, M. A. (1956b). Theory of propagation of elastic waves in a fluid-saturated porous solid. II. Higher frequency range. The Journal of the Acoustical Society of America, 28(2),
179–191.
Biot, M. A. (1962). Mechanics of deformation and acoustic propagation in porous media. Journal of Applied Physics, 33(4), 1482–1498.
Birmingham, E., Grogan, J. A., Niebur, G. L., McNamara, L. M., & McHugh, P. E. (2013). Computational modelling of the mechanics of trabecular bone and marrow using fluid
structure interaction techniques. Annals of Biomedical Engineering, 41(4), 814–826.
Blanchard, R., Morin, C., Malandrino, A., Vella, A., Sant, Z., & Hellmich, C. (2016). Patient-specific fracture risk assessment of vertebrae: A multiscale approach coupling x-ray
physics and continuum micromechanics. International Journal for Numerical Methods in Biomedical Engineering, 32(9).
Bleiler, C., Wagner, A., Stadelmann, V. A., Windolf, M., Köstler, H., Boger, A., Gueorguiev-Rüegg, B., Ehlers, W., & Röhrle, O. (2015). Multiphasic modelling of bonecement injection
into vertebral cancellous bone. International Journal for Numerical Methods in Biomedical Engineering, 31(1).
Carcione, J. M. (2001). Wave fields in real media: Wave propagation in anisotropic, Anelastic and porous media. Oxford: Pergamon-Elsevier.
Cardoso, L., & Cowin, S. C. (2011). Fabric dependence of quasi-waves in anisotropic porous media. The Journal of the Acoustical Society of America, 129(5), 3302.
Cardoso, L., Fritton, S. P., Gailani, G., Benalla, M., & Cowin, S. C. (2013). Advances in assessment of bone porosity, permeability and interstitial fluid flow. Journal of Biomechanics,
46(2), 253–265.
Chaffai, S., Padilla, F., Berger, G., & Laugier, P. (2000). In vitro measurement of the frequency dependent attenuation in cancellous bone between 0.2 and 2 mhz. Journal of the
Acoustical Society of America, 108, 1281–1289.
Coelho, P. G., Fernandes, P. R., & Rodrigues, H. C. (2011). Multiscale modeling of bone tissue with surface and permeability control. Journal of Biomechanics, 44(2), 321–329.
Coughlin, T. R., & Niebur, G. L. (2012). Fluid shear stress in trabecular bone marrow due to lowmagnitude high-frequency vibration. Journal of Biomechanics, 45(13), 2222–2229.
Cowin, S. C. (1985). The relationship between the elasticity tensor and the fabric tensor. Mechanics of Materials, 4(2), 137–147.
Cowin, S. C. (1986). Fabric dependence of an anisotropic strength criterion. Mechanics of Materials, 5(3), 251–260.
Cowin, S. C. (1999). Bone poroelasticity. Journal of Biomechanics, 32(3), 217–238.
Cowin, S. C. (2004). Anisotropic poroelasticity: Fabric tensor formulation. Mechanics of Materials, 36(8), 665–677.
Cowin, S. C., & Cardoso, L. (2011). Fabric dependence of wave propagation in anisotropic porous media. Biomechanics and Modeling in Mechanobiology, 10(1), 39–65.
Cowin, S. C., & Cardoso, L. (2014). Difficulties arising from different definitions of tortuosity for wave propagation in saturated poroelastic media models. ZAMM-Journal of Applied
Mathematics and Mechanics/Zeitschrift fu¨r Angewandte Mathematik und Mechanik, 94(9), 694–704.
De Paolis, A., Watanabe, H., Nelson, J. T., Bikson, M., Packer, M., & Cardoso, L. (2017). Human cochlear hydrodynamics: A high-resolution mct-based finite element study. Journal
of Biomechanics, 50, 209–216.
Fellah, Z. E. A., Chapelon, J. Y., Berger, S., Lauriks, W., & Depollier, C. (2004). Ultrasonic wave propagation in human cancellous bone: Application of the biot theory. The Journal of
the Acoustical Society of America, 116(1), 61–73.
explanation of bone strength. Journal of Theoretical Biology, 260(2), 230–252.
B. Fritzsche, B. Kirstein, and C. Mädler. On matrix-valued stieltjes functions with an emphasis on particular subclasses. arXiv preprint arXiv:1506.01600, 2015.
Fry, F. J., Barger, J. E., et al. (1978). Acoustical properties of the human skull. The Journal of the Acoustical Society of America, 63(5), 1576.
Gailani, G., & Cowin, S. (2011). Ramp loading in russian doll poroelasticity. Journal of the Mechanics and Physics of Solids, 59(1), 103–120.
Golden, K., & Papanicolaou, G. (1983). Bounds for effective parameters of heterogeneous media by analytic continuation. Communications in Mathematical Physics, 90(4),
473–491.
Grimm, M. J., & Williams, J. L. (1997). Measurements of permeability in human calcaneal trabecular bone. Journal of Biomechanics, 30(7), 743–745.
Harrigan, T. P., & Mann, R. W. (1984). Characterization of microstructural anisotropy in orthotropic materials using a second rank tensor. Journal of Materials Science, 19(3),
761–767.
Hellmich, C., & Ulm, F.-J. (2005). Microporodynamics of bones: Prediction of the Frenkel-biot slow compressional wave. Journal of Engineering Mechanics, 131(9), 918–927.
Hosokawa, A., & Otani, T. (1997). Ultrasonic wave propagation in bovine cancellous bone. The Journal of the Acoustical Society of America, 101, 558.
Johnson, D. L., Koplik, J., & Dashen, R. (1987). Theory of dynamic permeability and tortuosity in fluidsaturated porous media. Journal of Fluid Mechanics, 176(1), 379–402.
Kameo, Y., Ootao, Y., & Ishihara, M. (2016). Poroelastic analysis of interstitial fluid flow in a single lamellar trabecula subjected to cyclic loading. Biomechanics and Modeling in
Mechanobiology, 15(2), 361–370.
Kenneth, M., Golden, N. B. M., & Cherkaev, E. (2011). Spectral analysis and connectivity of porous microstructures in bone. Journal of Biomechanics, 44(2), 337–344.
Kohles, S. S., & Roberts, J. B. (2002). Linear poroelastic cancellous bone anisotropy: Trabecular solid elastic and fluid transport properties. Journal of Biomechanical Engineering,
124(5), 521–526.
Kohles, S. S., Roberts, J. B., Upton, M. L., Wilson, C. G., Bonassar, L. J., & Schlichting, A. L. (2001). Direct perfusion measurements of cancellous bone anisotropic permeability.
Journal of Biomechanics, 34(9), 1197–1202.
Kreipke, T. C., & Niebur, G. L. (2017). Anisotropic permeability of trabecular bone and its relationship to fabric and architecture: A computational study. Annals of Biomedical
Engineering, 1–12. https://doi.org/10.1007/s10439-017-1805-9.
Langton, C. M., & Njeh, C. F. (2003). The physical measurement of bone. Taylor & Francis.
Li, G., Bronk, J. T., An, K.-N., & Kelly, P. J. (1987). Permeability of cortical bone of canine tibiae. Microvascular Research, 34(3), 302–310.
McKelvie, M. L., & Palmer, S. B. (1991). The interaction of ultrasound with cancellous bone. Physics in Medicine and Biology, (10), 1331–1340.
Metzger, T. A., Schwaner, S. A., LaNeve, A. J., Kreipke, T. C., & Niebur, G. L. (2015). Pressure and shear stress in trabecular bone marrow during whole bone loading. Journal of
Biomechanics, 48(12), 3035–3043.
Miao-Jung Yvonne, O. (2014). On reconstruction of dynamic permeability and tortuosity from data at distinct frequencies. Inverse Problems, 30(9), 095002.
Milton, G. W. (2002). Section 18.6. The Theory of Composites. Cambridge University Press. Cambridge Books Online.
Morin, C., & Hellmich, C. (2014). A multiscale poromicromechanical approach to wave propagation and attenuation in bone. Ultrasonics, 54(5), 1251–1269.
Nauman, E. A., Fong, K. E., & Keaveny, T. M. (1999). Dependence of intertrabecular permeability on flow direction and anatomic site. Annals of Biomedical Engineering, 27(4),
517–524.
Odgaard, A., Kabel, J., van Rietbergen, B., Dalstra, M., & Huiskes, R. (1997). Fabric and elastic principal directions of cancellous bone are closely related. Journal of Biomechanics,
30(5), 487–495.
Ou, M. Y. (2012). Two-parameter integral representation formula for the effective elastic moduli of two-phase composites. Complex Variables and Elliptic Equations, 57(2–4),
411–424.
Pakula, M., Padilla, F., Laugier, P., & Kaczmarek, M. (2008). Application of biots theory to ultrasonic characterization of human cancellous bones: Determination of structural,
material, and mechanical properties. The Journal of the Acoustical Society of America, 123(4), 2415–2423.
Palacio-Mancheno, P. E., Larriera, A. I., Doty, S. B., Cardoso, L., & Fritton, S. P. (2014). 3D assessment of cortical bone porosity and tissue mineral density using high-resolution
mct: Effects of resolution and threshold method. Journal of Bone and Mineral Research, 29(1), 142–150.
Pride, S. (1992). Modeling the drag forces of porous media acoustics. Technical report. Massachusetts Institute of Technology. Earth Resources The Laboratory.
Sánchez-Palencia, E. (1980). Non-homogeneous media and vibration theory. In volume 127 of Lecture Notes in Physics. Springer-Verlag.
Sandino, C., Kroliczek, P., McErlain, D. D., & Boyd, S. K. (2014). Predicting the permeability of trabecular bone by micro-computed tomography and finite element modeling. Journal
of Biomechanics, 47(12), 3129–3134.
Scheiner, S., Pivonka, P., & Hellmich, C. (2016). Poromicromechanics reveals that physiological bone strains induce osteocyte-stimulating lacunar pressure. Biomechanics and
Modeling in Mechanobiology, 15(1), 9–28.
Souzanchi, M. F., Palacio-Mancheno, P., Borisov, Y. A., Cardoso, L., & Cowin, S. C. (2012). Microarchitecture and bone quality in the human calcaneus: Local variations of fabric
anisotropy. Journal of Bone and Mineral Research, 27(12), 2562–2572.
Souzanchi, M. F., Cardoso, L., & Cowin, S. C. (2013). Tortuosity and the averaging of microvelocity fields in poroelasticity. Journal of Applied Mechanics, 80(2), 020906.
Syahrom, A., Kadir, M. R. A., Harun, M. N., & Öchsner, A. (2015). Permeability study of cancellous bone and its idealised structures. Medical Engineering & Physics, 37(1), 77–86.
Tabor, Z. (2009). On the equivalence of two methods of determining fabric tensor. Medical Engineering & Physics, 31(10), 1313–1322.
Tabor, Z., & Rokita, E. (2007). Quantifying anisotropy of trabecular bone from gray-level images. Bone, 40(4), 966–972.
Tae-Hong, L., & Hong, J. H. (2000). Poroelastic properties of bovine vertebral trabecular bone. Journal of Orthopaedic Research, 18(4), 671.
Teo, J. C. M., & Teoh, S. H. (2012). Permeability study of vertebral cancellous bone using micro-computational fluid dynamics. Computer Methods in Biomechanics and Biomedical
Engineering, 15(4), 417–423.
Multiphase Porous Media Models for Mechanics in Medicine: Applications to
Transport Oncophysics and Diabetic Foot
Pietro Mascheroni and Raffaella Santagiuliana, University of Padova, Padova, Italy
Bernhard Schrefler, Technical University of Munich, Garching bei München, Germany; and Houston Methodist Research Institute,
Houston, TX, United States
Introduction 155
Multiphase Porous Media Models for Tumor Growth 156
Mathematical Model 157
Results From the Model 158
Tumor growing in vitro and ex vivo 158
Mechanical compression of tumor spheroids 158
Effects of interfacial tensions 159
Melanoma growth and angiogenesis 160
Diabetic Foot 162
Mathematical Model 163
Numerical Example for Gait Cycle 163
Conclusions 165
Further Reading 166
Glossary
Conservation equations In general, this expression denotes a set of laws describing the balance of mass, momentum, and
energy in a physical system. These equations are valid for a wide variety of systems and need to be closed by suitable
constitutive relations to address a specific problem.
Finite element method In the context of boundary value problems, the finite element method is a numerical technique for
computing approximate solutions to partial differential equations.
Mechanotransduction This term encompasses several mechanisms by which cells sense mechanical stimuli and convert them
into biochemical signals, producing specific cellular responses.
Partial differential equations These are mathematical equations that involve functions of multiple independent variables and
their partial derivatives.
Porous media A porous medium is a material containing a solid phase, denoted as the solid skeleton, and several
interconnected pores, that is, voids. These pores are typically filled with one or more fluid phases, which could be in a liquid or
gaseous state.
Introduction
Biological systems are very complex and span many orders of magnitude, ranging from molecules, cells, organs, and up to the whole
organism. These levels are tightly entangled, including multiple feedbacks and cross talks between the different parts. One way of
deconvolution of this complexity is the use of modeling based on physical and chemical principles. Further, the knowledge of many
quantities of interest is often not accessible through direct measurements, and some model is necessary to infer these from indirect
measurements. Hence, models are compulsory for aiding our understanding of biological processes. This is now well understood,
and a plethora of models have been developed. Here, we refer particularly to mathematical models, and, to restrict our field, we do
not discuss purely data-driven approaches (black box models). Our focus is on the modeling of physical and chemical processes for
which experimental data can be obtained. These models are usually cast in terms of partial differential equations. The field of appli-
cation of this approach is extraordinarily large. As a few key examples, we mention cardiovascular diseases, lifetime assessment of
stents, brain injuries, prosthetics, locomotion, injury biomechanics, and modeling of the intervertebral disk. Notably, in the last few
years, several models for drug delivery and tumor growth have been introduced in the research field. Starting from the analyses for
the design of injectable micro- and nano-devices for cancer therapy, additional methods have followed for the improvement of
immunotherapies, imaging of tumor lesions, and design of biomedical devices for tumor removal. The mechanics of cellular
processes, including mechanosensing and mechanotransduction, have been extensively described, and studies of cellular constitu-
tive behavior through the use of microfluidic systems and other physical approaches are still an active field of research. The list of
possible applications is far from being exhaustive, and new observations and discoveries appear almost every day. This endeavor

156 Biomechanics j Multiphase Porous Media Models for Mechanics in Medicine
brings together researchers in biology, medicine, engineering, physics, chemistry, material science, and applied mathematics, in
which numerical modeling plays an important role.
The successful development of such models is based on three major aspects: (i) the development of the nonlinear field theory of
mechanics during the 1950s and 1960s; (ii) the introduction of the finite element (FE) method in the 1950s; and, subsequently, (iii)
the rapid advances in computer technologies. Clearly, FE analysis is not the only one used in models dealing with mechanics in
medicine, but is by far the most popular and acknowledged one. It is in fact by now the most used discretization method in applied
sciences and mechanics. Proof of this is the sheer number of books that have been written or are still appearing, and the large
number of general purpose codes based on this method. They address all fields of solid and fluid mechanics, heat transfer, electro-
magnetics, computational chemistry, and physics, and, now in increasing number, also interaction problems between these fields.
This has made simulations possible that in the recent past were simply unthinkable.
To such interaction problems belong also the porous media theories discussed in this article. Such theories consider a generally
deformable solid skeleton and freely moving pore fluids, where the phases are fully connected. The nature of the fluid can be mani-
fold, ranging from water and air to biological fluids and even cell populations which also can be treated as particular fluid phases.
The kinematic quantities are a solid displacement vector, which tracks the movement of the porous solid with respect to a reference
configuration, and specific discharge vectors describing the motion of the fluid phases relative to the solid. The specific discharge is
defined as the rate of fluid volume crossing a unit area of porous solid. The interaction between the solid phase and the fluids is not
the only one considered. Interactions with thermal, chemical, and electromagnetic fields are also currently taken into account. It is
clear that the whole process is usually time dependent, and this aspect has to be taken into account in the development of the
computational implementations. The governing equations for such media are now generally obtained through averaging theories
such as the thermodynamically constrained averaging theory (TCAT) and are then discretized by means of the FE method.
Indeed, the TCAT framework provides a rigorous methodology for developing multiphase, continuum models at any scale of
interest. Larger scale variables are explicitly defined in terms of smaller variables at smaller scales. At the microscale, classical local
conservation equations and thermodynamic expressions can be written. However, as the domains of many problems are too large,
or the phase distributions are too complex, it is usually impossible to model the phenomena of interest directly on such a small
scale. In fact, simulations would be possible only for very small domains. To overcome such problems, many porous media models
are formulated at a larger scale, called the macroscale. Standard continuum mechanics techniques for the formulation of these
models rely on a direct approach, in which the conservation equations are written at the larger scale. Usually, rational thermody-
namic assumptions are enforced to obtain closure relations. Unfortunately, the use of such methods may fail to retain a connection
between larger scale variables and their microscale precursors. Through a set of averaging theorems applied to conservation and
thermodynamic equations at the small scale, TCAT avoids these shortcomings and leads to equations that are both thermodynam-
ically and physically consistent. The theory consistently transforms microscale conservation and thermodynamic equations to the
macroscale and converts averages of microscale derivatives into derivatives of macroscale average quantities. Note that a realistic
description of a multiphase system must include dynamic conservation and thermodynamic equations for all the phases and for
all the points in which they interact. Although TCAT has been primarily employed in hydrology, it impacts also biomechanical
modeling, since the underlying physics and mathematics are related.
Once the governing equations are obtained at the macroscale, their weak form is derived by means of the standard Galerkin
procedure and is then discretized in space by means of the FE method. For the particular type of problems that we discuss, integra-
tion in the time domain is carried out by the finite difference method adopting the q-Wilson procedure. Within each time step, the
equations are linearized by the Newton–Raphson algorithm. The system of equations has been implemented in the three-
dimensional FE code CAST3M (http://www-cast3m.cea.fr) of the French Atomic Energy Commission. A staggered scheme is adop-
ted, with iterations within each time step to preserve the coupled nature of the system.
In the following, we discuss two applications of these procedures. We report on a model for tumor growth and its interactions
with the surrounding environment. Then, we show results for the case of ulceration of the diabetic foot.
Multiphase Porous Media Models for Tumor Growth
Cancer is a collection of related diseases in which some cells of the body start to divide without stopping and eventually spread into
their surroundings. In normal tissues, healthy cells (HCs) grow and divide according to the needs of the organism. When cells grow
old or become damaged, they are eliminated, and new cells take their place. However, when cancer develops, this carefully
controlled process breaks down. As multiple alterations accumulate, old or damaged cells survive when they should die, and
new cells form even if they are not needed. These extra cells divide uncontrolled and may result in abnormal masses called tumors.
Malignant cancerous tumors can spread into surrounding tissues, displacing the neighboring HCs. In addition, as the tumor
develops, some cancer cells are able to detach from the original tumor mass and travel to distant organs in the body through
the circulation. Eventually, these cancerous cells may form metastases, that is, new tumors far from the original formation.
Nowadays, cancer figures among the leading causes of mortality worldwide, with approximately 14 million new cases and 8.2
million cancer-related deaths in 2012. Despite new technological advances and significant efforts (projected national expenditures
for cancer care are expected to total nearly $157 billion in 2020 just in the United States), the initial hopes put in the war on cancer
have been largely disillusioned. Since the 1950s, indeed, age-adjusted cancer mortality rates have decreased by only 11%. Preven-
tion, screening, and treatment success with some cancers have saved millions of lives, but the prognosis for many with metastatic
cancer remains still as gloomy as it was several years ago.
Biomechanics j Multiphase Porous Media Models for Mechanics in Medicine 157
Looking at these premises, researchers from quantitative disciplines such as physicists, mathematicians, and engineers have
contributed to cancer research over the last few years. One contribution results from discoveries and technological developments,
which have led to advances in medical imaging and radiation therapy for the diagnosis and treatment of tumors. A second impor-
tant contribution is brought by bioinformatics, providing the tools to handle large datasets of genome sequences, gene expression
patterns, and cell-signaling networks. Finally, a third contribution has recently gained interest. This direction involves a more quan-
titative investigation of the physical processes underlying the evolution of a tumor. Indeed, it is now recognized that the physico-
chemical properties of several biological barriers are responsible for the transport of cells, particles, and molecules across the tissues.
Remarkably, this transport and its deregulation play a predominant role in cancer physics. Tissue invasion can be considered as
mass transport deregulation at the interface between the cell and the microenvironment; metastasis is a deregulation of local
and distant cellular transport at the scale of the organism; tumor angiogenesis completely alters mass and fluid exchange across
the microcirculation; abnormalities in the signaling pathways that accompany the evasion of apoptosis, growth signal dependence,
and growth inhibitory messages from the microenvironment are also disruptions in molecular transport, since molecular signaling
directly depends on the transport of signaling molecules. In addition, several aspects related to transport control the delivery of ther-
apeutic agents, such as chemotherapeutics or molecularly targeted therapies. To be effective, these substances must pass through
different and heterogeneous tumor and healthy compartments (e.g., vascular, stroma) with distinct physical properties. In fact,
drug delivery is an extremely complex process involving different spatial and temporal scales. It involves a series of events that
take place over several levels, ranging from the organism to the intercellular environment. Throughout these levels, different
phenomena may act as transport barriers, possibly contributing to poor survival rates in cancer therapy. As a first example, the
mononuclear phagocyte system belongs to these barriers, removing foreign substances from the body such as nanoparticles
(NPs) in the blood plasma. A second obstacle is given by the tumor neovasculature, resulting from tumor angiogenesis. The
new vascular network is tortuous and distorted and shows large fenestrations, leading to poor perfusion of the tumor tissue, chaotic
blood flow, and uneven supply of nutrients. However, these fenestrations also allow for the enhanced permeability and retention
effect (EPR), discovered in the 1980s by Maeda and colleagues. Indeed, the increased accumulation of long-circulating macromol-
ecules and NP by extravasation through the tumor blood vessels has been carefully characterized in the last few years. Nevertheless,
the EPR effect may still be hindered because of the altered gradients of the interstitial fluid (IF) pressure in the local tumor environ-
ment. Furthermore, limited drainage of IF (due to the lack of functional lymphatics and extensive fibrosis in the tumor) brings
about pressure rises, reducing the extravasation of therapeutic molecules through advection. Hence, poor perfusion through the
local environment hinders the diffusion of therapeutic agents in the tumor interstitium and affects ultimately the cellular uptake
of NPs. In general, NP uptake by the cells follows endocytosis; only in a second step the payload of the NP might be released
into the cytoplasm.
The concept of biological barriers has brought a new understanding of how transport can modulate cancer biology and efficacy
of therapies. Indeed, several studies have confirmed that altered transport plays a crucial role in cancer and drug delivery, including
the phenomenon of resistance. Transport oncophysics views hence cancer as a disease of multiscale mass transport deregulation,
involving distinct biological barriers at different levels. Computational transport oncophysics provides a set of computational tools
that, together with imaging, data analysis, and quantification, can contribute to rationalize the development of the tumor and opti-
mize the delivery of therapies. This framework should complement classical tools used to study pharmacokinetic and efficacy rela-
tions, with the final aim of creating novel precision tools to rationally tailor individual treatments.
Mathematical Model
We concentrate here on mathematical models for tumor growth. Such models describe the behavior of several tumor constituents,
such as tumor cells (TCs), both viable and necrotic, HCs, extracellular matrix (ECM), IF, neovasculature and co-opted blood vessels,
nutrients, and waste products. A great variety of models have been developed, often based on different starting assumptions and
fields of application. Some of the components mentioned above may be neglected, resulting in different types of models accounting
for diffusion, single-phase flow, and multiphase flow with or without a solid phase. Three major classes of models can be distin-
guished as evidenced in several review papers: discrete, continuum, and hybrid models. Discrete models follow the fate of a single
cell, or a cohort of cells, over time. As such, this modeling framework is not able to capture aspects of tissue mechanics, nor are the
modeled subdomains representative of the whole tumor. However, discrete models are suitable for explaining cell-to-cell cross
signaling and cell response to therapeutic molecules. On the other hand, in continuum models, cancerous tissues are regarded
as domains composed of multiple fluid and solid phases interacting one with the other. Partial differential equations based on
conservation laws and thermodynamics describe the spatiotemporal evolution of the system, but no direct information is provided
at the single-cell level. Finally, hybrid models incorporate different aspects of discrete and continuum models, according to the
problem of interest. For instance, cells may be represented individually, and the IF may be described as a continuum. Within
continuum models, we encounter biphasic solid-fluid models in which the ECM is lumped with the cells and the resulting porous
scaffold is permeated by the IF. An alternative is multiphase flow models, sometimes in a deforming porous material. Among this
last kind, we have developed a general multiphase flow model in an ECM, considered as a deforming porous solid which may
undergo remodeling. This model comprises three fluid phases, namely TCs, divided into living and necrotic cells, HCs, and IF.
The IF transports chemical species such as tumor angiogenic factors (TAFs), nutrients, and therapeutic agents. Transport of these
substances within extravascular space takes place by convection and diffusion. Co-opted blood vessels are included as line elements,
across which blood exchanges nutrients and therapeutic agents with the IF. Angiogenesis is represented by blood vessel density
(density of newly created endothelial cells). The model accounts not only for growth and necrosis, but also for migration of cells
through the ECM, for different stiffness of the cell population with respect to the ECM, buildup of cortical tension between healthy
and tumor tissues, and possible invasion of the tumor tissue by the healthy tissue or vice versa (mediated by these cortical tensions).
Further, it allows for modeling lysis and connected lymphatic outflow from the tumor, adhesion of the cells to their ECM, as well as
adhesion among cells (through dynamic viscosities) and possible detachment.
The mathematical model includes the mass balance equations of the four main constituents, namely the ECM, TCs, HCs, and IF.
Also, mass balance of the different species within the constituents is needed, resulting in advection–diffusion–reaction equations for
the transported substances in the IF (such as the nutrients). After that, the linear momentum balance equations of the phases are
stated, including the one for the solid. The model is finally closed with the necessary constitutive equations. In particular, the ECM is
considered to be Green-elastic or elasto-visco-plastic. The model has been extensively validated with respect to experiments either
from literature or carried out at the Houston Methodist Research Institute.
Results From the Model

Tumor growing in vitro and ex vivo
We show now a few results from the model, starting from the case of a tumor spheroid growing in vitro and ex vivo. Tumor spher-
oids are spherical aggregates of TCs that can be grown in the laboratory, with a careful control of the culture conditions. For tumor
spheroids grown in vitro, TCs deposit their own ECM during the tumor evolution. On the other hand, in the ex vivo case, the TCs are
seeded in a de-cellularized ECM to mimic the in vivo environment. This has been carried out successfully by Mishra and colleagues,
for an ex vivo 3D lung model in which it was possible to grow perfusable lung nodules. Fig. 1 shows the evolution of the solid
volume fraction for these two cases. In particular, the case of ECM deposited by TCs is shown on the top of Fig. 1, whereas the
case of a remodeling ECM scaffold is displayed at the bottom of the image. In the first case (spheroid grown in vitro), the initial
solid volume fraction is set to zero, since there is no solid phase. At the final stage, the solid volume fraction differs from zero, where
the TCs have grown and have deposited their ECM. In the second case (spheroid grown ex vivo), the initial and final average solid
volume fractions are fixed at 0.2, because the ECM scaffold is already present as a de-cellularized matrix.
Mechanical compression of tumor spheroids

In the second example, a reduced computational model is validated against data from tumor spheroid cultures. U87-MG cells, from
a human glioblastoma cell line, are cultured with a standard protocol. Cells are seeded at different initial numbers (1000, 5000,
10,000) and rapidly form spheroids suspended in standard culture medium. The evolution of the spheroid radii is then recorded
over time via optical microscopy, and the results are shown in Fig. 2. Here, points are experimental data, and error bars are the
Fig. 1 Solid volume fraction distribution at initial (A) and final (B) stages of a MTS growing in a ECM deposited by TCs and solid volume fraction
distribution at initial (C) and final (D) stages of a MTS growing in a remodeling ECM scaffold. Reprinted with permission under the CC-BY Attribution
License from Santagiuliana, R., Stigliano, C., Mascheroni, P. et al. (2015). The role of cell lysis and matrix deposition in tumor growth modeling.
Advanced Modeling and Simulation in Engineering Sciences, 2(1), 19. (http://creativecommons.org/licenses/).
Fig. 2 Growth curves recorded from the free growth experiments. Each curve represents a different initial condition in terms of seeded cells. In the
experiments, N 4 spheroids are considered for each condition. Points are experimental data, and error bars are the standard deviations of the
measurements; solid lines are the results of fits with the mathematical model. Reproduced from Mascheroni, P., Stigliano, C., Carfagna, M. et al.
(2016). Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media model. Biomechanics and Modeling in Mecha-
nobiology, 15(5), 1215–28. © Springer-Verlag Berlin Heidelberg 2016. With permission of Springer.
Fig. 3 Optical images of U-87 MG spheroids grown under the effect of the dextran solutions. The first row shows the control experiments and the
second row a spheroid under the highest compression (10 kPa). The scale bar is 200 mm, and the initial seeding is 5000 tumor cells. Reproduced from
Mascheroni, P., Stigliano, C., Carfagna, M. et al. (2016). Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media
model. Biomechanics and Modeling in Mechanobiology, 15(5), 1215–28. © Springer-Verlag Berlin Heidelberg 2016. With permission of Springer.
standard deviations of the measurements. The solid lines in Fig. 2 are the results of fits with the mathematical model. The model is
able to reproduce the results in the experiments, in which the spheroids reach a steady state after 20 days from cell seeding. As the
spheroids grow freely in the culture medium, the only mechanism to stop cell proliferation is nutrient deprivation. Lack of nutrients
in the inner regions of the spheroids provides a sufficient explanation for growth saturation and the following existence of an
asymptotic spheroid radius. After these preliminary experiments, the application of a constant mechanical stress on the surface
of the growing spheroids is then investigated. Following a technique developed by Montel and colleagues, dextran is added to
the cell culture medium producing an osmotic pressure on the outermost layer of cells located on the spheroid surface. Three pres-
sure conditions are explored, namely 1, 5, and 10 kPa, plus a control experiment with no external pressure. The growth of the spher-
oids is followed for 18 days after the addition of dextran. Fig. 3 shows optical images of sample spheroids referring to the control
and to the most compressed condition at different time instants. The comparison between experimental data and numerical values
is shown in Fig. 4. Points with error bars are experimental values while solid lines are the results of fits with the mathematical model.
The model results are in good agreement with the experimental values for all the different external mechanical pressures.
Effects of interfacial tensions

In a recent work, the computational model has been enriched to account for the features of fully developed three-phase flow, capturing
the interactions between the different cell populations. This required the introduction of suitable constitutive relationships, describing
the pressure differences between the cell phases and the IF. As a consequence, the interfacial tensions between the different fluids
appear explicitly. Note that these relationships allow for a more realistic modeling of adhesion between the cells and invasion of
different tissue compartments. Fig. 5 shows the last time step of a simulation in which a spherical tumor grows within an external
host tissue. The three panels display the volume fractions of the cellular phases over the radius of the spherical domain, for three
different TC-HC interfacial tensions. As shown in Fig. 5A, a rather high interfacial tension between TCs and HCs leads to a significant
spreading of the original tumor. Indeed, even if the tumor region was initially occupied only by TCs, their original distribution is not
sustained and no domain with only TCs is obtained. On the other hand, Fig. 5B refers to the case of a medium interfacial tension
Fig. 4 Comparison between experimental data (dots) and numerical results (solid lines) for the compression experiments. Error bars represent the
standard deviations of the measurements. The model is able to reproduce the experimental results for all the different compression regimes. For each
condition, N ¼ 5 spheroids are considered. Reproduced from Mascheroni, P., Stigliano, C., Carfagna, M. et al. (2016). Predicting the growth of glio-
blastoma multiforme spheroids using a multiphase porous media model. Biomechanics and Modeling in Mechanobiology, 15(5), 1215–28.
© Springer-Verlag Berlin Heidelberg 2016. With permission of Springer.
Fig. 5 Effect of different interfacial tensions between the cell populations. The three figures refer to the cases of high (A), medium (B), and zero
(C) interfacial tension between the TCs and the HCs. The model provides different profiles for the host tissue invasion, depending on the surface
interactions between the cells. Reproduced from Sciumè, G., Gray, W. G., Hussain, F. et al. (2016). Three phase flow dynamics in tumor growth.
Computational Mechanics, 53(3), 465–484. © Springer-Verlag Berlin Heidelberg 2013. With permission of Springer.
between TCs and HCs. The interactions between the two cell populations support the lateral displacement of the healthy tissue,
favoring a rapid growth of the malignant mass. In both the cases, a residual volume fraction of HCs remains present within the malig-
nant mass. However, the model predicts a higher density of TCs for lower values of the TC-HC interfacial tension.
Finally, in the limit case with zero interfacial tension between TCs and HCs (Fig. 5C), we observe the complete displacement of
the HCs by the TCs. In this case, it is possible to describe partial displacement of the host tissue by the tumor only in two ways: (i) by
considering different values for the dynamic viscosity of the two cell populations and (ii) by accounting explicitly for a heteroge-
neous adhesion to the ECM.
Melanoma growth and angiogenesis

As last application of the model, we show results for the growth of a melanoma in the presence of angiogenesis. Here, the endo-
thelial cell density is assumed proportional to the density of new vessels. The outer structure of skin is layered and three compart-
ments can be evidenced: (i) the epidermis, an outer epithelium of stratified cells; (ii) the dermis, an intermediate cushion of
vascularized connective tissue; and (iii) the hypodermis, the lowermost layer made of loose tissue and adipose cells. The dermis
is separated from the epidermis by the basement membrane, a tough sheet of ECM. Two well-defined clinical stages characterize
the progression of a melanoma. First, a radial expansion in the epidermis occurs, which may be followed in a second step by vertical
growth. In this second stage, the tumor grows perpendicularly to the skin surface and may eventually penetrate the basement
membrane. Angiogenesis occurs during this latter phase. Blood vessels are here assumed at the base of the dermis, as shown in
Fig. 6 which depicts the geometry of the simulated domain. Fig. 7 shows the resulting volume fraction of TCs after 10 and
20 days. At the beginning, the growing TCs deform the ECM and create a small bulge on the skin surface. Once the base membrane
is reached, the growth pattern changes into a more complex configuration. TCs consume oxygen due to cell proliferation and their
own metabolism. Oxygen consumption leads to a decrease of its mass fraction in the tumor area, as depicted in Fig. 8. As the cancer
develops, a region of necrotic cells forms in the tumor interior. Before undergoing necrosis, living TCs are subjected to hypoxic
conditions, in which the poor mass fraction of oxygen hinders cell replication. Living TCs under hypoxia produce TAFs that diffuse
into the domain. Fig. 9 shows the effects of TAF diffusion after 10 and 20 days. Then, diffusion of endothelial cells sensing the
chemotactic gradient of TAFs is depicted in Fig. 10. After 10 days, the distribution of the mass fraction of endothelial cells shows
a higher concentration near the tumor, where the TAF concentration is larger. This behavior is highlighted in the plot at 20 days.
Fig. 6 Growth of a melanoma in the presence of tumor angiogenesis. Skin structure and geometry of the simulated case. Reprinted from
Santagiuliana, R., Ferrari, M., Schrefler, B. A. (2016). Simulation of angiogenesis in a multiphase tumor growth model. Computer Methods in Applied
Mechanics and Engineering, 304, 197–216. Copyright 2016, with permission from Elsevier.
Fig. 7 Volume fraction of TCs after 10 (left) and 20 days (right). Reprinted from Santagiuliana, R., Ferrari, M., Schrefler, B. A. (2016). Simulation of
angiogenesis in a multiphase tumor growth model. Computer Methods in Applied Mechanics and Engineering, 304, 197–216. Copyright 2016, with
Fig. 8 Mass fraction of oxygen after 10 (left) and 20 days (right). Reprinted from Santagiuliana, R., Ferrari, M., Schrefler, B. A. (2016). Simulation
of angiogenesis in a multiphase tumor growth model. Computer Methods in Applied Mechanics and Engineering, 304, 197–216. Copyright 2016,
with permission from Elsevier.
Fig. 9 Mass fraction of TAF after 10 (left) and 20 days (right). Reprinted from Santagiuliana, R., Ferrari, M., Schrefler, B. A. (2016). Simulation of
angiogenesis in a multiphase tumor growth model. Computer Methods in Applied Mechanics and Engineering, 304, 197–216. Copyright 2016, with
Fig. 10 Mass fraction of endothelial cells after 10 (left) and 20 days (right). Reprinted from Santagiuliana, R., Ferrari, M., Schrefler, B. A. (2016).
Simulation of angiogenesis in a multiphase tumor growth model. Computer Methods in Applied Mechanics and Engineering, 304, 197–216. Copy-
right 2016, with permission from Elsevier.
Diabetic Foot
A severe complication of the diabetes mellitus is the pathology of diabetic foot. During the disease, the high levels of blood glucose
in diabetic patients can damage the nerves and blood vessels. The diabetic neuropathy, the peripheral nerve dysfunction associated
with diabetes, causes patients to have a reduced ability to feel pain. The loss of sensation leads to repetitive minor injuries from
internal (calluses, nails, foot deformities) or external causes (shoes, burns, foreign bodies) that remain undiscovered for a long
while and may eventually lead to ulcers and infections. Furthermore, associated damage to the blood vessels can also induce
low levels of blood and oxygen in the feet. This may cause vasoconstriction and decreased sweating, resulting in a loss of skin integ-
rity, and providing a site vulnerable to microbial infection. As a result, strong difficulties to feet healing arise, with serious cases of
ulcer infections that may lead to amputations.
Diabetic neuropathy is the common factor in almost 90% of diabetic foot ulcers. The prevention of diabetic foot is crucial,
considering the negative impact on a patient’s quality of life and the associated economic burden on the healthcare system.
The management of diabetic foot ulceration involves a multidisciplinary approach. To avoid ulcers and prevent amputation,
some diabetic treatments are adopted as offloading the wound by using appropriate therapeutic footwear, debridement when neces-
sary, antibiotic, optimal control of blood glucose, and evaluation and correction of peripheral arterial insufficiency.
The neuropathy causes loss of protective sensation and loss of coordination of muscle groups in the foot and leg, muscle weak-
ness, atrophy, and paresis. These effects increase mechanical stresses during ambulation. The excessive pressure in the foot tissue
may be measured, but due to the differences between the plantar pressure measurement devices, a universal threshold is not set
for ulceration. Furthermore, ulceration often starts at the interface between bone angularity and plantar tissue, probably due to
internal stresses, which are not directly measurable with these experimental tools.
In this context, an approach based on FE can be helpful, because it can predict the stress on the plantar tissue. FE models of the
foot have become even more faithfully realistic, including the actual geometry of the ligaments, muscles, and other foot substruc-
tures. As clearly shown in several studies from the recent literature, enhancement of FE models can aid the understanding of this
complex problem. However, most models have treated the plantar tissue, which is directly concerned with the foot ulceration, as
a hyperelastic material, whereas the other constituents (ligaments, bones, cartilage, and plantar fascia) have been considered as line-
arly elastic. This simplification has led to a poor predictive capability of the models. Moreover, in recent models, the duration of the
gait cycle has been taken into account by means of viscous constitutive laws for the plantar soft tissue. The model presented below
can simulate one or more gait cycles and predict the associated plantar pressure. It can integrate experimental data of patient-specific
foot kinematics into the modeling process, improving agreement between the computed and measured pressures. The model is time
dependent and includes information on the real microstructure of the plantar tissue. The latter is modeled as a porous medium, in
which cells and ECM constitute a solid skeleton that is permeated by IF.
Mathematical Model
The choice of adopting porous media mechanics for modeling the plantar tissue did not come about by chance. In fact, as observed
ultrasonographically, the plantar tissue consists of micro- and macro-chambers, filled by IF. Hence, theories developed for porous
materials can provide a realistic description of the mechanical behavior of this tissue: stress transfer from fluid to solid phase and
vice versa occurring inside the tissue can be taken into account, thus resulting in a global viscoelastic response of the material.
The model is a biphasic system in the large strain regime. The tissue cells and the ECM form the elastic porous solid skeleton. IF is
the fluid phase that completely fills the pore space. Transport of nutrients and possible drug delivery within the microvasculature
can be introduced as diffusion of chemical species within the IF. The diffusion coefficient can be estimated from the real degree of
vascularization of the zone of interest. In addition, the model can account for necrosis of the plantar tissue, depending on the stress
level and vasculopathies. The presence of the IF in the pores allows mimicking the real global viscoelastic behavior of the plantar soft
tissue. As in the tumor growth model presented before, the evolution of the solid and fluid phases is regulated by mass and
momentum balance equations.
Numerical Example for Gait Cycle

To mimic the evolution of the pressure in the foot by FE analysis, the gait cycle is modeled from the end of the contact phase (when
the heel is the first area of the foot in contact with the ground) to the active propulsion phase (when the heel lifts from the support
side and ends with opposite-side heel strike). Furthermore, the global forces measured experimentally by a force platform device,
translated and applied with the opposite sign to the ankle, are the input load for the numerical model. Applying a patient-specific
load history is essential because not only the foot morphology and tissue properties change because of diabetic diseases, but gait
alterations may also occur. For example, it has been observed that patients with diabetes commonly walk slower, tend to take
shorter steps with a wider base of support, and demonstrate a longer double support time.
In the next example, a healthy foot is modeled and four gait cycles are simulated. This numerical example is useful to show the
potentialities of the model and the interesting aspects that can be captured by modeling the plantar tissue as a fluid saturated
porous medium. Fig. 11 shows the geometry of the problem. For a real case, the geometry of a patient’s foot (obtained from
Fig. 11 Geometry and load conditions for the case of a healthy foot. Reproduced with permission from Sciumè, G., Boso, D.P., Gray, W.G., Cobelli,
C., Schrefler, B.A. (2014). A two-phase model of plantar tissue: a step toward prediction of diabetic foot ulceration. International Journal for Numer-
ical Methods in Biomedical Engineering, 30:1153–1169. © 2014 John Wiley & Sons, Ltd.
Fig. 12 (A) The black line is the input vertical force (P) and the gray line is the torque (C$d) (figure readapted from Natali, A. N., Forestiero, A.,
Carniel, E. L., Pavan, P. G., Dal Zovo, C. (2010). Investigation of foot plantar pressure: experimental and numerical analysis. Medical and Biological
Engineering and Computing, 48:1167–1174). (B and C) Vertical stresses, SMYY, and interstitial fluid pressures, PF, computed in the points T1 and
T2 (these points are represented in Fig. 11). In (B and C), the markers (disks) represent solutions obtained with ABAQUS (using a monolithic solver)
while the continuous lines are those obtained with Cast3M using the presented staggered procedure. Reproduced with permission from Sciumè, G.,
Boso, D.P., Gray, W.G., Cobelli, C., Schrefler, B.A. (2014). A two-phase model of plantar tissue: a step toward prediction of diabetic foot ulceration.
International Journal for Numerical Methods in Biomedical Engineering, 30:1153–1169. © 2014 John Wiley & Sons, Ltd.
an MR image) and patient-specific parameters must be used to obtain predictive results. Moreover, in vivo measurements of
displacements and rotations of the bone segments can be useful to confirm that the kinematics of the foot obtained numerically
is sound.
Fig. 12A shows the evolution with time of the vertical force and of the torque. This can be computed from the experimental
position of the plantar pressure resultant during the gait analysis and is applied to the foot as shown in Fig. 11. Fig. 12B and C
displays the evolution of the vertical total stress and of the IF pressure, during the four gait cycles for the points T1 and T2 repre-
sented in Fig. 11. Note that the IF pressure reverses sign during a gait cycle, which would imply flow reversal. The intrinsic perme-
ability has a dominant role because it governs the efficiency of the tissue in absorbing quite important loads; the loads initially
supported by the IF are then progressively transferred to the solid phase.
Fig. 13 shows the vertical total stress in the tissue and in the supporting surface at the same time. We can see how in the first part
of the midstance phase (4.0 s), the load is mostly supported by the heel, while at the end of the midstance phase (4.4 s), it is mostly
supported by the forefoot.
Fig. 13 Vertical stress during the last gait cycle at 4.0, 4.2, and 4.4 s. Reproduced with permission from Sciumè, G., Boso, D.P., Gray, W.G.,
Cobelli, C., Schrefler, B.A. (2014). A two-phase model of plantar tissue: a step toward prediction of diabetic foot ulceration. International Journal for
Numerical Methods in Biomedical Engineering, 30:1153–1169. © 2014 John Wiley & Sons, Ltd.
Conclusions
For a long time, the mechanics community has focused on complex engineering problems in a wide set of fields, such as transpor-
tation, material behavior, and defense. A broad collection of theoretical, numerical, and experimental tools have been validated,
following prior development and testing. In the last few years, this collection of techniques and methods has been exploited to
answer open questions in medicine. With the tools from mechanics, intricate problems involving many degrees of freedom have
been solved, and answers are on their way to the clinic. In this work, we reported on computational models developed in the frame-
work of porous media mechanics and applied to biomedical topics of significant relevance. We started from the case of tumor
growth, describing the evolution of the tumor mass under different conditions. We analyzed the evolution of tumor spheroids, first
as freely growing in the culture medium and then subjected to an external compression. After that, we focused on the coupled
dynamics of tumor and host cells, analyzing the effects of different surface interactions between the two cellular species. Finally,
the case of a melanoma was investigated, with a few remarks about tumor angiogenesis and oxygen consumption. The second
part of the work was devoted to the study of the diabetic foot. Contrary to the usual approach, which employs the hypothesis
of hyperelasticity, we modeled the plantar tissue as a porous material. This enabled a more realistic description of the tissue mechan-
ical properties, which were directly influenced by the time evolution of the loading conditions. The vertical stress and fluid pore
pressure were analyzed at different times. Faced with the surge in cancer incidence and occurrence of diabetic pathologies, the
approaches presented here should stimulate novel therapeutic strategies and treatment optimizations, with the aim of improving
the prognosis, outcome of intervention, and quality of life for patients.
Further Reading
Alexiadou, K., & Doupis, J. (2012). Management of diabetic foot ulcers. Diabetes Therapy, 3, 4.
Araujo, R. P., & McElwain, D. L. S. (2004). A history of the study of solid tumour growth: the contribution of mathematical modelling. Bulletin of Mathematical Biology, 66(5),
1039–1091.
Bellomo, N., De Angelis, E., & Preziosi, L. (2003). Multiscale modeling and mathematical problems related to tumor evolution and medical therapy. Journal of Theoretical Medicine,
5(2), 111–136.
Blanco, E., Shen, H., & Ferrari, M. (2015). Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nature Biotechnology, 33(9), 941–951.
Byrne, H. M. (2010). Dissecting cancer through mathematics: from the cell to the animal model. Nature Reviews. Cancer, 10(3), 221–230.
Carson, E., & Cobelli, C. (2014). Modelling methodology for physiology and medicine (2nd edn). London: Elsevier.
Chen, W. M., Lee, T., Lee, P. V. S., Lee, J. W., & Lee, S. J. (2010). Effects of internal stress concentrations in plantar soft-tissue – a preliminary three-dimensional finite element
analysis. Medical Engineering & Physics, 32, 324–331.
Cheng, A. H.-D. (2016). Poroelasticity. Switzerland: Springer International Publishing.
Ferrari, M. (2010). Frontiers in cancer nanomedicine: directing mass transport through biological barriers. Trends in Biotechnology, 28(4), 181–188.
Gefen, A., Megido-Ravid, M., & Itzchak, Y. (2001). In vivo biomechanical behavior of the human heel pad during the stance phase of gait. Journal of Biomechanics, 34,
1661–1665.
Gray, W. G., & Miller, C. T. (2014). Introduction to the thermodynamically constrained averaging theory for porous medium systems. Switzerland: Springer International Publishing.
Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674.
Jain, R. K., & Stylianopoulos, T. (2010). Delivering nanomedicine to solid tumors. Nature Reviews. Clinical Oncology, 7(11), 653–664.
Lewis, R. W., & Schrefler, B. A. (1998). The finite element method in the static and dynamic deformation and consolidation in porous media. Chichester: Wiley.
Mascheroni, P., Stigliano, C., Carfagna, M., et al. (2016). Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media model. Biomechanics and
Modeling in Mechanobiology, 15(5), 1215–1228.
Michor, F., & Beal, K. (2015). Improving cancer treatment via mathematical modeling: surmounting the challenges is worth the effort. Cell, 163(5), 1059–1063.
Michor, F., Liphardt, J., Ferrari, M., & Widom, J. (2011). What does physics have to do with cancer? Nature Reviews. Cancer, 11(9), 657–670.
Moore, N. M., Kuhn, N. Z., Hanlon, S. E., Lee, J. S. H., & Nagahara, L. A. (2011). De-convoluting cancer’s complexity: using a “physical sciences lens” to provide a different
(clearer) perspective of cancer. Physical Biology, 8(1), 10302.
Pai, S., & Ledoux, W. R. (2011). The quasi-linear viscoelastic properties of diabetic and non-diabetic plantar soft tissue. Annals of Biomedical Engineering, 39(5), 1517–1527.
Quaranta, V., Weaver, A. M., Cummings, P. T., & Anderson, A. R. A. (2005). Mathematical modeling of cancer: the future of prognosis and treatment. Clinica Chimica Acta, 357(2),
173–179.
Risler, T. (2015). Focus on the physics of cancer. New Journal of Physics, 17(5), 55011.
Santagiuliana, R., Stigliano, C., Mascheroni, P., et al. (2015). The role of cell lysis and matrix deposition in tumor growth modeling. Advanced Modeling and Simulation in
Engineering Sciences, 2(1), 19.
Santagiuliana, R., Ferrari, M., & Schrefler, B. A. (2016). Simulation of angiogenesis in a multiphase tumor growth model. Computer Methods in Applied Mechanics and
Sciumè, G., Gray, W. G., Ferrari, M., Decuzzi, P., & Schrefler, B. A. (2013). On computational modeling in tumor growth. Archives of Computational Methods in Engineering, 20(4),
327–352.
Sciumè, G., Gray, W. G., Hussain, F., et al. (2013a). Three phase flow dynamics in tumor growth. Computational Mechanics, 53(3), 465–484.
Sciumè, G., Shelton, S., Gray, W., et al. (2013b). A multiphase model for three-dimensional tumor growth. New Journal of Physics, 15(1), 15005.
Sciumè, G., Ferrari, M., & Schrefler, B. A. (2014). Saturation–pressure relationships for two- and three-phase flow analogies for soft matter. Mechanics Research Communications,
62, 132–137.
Sciumè, G., Boso, D. P., Gray, W. G., Cobelli, C., & Schrefler, B. A. (2014a). A two-phase model of plantar tissue: a step toward prediction of diabetic foot ulceration. International
Journal for Numerical Methods in Biomedical Engineering, 30, 1153–1169.
Sciumè, G., Santagiuliana, R., Ferrari, M., Decuzzi, P., & Schrefler, B. A. (2014b). A tumor growth model with deformable ECM. Physical Biology, 11(6), 65004.
Multiscale Bone Mechanobiology
Stefan Scheiner, Vienna University of Technology, Vienna, Austria
Maria-Ioana Pastrama, KU Leuven, Leuven, Belgium
Peter Pivonka, Queensland University of Technology, Brisbane, Australia
Christian Hellmich, Vienna University of Technology, Vienna, Austria
Introduction to Bone Remodeling 168

Basics and Involved Key Players 168
Implications of Bone Remodeling Disorders 169
Mechanobiological Regulation 169
Major Regulatory Pathways in a Nutshell 169
Mechanical Stimulation 170
Regulatory Interplay Across Several Orders of Magnitude 170
Mathematical Modeling of Bone Remodeling 171
Expected Benefits 171
Historical Overview 172
New Development: Pore Space-Specific, Multiscale Modeling 173
Summary and Outlook 177
References 178
Further Reading 178
Glossary
Bone cell population model A model taking into account the behavior of cell populations, quantified in terms of cell numbers
of molar concentrations, within a defined volume of interest.
Cortical bone The compact type of bone forming the outer shell of most bones, exhibiting superior mechanical properties (as
compared to trabecular bone).
Deterministic modeling A system, for example concerning the behavior of cells, is modeled as detailed as possible, explicitly
taking into account all known subprocesses and contributing factors.
Homogenization The utilization of mathematical methods for finding estimates of the overall behavior of composite
materials, in terms of specific physical quantities.
Mechanobiology The neologism made up for expressing that related phenomena are governed by both mechanical stimuli
and biological processes, in coupled fashion.
Model validation The process of testing the soundness of a model, for example through comparison of model predictions with
independent experimental data (the more validation attempts a model survives without significant discrepancies to the
respective reference data, the better the model).
Phenomenological modeling A modeling strategy where only the overall behavior of a system is taken into account
empirically, that is based on observations, without considering subprocesses and how additional factors influence the system’s
behavior.
Representative volume element A microheterogeneous domain that can be considered as macroscopically homogeneous if
the characteristic length of the heterogeneities is significantly smaller than the characteristic length of the representative volume
element.
Tissue Engineering A field at the interface of engineering and medicine, where malfunctioning or pathological biological
tissues are replaced by biocompatible, synthesized materials, engineered in terms of morphology, biological characteristics,
and mechanical properties.
Trabecular bone The spongious, highly porous type of tissue usually found in the interior of bones (particularly at the ends of
long bones).
Nomenclature
Abbreviations
CT Computed tomography
FE Finite Element

168 Biomechanics j Multiscale Bone Mechanobiology
lac Lacunar pore space

M-CSF Macrophage colony stimulating factor
OBA Active osteoblasts
OBP Osteoblast precursor cells
OBU Uncommitted osteoblast progenitor cells
OCA Active osteoclasts
OCP Osteoclast precursor cells
OCY Osteocytes
OPG Osteoprotegerin
PTH Parathyroid hormone
RANK Receptor activator of nuclear factor kappa b
RANKL Ligand of RANK
RVE Representative volume element
TGFb Transforming growth factor b
vas Vascular pore space
Wnt Wingless gene
Latin symbols
A vas
OBA Constant apoptosis rate of active osteoblasts
Cij Molar concentration of species i within domain j
CTGFb
vas
Molar concentration of TGFb in the vascular pore space
COCY
exvas
Concentration of osteocytes across extravascular bone matrix
DvasOBP Maximum differentiation rate of OBPs in the vascular pore space
fj Volume fraction of domain j
fvas Vascular porosity
f Frequency at which loading peaks occur
Krep,TGFb
OBP/OBA
Repression coefficient of OBP differentiation due to TGFb
Ni Amount of species i
plac
peak
Lacunar pore pressure at peak loading
pvas
peak
Vascular pore pressure at peak loading
P vas
OBP Maximum proliferation rate of OBPs in the vascular pore space
Vj Volume j
t Time variable
Greek symbols
prep,TGFb
OBP/OBA
Repression function for differentiation of OBPs to OBAs, due to the presence of TGFb
Pact,OBP
mech,vas
Activation function of OBP proliferation in the vascular pore space, due to mechanical excitation
mech;vas
b
P Baseline value of Pact,OBP
mech,vas
act;OBP
mlac Mechanical stimulus in the lacunar pore space
mlac
ref
Mechanical stimulus in the lacunar pore space at reference loading
mvas Mechanical stimulus in the vascular pore space
mvas
ref
Mechanical stimulus in the vascular pore space at reference loading
lOBP
vas
Anabolic strength parameter related to direct excitation of the OBPs
lWnt/scl
vas
Anabolic strength parameter related to excitation of the OCYs
Introduction to Bone Remodeling

Basics and Involved Key Players
Nature has equipped bone with intriguing features, which allow for fulfillment of its vital roles for the human body, such as provi-
sion of sufficient load-carrying capacity, protection of organs, locomotion, and calcium homeostasis. For these purposes, contin-
uous maintenance of the microstructural integrity of the bone tissue is crucial, to avoid macroscopic bone fractures. The
mechanism concerned with this important task is referred to as bone remodeling (Robling et al., 2006).
Biomechanics j Multiscale Bone Mechanobiology 169
In short, bone remodeling involves numerous biochemically and mechanically stimulated mechanisms (which are briefly dis-
cussed in the following sections of this article), together leading to removal of bone tissue by multinuclear cells deriving from hema-
topoietic stem cells, called osteoclasts, and to concurrent addition of bone tissue by mononuclear cells deriving from mesenchymal
stem cells, called osteoblasts. More precisely, osteoclasts attach themselves to the bone surface, and create an acidic environment in
order to dissolve the bone matrix, thus leaving behind a resorption cavity. Osteoblasts lay down osteoid to fill this cavity, a substance
consisting mainly of collagen type I. Over time, osteoid gets mineralized, with the bone mineral mainly consisting of carbonated
hydroxyapatite. Since the greater part of bone mineral is formed very fast (within a few days), osteoblasts are often, for simplicity,
referred to as bone-forming cells, without explicitly mentioning the intermediate step of osteoid secretion. Moreover, osteocytes,
a third cell type found in bone, have been identified as bone remodeling “conductor.” In particular, osteocytes are former osteo-
blasts that have been “buried” in newly secreted osteoid. They are the most abundant cell type in bone tissue, and they are consid-
ered capable of sensing changes in their environment, particularly concerning the mechanical loading. Subsequently, osteocytes
transduce these changes into biochemical signals, which, in turn, lead to corresponding changes of cellular activities in the bone
microenvironment.
Implications of Bone Remodeling Disorders

In healthy bone, the processes involved in bone remodeling are finely tuned such that the amounts of removed and added bone
tissue are balanced, and that the bone tissue composition, that is the proportions of collagen, mineral, and water across several
length scale orders of magnitude, is constant, thereby entailing the required bone stiffness. However, a range of circumstances
may induce disturbances of this balance.
Bone disorders are usually caused by the dysfunction or dysregulation of one or several factors governing bone remodeling
(Robling et al., 2006). This concerns both sudden perturbations of the hormonal balance (e.g., occurring at and after menopause)
and hereditary disorders (being the cause for pathologies such as osteogenesis imperfecta). A prominent example of the former is
postmenopausal osteoporosis, where a reduced estrogen concentration initiates a cascade of biochemical processes leading to
increased osteoclast activity, and, eventually, to more porous bone tissue. On the other hand, also long-term changes of the physical
activity are potentially effective in terms of triggering corresponding changes of osteoclast and/or osteoblast activities; for example,
bed rest and a sedentary lifestyle have been associated with significant bone loss.
Exhaustively unraveling the regulatory intricacies behind these effects would have huge impacts, both in terms of the well-being
of affected people, and on a commercial leveldwhen considering, for example, the fields of tissue engineering or pharmacology.
Mechanobiological Regulation
Major Regulatory Pathways in a Nutshell
Ever since the discovery of RANKL, which is the protein acting as ligand for RANK, the receptor activator of nuclear factor kappa
b, the RANK-RANKL-OPG-pathway is considered to be the regulatory pathway directing bone remodeling. In particular,
binding of RANKL, which is produced by both osteoblasts and osteocytes, to RANK, is a transmembrane protein found on oste-
oclasts, is required for osteoclastogenesis. Osteoprotegerin, another protein produced by osteoblasts, is capable of preventing
the binding of RANKL to RANK, by binding to RANKL itself; thus it is considered to be a so-called decoy receptor for RANKL.
Furthermore, PTH, a hormone produced by the parathyroid glands, is able to influence the RANK-RANKL-OPG pathway in
catabolic fashion as it promotes the production of RANKL and inhibits the production of OPG, respectively. Another important
protein for the signaling pathways in bone remodeling is the cytokine usually referred to as transforming growth factor b, stan-
dardly abbreviated as TGFb. Besides the prominent roles TGFb plays in tumor growth and cardiovascular diseases, it is also
known to influence the differentiation tendencies of osteoblasts and osteoclasts. In particular, TGFb has turned out to
(i) promote the differentiation of uncommitted osteoblast progenitor cells (derived from mesenchymal stem cells) to osteo-
blast precursor cells, to (ii) inhibit the further differentiation of osteoblast precursor cells to active osteoblasts, which are
the cells which actually secrete osteoid, and to (iii) promote the apoptosis (or, programmed death) of active osteoclasts.
Furthermore, macrophage colony stimulation factor (standardly abbreviated to M-CSF), which is produced by osteoblasts,
contributes to the regulatory pathways of bone remodeling as it has a positive effect on the differentiation behavior of
early-stage osteoclasts. Apart from the abovementioned factors, bone remodeling is known to be influenced by a large number
of further proteins and hormones (such as nitric oxide, prostaglandins, vitamin D, corticosteroids, interleukins, calcitonin, just
to mention a few). More details on this matter can be found in respective review articles (Robling et al., 2006), some of which
are listed in the further reading lists at the end of this article.
Importantly, osteocytes are known to somehow sense changes of the mechanical loading, see the next subsection (on mechan-
ical stimulation of bone remodeling) for more details in this regard. In case of increased mechanical loading, osteocytes accordingly
downregulate the production of sclerostin, a glycoprotein, which itself inhibits the Wntb-catenin pathway, Wnt being the common
abbreviation for wingless gene. The latter pathway upregulates osteoblast proliferation; thus osteocytes are able to indirectly
promote bone formation. The production of RANKL by osteocytes is also modulated by the prevailing mechanical loading. In
particular, the amount of RANKL produced by osteocytes is indirectly proportional to the mechanical loading; thus increased
mechanical loading causes osteocytes to produce less RANKL, and vice versa.
Mechanical Stimulation
It has already been mentioned above that specific subprocesses (e.g., proliferation of osteoblasts and production of RANKL)
occurring in the course of bone remodeling are modulated by the prevailing mechanical loading. Thus, scrutinizing how and
to which extent mechanical loading is capable of influencing the aforementioned processes is key for understanding the related
effects on a macroscopic level. The overall response of bone tissue to mechanical loading is well known and undisputed; usually,
increased mechanical loading, with respect to a certain “normal” physiological loading relating to everyday activities, leads to
gain of bone mass (or volume), while decreased mechanical loading leads to bone loss. It is also well accepted that changes
of the prevailing mechanical loading induce corresponding changes of the activities of osteoblasts and osteoclasts. The under-
lying mechanisms of mechanosensing (i.e., how cells sense the mechanical loading and changes thereof) and mechanotransduc-
tion (i.e., how cells process changes of the mechanical loading) have been studied intensely. As a result, the eventual effects of
changed mechanical loading are (at least qualitatively) known; for example, an increased mechanical loading causes, via the Wnt-
signaling pathway, increased proliferation of osteoblast precursor cells (and in further consequence elevated osteoblast activity),
while a decreased mechanical loading causes increased production of RANKL (and in further consequence elevated osteoclasto-
genesis). However, the true routes along which mechanobiological regulation of bone remodeling occurs, including the question
of how exactly the involved cells are able to perceive changes in their mechanical environment, still remain to be revealed. Never-
theless, it is instructive to briefly review the state of the art concerning the mechanical stimuli that have been hypothesized to
effectively influence the activities of the bone remodeling-driving cells (Klein-Nulend et al., 2005; Robling et al., 2006; Sugiyama
et al., 2012; Galea et al., 2013). For the latter task, a few specific mechanical stimuli have turned out as particularly promising
candidates; these are discussed next.
Undoubtedly, subjecting bone to mechanical loading causes, in some form, pressurization of the fluid filling the various pore
spaces of bone tissue. In vitro studies have confirmed that bone cells (i.e., osteocytes, and also osteoblasts, osteoclasts, as well as
their progenitors) exhibit altered activities when subjected to hydrostatic pressure at frequencies of up to 1 Hz, with amplitudes
of several tens (to hundreds) of kilopascals. Remarkably, it could be shown, through poromicromechanical modeling, that phys-
iological macroscopic loading implies lacunar pore pressures of adequate amplitudes, thus corroborating the potential relevance of
pore pressures for mechanosensation of osteocytes. On the other hand, a heterogeneous distribution of stresses across the bone
tissue (as, for instance, due to exposure to bending moments, or a heterogeneously distributed bone composition) leads to the
occurrence of pore pressure gradients, which, in turn, induces the movement of the pore fluid. In the late 1980s, the hypothesis
was brought forth that this flow of the fluid contained in the network made up by lacunar and canalicular pores leads to correspond-
ing shear stresses acting on osteocytes and their cell processes, and that these shear stresses may be effective mechanical triggers of
mechanotransduction. The bone research community has readily embraced this idea ever since. Direct experimental evidence is not
available for the two pore pressure-related mechanisms of mechanosensation. It is thus instructive to analyze the characteristic
loading times of bone when subjected to physiological loading regimes. According to a recent study (Scheiner et al., 2016), it turns
out that for most physiological loading conditions, undrained conditions are to be expected in the lacunar–canalicular pore
network, that is the pore fluid remains “trapped” in the respective pore spaces when exposed to the typically fast-occurring, dynam-
ical loading. Thus, while the exact roles of both mechanisms for the mechanobiology of bone remodeling are not yet fully under-
stood, it is likely that in the past the importance of pore pressures may have been underestimated, while the importance of fluid
flow-induced shear stresses may have been overestimated.
Apart from the aforementioned pore fluid pressure-related mechanical stimuli, several alternatives have been suggested. Firstly,
once cells are attached to bone surfaces, they may (partly) undergo the same deformation as the surrounding bone tissue. This defor-
mation was also hypothesized to trigger mechanotransductive responses of such cells to mechanical loading and changes thereof.
However, as in vitro studies have shown, the strains required to induce effective responses in terms of cellular activities are quite
high, reaching up to 10%. These high strains may be explained by amplification effects occurring in the immediate vicinity of holes,
such as lacunar pores. Another potential factor contributing to the mechanical regulation of bone remodeling are microcracks occur-
ring as consequence of (mild) overloading. It was suggested that, on the one hand, microcracks cause osteocyte apoptosis and
disrupt the communication pathways of the lacunar–canalicular network. Interestingly, it has also been shown that nonapoptotic
osteocytes surrounding the apoptotic ones upregulate the expression of RANKL and of VEGF (which is short for vascular endothelial
growth factor) and decrease the expression of OPG. On the other hand, it has also been hypothesized that microcracks alter the fluid
flow characteristics. However, to date, no conclusive experimental evidence could be found confirming the suggested effects of
microcracks. Additionally, the piezoelectricity of collagen was suspected to be related to the mechanosensitivity of osteocytes.
Importantly, not only the amplitude of the mechanical stimuli but also other aspects of load application (Turner, 1999) are key
for mechanical regulation of bone remodeling. These include the load frequency, the number of load cycles, the rate of load appli-
cation, the duration and amplitude of the dynamic part of the loads, as well as the particularity of the mechanical stimulus (with
respect to “normal” loading).
Regulatory Interplay Across Several Orders of Magnitude

An additional intricacy related to the regulation of bone remodeling manifests itself in the hierarchical organization of bone,
spanning several orders of length scale magnitude. Taking into account the wealth of experimental data available in literature
concerning the structural biology of bone, a multilevel organization emerges (Vass et al., 2018). At an observation scale of
several tens of nanometers, a matrix of cross-linked collagen molecules hosts intermolecular pore space (the latter being filled
with pore fluid), together forming wet collagen. One level up, wet collagen and crystals of impure hydroxyapatite form the
so-called mineralized fibrils, with a characteristic length of several hundreds of nanometers. These fibrils, in turn, are
embedded in a conglomerate of hydroxyapatite crystals and intercrystalline pore space, typically referred to as extrafibrillar
space; transmission electron micrographs show the arrangement of collagen-free extrafibrillar space and collagen-rich fibrils,
where the former is visible as dark regions and the latter as light regions. Together, extrafibrillar space and mineralized
collagen fibrils form the bone ultrastructure, optionally known as extracellular bone matrix, which exhibits a characteristic
length of a few micrometers. In extracellular bone, pore spaces are embedded, which are of great importance for the metab-
olism of bone. These are the osteocytes-hosting lacunar pores (with a characteristic length of approximately 10 mm) and the
lacunae-connecting canalicular pores (being typically several hundreds of nanometers long, with a diameter of approximately
35 nm), together forming a dense pore network. On an observation scale of several hundreds of micrometers, extravascular
bone is composed of extracellular bone and the lacunae–canaliculi network. Extravascular bone, in turn, is the matrix in
which vascular pores can be found, with a characteristic length of 50–80 mm. In cortical bone, the vascular pores are synon-
ymous to the network of Haversian and Volkmann canals, and in trabecular bone to intertrabecular pore space. In any case,
the vascular space hosts many cells and biochemical factors initiating and driving bone remodeling. Importantly, exchange of
factors between osteocytes and the vascular pore space is enabled through canaliculi extending to the surfaces of extravascular
bone. Finally, bone microstructure, that is the tissue building up the substance of bone organs on a macroscopic observation
scale, is the composite of extravascular bone and vascular pore space.
Considering the multilevel organization of the bone tissue, it becomes apparent that for understanding the effects of specific
influences (of both mechanical and biochemical nature) acting upon cells and other factors on the progress of bone remodeling,
it is of utmost importance to take into account that the key players of bone remodeling are located in pore spaces of distinctively
different characteristic lengths. In the following, this statement is underpinned by describing the cascade of events occurring in
response to the mechanical loading to which a bone organ is subjected. The mechanical loading is applied macroscopically
onto the respective bone. Then the loading is transferred, depending on the actual, site-specific bone composition, to the mecha-
nosensitive osteocytes. The osteocytes then transduce the mechanical signal into biochemical signals that affect the behavior of oste-
oblasts and osteoclasts. The extent of bone formation by osteoblasts and of bone resorption by osteoclasts governs the bone
composition evolution, that is whether the bone mass remains constant, increases, or decreases. Eventually, the bone composition
determines the transfer of the mechanical loading from the macroscopic to the osteocytic length scale (Fritsch and Hellmich, 2007;
Scheiner et al., 2013). Thus, the progress of bone remodeling is governed by several biochemical, mechanical, and bone
composition-related factors, acting time-dependently, site-specifically, and length scale-specifically; many of these factors being
actually linked with each other, in feedback-type fashion.
Covering all these aspects in the framework of experimental studies is difficult, if not impossible. Typically, two kinds of exper-
imental studies are performed: On the one hand, in vitro studies focus on exploring the specific aspects, for example related to the
differentiation or expression behavior of cells, when subjected to specific factors. However, the “bigger picture” is lacking in such
small-scale tests, mainly concerning an adequate physiological environment including the multiple interactions of the studied cell
with factors other than the few which can be considered in the laboratory, and the embedding of the cells and factors in the respec-
tive pore spaces. On the other hand, in vivo tests, typically carried out in the form of animal studies (Sugiyama et al., 2012) or by
making use of bioreactors, cannot provide continuous information on cell activities or morphological changes of the bone tissue,
but only, if at all, at distinct time points, which is usually accompanied with termination of the respective test. Mathematical
modeling of bone remodeling, making use of the wealth of experimental data collected in in vitro and in vivo studies, seems to
be a reasonable remedy to the predicament sketched above. Describing the historical development and the current state of such
modeling approaches will be the subject of the following section.
Mathematical Modeling of Bone Remodeling

Expected Benefits
Utilization of mathematical modeling for replication and prediction of the bone remodeling progress in response to specifically
prescribed conditions holds a number of promises: First of all, simulation of bone remodeling based on a physically, biologically,
and mechanically substantiated mathematical model is expected to significantly improve the understanding of the bone remodeling
process itself. Ideally, computer simulations allow to study the effects of variations of specific factors on the bone metabolism.
Considering that the underlying mathematical models take into account the results of experimental studies extensively, they provide
some kind of sophisticated inter- and extrapolations of experimental data. Due to the complexity and nonlinearity of the bone
remodeling process, this cannot be achieved by intuition or experience-guided guessing, at least not in a quantitatively reliable way.
Once such a mathematical model has been established and validated, it can be applied as decision-support utility for a large
variety of practically relevant problems. Examples include
• Pharmaceutical engineering, where the number of animal studies could be dramatically reduced by assessing the efficacy of
drugs in silico (for example through predicting the effects of varying drug doses and administration regimes);
• Tissue engineering, where the effects of many different parameters of the respective tissue engineering materials can be studied
by means of computer simulations, such as different morphologies of the material, or the effects of seeding cells and other
factors on tissue engineering scaffolds made up by such materials; and
• Design of patient-specific treatment regimes, not only based on combining the aforementioned two fields, namely pharma-
ceutical and tissue engineering, but also based on recommendation of specifically tailored regimes of physical exercise.
Historical Overview
Very often, until today, the seminal work of Julius Wolff is considered to be the basis for mathematical models of bone remodeling,
at least when focusing on adaptation of bone to the prevailing mechanical loading. Julius Wolff summarized the main findings of
his work as surgeon in the book “The Law of Transformation of Bone,” first published in 1892, where he postulated that “as a conse-
quence of primary shape variations and continuous loading, or even due to loading alone, bone changes its inner architecture according to math-
ematical rules and, as a secondary effect and governed by the same mathematical rules, also changes its shape.”dnotably, a similar (yet more
general) proposition was published already in 1638 by Galileo Galilei. Wolff’s hypothesis was warmly welcomed by the bone
research community, and refined in the course of the 20th century. The 1960’s can be considered as formative years in the search
for suitable (mathematical) formulations relating mechanical loading and bone metabolism-driven changes of the shape and
morphology of bone (organs). Namely, Harold M. Frost published his famous “Utah Paradigm of Bone Physiology,” including
the so-called mechanostat theory. The latter established a simple relation between the strain state bone is experiencing and the cor-
responding bone turnover. Due to its simplicity, the mechanostat theory has shaped the field of computational simulation of bone
remodeling ever since.
The development of mathematical models for simulating the (mechanical) behavior of complex structures is traditionally the
specialty of civil and mechanical engineers. It is thus not surprising that mathematical modeling and computational simulation
of bone remodeling phenomena was pioneered and expedited by researchers coming from the aforementioned engineering disci-
plines, and that related efforts first focused on the structural scale, that is the scale of whole bone organs. Facilitated by the ever-
increasing power and availability of computers systems, the respective state of the art between the 1970s and 1990s was dominated
by large-scale Finite Element (FE) simulations (Webster and Müller, 2011; Schulte et al., 2013), complemented by simplified (and
purely phenomenological) bone remodeling rules governed by mechanical stimuli, according to Frost’s mechanostat theory, while
biological details were left aside. Clearly, the hierarchical organization of bone, spanning several orders of magnitude, could not
(and still cannot) be considered in these FE simulations, due to the unmanageable computational effort implied by a sufficiently
fine discretization of bone tissue. Hence, also the mechanical stimulus for bone remodeling could not (and still cannot) be chosen
according to physiological insights on mechanical stimulation of bone remodeling. Instead, FE simulations of mechanically modu-
lated bone remodeling events typically consider simplified strain-representative measures, such as the strain energy density occur-
ring in the macroscopic bone tissue in response to specifically prescribed loading boundary conditions, or scalar damage variables.
The flexibility of the FE method, in terms of studying structures of arbitrary, optionally changing shapes, and the over the years more
and more increasing availability of commercial FE software (or, alternatively, of open-source FE codes) have led to the long-term
establishment of the FE technique as the gold standard in the field. Recently, more and more detailed models, based on high-
resolution imaging techniques, could be successfully implemented, allowing even to take into account the exact mechanism by
which osteocytes have been hypothesized to be mechanically stimulated, for example via the primary cilia, or through the response
of the cell cytoskeleton to mechanical forces. However, due to the substantial computational effort related to FE simulations, organ-
scale models mostly consider macroscopic features of the studied bone organ, entailing the danger of (quantitatively) inaccurate
model predictions, see Fig. 1.
The comparison of model-predicted and experimentally observed sites of bone formation and bone resorption due to mechan-
ical stimulation of the 6th caudal vertebra of a mouse, as illustrated in Fig. 1, shows that the numerical model fails to predict the
distribution of these sites correctly, while other model-predicted parameters, such as formation and resorption rates, agree reason-
ably well with the experimental data (Schulte et al., 2013). The reasons for this discrepancy could be that, on the one hand, the exact
distribution of the (anisotropic) mechanical properties of the bone tissue was neglected, and, on the other hand, the exact
(A) (B)
Resorbed
bone
Formed bone
Fig. 1 Exemplary results from bone remodeling studies on the 6th caudal vertebra of a mouse, performed (A) by means of the Finite Element
method, and (B) experimentally (by means of micro-CT), highlighting that the spatial distribution of resorption and formation sites following from
numerical simulations differs from the one obtained experimentally. Reprinted from (Schulte et al., 2013), with permission from Elsevier B.V.
Proliferative Terminally differentiated

Sclerostin
Strain E2 E2
Wnt
Strain E2 Strain
IGF-1R
LRP
ERα ERα
ERβ ERα /β
AKT
ERα ERα /β
ERα ?
ERβ β-cat ERα ERβ
?
ERα
Proliferation ERK
Differentiation
β-cat β-cat SOST
E2
β-cat
LRP Proliferation Migration
Strain Differentiation Antiapoptosis ERα
Wnt Matrix production
Antiapoptosis
Sclerostin Proapoptosis
Preosteoblast Osteoblast Osteocyte Osteoclast
Fig. 2 Illustration of the manifold ways mechanical stimulation of osteoblasts and osteocytes influences the role of estrogen receptors ERa and
ERb, which, in turn, influence the behaviors of these cells; E2 stands for estrogen, LRP for low-density lipoprotein receptor, b-cat for beta-catenin,
Wnt for wingless gene, ERK for extracellular signal-regulated kinase, IGF-1R for insulin-like growth factor 1 receptor. Reproduced from (Galea et al.,
2013), by permission from Macmillan Publishers Ltd, BonekEy Reports, ©2009
biochemical and biomechanical regulation of bone remodeling was not taken into account. Particularly the latter is a challenging
task, as the involved pathways are manifold and intricate, see for example Fig. 2 for the various ways mechanical stimulation acts on
the estrogen receptors in osteoblasts and osteocytes, which, in turn, are important regulatory components for the behaviors of these
cells.
Mathematically modeling the multiply interrelated actions of different types of bone cells (osteoclasts, osteoblasts, and osteo-
cytes) and the large number of biological factors, overall constituting the process of bone remodeling, was tackled in the early 2000s.
In particular, the tool of bone cell population kinetics was discovered for quasi-deterministic simulation of bone remodeling in
a temporarily evolving manner, within macroscopic representative volume elements of (cortical or trabecular) bone tissue. Taking
into account only the major regulatory pathways of bone remodeling, that is explicitly considering only RANK, RANKL, OPG, PTH,
and TGFb, systems of ordinary differential equations were compiled allowing for predicting the temporal evolutions of bone cell
populations (quantified in terms of molar concentrations), in response to dynamically prescribed biochemical boundary condi-
tions (relating e.g., to bone pathologies, such as osteoporosis). Thereby, the regulatory effects of the aforementioned biochemical
factors were considered by means of respective (Hill-type) activation or repression functions. Additionally, efforts were undertaken
to extend the merely temporal approaches to spatio-temporal models, by considering also spatial aspects (leading to sets of partial
differential equations) see (Lemaire et al., 2004; Pivonka et al., 2008; Scheiner et al., 2013).
So far, two very contrary, isolated approaches have been introduced, shaping the state of the art in computational simulation of
bone remodeling over the past decades; on the one hand, the mostly mechanically driven large-scale numerical simulations, and, on
the other hand, the biochemically driven bone cell population models. Thus, the obvious next step was to develop models consid-
ering both mechanical and biochemical regulation of bone remodeling, thus mathematically describing bone remodeling as
a mechanobiologically driven process. This was achieved by coupling bone cell population models with continuum microme-
chanics models. The latter allow for downscaling the macroscopically applied mechanical loading bone is experiencing to the level
of the mechanosensitive osteocytes. Such calculated local mechanical stimuli were then fed into a bone cell population model,
according to experimental evidence. However, despite the conceptual advantages these models exhibit (compared to “conven-
tional” modeling approaches), they do not take into account the fact that the players (cells and biochemical factors) are actually
situated in pore spaces of separated characteristic length, namely in the vascular and lacunar pore spaces. This deficit provides
the incentive for further developments, described next.
New Development: Pore Space-Specific, Multiscale Modeling

In order to demonstrate how comprehensive multiscale modeling of bone remodeling can actually be achieved, this section is
devoted to presenting a novel modeling approach for computational simulation of bone remodeling, developed recently by the
authors of this article, and thoroughly obeying the multiscale paradigm, that is taking into account all relevant mechanisms on
the appropriate length scales (Pastrama et al., 2018).
The development of mathematical models is fundamentally based on reasonably simplifying the “real” material or structure that
is to be modeled. Thereby, the guiding principle should be to keep the model representation as simple as possible, but at the same
time as complex as necessary. In the present context aiming for a mathematical model allowing for simulation of bone remodeling,
such model representation preferably spans three distinct observation scales, see Fig. 3A and B (Cardoso et al., 2013):
• On the bone organ scale, macroscopically homogeneous regions of cortical or trabecular bone tissue build up whole organs.
Within the organ-scale bone tissue, no further distinction between different material phases is made. This scale is particularly
relevant for imposition of the mechanical loading conditions.
• On the scale of macroscopic bone tissue, two constituents can be distinguished, extravascular bone matrix and vascular pores.
The latter pores, typically exhibiting a characteristic length of 50–80 10 6 m, can be found in cortical bone in the form of
Haversian and Volkmann canals. Under normal physiological conditions, the cortical vascular porosity amounts to approxi-
mately 3%–5%, while in pathological bone, the cortical vascular porosity can be as high 35% (the porosity values relate to the
respective pore volume fractions within macroscopic bone tissue). In trabecular bone, the intertrabecular pores exhibit porosities
ranging from 50% to 90%. Vascular pores contain osteoblasts and osteoclasts (as well as their progenitor cells), and numerous
biochemical factors affecting the behavior of these cells.
• On the scale of extravascular bone tissue, the extralacunar bone matrix hosts the lacunar pores, in which the mechanosensitive
osteocytes reside. The lacunar pores exhibit a characteristic length of approximately 10 5 m. The exact value of the lacunar
(A) Bone organization (C) Micromechanical representation

5 mm
OBU Vascular pore space
on
tiati (pressurized)
en
fer
Dif OCP
OBP
n
Di
Proliferation
tio
Apoptosis
ffe
Apoptosis
ia
re
nt
nt
e
re
ia th ay
on v pathw
ffe
iat
racti G
Di
Inte KL-OP y PTH)
ion
AN b OCA
K-R nced Wnt signalling pathway
OBA RAN o influe
(als
Release of TGFβ
RANKL production
Cross section A-A
OCY
A A
Extravascular bone matrix Lacunar pores

(pressurized)
(B)
dvas
10 μm 2 mm
Lexvas
Lmacro
dlac
10 μm
1 μm Vascular pores Extralacunar bone matrix
Micromechanical representation
Fig. 3 (A) Hierarchical organization of cortical and trabecular bone, illustrated via an X-ray of a human femur showing a cross-section in the mid-
shaft region acquired by means of microradiography, with the zoom into the cortical part showing a scanning electron microscopy (SEM) image
highlighting the arrangement of vascular and lacunar pores, with the latter containing osteocytes, see the respective SEM image, while trabecular
bone, illustrated by means of computed tomography (CT), has a differing microstructure, as shown by photomicrography, however also containing
lacunae; (A) allows to deduce a respective micromechanical representation (B), spanning from macroscopic bone tissue to the lacunar pores; and
(C) the cell population model taking into account cells and biochemical factors, as well as mechanical stimulation in the lacunar and vascular pore
spaces (orange arrows indicate where mechanical loading eventually intervenes with specific, cell development-influencing processes; the X-ray
image of a human femur and the photomicrograph of trabecular bone have been reproduced from Sinclair et al. (2013) with permission of Elsevier
B.V.; the cross-section of the femur has been reproduced by courtesy of John G. Clement and David Thomas (taken from the Melbourne Femur
Collection); the SEM image of cortical bone has been reprinted from Kessel and Kardon (1979), by courtesy of Randy H. Kardon; the CT image of
trabecular bone has been reproduced from Padilla et al. (2008), with permission of Elsevier B.V.; the SEM of a single osteocyte has been reproduced
from Pajevic (2009), by permission from Macmillan Publishers Ltd, IBMS BonekEy, ©2009.
porosity is still a somewhat open issue; depending on the chosen imaging technique, porosities between 2% and 10% have been
identifieddboth values relate to the pore volume fraction within the extravascular bone matrix.
Morphological features discernible on lower observation scales, such as the canalicular pores, or the different arrangements of
the extralacunar bone matrix components collagen, hydroxyapatite crystals, and water are not explicitly resolved in this
model, but implicitly taken into account through an appropriate choice of the extralacunar bone matrix’s mechanical
properties. It should be emphasized that the chosen model representation has been extensively and successfully validated
based on comparing model predictions (in terms of stiffness, strength, poroelastic properties, and viscoelastic behavior)
with corresponding experimental data, considering different types of bone, anatomical locations, species, and experimental
protocols.
Tying in with the aforementioned bone cell population models, the alluded-to modeling approach deals with temporal evolu-
tions of cell numbers, or, when relating the cell numbers to the cell-hosting volumes, of molar cell concentrations, defined as
j Ni
Ci ¼ ; (1)
Vj
with Cij denoting the molar concentration of species i, quantified with respect to the hosting domain j, Ni is the number or the
amount of species i, and Vj is the volume of domain j. It is now instructive to differentiate Eq. (1) with respect to time, in order to
obtain a mathematical expression describing how Cij changes with time, yielding
j
dCi d Ni 1 dNi dVj
¼ ¼ 2 Vj Ni (2)
dt dt Vj Vj dt dt
Multiplying the right-hand side of Eq. (2) with the term (Vmacro/Vmacro), as well as considering the definition of pore volume
fraction fj, fj ¼ Vj =Vmacro , leads to
j j
dCi 1 dNi Ci dfj
¼ : (3)
dt Vmacro fj dt fj dt
Eq. (3) expresses the fact that the concentration Cij changes, on the one hand, due to an increase of the amount of species i, and, on
the other hand, due to the change of the species-hosting volume j.
For the sake of demonstration, Eq. (3) is now applied to a specific subpopulation of osteoblasts, namely the active osteoblasts.
The amount of these cells, NOBA, is known to increase due to differentiation from the committed osteoblast precursor cells,
inhibited by the presence of TGFb, and to decrease due to cell apoptosis. Hence, the following mathematical expression emerges:
dNOBA OBP=OBA vas
¼ Dvas
OBP prep;TGFb NOBP A OBA NOBA ; (4)
dt
where Dvas
OBP denotes the maximum differentiation rate of osteoblast precursors in the vascular pore space; prep,TGFb , the repression
OBP/OBA
function of osteoblast precursor differentiation, governed by TGFb; NOBP, the amount of osteoblast precursor cells; and A vasOBA the
constant apoptosis rate of active osteoblasts in the vascular pore space. Inserting Eq. (4) into Eq. (3) yields then
dCvas OBP=OBA vas vas Cvas
OBA dfvas
OBA
¼ Dvas vas
OBP prep;TGFb COBP A OBA COBA ; (5)
dt fvas dt
with fvas as the vascular porosity. Repression function prep,TGFb
OBP/OBA
can be straightforwardly defined as so-called Hill-type function,
reading as
OBP=OBA
OBP=OBA Krep;TGFb
prep;TGFb ¼ OBP=OBA
; (6)
Krep;TGFb þ Cvas
TGFb
OBP/OBA vas
where Krep,TGFb is the repression coefficient of osteoblast precursor differentiation due to TGFb, and CTGFb is the concentration of
TGF b in the vascular pore space.
Analogously, all other relevant cell populations, biochemical factors, and mechanical stimuli can be considered. In line with the
pioneering contributions in the field, see for example references (Lemaire et al., 2004; Pivonka et al., 2008), the mathematical
framework of the multiscale bone cell population model briefly elaborated here includes the following processes and features,
see also Fig. 3C:
• The osteoblast lineage comprises uncommitted osteoblast progenitor cells, committed osteoblast precursors, and active
osteoblasts.
– Due to their abundance, the concentration of the progenitor cells can be assumed constant.
– The amount of osteoblast precursors increases due to differentiation from the progenitor cells (promoted by the presence of
TGFb), increases due to proliferation (promoted by mechanical stimulation), and decreases due to differentiation to active
osteoblasts (inhibited by the presence of TGFb).
– The concentration of active osteoblasts is governed as described above, see Eqs. (4)–(6).
• The osteoclast lineage comprises committed osteoblast precursors and active osteoblasts.
– Due to their abundance, the concentration of osteoclast precursors can be assumed constant.
– The amount of active osteoclasts increases due to differentiation from the precursor cells (promoted by the binding of RANK
to RANKL, with the availability of RANKL being inhibited by mechanical stimulation) and decreases due to apoptosis
(promoted by the presence of TGFb).
While the mathematical implementation of these aspects is described elsewhere, see references (Lemaire et al., 2004; Pivonka et al.,
2008; Scheiner et al., 2013) , explaining how mechanical stimulation of bone remodeling can be efficiently considered is still
pending. For this purpose, the proliferation of osteoblast precursors is considered. Similarly to the Hill-type repression function
defined in Eq. (6), upregulation of osteoblast precursor proliferation can be considered by function Pmech,vas
act,OBP ; taking into account
only the change in the amount of the osteoblast precursors due to proliferation, one would obtain
vas
dNOBP
OBP Pact;OBP NOBP ;
mech;vas vas
¼ P vas (7)
dt prolif
OBP is the maximum proliferation rate of osteoblast precursor cell in the vascular pore space. For definition of Pact,OBP , it is
where P vas mech,vas
considered that the promotion of osteoblast precursor proliferation occurs in two ways: On the one hand, it has been shown
experimentally that mechanical stimulation of osteoblasts leads to an increase of their proliferation, by up to 100%. On the other
hand, it is also known that mechanical stimulation of osteocytes leads to downregulation of sclerostin expression, which, in turn,
somehow causes upregulation of osteoblast precursor proliferation via the Wnt/b-catenin pathway. For capturing these processes
mathematically, the following formulation has proven adequate:
" ! !#
b mech;vas mvas exvas 1 fvas mlac
Pact;OBP ¼ P act;OBP 1 þ lOBP ref 1 þ lWnt=scl COCY
mech;vas vas vas
1 1: (8)
mvas fvas mref
vas
In Eq. (8), P b mech;vas is the baseline value of Pmech,vas (thus indicating systemic precursor proliferation, independent of the
act;OBP act,OBP
mechanical loading), lOBP vas
is the anabolic strength parameter related to direct excitation of the precursor cells (quantifying how
mechanical stimulation is translated into additional proliferation), lWnt/scl vas
is the anabolic strength parameter related to excitation of
exvas
the osteocytes, COCY is the concentration of osteocytes across the extravascular bone matrix, mvas and mlac are the mechanical stimuli
occurring in the vascular and in the lacunar pore spaces, while mvas ref
and mlac
ref
are the respective reference values, relating to steady-state
conditions. For further definition of the mechanical stimuli, it is taken into account that the hydrostatic pressure has turned out to
be a promising candidate as mechanoregulatory stimulus, that the prime properties of loading history are the peaks (and not the
average loading), and that not only the magnitudes of the loading peaks are decisive but also the frequency at which they occur; thus
peak peak
mvas ¼ pvas f and mlac ¼ plac f, where pvas peak peak
and plac are the vascular and lacunar pore pressures related to the loading peaks, and f is
their frequency. Notably, the model representation illustrated in Fig. 3B satisfies the so-called separation of scales condition for
definition of representative volume elements (RVEs), in a micromechanical sense, thus allowing to employ the theory of poro-
micromechanics. On this conceptual basis, it is possible to straightforwardly calculate the hydrostatic pressures in the vascular and
lacunar pore spaces as they occur in response to macroscopically applied physiological loading.
Next, the focus is on highlighting how such multiscale systems biology model of bone remodeling can be evaluated. Firstly,
formation and resorption of bone was simulated considering the mouse tibia, for which the mechanical loading at which the
bone composition remains constant was experimentally identified to be related to a macroscopic (compressive) peak strain of
Epeak ¼ 1056 106 , at a load frequency of 0.1 Hz, applied with 40 load cycles per day. Furthermore, both cortical bone,
with an initial vascular porosity of 0.05, and trabecular bone, with an initial vascular porosity of 0.75, were considered, while
for both types of bone a lacunar porosity of 0.1 was prescribed, quantified within the extravascular bone matrix. Then, the effects
of different loading regimes were studied, considering peak loads corresponding to macroscopic peak strains of 0, 500 106 ,
1000 106 , 1500 106 , and 2000 106 (all of which are initialized at t ¼ 0). Results are illustrated in Fig. 4, showing
how the bone composition (quantified in terms of the vascular porosity) adapts to the changed mechanical loading. In particular,
for both cortical bone, see Fig. 4A, and trabecular bone, see Fig. 4B, the vascular porosity increases over time if the prescribed
loading is lower than the reference value (Epeak ¼ 1056 106 ), while it decreases if the prescribed loading is higher than
the reference value. The relative change is proportional to the difference of the prescribed loading to the reference value, and to
the time span this adaptation process requires. Notably, in cortical bone, bone loss due to disuse loading turns out to be more
pronounced (with respect to trabecular bone), whereas in trabecular bone, bone gain due to overloading turns out to be more
pronounced (with respect to cortical bone). Moreover, the adaption to disuse loading occurs generally faster than to overloading.
Secondly, as shown in histomorphometric studies, the vascular porosity increases with increasing age, while the lacunar porosity
decreases with increasing age. Considering related experimental trends in a simulation of disuse loading reveals how the effect of
loading may influence mechanical regulation of bone remodeling. In particular, human bone was studied, with “normal” loading
resulting from walking (related to peak strains of Epeak ¼ 500 106 , occurring at a frequency of 2 Hz, and a disuse loading
represented by peak strains of Epeak;disuse ¼ 450 106 , occurring again at a frequency of 2 Hz). The disuse loading was initiated
at t ¼ 0, and after 5 years the loading was set back to normal loading conditions. Furthermore, two different starting ages were
considered, namely 18 and 60 years, in order to investigate whether and to which extent the effect of aging varies between signif-
icantly different age groups. The simulation results show that the vascular porosity is much less affected by changes in the
1.0
Epeak = 0 Epeak = −1000e–6 Epeak = −2000e–6
Epeak = −500e–6 Epeak = −1500e–6
0.8
Trabecular bone
Vascular porosity [-]

0.6
0.4
0.2
Cortical bone
0
0 20 40 60 80 100
Time after initialization of loading regime [days]
Fig. 4 Computed developments of the vascular porosity related to cortical bone (starting at an initial porosity of fvas ¼ 0.05) and trabecular bone
(starting at an initial porosity of fvas ¼ 0.75), for various disuse and overloading scenarios.
(A) (B) (C)

0.09 0.09 0.051
0.08 0.085 0.05075

Vascular porosity [-]
0.07 0.08 0.0505
0.06 0.075 0.05025
0.05 0.07 0.05

0 2 4 6 8 10 4.95 5 5.05 9.9 9.95 10
Time after initialization of the loading regime [years]
No aging Aging, 18-year old Aging, 60-year old
Fig. 5 Effects of aging on the mechanoresponsiveness of human (cortical) bone, when subjected to disuse conditions at t ¼ 0, returning to normal
physiological loading at t ¼ 5 years: comparison of nonaging bone and aging bone with disuse initiated at ages 18 and 60 years; (A) the development
of the vascular porosity over 10 years, (B) a zoom-out to the time span around 5 years, and (C) a zoom-out to the time span immediately before 10
years.
mechanical loading conditions in an older subject compared to a younger subject (see Fig. 5). However, it becomes also apparent in
these simulations that, in quantitative terms, the effect of porosity changes on the mechanoresponsiveness of bone is of minor
importance, and consideration of additional mechanisms, such as osteocyte apoptosis, may be required in order to explain why
in senescent bone the responses to changes in the mechanical loading differ significantly from the responses in young bone.
Besides the presented parameter studies, the multiscale systems biology model could be successfully validated considering
mechanical excitation of mouse tibiae (including both disuse and overuse), disuse in the tibiae of rats induced by tail suspension,
and disuse in humans due to exposure to microgravity.
Summary and Outlook
The intention of this article was to present an overview on the intricacies and various aspects that have to be considered when
attempting to simulate bone remodeling computationally. On the one hand, the value of numerical methods (mostly of the Finite
Element) stands undisputed, particularly in the context of studying the mechanobiological behavior of whole bone organs. The
ever-increasing accuracy of high-resolution imaging of bone allows nowadays to create three-dimensional numerical models which
are precise replicates of the bone organ under investigation. The challenges to be tackled in future research activities involve, to
a large extent, the conditioning of numerical models such that the governing field equations are solvable within a reasonable
amount of time.
On the other hand, coupling the aforementioned large-scale numerical models with multiscale material models, taking into
account the hierarchical organization of bone tissue, and also the biochemically as well as biomechanically driven dynamic
evolution of the bone composition (in response to the immediate environment of the respective organ, see the section on
“New Development: Pore Space-Specific, Multiscale Modeling” of this article), seems to be a promising strategy. In this way,
the various aspects of bone remodeling (being of structural, compositional, biochemical, and biomechanical kinds), perceptible
on distinctively separated characteristic lengths but together contributing to the mechanobiological behavior of bone, can be
reconciled.
References
Cardoso, L., Fritton, S. P., Gailani, G., Benalla, M., & Cowin, S. C. (2013). Advances in assessment of bone porosity, permeability, and interstitial fluid flow. Journal of Biomechanics,
46, 253–265.
Fritsch, A., & Hellmich, C. (2007). ‘Universal’ microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: Micromechanics-based prediction of
anisotropic elasticity. Journal of Theoretical Biology, 244, 597–620.
Galea, G. L., Price, J. S., & Lanyon, L. E. (2013). Estrogen receptors’ roles in the control of mechanically adaptive bone (re)modeling. BoneKEy Reports, 2, 413.
Klein-Nulend, J., Bacabac, R. G., & Mullender, M. G. (2005). Mechanobiology of bone tissue. Pathologie Biologie, 53, 576–580.
Lemaire, V., Tobin, F. L., Greller, L. D., Cho, C. R., & Suva, L. J. (2004). Modeling the interactions of osteoblast and osteoclast activities in bone remodeling. Journal of Theoretical
Biology, 229, 293–309.
Pastrama, M. I., Scheiner, S., Pivonka, P., & Hellmich, C. (2018). A mathematical multiscale model of bone remodeling, accounting for pore space-specific mechanosensation.
Bone, 107, 208–221.
Pivonka, P., Zimak, J., Smith, D. W., Gardiner, B. S., Dunstan, C. R., Sims, N. A., Martin, T. J., & Mundy, G. R. (2008). Model structure and control of bone remodelling: A
theoretical study. Bone, 43, 249–273.
Robling, A. G., Castillo, A. B., & Turner, C. H. (2006). Biomechanical and molecular regulation of bone remodeling. Annual Reviews of Biomedical Engineering, 8,
455–498.
Scheiner, S., Pivonka, P., & Hellmich, C. (2013). Coupling systems biology with multiscale mechanics, for computer simulations of bone remodeling. Computer Methods in Applied
Mechanics and Engineering, 254, 181–196.
Scheiner, S., Pivonka, P., & Hellmich, C. (2016). Poromicromechanics reveals that physiological strains induce osteocyte-stimulating lacunar pressure. Biomechanics and Modeling
in Mechanobiology, 15, 9–28.
Schulte, F. A., Zwahlen, A., Lambers, F. M., Kuhn, G., Ruffoni, D., Betts, D., Webster, D. J., & Müller, R. (2013). Strain-adaptive in silico modeling of bone adaptationdA computer
simulation validated by in vivo micro-computed tomography data. Bone, 52, 485–492.
Sugiyama, T., Meakin, L., Browne, W. J., Galae, G. L., Price, J. S., & Lanyon, L. E. (2012). Bones’ adaptive responses to mechanical loading is essentially linear between low strains
associated with disuse and the high strains associated with the lamellar/woven bone transition. Journal of Bone and Mineral Research, 27, 1784–1793.
Turner, C. H. (1999). Toward a mathematical description of bone biology: The principle of cellular accommodation. Calcified Tissue International, 65, 466–471.
Vass, V., Morin, C., Scheiner, S., & Hellmich, C. (2018). Review of “universal” rules governing bone composition, organization, and elasticity across organizational hierarchies. In
P. Pivonka (Ed.), CISM Courses and Lecture Series, Vol. 578: Multiscale Mechanobiology of Bone Remodeling and Adaptation (pp. 175–229). Berlin: Springer.
Webster, D., & Müller, R. (2011). In silico models of bone remodeling from macro to nanodFrom organ to cell. Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 3,
241–251.
Further Reading
Aubin, J. E. (1998). Bone stem cells. Journal of Cellular Biochemistry Supplements, 30(31), 73–82.
Buckwalter, J. A., Glimcher, M. J., Cooper, R. R., & Recker, R. (1995a). Bone biology. Part I. Structure, blood supply, cells, matrix, and mineralization. Journal of Bone and Joint
SurgerydSeries A, 77, 1256–1275.
Buckwalter, J. A., Glimcher, M. J., Cooper, R. R., & Recker, R. (1995b). Bone biology. Part II. Formation form, modeling, remodeling, and regulation of cell function. Journal of Bone
and Joint SurgerydSeries A, 77, 1276–1289.
Busse, B., Djonic, D., Milovanovic, P., Hahn, M., Püschel, K., Ritchie, R. O., Djuric, M., & Amling, M. (2010). Decrease in the osteocyte lacunar density accompanied by
hypermineralized lacunar occlusion reveals failure and delay of remodeling aged human bone. Aging Cell, 9, 1065–1075.
Colloca, M., Blanchard, R., Hellmich, C., Ito, K., & van Rietbergen, B. (2014). A multiscale approach for bone remodelling simulations: Linking scales from collagen to trabeculae.
Bone, 64, 303–313.
Cooper, D. M. L., Thomas, D. L., Clement, J. G., Turinsky, A. L., Sensen, C. W., & Hallgrímson, B. (2007). Age-dependent change in the 3D structure of cortical porosity at the
human femoral midshaft. Bone, 40, 957–965.
Coussy, O. (2004). Poromechanics. Chichester: John Wiley & Sons.
Dormieux, L., Kondo, D., & Ulm, F.-J. (2006). Microporomechanics. Chichester: John Wiley & Sons.
Jacobs, C. R., Temiyasathit, S., & Castillo, A. B. (2010). Osteocyte mechanobiology and pericellular mechanics. Annual Reviews of Biomedical Engineering, 12, 369–400.
Kessel, R. G., & Kardon, R. H. (1979). Tissues and Organs: A Text-atlas of Scanning Electron Microscopy. San Francisco: W.H. Freeman and Co.
Khayyeri, H., Isaksson, H., & Prendergast, P. J. (2015). Corroboration of computational models for mechanoregulated stem cell differentiation. Computer Methods in Biomechanics
and Biomedical Engineering, 18, 15–23.
Lauffenburger, D. A., & Linderman, J. J. (1993). ReceptorsdModels for Binding, Trafficking, and Signaling. New York: Oxford University Press.
Lerebours, C., Buenzli, P. R., Scheiner, S., & Pivonka, P. (2016). A multiscale mechanobiological model of bone remodeling predicts site-specific bone loss in the femur during
osteoporosis and mechanical disuse. Biomechanics and Modeling in Mechanobiology, 15, 43–67.
Ozcivici, E., Luu, Y. K., Adler, B., et al. (2010). Mechanical signals as anabolic agents in bone. Nature Reviews. Rheumatology, 6, 50–59.
Padilla, F., Jenson, F., Bousson, V., Peyrin, F., & Laugier, P. (2008). Relationships of trabecular bone structure with quantitative ultrasound parameters: In vitro study on human
proximal femur using transmission and backscatter measurements. Bone, 42(6), 1193–1202.
Pajevic, P. D. (2009). Regulation of bone resorption and mineral homeostasis by osteocytes. IBMS BoneKEy, 6, 63–70.
Rubin, C. T., & Lanyon, L. E. (1985). Regulation of bone mass by mechanical strain magnitude. Calcified Tissue International, 37, 411–417.
Sinclair C, Birch HL, Smith RKW, and Goodship AE (2013) Skeletal physiology: Responses to exercise and training. In: Equine Sports Medicine and Surgery, 2nd edn., pp.
145–165.
Turner, C. (1992). Three rules for bone adaptation to mechanical stimuli. Bone, 23, 399–407.
Multiscale Mechanical Behavior of Large Arteries
Claire Morin, Ecole Nationale Supérieure des Mines de Saint-Etienne, CIS-EMSE, SAINBIOSE, Saint Etienne, France; INSERM, Saint
Etienne, France; and Université de Lyon, SAINBIOSE, Saint Etienne, France
Witold Krasny, Ecole Nationale Supérieure des Mines de Saint-Etienne, CIS-EMSE, SAINBIOSE, Saint Etienne, France; INSERM,
Saint Etienne, France; Université de Lyon, SAINBIOSE, Saint Etienne, France; and Université de Lyon, Ecole Centrale Lyon, France
Stéphane Avril, Ecole Nationale Supérieure des Mines de Saint-Etienne, CIS-EMSE, SAINBIOSE, Saint Etienne, France; INSERM,
Saint Etienne, France; and Université de Lyon, SAINBIOSE, Saint Etienne, France
Introduction 180
General Information 180
Structure–Property Relationships 181
Characterization of the Arterial Tissue Structure 181
Multiscale Observation Techniques of the Arterial Structure 182
Macrostructure (100 mm–5 mm) 182
Microstructure (5–100 mm) 182
Ultrastructure 184
Hierarchical Organization of the Arterial Tissue 184
Macrostructure 184
Microstructure 184
Ultrastructure 186
Quantitative Characterization Techniques of the Arterial Microstructure 186
Collagen and elastin mass fractions at the macroscopic scale 186
Volume fraction of fibers at the macroscopic scale 187
Quantitative Assessment of the Arterial Microstructure 187
Universal pattern for the arterial composition 187
Quantitative parameters related to the fiber network arrangement 188
Characterization of the Arterial Tissue’s Mechanical Function 188
Multiscale Characterization Techniques of the Arterial Mechanical Response 189
At the macrostructural scale 189
At the microstructural scale 189
At the ultrastructural scale 190
Multiscale Mechanical Response of the Arterial Tissue 190
At the macroscopic scale 190
At the microstructural scale 191
At the ultrastructural scale 195
Conclusion 197
References 198
Further Reading 202
Glossary
Autoclave pressure chamber used to carry out processes requiring elevated temperature and pressure different from
ambient air pressure. In the sequel, autoclaving is used to separate soluble proteins (such as collagen) from nonsoluble
proteins (such as elastin). Constitutive relation in mechanics, material-specific relation between stress and strain that
characterizes the response of the material to an applied mechanical loading.
In vivo pre stretch and pre stress The in vivo state of the arterial tissue is not free of mechanical loading; in particular, it is
subjected to an axial elongation and residual stresses. Staining auxiliary technique used in microscopy to highlight
structures in materials. For biological tissues, it involves the use of structure-specific dyes.
Introduction
General Information
In the cardiovascular system, the main role of arteries is to ensure the circulation of blood from the heart to peripheral capillaries, in
order to deliver oxygen and nutrients to all tissues of the body. This physiological function can be maintained as long as the arterial

Biomechanics j Multiscale Mechanical Behavior of Large Arteries 181
system can receive spurts of blood from the left ventricle and distribute them as steady flow through peripheral capillaries. This
implies that flow and pressure pulsations remain confined to the larger arteries which act as cushions through elastic dilatation
and recoiling at every cardiac cycle (O’Rourke and Hashimoto, 2007). In more details, during the systolic phase, the blood pressure
rises and the large arteries distend and store blood; then, when the blood pressure falls during the diastolic phase, they recoil and
discharge the previously stored blood to the smaller (muscular) arteries. This specific mechanical property of the large elastic arteries
is usually referred to as the Windkessel effect (Wagner and Kapal, 1952). Fulfilling these different functions involves ensuring the
mechanical integrity of the arterial structure. Conversely, a failure in the mechanical integrity of arteries can have lethal consequences,
due to a lack in oxygen supply to different organs. As a matter of fact, the World Health Organization reports that cardiovascular
disorders are the first cause of death worldwide, killing 17.5 million of people yearly (i.e., 31% of deaths) (Sidloff et al., 2014; San-
tulli, 2013). Many of these cardiovascular disorders are directly related to dysfunctions of the arterial mechanical role. For instance, an
aneurysm is a permanent localized dilatation of an artery having at least a 50% increase in diameter compared to the expected normal
diameter of the artery in question (Johnston et al., 1991). It is characterized by a modified and generally damaged arterial microstruc-
ture as well as by strongly modified mechanical load conditions applied on the arterial wall, jeopardizing the mechanical integrity of
the artery. When detecting an aneurysm, clinicians need to decide whether or not a surgical intervention has to be scheduled. Surgical
interventions consisting in the replacement of the aneurysmal aorta by a vascular prosthesis may have a mortality rate oscillating
between 3% and 5%. Therefore, taking a clinical decision in such circumstances requires reliable and patient-specific criteria about
the potential risks of both elective repair and conservative management of the aneurysm. Currently, surgery is clinically indicated by
an invariable, purely geometrical criterion combining diameter and growth rate thresholds, representing a trade-off between surgery
mortality and rupture (Davies et al., 2002; Fillinger, 2007). However, it is widely acknowledged that this criterion is not a reliable
predictor of rupture risk (Gasser et al., 2010). As another example of a common cardiovascular pathology, atherosclerosis manifests
as thickening and hardening of arteries, with the formation of a fatty, stiff plaque inside the arterial wall. Atherosclerotic plaques
progressively obstruct the lumen and therefore hamper oxygen delivery to the organs. In some cases, debris of plaque may detach
from the wall and may block the blood flow downstream, eventually causing a heart attack or stroke.
If one wants to adapt and improve the management of these cardiovascular disorders, there is a pressing need to develop
methods for assessing the mechanical integrity of blood vessels accounting for their loading conditions and composition (deBotton
and Oren, 2013).
Structure–Property Relationships
The mechanical integrity of biological tissues primarily depends on: (1) the mechanical loading to which it is subjected, (2) its
specific geometry, (3) its mechanical properties. At the tissue scale, the mechanical properties of arteries appear as extremely scat-
tered and were shown to vary with aging and pathologies. They vary also across the considered organs or species (Lally et al., 2004;
Weizsäcker et al., 1983; Holzapfel et al., 2005). However, this variability may mainly result from the variability of microstructures
and compositions in the tested tissues (Holzapfel et al., 2005).
Due to its biological nature, the arterial composition and microstructure are not invariant over time but actually change due to
biological processes called growth and remodeling (Gibbons and Dzau, 1994; Humphrey, 1995). The latter depend not only on
biochemical conditions but also on the mechanical loading itself (Leung et al., 1976; Humphrey, 2006; Humphrey et al., 2014),
such that the tissue actively adapts to its bio-chemo-mechanical environment. Tissue adaptation is thought to be governed by
the different populations of cells in the arteries which try to reach a homeostatic bio-chemo-mechanical state (Humphrey,
2008). Cardiovascular pathologies or aging manifest with altered remodeling which may severely impact the mechanical integrity
of arteries at long term (Jacob et al., 2001). For instance, abnormally high blood pressure (hypertension) results in wall thickening
and stiffening (Berry and Greenwald, 1976; Jacob et al., 2001). These altered conditions may lead to accelerated fatigue and failure
of the different arterial wall constituents, such as the elastic lamellae, and therefore to an increased stiffening of the arterial wall. This
may end up with transmission of the pulsatile blood flow to smaller vessels and organs. Also, in the aorta, (mechanical) damage to
or (chemical) degradation of the elastic fibers, in combination with the loss of smooth muscle function, is a common early contrib-
utor to the formation or expansion of all aneurysms. Then, both remodeling of collagen (i.e., turnover) and inflammation (i.e.,
invasion of mononuclear cells such as lymphocytes T and B) play a fundamental role in aneurysm enlargement and rupture (Jacob
et al., 2001). All these examples show that the vascular pathologies may be translated at a given time instant into microstructural
changes, whether composition, arrangement, or mechanical properties of the individual constituents. Still, up to now, mechanical
and microstructural investigations were achieved separately and rarely bridged together. However, improving diagnosis and treat-
ment of cardiovascular pathologies necessarily implies to account for the structure–property relationships (Humphrey, 2009).
In this article, we review all relevant information about the structure–property relationships in healthy large elastic arteries. We
first review in “Characterization of the Arterial Tissue’s Structure” section their microstructure organization, their characterization,
and their composition. Then we review in “Characterization of the Arterial Tissue’s Mechanical Function” section their multiscale
mechanical properties, showing the large variability across species and organs.
Characterization of the Arterial Tissue Structure
Arteries are characterized by a great diversity and variability. Depending on the species or on the location in the organism, they have
to sustain and regulate different levels of blood pressure. Still, the hierarchical structure of the arterial wall remains unchanged
182 Biomechanics j Multiscale Mechanical Behavior of Large Arteries
throughout the vertebrate kingdom. The arterial wall is made up by three main elementary constituents, namely elastin (see
Fig. 1(J)), collagen (see Fig. 1(H)), and water, as well as by different other organic molecules (e.g., glycosaminoglycans, proteogly-
cans). These elementary constituents are arranged in morphological hierarchical structures occurring in most of the large arteries of
vertebrates and described by means of the following three levels:
- At a characteristic length of several millimeters, the macrostructure of the arterial wall is characterized by varying thicknesses and
mechanical properties depending on the precise biological function of the considered arterial wall (see Fig. 1(A)). This arterial
macrostructure is composed, at a few hundreds of micrometers scale, of three concentric tunicae: the intima, media, and
adventitia, see Fig. 1(B). Each of these layers has a specific morphology as well as a specific function within the arterial wall. This
scale will be referred to as the macrostructure of the arterial wall.
- At a characteristic length of some micrometers to some tens of micrometers, each layer is made up by an arrangement of cells
embedded in different imbricated fibrous elastic and collagenous networks (see Fig. 1(C–F)). This will be referred to as the
arterial microstructure.
- Zooming at these fibrous networks reveals the ultrastructure of the fibers, made of an arrangement of cross-linked fibrils, at
a hundred of nanometers scale, see Fig. 1(G, I).
Understanding the arrangement of the different constituents within the arterial wall has been the source of an impressive amount of
scientific publications; the emergence in the last 15 years of multiphoton microscopy has led to a better understanding of the
constituent arrangement and of the relationship to their mechanical function. After a brief overview of the diverse observation tech-
niques, the multiscale description of the arterial wall microstructure will be reviewed in details. As a second step, the qualitative
hierarchical description of arteries is completed by the extraction of quantitative parameters characterizing this microstructure,
such as arterial composition, fiber orientations, and shape.
Multiscale Observation Techniques of the Arterial Structure

The hierarchical character of arteries has been revealed by the use of different microscopy techniques, offering a wide range of reso-
lutions, as well as different imaging characteristics (in-depth resolution, need for prior staining, etc.).
Macrostructure (100 mm–5 mm)

The composite structure of the artery has been originally revealed by histological studies using optical microscopy. Optical micros-
copy is the gold standard to observe the composition of tissues. It uses visible light and a system of lenses to magnify images of
biological samples. The specimens are previously sectioned (cut into a thin cross section with a microtome), stained, and mounted
on a microscope slide.
Microstructure (5–100 mm)

Different microscopy techniques may be used to visualize the arrangement of the vascular microstructure at the micrometer scale.
One common optical sectioning method allowing in-depth imaging is called confocal microscopy or confocal laser scanning
(Voytik-Harbin et al., 2003). It consists in the installation of a spatial pinhole at the confocal plane of the lens, which acts as a spatial
filter and allows only the in-focus portion of the light to be imaged. Confocal microscopy is characterized by an increased spatial
resolution but decreased signal intensity, inducing long exposure times of the biological samples to the imaging beam and the use
of photomultipliers. More recently, multiphoton microscopy, also called nonlinear or two-photon microscopy, has been developed
in order to increase axial resolution and penetration depth (van Zandvoort et al., 2004). Under multiphoton microscopy, the
sample is illuminated at twice its normal excitation wavelength by a high-energy short-pulsed laser beam, allowing fluorescence
to be precisely localized to the illumination region. When imaging biological tissues, the signal of collagen is generated from
the second harmonic generation, while elastin signal on the other hand is recovered by autofluorescence, before being collected
by two bandpass filters. Multiphoton microscopy can also reveal vascular cells such as fibroblasts or smooth muscle cells (O’Con-
nell et al., 2008) upon prior staining for fluorescence. The both aforementioned techniques provide high optical resolution and
contrast while eliminating out-of-focus light. Eventually, they enable the reconstruction of three-dimensional structures from the
obtained images by collecting sets of images at different depths.
Diffusion tensor imaging (Vilanova et al., 2006) relies on a different technology: it uses the diffusion of water molecules to
generate contrast in magnetic resonance images. As the molecular diffusion of water in tissues is constrained, it can reflect interac-
tions with many obstacles when tracked, such as macromolecules, fibers, and membranes. This tracking approach provides
a mapping of diffusion patterns revealing microscopic details about the tissue architecture.
Tomographic imaging of biological tissues provides section views of organic components through the use of various penetrating
waves (e.g., X-ray). Tomographic slices can be reconstructed into 3D views by means of specific reconstruction techniques (Fujimoto
et al., 1999).
Finally, the vascular microstructure can also be imaged using polarized light microscopy (Gasser et al., 2012). Polarized light is
obtained by means of a polarizer oriented at 90 to filter out the directly transmitted light. When imaging biological tissues under
polarized light, previously stained collagen expresses different interference colors influenced not only by the fiber thickness but also
by the way collagen fibers are packed.
(A) (B) (C)

20 μm
Lumen
(D)
5 μm
1 mm
(E1) (F1)
50 μm 50 μm
Lumen
50 μm 50 μm
Longit. Adventitia Longit.

Intima
Circumf. Circumf.
(E2) (E3) (F2) (F3)
(G) (H) (I) (J)

1 μm 10 nm Lumen 10 μm 10 nm
Fig. 1 Hierarchical structure of large elastic arteries: (A) macroscopic view of large arteries of a mouse, taken from Keyes et al. (2011)); (B) at the
millimeter scale, the arterial wall is made of three concentric layers, the adventitia tunica (A), the tunica media (M) and the intima tunica (I); electron
micrograph of a rabbit femoral artery from Ratz (2016); (C) at the micrometer scale, the intima is made of a continuous layer of endothelial cells
(inverted phase microscope image of bovine aortic endothelial cells from Ives et al. (1986); the media is made of an arrangement of medial lamellar
units, as seen in (D) by means of scanning electron microscopy on a rat abdominal aorta (taken from O’Connell et al., 2008), and (F1) from multi-
photon microscopy stack of images; zooming further on the lamellar unit, multiphoton microscopy allows to distinguish (F2) the collagen fibers and
(F3) the elastin network; the adventitia is made of (E1) an arrangement of collagen bundles (also E2) and elastin fibers (also E3); finally, the collagen
fibers are made of a (G) staggered arrangement of collagen fibrils (scanning electron microscope image taken from Ushiki, 2002), themselves made
of an (H) arrangement of cross-linked collagen molecules (scanning electron microscope image taken from Ushiki, 2002); while the elastin network is
made of (I) elastic fibers, lamellae, and struts, themselves made of an (J) arrangement of elastin and cross-linking molecules (scanning electron
microscope image taken from Ushiki, 2002). Images (E1)–(E3, (F1)-(F3) were obtained by a multiphoton microscope (IVTV Platform, ANR-10-EQPX-
06-01, FR) imaging a rabbit carotid artery.
Ultrastructure
At the scale of a few tens to hundreds of nanometers, the resolved structure of the different fiber networks can be revealed by means
of scanning electron microscopy. This technique uses a beam of accelerated electrons as a source of illumination. The higher
resolving power of scanning electron microscopes originates in the wavelength of electrons being up to 100,000 times shorter
than that of visible light photons used in optical microscopy. Prior acid and elastase digestion of the tissue can be performed in
order to highlight specific cells.
Hierarchical Organization of the Arterial Tissue

Use of these different microscopy techniques allowed revealing the hierarchical structure of arteries.
Macrostructure
At the macroscopic scale, the artery is a composite cylindrical structure made of three concentric layers: the adventitia, the media,
and the intima, as seen in Fig. 1(B), an electron micrograph obtained by Ratz (2016). Each of these layers is characterized by specific
microstructures, specific thicknesses (Wolinsky and Glagov, 1967b), different mechanical properties (Holzapfel et al., 2005), and
different structural and biological functions (O’Connell et al., 2008). The relative proportions of layer thicknesses differ in particular
between proximal and distal regions (Canham et al., 1989).
First, the most inner layer of the arterial wall is the tunica intima, made of the endothelium and of an internal elastic lamina. The
endothelium is a monolayer of endothelial cells, lining the luminal surface of blood vessels, as shown in Fig. 1(C), obtained from
(Ives et al., 1986) by means of an inverted phase optical microscope. It plays the role of an interface between the vessel wall and the
blood flow (Ives et al., 1986). The endothelium is subjected to both fluid shear stresses and pressure-induced strains (Ives et al.,
1986). More specifically, shear stresses affect the morphology and function of endothelial cells morphology. Different studies high-
lighted the tendency of the endothelium to align parallel to the principal axis of strain. It was also shown that the morphology of
endothelial cells is closely related to its cytoskeletal structure (Ohashi and Sato, 2005). Moreover, the mechanical forces applied by
the blood flow are essential factors in cardiovascular diseases, such as atherosclerosis, as they can alter the morphology and function
of endothelial cells (Ives et al., 1986). The internal elastic lamina (not shown in Fig. 1) follows the endothelium and is known to
provide structural cohesion and support for axial pretension (Farand et al., 2007; Timmins et al., 2010).
Then, the tunica media, the second concentric layer of the arterial wall, is a concentric set of superimposed medial lamellar units,
as reconstructed in Fig. 1(F1) from a stack of multiphoton microscope images. A single medial lamellar unit, as shown in Fig. 1(D),
obtained from O’Connell et al. (2008), by means of scanning electron microscopy, consists in a row of overlapping smooth muscle
cells surrounded on the upper and lower sides by two concentric elastic lamellae. The overlapping smooth muscle cells lie parallel to
tangential planes of the circumferential direction. The number of lamellar units in the media of adult mammalian aortas has been
shown to be nearly proportional to the aortic radius regardless of species or of variations in measured wall thickness (Wolinsky and
Glagov, 1967a, 1967b). The tunica media is therefore, from a morphological point of view, a composite material organized peri-
odically: the radial transmural disposition of cells and matrix fibers on transverse sections of the media in well-developed aortas is
proved to be cells, elastic lamellae, cells.
Finally, the tunica adventitia, the third and most outer concentric layer of the arterial wall, is made of an arrangement of collagen
bundles and few elastic fibers, as seen in Fig. 1(E1) obtained from reconstruction of a stack of multiphoton microscope images,
together with embedded fibroblasts.
Microstructure
At the micrometer scale, microscopy techniques reveal the precise morphology of the different fiber networks that exist in each arte-
rial layer. Starting from the arterial lumen, in the endothelium, the actin filaments (F-actin) are one of the major cytoskeletal struc-
tures of the endothelial cells (Ookawa et al., 1992); this actin network also exists in the smooth muscle cells. They are organized in
bundles and are usually grouped in the central part of the cells. Noticeably, the redistribution of F-actin filaments within the cells is
one of the early cellular responses to the onset of shear stress: when experiencing low-shear forces coming from the blood flow, F-
actin filaments localize at the periphery of the endothelial cells (Ookawa et al., 1992); whereas when experiencing high shear forces,
F-actin bundles are observed in the central part of the elongated cells. In parallel, the cells orient in the direction of the applied flow.
A continuous sheet of elastin, called the internal elastic lamina (Farand et al., 2007; Timmins et al., 2010), surrounds the endothe-
lium and marks a frontier between the endothelium and the tunica media. It takes the form of a dense elastin sheet equipped with
a longitudinal network of elastic fibers coated on its continuous surface.
Then, as mentioned earlier, the medial lamellar unit is made of an arrangement of elastic fibers, vascular smooth muscle cells,
and collagen fibers, which are now successively described. From a morphological point of view at the micrometer scale, medial
elastin takes three different forms:
- First, lamellae, composed of a dense meshwork of elastic fibers oriented circumferentially (Farand et al., 2007; Timmins et al.,
2010). They form a periodical concentric separation between medial lamellar units containing the vascular smooth muscles cells,
see Fig. 1(F1) and Fig. 2(E1). These lamellae show large, round, reinforced fenestrations (see Fig. 2(E2), also visible on the upper
lamella of Fig. 1(F1)), and house the superior and inferior anchorages of smooth muscle cells, allowing them to weave through
the tunica media (Dingemans et al., 2000);
E1 E2
C1
C2
C3
SMC
r
z 5 μm
θ
E3 E4
Fig. 2 Schematic representation of the medial lamellar unit morphology. (E1) elastin lamella, (E2) fenestration of the elastin lamella, (E3) elastin
interlamellar struts, (E4) elastin surface ridges, (SMC) smooth muscle cells, (C1) collagen fibers, (C2) cohesive collagen microfibril bundles, (C3)
collagen envelope of SMCs. Inspired from illustrations by Dingemans, K. P., et al. (2000). Extracellular matrix of the human aortic media: An ultra-
structural histochemical and immunohistochemical study of the adult aortic media. Anatomical Record 258 (1), 1–14; O’Connell, M. K., et al. (2008).
The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging.
Matrix Biology 27 (3), 171–181.
- Second, thick radial elastin struts provide structural cohesion to the overall medial lamellar unit, while preserving an angular 20
tilt with respect to the smooth muscle cell orientation, see Fig. 2(E3) (Dingemans et al., 2000; Koch et al., 2014; Tsamis et al.,
2013a);
- Finally, thin radial elastic fibers take the form of ridges (Dingemans et al., 2000) or protruding ribs (Raspanti et al., 2006),
connecting the smooth muscle cells to both lamellae, see Fig. 2(E4).
Through their actively adaptive plasticity, the vascular smooth muscle cells are responsible for the regulation and maintenance of
blood flow and for the regulation of stress across the arterial wall thickness. For this reason, vascular smooth muscle cells harbor
actin–myosin filaments (myofilament) that permit rapid stress development and sustain stress maintenance and vessel constriction
(Ratz, 2004). The relaxed smooth muscle cell is a very long and thin (fusiform) structure (Ratz, 2004) with a high surface area, and
an ellipsoidal shape of its nucleus (O’Connell et al., 2008). Upon contraction, the vascular muscle cells can undergo dramatic short-
ening accompanied by shape change, surface rearrangements, and a loss of volume that is recovered upon relengthening. As for their
spatial positioning among the constituents of the medial lamellar unit, it shows noticeable characteristics, namely, the cells weaving
throughout the interlamellar elastin framework, resulting in approximately 20 radial tilt (O’Connell et al., 2008).
Collagen is also organized in different morphologies within the tunica media. First, interlaced bundles of type IV collagen micro-
fibrils (oxytalan fibers) of the immediate pericellular matrix contribute, along with elastin radial struts and interlamellar elastin
protrusions, to smooth muscle cell cohesion and fixation on the lamellae, see Fig. 2(C2) (Clark and Glagov, 1985; Dingemans
et al., 2000). It is understood that the smooth muscle cells preferentially adhere to these ill-defined streaks rather than directly
to the solid lamellae, see Fig. 2(SMC) (Dingemans et al., 2000); second, wavy collagen fiber bundles are interposed between the
facing elastin systems within the fibrous regions between cell layers, see Fig. 2(C1) and Fig. 1(F2) (Clark and Glagov, 1985). These
collagen fibers are oriented circumferentially (O’Connell et al., 2008; Timmins et al., 2010; Roy et al., 2010; Hill et al., 2012) and are
closely associated with the elastic lamellae (Dingemans et al., 2000) but not with the smooth muscle cells. Upon pressure, these
medial collagen fibers decrimp and stretch to prevent over distension of the vessel. Collagen takes also the form of membranes
enveloping the smooth muscle cells (see Fig. 2(C3)).
Finally, the most outer arterial layer, the tunica adventitia, is made of an arrangement of networks of elastin and collagen with
embedded fibroblasts. In the adventitia, elastin takes the form of a low-density meshwork made of variously oriented fibers showing
bifurcations (transversely oriented segments), with a dominant longitudinal direction (Chen et al., 2011, 2013; see Fig. 1(E3)).
Contrarily, collagen fibers pack into thick bundles of 10–30 fibers, folded (crimped) under the in vivo prestress and prestretch
conditions. These wavy bundles are oriented helicoidally about the 45 angle and show a negligible transmurality (Roy et al.,
2010; Rezakhaniha et al., 2012; Schrauwen et al., 2012; see Fig. 1(E1) and (E3)). For a flat piece of arterial tissue, the crimping
appears considerably more important than for a cylindrical piece of arterial tissue, and the bundles are oriented closer to the axial
direction (Tsamis et al., 2013a; Phillippi et al., 2014; Koch et al., 2014; D’Amore et al., 2010). The adventitial collagen network is
capable of undergoing important morphology rearrangements under mechanical load, namely, decrimping, stretching, and re-
orientation in the load direction, with amplitudes that can exceed affinely predicted reorientation (Billiar and Sacks, 1997; Chan-
dran and Barocas, 2006). These changes in morphology are known to be linked to the significant nonlinearity of the mechanical
response and give rise to a very pronounced material stiffening under large strain (Chen et al., 2011; Schrauwen et al., 2012). These
aspects are further detailed in section “Characterization of the Arterial Tissue’s Mechanical Function” of this article. The adventitial
microstructure is also composed of fibroblasts (not shown on Fig. 1), which are mechanosensitive cells, participating to arterial
remodeling and arranged circumferentially about collagen bundles (Esterly et al., 1968).
Ultrastructure
At the hundreds of nanometers scale, the structure of elastic and collagen fibers is revealed, as well as the existence of cross-links
between and within these fibers. The morphology of the elastic lamellae presents a fibrous texture suggestive of a “criss-crossed”,
delicate filamentous scaffold (Ushiki, 2002; Raspanti et al., 2006). The elastic fibers and elastin meshwork however are made of
0.2 mm thick elastin fibrils and microfibrils that run in various directions (see Fig. 1(I) and (J)). Those microfibrils are coated
together within an elastic fiber by proteoglycans and glycosaminoglycans (Ushiki, 2002). At the same scale, collagen bundles
are made of closely packed, parallel, thin collagen fibrils, with a characteristic diameter of 30–100 nm, with altering number of
coated fibrils depending on the region in the bundle (Ushiki, 2002; Raspanti et al., 2006). In the adventitia, these bundles are
thicker than in the media due to a higher number of constituting fibrils, see Fig. 1(G). The fibrils present a regular, orthogonal
lattice of surface-bound proteoglycans (Raspanti et al., 2006; Berillis, 2013) (see Fig. 1(H)). Finally, at the nanometer scale,
the cohesion between fibrils constituting elastic or collagen fibers is realized by cross-linking proteoglycans, formed by covalent
bonding between acid glycosaminoglycans and proteins (Eisenstein et al., 1975). At the microfibrillar level, collagen and elastin
are cross-linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysine side chains (Eyre et al., 1984;
Sáez et al., 2016).
Quantitative Characterization Techniques of the Arterial Microstructure

Many tests were performed over the last 70 years to determine the relative amount of elastin, collagen, and water, among different
arteries, species, and at different ages, resulting in a broad variety of arterial compositions. The relative mass or volume fractions of
collagen and elastin can be determined at different scales, depending on the chosen technique: chemical methods provide average
weight fractions of collagen and elastin over a millimeter-sized dehydrated defatted arterial sample, while histochemical methods
allow determining the volume fractions of collagen and elastin fibers in the arterial tissue.
Collagen and elastin mass fractions at the macroscopic scale

Associated experimental endeavors involve primarily the following steps:
- The arterial samples are generally excised from freshly sacrificed animals and analyzed directly after excision, or wrapped airtight
in plastic films and stored at 20 C (Brüel and Oxlund, 1996; Looker and Berry, 1972). In some cases (Leung et al., 1977; Berry
and Greenwald, 1976; Myers and Lang, 1946), the adventitia is removed by careful dissection, and only the compositions of the
media and the intima are determined.
- Defatting the arterial wall is performed by means of successive immersions in acetone and ether, for different durations
depending on the precise protocol (Fischer and Llaurado, 1966; Harkness et al., 1957; Grant, 1967; Neuman and Logan, 1950;
Feldman and Glagov, 1971);
- The dehydration procedure consists in either drying to constant weight in vacuum (Harkness et al., 1957; Looker and Berry, 1972;
Farrar et al., 1965; Hosoda et al., 1984; Berry and Greenwald, 1976; Feldman and Glagov, 1971; Spina et al., 1983) or drying to
constant weight in an oven for few hours at a temperature higher than 50 C (Grant, 1967; Fischer and Llaurado, 1966; Lowry
et al., 1941; Neumann and Logan, 1950; Leung et al., 1977). As a result, water represents about 70%–80% of the wet weight of
the aortic tissue (Looker and Berry, 1972; Fischer and Llaurado, 1966; Dahl et al., 2007).
- Although different methods exist for the detection and estimation of collagen mass fraction in a biological sample, the deter-
mination of hydroxyproline in the tissue seems to be the most widely used method. Hydroxyproline is an amino acid, whose
content varies between 13.1% in collagen type I to 17.4% in collagen type III (Etherington and Sims, 1981). As elastin also
contains hydroxyproline, collagen needs first to be removed from the tissue by autoclaving. This separation method between
elastin and collagen accounts for the nonsoluble property of elastin. More precisely, a small piece of the dry defatted tissue is
autoclaved twice for 3 h at 1 bar, [Pressure and duration of autoclaving were varied by different experimentalists: opted for 6 h at
1 bar (Grant, 1964; Grant, 1967); for 6 h at 2.75 bar (Feldman and Glagov, 1971); for 18 h at 1–1.4 bar (Fischer and Llaurado,
1966) followed by a second for 3 h; for 6 h at 2 bars (Harkness et al., 1957; Looker and Berry, 1972; Farrar et al., 1965; Berry and
Greenwald, 1976); for 4 h at 3.5 bars (Lowry et al., 1941).] washed with water and centrifuged, and the resulting extracts are dried
out (Neuman and Logan, 1950). Then, the following steps (with possible slight adaptations) are followed for hydroxyproline
determination: (i) hydrolysis at 3.4 bar for 3 h with 6 mol L 1 hydrochloric acid to release the hydroxyproline from peptide
linkage, (ii) oxidation with sodium peroxide, and (iii) formation of a reddish purple complex with p-dimethylamino-
benzaldehyde (Harkness et al., 1957; Looker and Berry, 1972; Farrar et al., 1965; Berry and Greenwald, 1976; Neumann and
Logan, 1950). Chloramine T together with Ehrlich’s reagent for colorimetric determination was preferred for use as an oxidant
instead of sodium peroxide (Stegemann and Stalder, 1967; Hosoda et al., 1984; Brüel and Oxlund, 1996; Han et al., 2009). The
collagen content follows from a multiplicative correction of the hydroxyproline content; this correction factor varies among the
sources between 7.14, 7.46, or 7.8 depending on the exact content of hydroxyproline in the different collagen types (Neuman and
Logan, 1950; Feldman and Glagov, 1971; Fischer and Llaurado, 1966; Grant, 1964, 1967; Harkness et al., 1957; Berry and
Greenwald, 1976).
- After autoclaving the arterial sample, the insoluble elastin remains in the residue, and its content is determined by a gravimetric
method (Looker and Berry, 1972; Berry and Greenwald, 1976; Farrar et al., 1965; Leung et al., 1977; Hosoda et al., 1984; Lowry
et al., 1941; Myers and Lang, 1946; Feldman and Glagov, 1971). In short, this method consists in purifying the residue remaining
after collagen extraction, first by heating for 45 min at 100 C with a 0.1 mol L 1 sodium hydroxide solution (Lansing et al.,
1952) and then by washing with water, dehydrating with acetone, and finally drying to constant weight in a hot air oven (Grant,
1967) or in vacuum over phosphorous pentoxide (Harkness et al., 1957; Looker and Berry, 1972) and weighing. Other methods
for elastin determination include the same procedure of hydroxyproline determination as for collagen (Fischer and Llaurado,
1966; Harkness et al., 1957; Grant, 1964, 1967; Leung et al., 1977) and the determination of elastin-specific cross-linking amino
acids (desmosine and isodesmosine) (Han et al., 2009).
Finally, the volume fractions of cells and/or the cell content were also determined in several studies: dosage of DNA gives access to
the total cell content of arterial tissue, being of 200 million of cells per milligram of dry defatted tissue in adult rats (Dahl et al.,
2007), while histological observations with prior cell staining provide access to the volume fraction of some specific cell popula-
tions (O’Connell et al., 2008; Tonar et al., 2008).
Volume fraction of fibers at the macroscopic scale

Histochemistry consists in studying the chemical constituents of a tissue by means of staining reagents. The arterial tissue is first
fixed with paraffin and then stained with different dyes. In particular, collagen stains include (blue or green) Masson’s trichrome
(Phillippi et al., 2014; Anidjar et al., 1990; Dahl et al., 2007), Picrosirius red (Phillippi et al., 2014), (yellow) Safranin-O staining
(Azeloglu et al., 2008), (pink) eosin (Carmo et al., 2002), Van Gieson staining, which is a mixture of picric acid and acid fuchsin,
staining collagen in red, nuclei in black, and cytoplasm in yellow (Carmo et al., 2002; Cattell et al., 1996). Elastin stains include
(blue-black) Verhoeff’s staining (Tonar et al., 2003; Phillippi et al., 2014; Dahl et al., 2007), permanganate–bisulfite–toluidine
blue reaction (Clark and Glagov, 1985; Fischer, 1979), (red) orcein staining (Hosoda and Minoshima, 1965; Anidjar et al.,
1990; Scarselli and Repetto, 1959; Scarselli, 1959), Weigert’s (blue-black) resorcin-fuchsin staining (Hayashi et al., 1974). Histo-
chemical methods also allow cells staining: toluidine blue stains nucleic acids in blue, hematoxylin (also called Weigert’s iron
hematoxylin stain) stain cells nuclei in dark blue, eosin stains in pink the cell’s cytoplasm (Carmo et al., 2002); finally, Movat’s
stain allows to identify glycosaminoglycans in blue (Dahl et al., 2007).
The stained histological section is observed under a microscope and image processing techniques provide access to quantitative
parameters. For example, several methods have been proposed to analyze the statistical fiber orientation based on microstructure
imaging; they involved Hough transforms (Chaudhuri et al., 1993; Karlon et al., 1998), structure tensor-based texture analysis
(Rezakhaniha et al., 2012), direct fiber tracking (Pourdeyhimi, 1999; Mori and van Zijl, 2002; Rezakhaniha et al., 2012; Hill
et al., 2012; Ghazanfari et al., 2012), or 2D Fast Fourier transform (Ayres et al., 2006; Ayres et al., 2008; Timmins et al., 2010; Schriefl
et al., 2012; Polzer et al., 2013).
Quantitative Assessment of the Arterial Microstructure

Universal pattern for the arterial composition
The mechanical properties of arterial tissues depend primarily on the tissue composition and on the arrangement of the constit-
uents of its microstructure. Since access to the arterial composition is complex in vivo, we here propose to seek for such a general
composition rule, valid over a wide variety of organs, species, and ages. To our best knowledge, no such relationship governing the
arterial composition has ever been proposed. At most, correlations between the arterial composition and some other characteris-
tics of the tissue have been established, but generally limited to very restricted sets of data. We here collected the mass fractions of
elastin and collagen in various large elastic arteries from a great variety of species and ages: from rats to human, from the abdom-
inal to the carotid arteries, etc. However, since important modifications in the arterial composition occur in the perinatal and early
childhood periods (Bendeck and Langille, 1991), as well as in aging organisms (Tsamis et al., 2013b; Schlatmann and Becker,
1977), we restricted our selection to adult organs with no aging effects. In the perinatal period, a rapid accumulation of both
elastin and collagen has been observed followed by a marked postnatal increase in arterial pressure, see (Bendeck and Langille,
1991). Upon aging, the absolute collagen content is increased, while the absolute elastin content remains constant, although
the elastin becomes fragmented (Tsamis et al., 2013b; Schlatmann and Becker, 1977). We plot the collagen mass fraction as a func-
tion of the elastin mass fraction, for cases where those contents are related to the media and intima only (see Fig. 3) or to the whole
arterial thickness (see Fig. 4). It is interesting to notice that collagen and elastin contents strongly correlate mutually, whether only
the intima–media is considered or the entire thickness of the arteries. A closer look into the resulting graphs shows that, as a rule,
the thoracic aortas are characterized by a larger content in elastin as compared to the abdominal aortas, which contain more
collagen (compare the filled markers in Figs. 3 and 4 to the empty ones). This is in agreement with observations made on
more restricted arterial tissue origins and numbers by Harkness et al. (1957), Sokolis (2007), Halloran et al. (1995), Cheuk
and Cheng (2005): the elastin content decreases from proximal to distal regions. In parallel, it was also observed that the number
of lamellar units also decreases in more distal aortas (Wolinsky and Glagov, 1969; Sokolis et al., 2002a,b). These changes in the
arterial composition affect the mechanical behavior, as reviewed in section “Characterization of the Arterial Tissue’s Mechanical
Function”.
65
60
55
50
45
WF collagen
40
35
30
25
20
15
10
10 15 20 25 30 35 40 45 50 55 60 65
WF elastin
Fig. 3 Composition of the media and intima layers of different arteries stemming from rabbit (upward pointing triangles), rats (diamonds), and
human (right pointing triangles). Collagen and elastin weight fractions (WF) are related to the dry defatted weight of the arterial tissue. Filled markers
correspond to thoracic arterial segments, while empty markers correspond to abdominal arterial segments as well as one pulmonary trunk tissue and
one arch tissue. Data were taken from Leung, D. Y., Glagov, S., Mathews, M.B. (1977). Elastin and collagen accumulation in rabbit ascending aorta
and pulmonary trunk during postnatal growth. Correlation of cellular synthetic response with medial tension. Circulation Research 41 (3), 316–323;
Berry, C. L., Greenwald, S. E. (1976). Effects of hypertension on the static mechanical properties and chemical composition of the rat aorta. Cardio-
vascular Research 10 (4), 437–451; Brüel, A., Oxlund, H. (1991). Biosynthetic growth hormone changes the collagen and elastin contents and biome-
chanical properties of the rat aorta. Acta Endocrinologica 125 (1), 49–57; Han, W.-Q., et al. (2009). Changes in the composition of the thoracic aortic
wall in spontaneously hypertensive rats treated with losartan or spironolactone. Clinical and Experimental Pharmacology and Physiology 36 (5–6), 583–
588; Andreotti, L., et al. (1985). Aortic connective tissue in ageing – A biochemical study. Angiology 36 (12), 872–879; Feldman, S. A., Glagov, S.
(1971). Transmedial collagen and elastin gradients in human aortas: Reversal with age. Atherosclerosis 13 (3), 385–394; Apter, J. T., Rabinowitz, M.,
Cummings, D. H. (1966). Correlation of visco-elastic properties of large arteries with microscopic structure. Circulation Research 19 (1), 104–121;
Tonar, Z., et al. (2003). Microscopic image analysis of elastin network in samples of normal, atherosclerotic and aneurysmatic abdominal aorta and
its biomechanical implic ations. Journal of Applied Biomedicine 1 (3), 149–159; Spina, M., et al. (1983). Age-related changes in composition and
mechanical properties of the tunica media of the upper thoracic human aorta. Arteriosclerosis, Thrombosis, and Vascular Biology 3 (1), 64–76.
Such a good correlation between elastin and collagen content could be used to determine the composition of healthy adult
arteries, or to verify if a given arterial segment is healthy.
Quantitative parameters related to the fiber network arrangement

Along with a detailed assessment of the vascular biological composition, many studies have focused on the extraction of particular
geometrical characteristics of the microstructure (Humphrey and Holzapfel, 2012). Nonexhaustively, the implemented image anal-
ysis methods focused on the analysis of the fiber diameters (D’Amore et al., 2010; Phillippi et al., 2014), the fiber lengths (D’Amore
et al., 2010; Rezakhaniha et al., 2011; Hill et al., 2012; Tsamis et al., 2013a; Cicchi et al., 2014), fiber volume fractions (Tonar et al.,
2003; Hayashi et al., 1974; O’Connell et al., 2008; Verheyen et al., 1987), as well as on the evaluation of fiber waviness (Hill et al.,
2012; Roy et al., 2010; Rezakhaniha et al., 2012; Schrauwen et al., 2012), and orientations (Holzapfel, 2006). More recently, for
modeling purposes, more complex morphological features were quantified, such as fiber tortuosity (Koch et al., 2014), node
connectivity and spatial intersections density (D’Amore et al., 2010; Koch et al., 2014), or density of transversely oriented segments
(Koch et al., 2014). The number of elastic lamellae and of fenestrations inside the lamellae (Brüel and Oxlund, 1996) and appear-
ance of collagen fibers (Wolinsky and Glagov, 1964) have also been investigated using image processing techniques.
Characterization of the Arterial Tissue’s Mechanical Function
Arteries exhibit a highly nonlinear mechanical behavior, which has long been investigated from different point of views. As a first
approach to arterial biomechanics, the macroscopic response of the arterial wall has been characterized through different uniaxial,
biaxial, and tension–inflation tests. More recently, experimental setups coupling mechanical testing with live microscopy have been
developed to decipher the microstructural mechanisms that are behind the nonlinear character of the response. Finally, at the
100 nm scale, the constitutive responses of collagen and elastic fibers have been characterized. We here restrict the review to
in vitro mechanical testing techniques.
60
55
50
45
WF collagen
40
35
30
25
20
15
10
10 15 20 25 30 35 40 45 50 55 60
WF elastin
Fig. 4 Composition of the whole arterial segment of different arteries stemming from pigs (hexagrams), dogs (circles), sheep (downward pointing
triangles), rats (diamonds), goats (upward pointing triangles), bovines (squares), puppies (pentagrams), and humans (right pointing triangles).
Collagen and elastin weight fractions (WF) are related to the dry defatted weight of the arterial tissue. Filled markers correspond to thoracic and arch
arterial segments, while empty markers correspond to abdominal and other arterial segments. Data were taken from Grant, R. A. (1967). Content and
distribution of aortic collagen, elastin and carbohydrate in different species. Journal of Atherosclerosis Research 7 (4), 463–472; Grant, R. A. (1964).
Estimation of hydroxyproline by the autoanalyser. Journal of Clinical Pathology 17, 685–686; Neuman, R. E., Logan, M. A. (1950). The determination
of collagen and elastin in tissues. Journal of Biological Chemistry 186 (2), 549–556; Fischer, G. M., Llaurado, J. G. (1966). Collagen and elastin
content in canine arteries selected from functionally different vascular beds. Circulation Research 19 (2), 394–399; Harkness, M. L. R., Harkness, R.
D., McDonald, D. A. (1957). The collagen and elastin content of the arterial wall in the dog. Proceedings of the Royal Society B: Biological Sciences 146
(925), 541–551; Looker, T., Berry, C. L. (1972). The growth and development of the rat aorta. II. Changes in nucleic acid and scleroprotein content.
Journal of Anatomy 113 (Pt 1), 17–34; Farrar, J. F., Blomfield, J., Reye, R. D. K. (1965). The structure and composition of the maturing pulmonary
circulation. Journal of Pathology and Bacteriology 90 (1), 83–96; Hosoda, Y., et al. (1984). Age-dependent changes of collagen and elastin content in
human aorta and pulmonary artery. Angiology 35 (10), 615–621.
Multiscale Characterization Techniques of the Arterial Mechanical Response

At the macrostructural scale
Due to the biological nature of arterial tissue, the storage conditions and the temperature prevailing during the mechanical tests may
have an impact on the in vitro mechanical response of the arterial tissue.
Concerning storage procedures, the arteries are usually harvested in freshly sacrificed animals or shortly after death. After exci-
sion, the arterial sample may be stored for 2–3 days in a saline solution at 4 C, or kept frozen at 20 C or at 80 C (Collins and
Hu, 1972; Pham et al., 2013), to avoid sample drying and accelerated sample degradation. The impact of these different protocols
on the mechanical properties was investigated by comparing the uniaxial tensile response of specimens stemming from the same
tissue sample but subjected to different preparation protocols. Concerning the temperature of the mechanical test, two choices are
classically made: either the ambient temperature of the room or the physiological temperature. Again, the uniaxial tensile responses
relative to different temperatures of tests are compared.
In order to characterize the arterial constitutive behavior, biomechanics imported the well-established uniaxial and biaxial
tensile tests existing for metallic or inert materials to the arterial tissues: across the last 17 years, different arterial tissues stemming
from different species and organs, at different ages, and different healthy or pathological states have been mechanically character-
ized. More precisely, for uniaxial tests, a dog bone-shaped sample is cut from the excised arterial segment, with the circumferential
or longitudinal direction as main axis. Because uniaxial tensile tests do not reproduce the in vivo load conditions to which arteries
are subjected, biaxial tests on flat squared samples have been developed. The load is applied simultaneously in both directions,
maintaining a constant stress ratio between the two directions. Finally, tension inflation tests make use of a cylindrical arterial
segment, subjected to a fixed axial stretch and a variable internal pressure.
At the microstructural scale

Several studies have dealt with the possible relation between the observed variability in the macroscopic mechanical response and
the variability in the arterial wall composition or organization. By combining mechanical tests to histology or to the chemical anal-
ysis of the arterial composition, such studies seek for relations between arterial composition/morphology and subsequent mechan-
ical response. The influence of each fiber network on the overall mechanical response has also been investigated: enzymes such as
collagenase or elastase are used to (partially) degrade one constituent, and the mechanical response of the degraded system is then
compared to that of the original system, and to those of systems with different degrees of degradation.
Besides, different studies proposed to physically separate the three arterial layers, so as to study the contribution of each arterial
layer on the overall mechanical response (Weisbecker et al., 2012; Holzapfel et al., 2005; Sommer et al., 2010).
Aiming at deciphering the microstructural mechanisms that lead to the nonlinear mechanical response, original testing devices
have been developed, permitting live imaging of the arterial microstructure with a multiphoton microscope during the application
of the load. Such tests, coupled to the previously described image processing techniques, permit the characterization of the rear-
rangements of the different fiber networks.
At the ultrastructural scale

At the ultrastructural scale, attempts were made to characterize the mechanical behavior of the elementary constituents of the arterial
wall. We here focus on collagen and elastin.
Concerning collagen, this task is more complex because of the existence of different collagen types, with different mechanical
properties. Our review is restricted to the characterization of collagen type I. This is the most abundant collagen type in bone,
tendon, as well as in the tunica adventitia of arteries. Different characterization techniques have been employed for retrieving its
mechanical characteristics at different length scales. Brillouin light scattering (Harley et al., 1977; Cusack and Miller, 1979) gives
access to the velocity of elastic waves having a wavelength of several hundreds of nanometers; hence, it allows for characterizing
the arrangement of several collagen molecules within fibrils. At a higher scale, nanoindentation tests performed by means of an
Atomic Force Microscope provide access to the mechanical properties of collagen fibrils (Yadavalli et al., 2010; Strasser et al.,
2007; Wenger et al., 2007; Graham et al., 2004; van der Rijt et al., 2006). Finally, the mechanical response of collagenous tissues
at higher scales, from the fibrils to the tissue scales, is characterized by tensile tests, with different types and characteristic sizes of
samples. The mechanical properties of collagen fibrils and fibers primarily depend on the hydration degree of the tissue under
consideration.
The picture is quite different regarding the mechanical characterization of elastin or elastic fibers. Elastic fibers are often charac-
terized by the mechanical properties of collagen-free biological tissues (such as arterial tissue or nuchal ligaments), considering that
the ground substance surrounding the elastic fibers is not mechanically active. Collagen is removed either by autoclaving (Aaron
and Gosline, 1981) or by alkali treatment (Hass, 1942b). Then, uniaxial tensile tests are performed on the extracted elastin fibers.
Multiscale Mechanical Response of the Arterial Tissue

The biological nature and the physiological functions of the arterial tissue render the characterization of the mechanical properties
as a delicate task: harvesting the tissue implies the death of cells, with different consequences: in vitro characterization only inves-
tigates the passive response of the tissue and therefore cannot account for the active role of smooth muscle cells in distributing the
load across the tissue thickness; the termination of the constituent turnover and the progressive degradation of the organic constit-
uents making up the tissue have consequences on the mechanical properties of the tissue; harvesting the tissue also implies the relax-
ation of some of the prestress and prestretch existing in the arterial tissue in vivo, leading to fiber rearrangements in the
microstructure with consequences on the macroscopic mechanical response.
At the macroscopic scale

Before reviewing the macroscopic mechanical properties of arteries, efforts were made toward revealing the influence of sample
freezing or refrigerating on the resulting mechanical behavior. Several studies (Zemánek et al., 2009; Adham et al., 1996; Armentano
et al., 2006) reported no significant variations of the mechanical response after storage, while, in other studies (Venkatasubrama-
nian et al., 2006; Chow and Zhang, 2011; Stemper et al., 2007), the variations in the mechanical behavior encompass variations in
the initial and final stress–strain slopes, as well as changes in the knee point of stress strain curves, and in the ultimate stress. These
variations may be explained by some damage occurring in the sample during freezing or refrigerating: formation of ice crystals, bulk
water movement (Venkatasubramanian et al., 2006) can induce fiber cracking, loss of cross-links, networks disruption, and death of
cells. These variations may also be explained by the decrease in the collagen content after 48 h cold storage (Chow and Zhang,
2011), as well as by the exact procedure followed to freeze the sample. Still, it is impossible to decide on the directions of variations,
since the different relevant studies come to apparently contradictory results. It is however generally admitted that freezing better
maintains the mechanical properties of arterial samples than refrigeration (Chow and Zhang, 2011; Stemper et al., 2007). Zemánek
et al. (2009) studied the influence of the temperature choice on the mechanical response of the arterial wall and showed that
samples are stiffer at ambient temperature than at in vivo temperature, since a temperature increase by 1 C results in a 5% stiffness
decrease; this result is in good agreement with Fung (1993).
Furthermore, arteries are subjected in vivo to residual stresses and prestretch, as evidenced by Bergel (1961), Fung (1993), and
Vaishnav and Vossoughi (1987), the amount of which depends on the organ and species under consideration; this in vivo stress–
strain state originates in the growth and remodeling processes undergone by arteries and allows achieving a homeostatic stress state,
being nearly uniform and equibiaxial across the arterial wall thickness (Humphrey, 2009). As a consequence, excising and cutting
open arterial segments partially release this existing in vivo stress–strain state, but there is no certainty that the load free configu-
ration corresponds to a stress- and strain-free configuration.
Another important feature of the arterial constitutive behavior is the existence of a transient mechanical response: during the first
mechanical cycles, the mechanical response of the arterial wall exhibits an important hysteresis which is reduced after several load
cycles; the stabilized mechanical response barely shows any hysteresis. Experimentalists usually get rid of this transient response by
performing several preconditioning cycles. The number of preconditioning cycles varies with the precise protocol, and the loading
path and maximum load generally coincide with the further applied loading (see column 6 of Table 1). The microstructural under-
lying mechanisms occurring during preconditioning have not been elucidated yet. The mechanisms are probably related to viscous
effects, since the transient response is observed after a prolonged stop of the mechanical loading. Interestingly, Zemánek et al.
(2009) noticed that no preconditioning was necessary for equibiaxial tensile tests.
The previously described macroscopic testing procedures allow characterizing the mechanical response of a millimeter-sized
arterial sample (see Table 1 for a literature review). The arterial wall exhibits a highly nonlinear mechanical response, which was
already described in the 1880s (Roy, 1881): while at low applied stresses arteries are very easily deformed, the arterial response
becomes much stiffer at higher applied stresses. This nonlinear response occurs in any load direction. As many other biological
tissues, arteries exhibit an important variability in their mechanical response, both across species, organs, location, or interindi-
vidual (see Fig. 5). As a result, there is an important variability in the stretch and stress levels at which the change of stiffness reaches
its maximum.
Concerning tension–inflation tests, the arterial response is characterized by the variations of the arterial diameter as a function of
the applied pressure as well as by the variations of the axial reaction force (Cox, 1975; Weizsäcker et al., 1983; Dobrin, 1986). The
tension–inflation tests evidence the salient feature of the in vivo prestretch level; indeed, for axial prestretches being smaller (resp.
larger) than the in vivo prestretch, the axial reaction force decreases (resp. increases) when the pressure increases. But, when the axial
prestretch is equal to the in vivo prestretch, the axial reaction force does not depend on the applied inner pressure (Weizsäcker et al.,
1983; Sommer et al., 2010) and remains constant during the pressure cycle (see Fig. 6, 4th curve from above). Similar results exist
for isotonic tests, in which the axial force is kept constant but the axial prestretch varies with the applied pressure (Cox, 1975).
Anisotropy of arterial walls has also been investigated through these experimental setups (Vosshougi and Weizsäcker, 1985; Lally
et al., 2004). But no general conclusion can be drawn regarding the anisotropy of the arterial tissue. Still, carotid arteries are found
stiffer in the circumferential direction (Cox, 1975; Patel and Janicki, 1970), while coronary arteries are stiffer in the longitudinal
direction (Papageorgiou and Jones, 1988; Patel and Janicki, 1970). It is however remarkable that there is no statistical difference
in the mechanical responses of arteries whether tested in the circumferential or in the longitudinal directions, when the tests are
performed in conditions close to the physiological ones: this was observed, e.g., by Sato et al. (1979) on dog abdominal aortas,
by Dobrin (1986) on dog carotid arteries, and by Sommer et al. (2010) on human carotid arteries.
Another much debated feature is the strain-rate dependence of the arterial mechanical response. It is now admitted that at low
strain rates, the mechanical response of arteries does not vary with the strain rate (Sato et al., 1979; Zemánek et al., 2009; Tanaka and
Fung, 1974). The viscous character of arteries is however a much more complex question, since also creep and relaxation tests
should be investigated.
Finally, these macroscopic mechanical tests also allow for checking the widely accepted assumption of incompressibility of arte-
rial tissues. Incompressibility implies that the changes in sample thickness can be deduced from the changes in the circumferential
and axial dimensions. To our best knowledge, Carew et al. (1968) was the only study that experimentally checked this assumption,
by evaluating the ratio between bulk and shear moduli based on a tension–inflation test. As a result, the arterial bulk modulus was
about three times larger than the Young’s modulus determined by Bergel (1961), while the hydrostatic and deviatoric stresses were
of the same order of magnitude. They could therefore conclude that arteries are only slightly compressible.
At the microstructural scale

As a transition between purely mechanical characterization and mechanical testing coupled with live imaging, we here focus on
correlations existing between mechanical characteristics of arteries and their composition. In this respect, we report two investiga-
tion results.
First, several studies (Sato et al., 1979; Hayashi et al., 1974) emphasized the dependence of the mechanical response on the
sample location along the aortic tree. The aortic stiffness is found to be higher in distal regions than in proximal regions
(Zeinali-Davarani et al., 2015; Sokolis, 2007; Haskett et al., 2010; Tanaka and Fung, 1974), which correlates with a higher collagen
content in the distal aortic regions. This property is directly related to the difference in mechanical function between proximal and
distal regions: proximal regions of the aortic tree directly receives blood from the heart and therefore needs to exhibit larger damping
properties, which is microstructurally translated through a larger elastin content and more undulated collagen bundles in the prox-
imal aortas (Zeinali-Davarani et al., 2015).
Second, several mechanical tests were performed after partial enzyme degradation of either elastin or collagen. By imparting
compressive stresses to the collagen (Chow et al., 2014), the presence of elastin increases collagen folding, resulting in a more
compliant response of the tissue (Ferruzzi et al., 2011), since straightened collagen fibers are stiffer than elastin fibers. As a conse-
quence, degrading elastin leads to vessel enlargement, by relaxation of the internal compressive stress. This results in a mechanical
response being softer in the low stress regime, and stiffer in the large stress regime (Fonck et al., 2007; Weisbecker et al., 2013).
Elastin degradation also leads to an earlier recruitment of collagen fibers, the mechanical response being sooner stiffer (Rezakhaniha
et al., 2011; Zeinali-Davarani et al., 2013). On the other hand, collagen degradation leads to the disappearance of the progressive
stiffening of the mechanical response, while the initial slope of the stress–strain curves remains unchanged (Weisbecker et al.,
2013).
Uniaxial, biaxial, and tension inflation tests on large elastic arteries. LONG (resp. CIRC) stands for the longitudinal (resp. circumferential) direction
192
Table 1
Reference Animal Artery Layer Sollicitation Preconditioning Direction Number of tests
Biomechanics j Multiscale Mechanical Behavior of Large Arteries

(Balzani et al., 2006) Human Abdominal aorta Media Uniaxial tensile test on strips in 5 loading-unloading cycles at Long 1
saline solution at 37 C 1 mm min 1
(Balzani et al., 2006) Human Abdominal aorta Media Uniaxial tensile test on strips in 5 loading-unloading cycles at Circ 1
saline solution at 37 C 1 mm min 1
(Choudhury et al., Human Ascending thoracic aorta All Equibiaxial test on flat square 5 healthy
2009) samples
(Chuong and Fung, Rabbit Thoracic aorta All Compression on square flat Loading until 600 g and then Radial 4
1984) samples in air unloading
(Cox, 1975) Dog Carotid arteries All Inflation tests at constant tensile Various number of inflation/ Circ at fixed Long 10 animals
stretch or constant tensile load, deflation cycles until a stabilized stretch
controlled temperature response is found And Circ at fixed
Long force
(Dobrin, 1986) Dog Carotid arteries All Inflation test at fixed tensile 5 inflation/deflation tests until Circ at fixed Long 120 tests
stretch stabilization of the response stretch
(Hill et al., 2012) Rabbit Carotid arteries All Tension of flat dog bone samples 5 cycles at 1 N under quasistatic Circ 8 samples
in a saline solution loading
(Holzapfel, 2006) Human (80 years Abdominal aorta Adventitia/ Tensile test in a saline solution on 5 cycles preconditioning Circ and Long 2 per layer
old woman) media/intima strips
(Holzapfel et al., Human Left anterior coronary artery Adventitia/ Tensile test in a saline solution on 5 cycles at 1 mm. min 1 Circ and Long 78 strips
2005) media/intima strips (6 13)
(Keyes et al., 2013) Porcine Descending coronary arteries All Planar biaxial testing at different 10 cycles tensile inflation tests Long/Circ 5
stress ratios and tubular testing
(Kim and Baek, Porcine Thoracic Aorta All Inflation tests under fixed axial In the longitudinal and Circ at prescribed 7
2011) stretch circumferential direction long. stretch
(Lally et al., 2004) Porcine Coronary arteries All Tensile tests on strips in a saline 5 cycles at 1 N at a strain rate of Long 21
bath 60%/min
(Lally et al., 2004) Porcine Coronary arteries All Equibiaxial tests on square 5 cycles at 0.5 N at a strain rate of Long/Circ 8
samples in a saline bath 60%/min
(Mohan and Melvin, Human Mid-thoracic descending All Tensile tests at room temperature 5 cycles Long and Circ 18 each
1982) aorta sprayed with saline solution
(Patel and Janicki, Dog Coronary and carotid artery All Inflation test at in vivo stretch Response stabilizes after two load Long/Circ 14 animals
1970) cycles
(Pandit et al., 2005) Porcine Coronary arteries All Inflation test in saline solution at Not reported Circ 12
imposed axial stretches
(Papageorgiou and Human Iliac arteries All Inflation test at in vivo length, One cycle pressure up to 160 kPa Circ followed by 18
Jones, 1988) static tests, kept wet; followed Long
by axial tensile test on the whole
sample
(Samila and Carter, Human Carotid arteries Media and Uniaxial tensile tests in saline Not reported Long and Circ 20
1981) Intima solution
(Sato et al., 1979) Dog Abdominal aorta, carotid All Tensile test at fixed pressure, Repeated pressure loading and Long at fixed Circ 5 animals
arteries, femoral arteries 37 C, sample kept wet cyclic stretching pressure
(Schmid et al., 2005) Human aorta adventitia Tensile test immerged in a saline 3 quasistatic loading/unloading Long and Circ 2 samples
bath cycles
(Silver et al., 2003) Porcine Aorta, carotid and vena cava All Tensile test in a saline bath on Not reported Long and Circ 6 each
strips
(Sokolis et al., Rabbit Abdominal aorta media Tensile test on strips at 37 C 10 tensile cycles to the maximum Long 15
2002b) immerged in a saline solution tensile strain
(Sokolis et al., Porcine Abdominal aorta media Tensile test on strips at 37 C 10 tensile cycles to the maximum Long 20
2002b) immerged in a saline solution tensile strain
(Sokolis et al., 2006) Rabbit Descending thoracic aorta all Tensile test on strips at 37 C 10 tensile cycles to the maximum Long 35
immerged in a saline solution tensile strain
(Sommer et al., Human Carotid arteries All and layer Inflation under controlled axial 5 cycles axial preconditioning Circ 20
2010) specific force followed by 5 inflation–deflation
test
(Storkholm et al., Porcine Abdominal aorta All Inflation test under controlled axial Cyclic loading/unloading test Circ 5
1997) stretch between 0 and 25 kPa until
stable response
(Tanaka and Fung, Dog Aorta All Uniaxial tensile test on strips in Cyclic loading unloading until Long and Circ
1974) a saline solution stabilization
Biomechanics j Multiscale Mechanical Behavior of Large Arteries

(van de Geest et al., Human Abdominal aorta All Flat biaxial tests at different stress 9 loading/unloading cycles for Long/Circ 8 healthy
2006) ratios each tension ratio specimens
(von Maltzahn et al., Bovine Carotid arteries Intact and Inflation tests under different Inflation/deflation tests between Circ 18
1984) media/intima stretch ratios 0 and 250 mmHg at 1 kPa s 1
only
(Vorp et al., 2003) Human Ascending aorta All Uniaxial tensile in a saline solution Not reported Long and Circ 7 each
(Vosshougi and Rat and pig Aorta Media and Uniaxial tensile test in a saline Three loading/unloading cycles at Long and Circ 13 rats and 7
Weizsäcker, 1985) Intima solution at 37 and 24 C constant extension rate pigs
(Weisbecker et al., Human Thoracic aorta Media Uniaxial tensile test in a saline Not reported Circ 1 sample not
2013) solution at 37 C elastase treated
(Weizsäcker et al., Rat Carotid arteries All Inflation test at constant axial 10 inflation/deflation tests Long and Circ 6 rats
1983) stretch at 37 C, in a saline bath
and axial stretch at different
pressures
(Zeinali-Davarani Porcine Descending thoracic aorta All Planar biaxial tests at different Eight loading cycles up to 30 N m Long/Circ 4 locations, 1
1
et al., 2015) stress ratios sample each
(Zemánek et al., Porcine Thoracic aorta all Equibiaxial tensile test in a saline Cyclic loading of the specimen at Long/Circ 4 locations, 1
2009) solution 0.5 N until stabilization of the sample each
response
193
(A) (B)
1 3
0.9
2.5
0.8
0.7
2
Stress [MPa]
Stress [MPa]
0.6
0.5 1.5
0.4
1
0.3
0.2
0.5
0.1
0 0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.2 0.4 0.6 0.8 1 1.2
Uniaxial stretch Uniaxial stretch
Fig. 5 Mechanical response of different arterial tissues subjected to uniaxial tension in the (A) circumferential and (B) longitudinal direction: porcine
coronary arteries (crosses, Lally et al., 2004), human ascending aorta (circles, Choudhury et al., 2009), human mid-thoracic descending aortas (dia-
monds, Mohan and Melvin, 1982), and rabbit and pig aortas (triangles, Sokolis et al., 2002b, 2006).
80
70
60
Axial force [10–3 N]
50
40
30
20
10
0
0 5 10 15 20 25
Intraluminal pressure [kPa]
Fig. 6 Variation of the axial reaction force as a function of the applied luminal pressure, for different prescribed axial prestretches. Experimental
data from Weizsäcker, H. W., Lambert, H., Pascale, K. (1983). Analysis of the passive mechanical properties of rat carotid arteries. Journal of Biome-
chanics 16 (9), 703–715.
Besides, arteries exhibit a layer-specific mechanical response, which depends on the layer morphology. Mechanical tests on the
tunica intima were performed by Holzapfel et al. (2005) and Weisbecker et al. (2012): intima exhibits a stiffer mechanical response
when loaded in the longitudinal direction than in the circumferential one, which is in good agreement with the longitudinal orien-
tation of the fibers of the internal elastic lamina (Farand et al., 2007). However, it is generally agreed on that the tunica intima barely
contributes to the mechanical response of arteries. As regards the tunica media, the circumferential direction shows a stiffer uniaxial
response than the longitudinal direction, which correlates with the preferred circumferential orientation of the fiber networks in the
media. Contrarily, in the tunica adventitia, the uniaxial mechanical response is stiffer in the longitudinal direction than in the
circumferential one (Holzapfel et al., 2005; Weisbecker et al., 2012) because of the longitudinal orientation of the fibers of the strips
in the load-free configuration (Chen et al., 2013), see Fig. 7. Finally, the larger elastin content in the media as compared to the
adventitia makes the media more compliant, while the adventitia resists to larger loads.
At the scale of fiber networks, the microstructural origin of the nonlinear mechanical behavior could be evidenced using multi-
photon microscopy: namely, in uniaxial tensile tests, when the load is increased, fiber networks tend to rearrange significantly. In
situ mechanical testing showed the ability of the different fiber networks to resist the applied mechanical loading by progressively
(A) (B)
0.15 0.15
0.1 0.1
Stress [MPa]
Stress [MPa]
0.05 0.05
0 0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Uniaxial stretch Uniaxial stretch
Fig. 7 Mechanical response of the tunica media (A) and adventitia (B) for uniaxial tests performed in the circumferential direction (crosses) and in
the longitudinal direction (circles). The engineering stress is plotted against the uniaxial stretch, for human descending coronary arteries (Holzapfel
et al., 2005), human and porcine aortas (Balzani et al., 2006; Schmid et al., 2005).
aligning with the load direction (see Fig. 8). At rest, the collagen bundles of the tunica adventitia form a dense and crimped network,
which is progressively straightened and then stretched. The straightening mechanism of fibers is generally referred to as the fiber
recruitment and coincides with the response at low stresses (Hill et al., 2012; Schrauwen et al., 2012; Sokolis et al., 2006): as
long as collagen fibers are not straight, they cannot sustain load and the softer constituents making up the arterial tissue sustain
the applied load. Furthermore, whatever the load direction in both uniaxial and biaxial tests, adventitial collagen bundles also
have the faculty to reorient and to align with the load direction (Chen et al., 2013); the stretching of collagen bundles takes place
after fiber realignment, as shown by tracking collagen bundle deformation, either by means of fluorescent microspheres (Chen et al.,
2011) or by means of X-ray diffraction (Schmid et al., 2005). Nevertheless, the mechanisms driving the reorientation of collagen
networks remain unclear. Two hypotheses are currently discussed: affine rotation, where the fibers follow the matrix deformation
(Wan et al., 2012), or larger rotations of the collagen network, where the collagen bundles are able to generate shear stresses to rotate
faster than the matrix (Jayyosi, 2015; Billiar and Sacks, 1997). It might well be that both assumptions are valid but in different strain
ranges, the affine rotations being validated over the physiological deformation range (Wan et al., 2012). The collagen bundles
recruitment is driven by the presence of the elastin network: in the adventitia, the elastin network is, at rest, aligned with the collagen
network (Chen et al., 2011), and it tends to reorient and align by application of a mechanical loading. Still, the realignment of the
adventitial elastin network is less pronounced than the collagen realignment (Chen et al., 2013).
In the media, elastin lamellae, collagen fibers, and smooth muscle cells also undergo load-induced reorientation. Under uniaxial
and biaxial load cases, the medial fiber networks and the collagen network tend to align with the load direction (Timmins et al.,
2010). Also, the engagement of collagen fibers occurs first in the media and starts later in the adventitia (Zeinali-Davarani et al.,
2015; Chow et al., 2014). However, fiber rotations are larger for the collagen bundles of the adventitia (see Fig. 8): rotations of
the medial constituents as well as of the adventitial elastin remain limited (Chow et al., 2014); this may be related to the very
different nature of the networks, the elastin network of the media being for instance a very dense and interconnected network,
with elastic segments oriented in all directions. At a larger scale, the elastic lamellae progressively unfold with load application
and then stretch, as observed by means of polarized light microscopy by (Sokolis et al., 2006). The cohesive pericellular interlaced
bundles also straighten and reorient by application of a load (Sokolis et al., 2006), and this recruitment process was shown to be
faster than the recruitment of the circumferentially oriented, parallel bundles covering the elastic lamellae (Sugita and Matsumoto,
2016); the latter authors propose the stiffness difference between elastin and smooth muscle cells as the possible explanation, the
more compliant surrounding medium allowing faster recruitment of the initially crimped fibers.
At the ultrastructural scale

In this section, the mechanical properties of the collagen and elastin structures are reviewed.
Concerning collagen, the literature reports a very broad range of variations for the mechanical properties of collagenous tissues:
from several hundreds of MPa for a hydrated arrangement of collagen fibers to a few tens of GPa for an arrangement of air-dried
collagen molecules (see Table 2 for a literature review). At an intermediate scale, collagen fibrils have an elastic modulus of several
GPa, whereby this exact value depends on the hydration degree of the sample.
Concerning elastin, its mechanical behavior is purely elastic, with neither hysteresis nor transient effect up to more than 100%
strain, eventually showing a brittle failure mode (Aaron and Gosline, 1981). Young’s moduli reported in the literature exhibit less
variability: the elastic modulus of intact purified elastic fibers from aortas was reported to be 0.4 MPa (Hass, 1942a, 1942b, 1943;
λ = 1.7
(A) λ=1 (B)
Collagen Elastin Collagen Elastin
Adventitia
(C) (D)
Collagen Elastin Collagen Elastin

Media
Longit.
Circumf. 30 μm
Fig. 8 Load-induced changes in the morphology of the fiber networks under a circumferential uniaxial load case. Top: adventitial networks; Bottom:
medial networks; Left: load-free configuration; Right: uniaxial stretch of 1.7. Images taken on a rabbit carotid artery with a multiphoton microscope
(IVTV Platform, ANR-10-EQPX-06-01, FR) (Krasny et al., 2017).
Table 2 Mechanical properties of collagen: from molecular arrangement to collagenous tissues
Origin of the
Reference tissue Lengthscale Method Hydration state Measured stiffness
(Harley et al., 1977) Rat tail tendon Molecular Brillouin light scattering Variable hydration 9–21.5 GPa
states
(Cusack and Miller, Rat tail tendon Molecular Brillouin light scattering Variable hydration 7.8–17.9 GPa
1979) states
(Eppell et al., 2006) Dermis of sea Molecular Tensile test using a microdevice Hydrated 6 GPa
cucumber (nanofibril)
(Lorenzo and Molecular Molecular Molecular dynamics 4.8 1 GPa
Caffarena, 2005) dynamics
(Strasser et al., Calfskin Fibrils Nanoindentation by AFM Dried 1.2 GPa (transverse direction)
2007)
(Yadavalli et al., Calfskin Fibrils Nanoindentation by AFM Dried 1.03 0.31 GPa (tranverse
2010) direction)
(van der Rijt et al., Bovine Achilles Fibrils Tensile test using AFM Dried 2–7 GPa (Young’s modulus)
2006) tendon Hydrated 0.2–0.8 GPa (Young’s
modulus)
(Kato et al., 1989) Rat tail tendon Fibrils Tensile test Air-dried overnight 2.200 0.5 GPa
Wet state 500 150 MPa
Table 2 Mechanical properties of collagen: from molecular arrangement to collagenous tissuesdcont'd
Origin of the
Reference tissue Lengthscale Method Hydration state Measured stiffness
(Sasaki and Bovine Achilles Fibrils X-ray diffractometry In a saline solution 2.9 GPa (Young’s modulus)
Odajima 1996b) tendon
(Shen et al., 2008) Dermis of sea Fibrils Uniaxial tension using Hydrated 0.86 0.45 GPa (Young’s
cucumber a microelectromechanical system modulus)
(Svendsen and Rat tail tendon Fibrils Tensile test Hydrated 1.2–1.8 GPa
Thomson, 1984)
(Wenger et al., Rat tail tendon Fibrils Nanoindentation by AFM Different hydration 3.75–11.5 GPa
2007) states
(Yang et al., 2008) Bovine Achilles Fibrils Bending experiment by AFM Hydrated 1.0–3.9 GPa
tendon
(Sasaki and Bovine Achilles Fibrils Tensile test Hydrated 445 MPa
Odajima, 1996a) tendon
(Silver et al., 2001) Turkey Tendon Tensile test Hydrated 62 MPa
tendons
Krafka, 1937; Burton, 1954; Faury, 2001), while the water-swollen, single elastin fiber of Aaron and Gosline (1981) has a Young’s
modulus of 1.2 MPa. According to Gundiah et al. (2007), the difference in the Young’s modulus could be explained by the method
for elastin extraction, the autoclaving method, as used in Aaron and Gosline (1981), providing higher stiffness values as compared
to alkali-treated elastin, as proposed by Hass (1942b).
Finally, smooth muscle cells do not contribute at rest to the mechanical properties of arteries, with an elastic modulus of a few
tens of kPa and a high (up to 150%) distensibility (Faury, 2001; Nagayama and Matsumoto, 2004). Their functional role is however
of prime importance, since they are responsible for the regulation and maintenance of blood flow, to permit a more constant
peripheral blood flow (Ratz, 2004; Faury, 2001). There is also a broad consensus that smooth muscle cells distribute the load across
the arterial thickness: single lamellar units are able to bear a certain tension of 1–3 N.m 1 (as calculated by the product of the intra-
luminal pressure by the vessel radius), independently of organs or species under consideration (Faury, 2001; Humphrey, 2008;
Wolinsky and Glagov, 1967b). At the nanoscale, several proteins are considered as influential to vascular smooth muscle cells
mechanics, namely, (i) the motor protein, called myosin II, assembled into thick filaments; (ii) filamentous actin, which can be
seen as a cable on which myosin heads bind and can “walk” on (Ratz, 2016); and (iii) smooth muscle titin and microtubules
of the cytoskeleton. Actin filaments weave through the smooth muscle cell and are part of its contractile apparatus. They consist
in semiflexible polymers that can, in conjunction with myosin, act as biological active springs able to exert or resist against force
in a cellular environment (Blanchoin et al., 2014). They therefore participate in arterial acute adaptive plasticity (Bednarek et al.,
2011). It has also been shown that smooth muscle cell filamentous actin is in a continuous state of remodeling (Bursac et al.,
2007). Other studies about myosin thick filaments of smooth muscle cells have revealed their partial dissociation and reformation
during, respectively, relaxation and contraction (Smolensky et al., 2005).
Conclusion
This article demonstrates the great effort which has been put to reveal the hierarchical character of arterial tissues, from both a struc-
tural and a mechanical point of view. Healthy arterial tissue appears as a very complex tissue, with a local composition and structure
perfectly suited to the mechanical and physiological function of the tissue at this precise location: cells are able to maintain homeo-
static mechanical properties by adjusting the local concentrations of the diverse constituents. Interestingly, the present study
demonstrates the existence of a strong correlation between elastin and collagen content in healthy arterial tissue. Still, the
complexity in the arterial structure and composition leads to an important variability of the mechanical response of arteries, as
sketched in the different tables and figures of this article. To which extent this variability may be explained by local variations in
the composition and microstructural arrangement? To answer this question, two strategies need to be developed in parallel, namely,
a vast experimental campaign and the development of an effectively multiscale constitutive model for arteries. From an experi-
mental point of view, there is a pressing need for experimental data coupling composition data with microscopic observations
and mechanical testing for the same arterial segment; such data are necessary not only for a better understanding of the different
mechanisms occurring within the arterial wall but also for validating multiscale constitutive model. These mechanisms may also
be better deciphered when focusing on the evolution of the arterial mechanical properties due to remodeling. From the theoretical
point of view, there already exist numerous constitutive models for arteries, with varying complexity. These models are very well
suited for powerful computational approaches of the arterial biomechanics. However, these models remain phenomenological
in nature and therefore are not meant to account for the microscopic mechanisms occurring in arteries. As a consequence, any
(compositional or structural) change in the microstructure can only be accounted for by a new determination of the model
parameters. A true multiscale approach, as well developed for hard tissues (Fritsch and Hellmich, 2007; Fritsch et al., 2009; Hell-
mich et al., 2004; Vuong and Hellmich, 2011; Morin and Hellmich, 2014), and recently sketched for soft tissues (Morin et al., 2015;
Krasny et al., 2015), would involve a deep change of perspective: the arterial tissue is understood as a heterogeneous material; its
mechanical properties can then be estimated from the mechanical behavior of the inhomogeneities, considered as homogeneous
phases, their fractions, their characteristic shapes, and their interactions within a well-chosen volume called the representative
volume element. The mechanical properties of each phase can themselves arise from a homogenization process over a representative
volume element containing heterogeneities with smaller characteristic dimensions. Such an approach would allow to effectively
relate the arterial composition and structure to the mechanical behavior, as reviewed in this article, and therefore to discriminate
between variability and effect of the composition and structural arrangements. Such a multiscale approach could also be especially
useful when focusing on damage and strength of arterial wall, which is of prime importance for medical applications.
References
Aaron, B. B., & Gosline, J. M. (1981). Elastin as a random-network elastomer: A mechanical and optical analysis of single elastin fibers. Biopolymers, 20(6), 1247–1260.
Adham, M., et al. (1996). Mechanical characteristics of fresh and frozen human descending thoracic aorta. Journal of Surgical Research, 64(1), 32–34.
Anidjar, S., et al. (1990). Elastase-induced experimental aneurysms in rats. Circulation, 82(3), 973–981.
Armentano, R. L., et al. (2006). An in vitro study of cryopreserved and fresh human arteries: A comparison with ePTFE prostheses and human arteries studied non-invasively in vivo.
Cryobiology, 52(1), 17–26.
Ayres, C., et al. (2008). Measuring fiber alignment in electrospun scaffolds: A user’s guide to the 2D fast Fourier transform approach. Journal of Biomaterials Science, Polymer
Edition, 19(5), 603–621.
Ayres, C., et al. (2006). Modulation of anisotropy in electrospun tissue-engineering scaffolds: Analysis of fiber alignment by the fast Fourier transform. Biomaterials, 27(32),
5524–5534.
Azeloglu, E. U., et al. (2008). Heterogeneous transmural proteoglycan distribution provides a mechanism for regulating residual stresses in the aorta. American Journal of Physiology
– Heart and Circulatory Physiology, 294(3), H1197–H1205.
Balzani, D., et al. (2006). A polyconvex framework for soft biological tissues. Adjustment to experimental data. International Journal of Solids and Structures, 43(20), 6052–6070.
Bednarek, M. L., et al. (2011). Active tension adaptation at a shortened arterial muscle length: Inhibition by cytochalasin-D. American Journal of Physiology – Heart and Circulatory
Physiology, 300(4), H1166–H1173.
Bendeck, M. P., & Langille, B. L. (1991). Rapid accumulation of elastin and collagen in the aortas of sheep in the immediate perinatal period. Circulation Research, 69(4),
1165–1169.
Bergel, D. H. (1961). The static elastic properties of the arterial wall. Journal of Physiology, 156(3), 445–457.
Berillis, P. (2013). The role of collagen in the aorta’s structure. Open Circulation And Vascular Journal, 6, 1–8.
Berry, C. L., & Greenwald, S. E. (1976). Effects of hypertension on the static mechanical properties and chemical composition of the rat aorta. Cardiovascular Research, 10(4),
437–451.
Billiar, K. L., & Sacks, M. S. (1997). A method to quantify the fiber kinematics of planar tissues under biaxial stretch. Journal of Biomechanics, 30(7), 753–756.
Blanchoin, L., et al. (2014). Actin dynamics, architecture, and mechanics in cell motility. Physiological Reviews, 94(1), 235–263.
Brüel, A., & Oxlund, H. (1996). Changes in biomechanical properties, composition of collagen and elastin, and advanced glycation endproducts of the rat aorta in relation to age.
Atherosclerosis, 127(2), 155–165.
Bursac, P., et al. (2007). Cytoskeleton dynamics: Fluctuations within the network. Biochemical and Biophysical Research Communications, 355(2), 324–330.
Burton, A. C. (1954). Relation of structure to function of the tissues of the wall of blood vessels. Physiological Reviews, 34(4), 619–642.
Canham, P. B., et al. (1989). Measurements from light and polarised light microscopy of human coronary arteries fixed at distending pressure. Cardiovascular Research, 23(11),
973–982.
Carew, T. E., Vaishnav, R. N., & Patel, D. J. (1968). Compressibility of the arterial wall. Circulation Research, 23(1), 61–68.
Carmo, M., et al. (2002). Alteration of elastin, collagen and their cross-links in abdominal aortic aneurysms. European Journal of Vascular and Endovascular Surgery, 23(6),
543–549.
Cattell, M. A., Anderson, J. C., & Hasleton, P. S. (1996). Age-related changes in amounts and concentrations of collagen and elastin in normotensive human thoracic aorta. Clinica
Chimica Acta, 245(1), 73–84.
Chandran, P. L., & Barocas, V. H. (2006). Affine versus non-affine fibril kinematics in collagen networks: Theoretical studies of network behavior. Journal of Biomechanical
Engineering, 128(2), 259–270.
Chaudhuri, B. B., Kundu, P., & Sarkar, N. (1993). Detection and gradation of oriented texture. Pattern Recognition Letters, 14(2), 147–153.
Chen, H., et al. (2013). Biaxial deformation of collagen and elastin fibers in coronary adventitia. Journal of Applied Physiology, 115(11), 1683–1693.
Chen, H., et al. (2011). The layered structure of coronary adventitia under mechanical load. Biophysical Journal, 101(11), 2555–2562.
Cheuk, B. L. Y., & Cheng, S. W. K. (2005). Expression of integrin alpha5beta1 and the relationship to collagen and elastin content in human suprarenal and infrarenal aortas.
Vascular and Endovascular Surgery, 39(3), 245–251.
Choudhury, N., et al. (2009). Local mechanical and structural properties of healthy and diseased human ascending aorta tissue. Cardiovascular Pathology, 18(2), 83–91.
Chow, M.-J., et al. (2014). Arterial extracellular matrix: A mechanobiological study of the contributions and interactions of elastin and collagen. Biophysical Journal, 106(12),
2684–2692.
Chow, M.-J., & Zhang, Y. (2011). Changes in the mechanical and biochemical properties of aortic tissue due to cold storage. Journal of Surgical Research, 171(2), 434–442.
Chuong, C. J., & Fung, Y. C. (1984). Compressibility and constitutive equation of arterial wall in radial compression experiments. Journal of Biomechanics, 17(1), 35–40.
Cicchi, R., et al. (2014). Characterization of collagen and cholesterol deposition in atherosclerotic arterial tissue using non-linear microscopy. Journal of Biophotonics, 7(1–2),
135–143.
Clark, J. M., & Glagov, S. (1985). Transmural organization of the arterial media. The lamellar unit revisited. Arteriosclerosis, Thrombosis, and Vascular Biology, 5(1), 19–34.
Collins, R., & Hu, W. C. L. (1972). Dynamic deformation experiments on aortic tissue. Journal of Biomechanics, 5(4), 333–337.
Cox, R. H. (1975). Anisotropic properties of the canine carotid artery in vitro. Journal of Biomechanics, 8(5), 293–300.
Cusack, S., & Miller, A. (1979). Determination of the elastic constants of collagen by Brillouin light scattering. Journal of Molecular Biology, 135(1), 39–51.
D’Amore, A., et al. (2010). Characterization of the complete fiber network topology of planar fibrous tissues and scaffolds. Biomaterials, 31(20), 5345–5354.
Dahl, S. L. M., et al. (2007). Mechanical properties and compositions of tissue engineered and native arteries. Annals of Biomedical Engineering, 35(3), 348–355.
Davies, R. R., et al. (2002). Yearly rupture or dissection rates for thoracic aortic aneurysms: Simple prediction based on size. Annals of Thoracic Surgery, 73(1), 17–28.
deBotton, G., & Oren, T. (2013). Analytical and numerical analyses of the micromechanics of soft fibrous connective tissues. Biomechanics and Modeling in Mechanobiology, 12(1),
151–166.
Dingemans, K. P., et al. (2000). Extracellular matrix of the human aortic media: An ultrastructural histochemical and immunohistochemical study of the adult aortic media. The
Anatomical Record, 258(1), 1–14.
Dobrin, P. B. (1986). Biaxial anisotropy of dog carotid artery: Estimation of circumferential elastic modulus. Journal of Biomechanics, 19(5), 351–358.
Eisenstein, R., et al. (1975). The ground substance of the arterial wall. Part 1. Extractability of glycosammoglycans and the isolation of a proteoglycan from bovine aorta.
Atherosclerosis, 22(1), 1–17.
Eppell, S. J., et al. (2006). Nano measurements with micro-devices: Mechanical properties of hydrated collagen fibrils. Journal of the Royal Society, Interface, 3(6), 117–121.
Esterly, J. A., Glagov, S., & Ferguson, D. J. (1968). Morphogenesis of intimal obliterative hyperplasia of small arteries in experimental pulmonary hypertension. An ultrastructural
study of the role of smooth-muscle cells. American Journal of Pathology, 52(2), 325–347.
Etherington, D. J., & Sims, T. J. (1981). Detection and estimation of collagen. Journal of the Science of Food and Agriculture, 32(6), 539–546.
Eyre, D. R., Paz, M. A., & Gallop, P. M. (1984). Cross-linking in collagen and elastin. Annual Review of Biochemistry, 53(1), 717–748.
Farand, P., Garon, A., & Plante, G. E. (2007). Structure of large arteries: Orientation of elastin in rabbit aortic internal elastic lamina and in the elastic lamellae of aortic media.
Microvascular Research, 73(2), 95–99.
Farrar, J. F., Blomfield, J., & Reye, R. D. K. (1965). The structure and composition of the maturing pulmonary circulation. Journal of Pathology and Bacteriology, 90(1), 83–96.
Faury, G. (2001). Function–structure relationship of elastic arteries in evolution: From microfibrils to elastin and elastic fibres. Pathologie Biologie, 49(4), 310–325.
Feldman, S. A., & Glagov, S. (1971). Transmedial collagen and elastin gradients in human aortas: Reversal with age. Atherosclerosis, 13(3), 385–394.
Ferruzzi, J., et al. (2011). Mechanical assessment of elastin integrity in fibrillin-1-deficient carotid arteries: Implications for Marfan syndrome. Cardiovascular Research, 92(2),
287–295.
Fillinger, M. (2007). Who should we operate on and how do we decide: Predicting rupture and survival in patients with aortic aneurysm. Seminars in Vascular Surgery, 20(2),
121–127.
Fischer, G. M., & Llaurado, J. G. (1966). Collagen and elastin content in canine arteries selected from functionally different vascular beds. Circulation Research, 19(2), 394–399.
Fischer, J. (1979). Ultrastructure of elastic fibers as shown by polarization optics after the topo-optical permanganate-bisulfite-toluidine blue (PBT) reaction. Acta Histochemica,
65(1), 87–98.
Fonck, E., et al. (2007). Effect of elastin degradation on carotid wall mechanics as assessed by a constituent-based biomechanical model. American Journal of Physiology Heart and
Circulatory Physiology, 292(6), H2754–H2763.
Fritsch, A., & Hellmich, C. (2007). ‘Universal’ microstructural patterns in cortical and trabecular, extracellular and extravascular bone materials: Micromechanics-based prediction of
anisotropic elasticity. Journal of Theoretical Biology, 244(4), 597–620.
explanation of bone strength. Journal of Theoretical Biology, 260(2), 230–252.
Fujimoto, J. G., et al. (1999). High resolution in vivo intra-arterial imaging with optical coherence tomography. Heart (British Cardiac Society), 82(2), 128–133.
Fung, Y. (1993). Biomechanics: Mechanical properties of living tissues. New York: Springer-Verlag.
Gasser, T. C., et al. (2010). Biomechanical rupture risk assessment of abdominal aortic aneurysms: Model complexity versus predictability of finite element simulations. European
Journal of Vascular and Endovascular Surgery, 40(2), 176–185.
Gasser, T. C., et al. (2012). Spatial orientation of collagen fibers in the abdominal aortic aneurysm’s wall and its relation to wall mechanics. Acta Biomaterialia, 8(8), 3091–3103.
Ghazanfari, S., et al. (2012). A comparative analysis of the collagen architecture in the carotid artery: Second harmonic generation versus diffusion tensor imaging. Biochemical and
Biophysical Research Communications, 426(1), 54–58.
Gibbons, G. H., & Dzau, V. J. (1994). The emerging concept of vascular remodeling. New England Journal of Medicine, 330(20), 1431–1438.
Graham, J. S., et al. (2004). Structural changes in human type I collagen fibrils investigated by force spectroscopy. Experimental Cell Research, 299(2), 335–342.
Grant, R. A. (1967). Content and distribution of aortic collagen, elastin and carbohydrate in different species. Journal of Atherosclerosis Research, 7(4), 463–472.
Grant, R. A. (1964). Estimation of hydroxyproline by the autoanalyser. Journal of Clinical Pathology, 17, 685–686.
Gundiah, N., B Ratcliffe, M., & A Pruitt, L. (2007). Determination of strain energy function for arterial elastin: Experiments using histology and mechanical tests. Journal of
Biomechanics, 40(3), 586–594.
Halloran, B. G., et al. (1995). Localization of aortic disease is associated with intrinsic differences in aortic structure. Journal of Surgical Research, 59(1), 17–22.
Han, W.-Q., et al. (2009). Changes in the composition of the thoracic aortic wall in spontaneously hypertensive rats treated with losartan or spironolactone. Clinical and Experimental
Pharmacology and Physiology, 36(5–6), 583–588.
Harkness, M. L. R., Harkness, R. D., & McDonald, D. A. (1957). The collagen and elastin content of the arterial wall in the dog. Proceedings of the Royal Society B: Biological
Sciences, 146(925), 541–551.
Harley, R., et al. (1977). Phonons and the elastic moduli of collagen and muscle. Nature, 267(5608), 285–287.
Haskett, D., et al. (2010). Microstructural and biomechanical alterations of the human aorta as a function of age and location. Biomechanics and Modeling in Mechanobiology, 9(6),
725–736.
Hass, G. M. (1942a). Elasticity and tensile strength of elastic tissue isolated from the human aorta. Archives of Pathology, 34, 971–981.
Hass, G. M. (1942b). Method for the isolation of elastic tissues. Archives of Pathology, 34, 807–819.
Hass, G. M. (1943). Relations between structure of the ageing aorta and properties of isolated aortic elastic tissue. Archives of Pathology, 35, 29–45.
Hayashi, K., et al. (1974). Biomechanical study of the constitutive laws of vascular walls. Experimental Mechanics, 14(11), 440–444.
Hellmich, C., Ulm, F.-J., & Dormieux, L. (2004). Can the diverse elastic properties of trabecular and cortical bone be attributed to only a few tissue-independent phase properties and
their interactions? Arguments from a multiscale approach. Biomechanics and Modeling in Mechanobiology, 2(4), 219–238.
Hill, M. R., et al. (2012). A theoretical and non-destructive experimental approach for direct inclusion of measured collagen orientation and recruitment into mechanical models of the
artery wall. Journal of Biomechanics, 45(5), 762–771.
Holzapfel, G. A., et al. (2005). Determination of layer-specific mechanical properties of human coronary arteries with nonatherosclerotic intimal thickening and related constitutive
modeling. American Journal of Physiology. Heart and Circulatory Physiology, 289(5), H2048–H2058.
Holzapfel, G. A. (2006). Determination of material models for arterial walls from uniaxial extension tests and histological structure. Journal of Theoretical Biology, 238(2), 290–302.
Hosoda, Y., et al. (1984). Age-dependent changes of collagen and elastin content in human aorta and pulmonary artery. Angiology, 35(10), 615–621.
Hosoda, Y., & Minoshima, I. (1965). Elastin content of the aorta and the pulmonary artery in the Japanese. Angiology, 16(6), 325–332.
Humphrey, J. (2006). Towards a theory of vascular growth and remodeling. In G. A. Holzapfel, & R. W. Ogden (Eds.), Mechanics of Biological Tissue (pp. 3–15). Berlin: Springer
Berlin Heidelberg.
Humphrey, J. D., et al. (2014). Cell biology. Dysfunctional mechanosensing in aneurysms. Science, 344(6183), 477–479.
Humphrey, J.D., 1995. Mechanics of the arterial wall: Review and directions. Critical Reviews in Biomedical Engineering, 23(1–2), pp.1–162.
Humphrey, J. D. (2008). Vascular adaptation and mechanical homeostasis at tissue, cellular, and sub-cellular levels. Cell Biochemistry and Biophysics, 50(2), 53–78.
Humphrey, J. D. (2009). Vascular mechanics, mechanobiology, and remodeling. Journal of Mechanics in Medicine and Biology, 9(2), 243–257.
Humphrey, J. D., & Holzapfel, G. A. (2012). Mechanics, mechanobiology, and modeling of human abdominal aorta and aneurysms. Journal of Biomechanics, 45(5), 805–814.
Ives, C. L., Eskin, S. G., & McIntire, L. V. (1986). Mechanical effects on endothelial cell morphology: In vitro assessment. In Vitro Cellular & Developmental Biology, 22(9),
500–507.
Jacob, M., et al. (2001). Extracellular matrix remodeling in the vascular wall. Pathologie Biologie, 49(4), 326–332.
Jayyosi, C., 2015. Caractérisation mécanique et microstructurale du comportement à rupture de la capsule de Glisson pour la prédiction du risque de lésions des tissus hépatiques
humains, Université de Lyon 1.
Johnston, K. W., et al. (1991). Suggested standards for reporting on arterial a

Biomedical Engineering Encyclopedia Vol-1-3

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biomedical Engineering Encyclopedia Vol-1-3

Uploaded by

Copyright:

Available Formats

ENCYCLOPEDIA OF

Amsterdam • Boston • Heidelberg • London • New York • Oxford

Copyright Ó 2019 Elsevier Inc. All rights reserved.

Library of Congress Cataloging-in-Publication Data

British Library Cataloguing-in-Publication Data

For information on all publications visit our website

Publisher: Oliver Walter

Printed and bound in the United States

Diego Mantovani, Ph.D., FBSE.

Tyler Ackley M Bohner

Tzahi Cohen-Karni Margaret A T Freeberg

Natalia Davidenko Vincenzo Guarino

Xingyu Jiang Dimitrios A Lamprou

Stefani Mazzitelli David A Puleo

Swaminathan Sethuraman Mian Wang

Biomaterials: Science and Engineering

Natural Biopolymers for Biomedical Applications 162

Biomaterials: In Vitro and in Vivo Studies of Biomaterials

Biomaterials: Biomaterial Applications and Advanced Medical Technologies

Microﬂuidics for Biomedical Applications 368

Human Parthenogenetic Pluripotent Stem Cells 608

Porous Biomaterials and Scaffolds for Tissue Engineering 188

Biomaterials: In Vitro and in Vivo Studies of Biomaterials

Biomaterials: Biomaterial Applications and Advanced Medical Technologies

Shape-Memory Polymer Medical Devices 394

Introduction to Regenerative Engineering 624

Mathematical Quantiﬁcation of the Impact of Microstructure on the Various Effective

Iron-Based Degradable Implants 374

Magnetic Resonance Imaging 574

Rehabilitation Engineering and Integrative Technologies

Mathematical Techniques in Biomedical Engineering

Single-Cell-Based In Silico Models: A Tool for Dissecting Tumor Heterogeneity 130

Bioinstrumentation and Bioinformatics

Machine Learning in Biomedical Informatics 389

Roger J Narayan, M.D., Ph.D.

Figure 1 On the Use of Population-Based Statistical Models in Biomechanics

Figure 8a Nanotechnology for Regenerative Engineering

Figure 1 Translational Bioinformatics for Personalised Medicine

Figure 7c Nanotechnology for Regenerative Engineering

Alternative Processing Techniques for CoCr Dental Alloys

Encyclopedia of Biomedical Engineering, Volume 1 https://doi.org/10.1016/B978-0-12-801238-3.11100-6 1

Table 1 Classiﬁcation of dental alloys

Precious alloys Nonprecious alloys

Conventional crown and bridge alloys Nickel-based

Manufacturing Technologies for CoCr Alloys

Manufacturing technologies currently available for CoCr alloys are:

Fig. 1 Milling of prosthetic elements according to CAD ﬁles.

Fig. 2 CoCr blank for milling.

Fig. 3 CoCr prosthetic elements obtained by milling.

Fig. 4 3D digital description of the computer-based program for a fabrication tray.

Fig. 5 The laser selectively fuses the CoCr metal powder.

Fig. 6 Laser sintered prosthetic elements.

• Electron beam melting (EBM), developed by ARCAM AB in Sweden-General Electric-2016.

Evaluation of CoCr Alloys Manufactured by Different Technologies

Table 2 Composition of the alloys (wt.%)

Element Cast Milled Ceramill Sintron SLS

Fig. 7 Metallographic observation of a cast CoCr alloy.

Fig. 8 Micrographical structure of the CoCr alloy without chemical attack.

Fig. 9 SEM observation of the CoCr alloy without chemical attack.

Fig. 10 Micrographical structure of the CoCr alloy after chemical attack.

Table 3 The chemical composition of the phases

Spectrum 1: matrix 0.85 28.45 0.85 64.46 5.38 100.00

Fig. 11 Details without chemical attack.

Fig. 12 Microstructure after sintering.