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Springer Theses
Recognizing Outstanding Ph.D. Research
Liliang Ouyang
Study on
Microextrusion-based
3D Bioprinting and
Bioink Crosslinking
Mechanisms
Springer Theses
Recognizing Outstanding Ph.D. Research

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More information about this series at http://www.springer.com/series/8790

Liliang Ouyang
Study
on Microextrusion-based 3D
Bioprinting and Bioink
Crosslinking Mechanisms
Doctoral Thesis accepted by
Tsinghua University, Beijing, China
123
Author Supervisors
Dr. Liliang Ouyang Prof. Wei Sun
Department of Mechanical Engineering Department of Mechanical Engineering
Tsinghua University Tsinghua University
Beijing, China Beijing, China
Department of Mechanical Engineering
and Mechanics
Drexel University
Philadelphia, USA
Prof. Jason A. Burdick

Department of Bioengineering
University of Pennsylvania
Philadelphia, USA
ISSN 2190-5053 ISSN 2190-5061 (electronic)

Springer Theses
ISBN 978-981-13-9454-6 ISBN 978-981-13-9455-3 (eBook)
https://doi.org/10.1007/978-981-13-9455-3
Jointly published with Tsinghua University Press, Beijing, China
The print edition is not for sale in China Mainland. Customers from China Mainland please order the
print book from: Tsinghua University Press.
© Tsinghua University Press, Beijing and Springer Nature Singapore Pte Ltd. 2019
This work is subject to copyright. All rights are reserved by the Publishers, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
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Singapore
Supervisor’s Foreword
It is my great pleasure to introduce Dr. Ouyang’s thesis work, which is recognized

as an Outstanding Doctoral Thesis by Tsinghua University. Dr. Ouyang joined my
laboratory and started his Ph.D. study in September 2012, working on
extrusion-based 3D bioprinting, novel bioink development, and embryonic stem
cell printing. In 2015, he received a scholarship from the China Scholarship Council
and conducted one-year visiting research with Prof. Jason A. Burdick at the
University of Pennsylvania. Dr. Ouyang completed his doctoral work in July 2017,
ending up with an impressive list of high-impact publications in the field of
biofabrication.
In this dissertation, Dr. Ouyang presents a comprehensive study on microex-
trusion 3D bioprinting as well as the mechanisms of bioink crosslinking. Three-
dimensional bioprinting is normally referred to using living cells as building blocks
for constructing cellular structures, where the cell-containing ink materials are
termed as bioinks. In the last decade, 3D bioprinting has been witnessed with
numerous progresses and applications, while the fundamentals of the printing
technology, as well as the effect of printing process and bioink crosslinking on the
structural printability and cellular function, have not been fully investigated. To
bridge this gap, Dr. Ouyang has conducted a comprehensive study on the
microextrusion-based cell-printing process, and the use of responsive bioinks and
their crosslinking. A number of novel printing/crosslinking strategies were devel-
oped and presented in this thesis work, for example, a dual-crosslinking strategy for
shear-thinning supermolecular bioink printing (Chap. 4), a synergetic optimization
method for thermo-responsive bioink printing (Chap. 5), and an in-situ crosslinking
strategy for printing non-viscous photo-crosslinkable hydrogels (Chap. 6). In
addition, this thesis also reports some fundamental studies on a signal pathway
activation, cell–matrix interaction, as well as a proliferation-induced embryoid body
(EB) formation mechanism as results of bioprinting (Chap. 7). Findings, as well as
the developed methodologies from those studies, have greatly enriched the field of
bioprinting.
v
vi Supervisor’s Foreword
I am proud of Dr. Ouyang for what he has accomplished during his Ph.D. study
and proud of presenting this thesis book to the bioprinting and biofabrication
community.
Beijing, China/Philadelphia, USA Prof. Wei Sun, Ph.D.

May 2019
Parts of this Thesis have been Published in the Following Documents:
Journal Publications:
Ouyang L, Highley CB, Sun W, Burdick JA. A Generalizable Strategy for the 3D
Bioprinting of Hydrogels from Nonviscous Photo-crosslinkable Inks. Advanced
Materials. 2017; 29(8):1604983.
Ouyang L, Yao R, Zhao Y, Sun W. Effect of bioink properties on printability and
cell viability for 3D bioplotting of embryonic stem cells. Biofabrication. 2016;
8(3):035020.
Ouyang L#, Highley CB#, Rodell CB, Sun W, Burdick JA. 3D Printing of
Shear-Thinning Hyaluronic Acid Hydrogels with Secondary Cross-Linking. ACS
Biomaterials Science & Engineering. 2016; 2(10):1743–1751. (# Equal contribution)
Ouyang L#, Yao R#, Mao S, Chen X, Na J, Sun W. Three-dimensional bioprinting
of embryonic stem cells directs high-throughput and highly uniform embryoid body
formation. Biofabrication. 2015; 7(4):044101. (# Equal contribution)
Ouyang L#, Yao R#, Chen X#, Na J, Sun W. 3D printing of HEK 293FT cell-laden
hydrogel into macroporous constructs with high cell viability and normal biological
functions. Biofabrication. 2015; 7(1):015010. (# Equal contribution)
Zhang T, Yan KC, Ouyang L, Sun W. Mechanical characterization of bioprinted
in vitro soft tissue models. Biofabrication. 2013; 5(4):045010.
vii
Acknowledgements
From the bottom of my heart, I am grateful to all of those who have helped me
throughout my Ph.D. program.
First and foremost, I would like to thank Prof. Wei Sun for his fantastic
supervision on my research work since I joined his laboratory at Tsinghua
University (THU) in 2012. I benefited a lot from his tremendous support to my
laboratory work as well as academic activities. His constructive input and insightful
vision have continuously driven me to move forward with confidence. I could only
hope that I have inherited a portion of his commitment and enthusiasm. I must
express my sincere appreciation to Prof. Jason A. Burdick as well for having me
visiting his laboratory at the University of Pennsylvania (UPenn) from 2015 to
2016. He provided me with the most supportive supervision as I could imagine. His
complete trust and encouragement have made me believe that I could make a
difference. He has been a continual source of inspiration to me and has been such a
scientist that I would like to become.
This thesis would not have been possible without the help and guidance from my
mentors in both laboratories. I thank Dr. Ting Zhang for introducing me to Prof.
Sun’s group and mentoring me at the beginning of my project. I genuinely thank
Dr. Rui Yao for instilling in me her passion for research during our close interaction
in the first few years of my Ph.D. I owe a significant debt of gratitude to
Dr. Christopher B. Highley as he selflessly provided me with tremendous help and
encouragement when I was in Prof. Burdick’s group.
Some of my Ph.D. work regarding embryonic stem cells is owed to the collab-
oration with Dr. Jie Na and Dr. Xi Chen from the School of Medicine at THU.
I sincerely appreciate their invaluable inputs and contributions to the projects. I also
would like to thank my friend and colleague, Dr. Yu Zhao, who has been just reliable
and supportive to my Ph.D. work and life. This thesis would not come to fruition
without the advice, comments, and tips from the colleagues (past and present): Prof.
Feng Lin, Prof. Renji Zhang, Dr. Lei Zhang, Mr. Mingfeng Li, Mr. Laquan Zhang,
ix
x Acknowledgements
Mr. Long Zhao, Mr. Yu Li, Mrs. Jia Wang, Mr. Yongyi Quan, Mrs. Shuangshuang
Mao, and Dr. Karen Chang Yan at THU, as well as Dr. Yi-cheun Yeh,
Dr. Christopher B. Rodell, and Dr. Kwang Hong Song at UPenn.
I am profoundly grateful to all the teachers and mentors who have helped me in
the last twenty-one years since my primary education. I am also very grateful to my
friends for accepting me with nothing but trust.
I would like to say thank you to my parents and my big family for their spiritual
support throughout my education and my life in general. I owe a unique debt of
gratitude to my grandma, who has deeply impacted me with her easy philosophy of
life. Last but not least, I would like to express my special appreciation to my
beautiful wife Li for her unreserved support and contribution to my work and our
family. She is a great wife and also a great mother.
This thesis work has received financial support from the National Natural
Science Foundation of China (No. 51235006 and No. 31500818), National High
Technology Research and Development Program of China—863 Program
(No. 2012AA020506), Beijing Municipal Science & Technology Commission Key
Project (No. Z141100002814003), National Science Foundation MRSEC grant
at the University of Pennsylvania, as well as an Established Investigator Award
(J.A.B) from the American Heart Association. I also acknowledge the scholarship
granted by the China Scholarship Council (File 201506210148).
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Scope and Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Chapter Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 3D Bioprinting and Bioink: Background . . . . . . . . . . . . . . . . . . . . . 7
2.1 Concepts and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.1 Bioprinting Technologies . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.2 Bioinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.3 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3 Challenges and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.3.1 Bioprinting Technologies . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.2 Bioinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3.3 Tissue Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1 Process Analysis and Questions Refining . . . . . . . . . . . . . . . . . . . 25
3.2 General Criteria for Bioinks and 3D Bioprinting Process . . . . . . . 27
3.2.1 Bioink Injectability and Smooth Extrusion . . . . . . . . . . . . 28
3.2.2 Gel Filament Generation . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.3 Structural Integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.4 Cell Damage Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3 General Design of Bioinks and 3D Bioprinting Process . . . . . . . . 30
3.3.1 Bioinks and the Crosslinking Mechanism . . . . . . . . . . . . . 30
3.3.2 3D Model Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3.3 Process Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
xi
xii Contents
3.4 Research Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.4.1 General Research Route . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.4.2 Rheological Characterization . . . . . . . . . . . . . . . . . . . . . . 33
3.4.3 3D Printability Characterization . . . . . . . . . . . . . . . . . . . . 34
3.4.4 Shear Stress Determination . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4.5 Other Experimental Methods . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4 3D Bioprinting of Shear-Thinning Self-assembly Bioink . ......... 43
4.1 Bioink Preparation and Characterization . . . . . . . . . . . ......... 44
4.1.1 Guest–Host Chemistry Modification
of Hyaluronic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.1.2 Methacrylation of Hyaluronic Acid . . . . . . . . . . . . . . . . . . 45
4.1.3 Preparation of Bioinks . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.1.5 3D Bioprinting Process Design . . . . . . . . . . . . . . . . . . . . . 48
4.2 Printability and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2.2 Structure Stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3 Cytocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3.1 Cell Seeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3.2 Direct Cell Printing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5 3D Bioprinting of Thermal-Sensitive Bioink . . . . . . . . . . . . . . . . . . . 63
5.1 Bioink Preparation and Characterization . . . . . . . . . . . . . . . . . . . . 63
5.1.1 Preparation of Gelatin-Based Bioink . . . . . . . . . . . . . . . . . 63
5.1.3 3D Bioprinting Process Design . . . . . . . . . . . . . . . . . . . . . 67
5.2 Printability and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5.2.2 Structure Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
5.3.1 Cell Viability of Different Cells . . . . . . . . . . . . . . . . . . . . 74
5.3.2 Effect of Printing Parameters . . . . . . . . . . . . . . . . . . . . . . 76
5.3.3 Effect of Shear Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.4 Conjunction of Structure Printability and Cell Viability . . . . . . . . 78
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6 3D Bioprinting of Non-viscous Bioink . . . . . . . . . . . . . . . . . . . . . . . . 81
6.1 Strategy Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
6.1.1 Crosslinking Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . 82
6.1.2 Light-Permeable Needle . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Contents xiii
6.1.3 Printing Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

6.1.4 Printing Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
6.2 3D Printability and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.2.2 3D Structure Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . 89
6.2.3 Structure Stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.4 Generalization to Other Bioinks . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.4.1 Bioink Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
6.4.3 Printability and Cytocompatibility . . . . . . . . . . . . . . . . . . . 99
6.5 Complex Filament Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
7 Biological Characterization and Applications . . . . . . . . . . . . . . . . . . 105
7.1 Comparison of Different Technologies . . . . . . . . . . . . . . . . . . . . . 105
7.2 Cell Activity and Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.2.1 Long-Term Cell Activity . . . . . . . . . . . . . . . . . . . . . . . . . 107
7.2.2 Cell Proliferation in 3D . . . . . . . . . . . . . . . . . . . . . . . . . . 109
7.3 Signal Pathway Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
7.3.1 Cell Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
7.3.2 Protein Expression of Activator Cells . . . . . . . . . . . . . . . . 111
7.3.3 Activation of Reporter Cells . . . . . . . . . . . . . . . . . . . . . . . 112
7.4 Embryoid Body Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
7.4.1 Embryoid Body Growth . . . . . . . . . . . . . . . . . . . . . . . . . . 114
7.4.2 Morphology Characterization of Embryoid Body . . . . . . . . 115
7.4.3 Maintenance of Pluripotency . . . . . . . . . . . . . . . . . . . . . . 117
7.4.4 Comparison with Conventional Methods . . . . . . . . . . . . . . 118
7.5 Cell Behavior Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.5.1 Material Cues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.5.2 Cell Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
8 Conclusions and Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
8.1 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
8.2 Future Research Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Abbreviations
3DP Three-dimensional Printing

3T3 NIH 3T3 Fibroblast
Ad Adamantane
Ad-HA Adamantane Hyaluronic Acid
Alg Alginate
BSA Bovine Serum Albumin
CAD Computer-Aided Design
DAPI 4′,6-diamidino-2-phenylindole
DC Dual Crosslinking
DMSO Dimethyl Sulfoxide
DTT DL-Dithiothreitol
EB Embryoid Body
ECM Extracellular Matrix
FBS Fetal Bovine Serum
FDM Fused Deposition Modeling
G′ Storage Modulus
G″ Loss Modulus
GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase
Gel Gelatin
GelMA Gelatin Methacryloyl
GH Guest–Host
HA Hyaluronic Acid
HEK 293FT Human Embryonic Kidney 293FT cell
I2959 Irgacure 2959 (4-(2-hydroxyethoxy)phenyl-(2-propyl)ketone)
LAP Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate
LIF Leukemia Inhibitory Factor
MeHA Methacrylated Hyaluronic Acid
mESC Mouse Embryonic Stem Cell
MMP Matrix Metalloproteinase
MMP-deg Matrix Metalloproteinase-degradable Crosslinker
xv
xvi Abbreviations
Nanog Homeodomain-bearing Transcriptional Factor

NorHA Norbornene-functionalized Hyaluronic Acid
Oct-4 Octamer-Binding Transcription Factor 4
PBS Phosphate-Buffered Saline
PCR Polymerase Chain Reaction
PEG Polyethylene Glycol
PEGDA Poly(ethylene Glycol) Diacrylate
RGD Arginine–Glycine–Aspartic Acid
SSEA1 Stage-Specific Embryonic Antigen 1
bCD b-Cyclodextrin
bCD-HA b-Cyclodextrin–Hyaluronic Acid
η Viscosity
s Shear Force
c_ Shear Rate
C Circularity
Pr Printability
vink Ink Velocity
vnoz Nozzle Velocity
Chapter 1
Introduction
3D printing (3DP), also known as additive manufacturing, is believed to be a rev-

olutionary manufacturing technology in the twenty-first century [1, 2]. Compared
with traditional processing techniques (e.g., turning, casting), 3DP benefits from the
rapid prototyping of complex and customized structure: based on a bottom-to-up
logic, 3DP allows for freeform manipulation of building blocks [3]. The properties
of the materials being processed are critical to such a manufacturing fashion. This
consideration has led to numerous progress in developing printable ink materials,
including metal, ceramics, plastics, and elastomers.
The applications of 3DP in biomedicine vary with the ink materials and thus final
products. For example, prototyping printed from non-biocompatible plastic could be
used in surgery planning or guidance, while the customized orthodontic appliance
would be another example. The next stage comes to the implantation of 3D printed
product that is biocompatible but nondegradable, such as titanium bone scaffold [4].
One more step further, researchers have applied degradable biomaterials to print
implantable scaffolds that would guide the native tissue regeneration and would thus
achieve the ideal repair [5]. What this thesis presents belongs to another stage, where
cells and other biological elements are directly incorporated in the 3DP process to
create a living product, which is termed as 3D bioprinting [6]. This technology is
supposed to lead to organ printing in the future, which might sound crazy but is
actually happening.
1.1 Motivation
Organ failure is one of the major problems affecting human health. Traditional organ
transplantation methods (e.g., autologous and allogeneic transplantation) experience
fundamental challenges such as donor shortage, immune rejection, and ethical dis-
putes. The number of people on the organ transplant waiting list continues to be
much larger than the number of donors, which grows slowly. The recent progress in
tissue engineering might provide new solutions to this problem.
© Tsinghua University Press, Beijing and Springer Nature Singapore Pte Ltd. 2019 1
L. Ouyang, Study on Microextrusion-based 3D Bioprinting and Bioink Crosslinking
Mechanisms, Springer Theses, https://doi.org/10.1007/978-981-13-9455-3_1
2 1 Introduction
As an interdisciplinary area, tissue engineering combines the fundamentals of

engineering and life sciences and is dedicated to the development of biological alter-
natives for maintaining, repairing, or improving tissue function [7]. A traditional
strategy for tissue engineering is to obtain a small number of patient-derived cells,
which are expanded in vitro and seeded on a polymeric scaffold together with growth
factors, followed by implantation [8]. Despite advances made in the past decades, a
successful repair is still hindered by some enduring challenges such as cell seeding,
complex scaffold and cellular heterogeneity [9]. Based on the idea of additive man-
ufacturing, 3D printing manipulates building blocks spatially and temporarily and
would be potential serving as a novel fabrication technology for tissue engineering
[10]. For example, multi-material, pore-controllable, and complex acellular scaffolds
have been fabricated.
Nevertheless, poor cell localization and mismatch with in vivo microenvironment
remain problems. In 2003, researchers directly printed living cells using inkjet print-
ing [11, 12], which led to a new research field—3D bioprinting. Since then, printing
a cellular construct has become a new strategy for tissue engineering. 3D bioprinting
combines the scaffold manufacturing with cell seeding, namely uses cell-containing
ink materials, also known as bioinks, to build up tissue constructs directly. One
of the most critical aspects in 3D bioprinting is the bioink, which not only deter-
mines the implementation of the printing technology but also defines the 3D cellular
microenvironment [13]. Though numerous bioinks have been developed and applied
in bioprinting practice, rarely few work has been done that could reveal the nature of
bioprinting and bioinks and could summarize a practical guideline. Given this, we
seek to carry out a comprehensive study on microextrusion 3D bioprinting from the
perspective of the bioink crosslinking mechanism.
1.2 Scope and Contents
The topic of this thesis is 3D bioprinting, a multidisciplinary field involving man-

ufacturing, biomaterials and regenerative medicine. The relevant keywords include
3D bioprinting, biofabrication, bioink, crosslinking and tissue engineering [14]. The
objective of this thesis is to investigate a generalizable route for microextrusion bio-
printing studies, which will be achieved by general analysis, comprehensive case
studies, and applications proof-of-concept.
Specifically, bioink and its crosslinking mechanism are highlighted throughout
the study. Different bioprinting technologies vary with bioink crosslinking strategies.
By analyzing the general bioprinting process from raw materials to final products, we
divide it into two parts, i.e., forming gel filament and building up 3D structure, both of
which are closely associated with the crosslinking of bioink. In this study, three dif-
ferent microextrusion bioprinting technologies are developed and optimized based
on different bioink crosslinking mechanisms, including guest-host self-assembly,
gelatin-based thermal crosslinking and photo-crosslinking. All these technologies
are studied following the same path, which includes process design, filament gener-
1.2 Scope and Contents 3
ation, structure fabrication, post-stabilization, cell damage control, and fundamental

biological characterization.
A dual-crosslinking strategy was first used to facilitate the 3D bioprinting of
hyaluronic acid (HA) based on guest-host chemistry and covalent crosslinking, where
gel filaments were maintained by supramolecular bonds immediately upon extru-
sion until covalent crosslinking resulted in further stabilization. The printed scaf-
folds allowed for cell attachment and performed excellent mechanical properties,
which suggested the potential in cartilage and cardiac tissue engineering consid-
ering the wide use of HA in these areas [15]. This approach does not require the
use of other support materials or printable components and can be generalized to
other hydrogels where supramolecular and covalent cross-linking chemistries can be
combined [16]. Secondly, we introduced a practical method to optimize the typical
gelatin-based printing process, where structure printability and cell viability were
investigated simultaneously. The results indicated time-dependence of gelatin-based
bioinks in addition to the commonly known thermal sensitivity. After optimization,
mouse embryonic stem cells were printed into well-defined 3D hydrogel constructs
with high cell viability (>90%) [17]. Thirdly, we developed a generalizable tech-
nology for the 3D bioprinting of photo-crosslinkable hydrogels without limitations
to ink viscosity (can be low to 2.5 mPa·s). Different from commonly used pre- or
post-crosslinking approaches, an in situ crosslinking strategy was established by
introducing a photopermeable capillary to crosslink bioink and print standard fil-
aments simultaneously. Various photo-crosslinkable hydrogels, both synthetic and
natural with either ultraviolet or visible light, were tested in this system with excel-
lent printability and embedded cell viability (>90%). Through incorporating a coaxial
nozzle with this method, we were able to print complex building blocks, including
core-shell, hollow tubular, and heterogeneous filaments where material composition
varied along the filament [18].
These three systems call for different rheological properties and crosslinking
mechanisms for bioinks, and yield products with varying properties, which could
be utilized in a specific application accordingly. In this study, we presented some
fundamental application examples with these systems. Firstly, Wnt signal pathway
was demonstrated to be successfully activated in 3D bioprinted structure based on
genetically engineered embryonic cells, which suggested potential in studying cell
interactions and tissue remodeling [19]. Then we investigated the generation of the
pluripotent embryoid body (EB) in 3D bioprinted microenvironment, where the EBs
were seen with comprehensively better homogeneity, quality, and higher yield when
compared to commonly used suspension and hanging-drop methods [20]. Further-
more, we used the generalizable 3D bioprinting system to tune cell behavior by intro-
ducing a peptide motif (Arginine-Glycine-Aspartic, RGD) or degradable crosslinker
in the bioink formulation, both of which turned out to be helpful for cell spreading
and migration [18].
This study not only adds to the technological options but also provides a general
research route for microextrusion 3D bioprinting. Also, this study may serve as a
guideline for people who need to develop a printing process for specific bioink.
4 1 Introduction
1.3 Chapter Outline
This thesis is articulated into eight chapters (Fig. 1.1), the remaining of which is as
follows:
Chapter 2 introduces the background and the recent progress of 3D bioprinting.
Specifically, the state of the art of bioprinting technologies, bioinks and application
areas are reviewed, along with comments on challenges and perspectives.
Chapter 3 presents the materials and methods used in this study. It comprehen-
sively analyzes the general bioprinting process and refines key questions accordingly,
which leads to the formation of general criteria and research route. Some of the gen-
eral experimental methods are also enclosed.
Chapter 4 presents the 3D bioprinting of shear-thinning and self-healing bioinks
[16]. Based on the analysis in Chap. 3, structure printability and stability are mainly
studied with the novel supramolecular hydrogel formulations. Corresponding solu-
tions are provided to address specific technical problems, such as oxygen inhibition
and cell attachment.
Chapter 5 presents the 3D bioprinting of gelatin-based thermo-sensitive bioinks
[17]. It mainly introduces a synergy optimization approach both before and after
bioprinting: the rheological characterization and bioprinting outcomes (e.g., structure
printability and cell viability) are systematically monitored and controlled. Some
unnoted questions are revealed here, such as time-dependence of this typical bioink.
Chapter 6 presents the 3D bioprinting of non-viscous photo-crosslinkable bioinks
[18]. Given the enduring limitation of bioink viscosity, the in situ crosslinking strategy
Fig. 1.1 Flow diagram of

this thesis, showing the
structure with all chapters
1.3 Chapter Outline 5
is developed to allow for versatile bioprinting of different bioinks at a low viscosity.

All the established criteria are met through optimization.
Chapter 7 presents comprehensive studies on biological characterization and
applications, including signal pathway activation [19], embryoid body formation
[20], and cell behavior control [18].
Chapter 8 concludes this thesis and provides some suggestions regarding future
work could be considered.
References
1. Murphy SV, Atala A (2014) 3D bioprinting of tissues and organs. Nat Biotechnol 32(8):773–785
2. Huang S, Liu P, Mokasdar A, Hou L (2013) Additive manufacturing and its societal impact: a
literature review. Int J Adv Manuf Technol 67(5–8):1191–1203
3. Mandrycky C, Wang Z, Kim K, Kim DH (2016) 3D bioprinting for engineering complex
tissues. Biotechnol Adv 34(4):422–434
4. Ryan GE, Pandit AS, Apatsidis DP (2008) Porous titanium scaffolds fabricated using a rapid
prototyping and powder metallurgy technique. Biomaterials 29(27):3625–3635
5. Bose S, Vahabzadeh S, Bandyopadhyay A (2013) Bone tissue engineering using 3D printing.
Mater Today 16(12):496–504
6. Moroni L, Boland T, Burdick JA, De Maria C, Derby B, Forgacs G, Groll J, Li Q, Malda
J, Mironov VA, Mota C, Nakamura M, Shu W, Takeuchi S, Woodfield TBF, Xu T, Yoo JJ,
Vozzi G (2018) Biofabrication: a guide to technology and terminology. Trends Biotechnol
36(4):384–402
7. Langer R, Vacanti J (1993) Tissue Engineering. Science 260(5110):920–926
8. Drury JL, Mooney DJ (2003) Hydrogels for tissue engineering: scaffold design variables and
applications. Biomaterials 24(24):4337–4351
9. Griffith LG, Naughton G (2002) Tissue engineering–current challenges and expanding oppor-
tunities. Science 295(5557):1009–1014
10. Mironov V, Reis N, Derby B (2006) Review: bioprinting: a beginning. Tissue Eng
12(4):631–634
11. Mironov V, Boland T, Trusk T, Forgacs G, Markwald RR (2003) Organ printing: computer-
aided jet-based 3D tissue engineering. Trends Biotechnol 21(4):157–161
12. Boland T, Mironov V, Gutowska A, Roth EA, Markwald RR (2003) Cell and organ printing
2: fusion of cell aggregates in three-dimensional gels. Anat Rec A Discov Mol Cell Evol Biol
272(2):497–502
13. Holzl K, Lin SM, Tytgat L, Van Vlierberghe S, Gu LX, Ovsianikov A (2016) Bioink properties
before, during and after 3D bioprinting. Biofabrication 8(3)
14. Groll J, Boland T, Blunk T, Burdick JA, Cho DW, Dalton PD, Derby B, Forgacs G, Li Q,
Mironov VA, Moroni L, Nakamura M, Shu W, Takeuchi S, Vozzi G, Woodfield TB, Xu T, Yoo JJ,
Malda J (2016) Biofabrication: reappraising the definition of an evolving field. Biofabrication
8(1):013001
15. Burdick JA, Prestwich GD (2011) Hyaluronic acid hydrogels for biomedical applications. Adv
Mater 23(12):H41–H56
16. Ouyang L, Highley CB, Rodell CB, Sun W, Burdick JA (2016) 3D printing of shear-
thinning hyaluronic acid hydrogels with secondary cross-linking. ACS Biomater Sci Eng
2(10):1743–1751
17. Ouyang L, Yao R, Zhao Y, Sun W (2016) Effect of bioink properties on printability and cell
viability for 3D bioplotting of embryonic stem cells. Biofabrication 8(3):035020
6 1 Introduction
18. Ouyang L, Highley CB, Sun W, Burdick JA (2017) A generalizable strategy for the 3D bio-
printing of hydrogels from nonviscous photo-crosslinkable Inks. Adv Mater 29(8)
19. Ouyang L, Yao R, Chen X, Na J, Sun W (2015) 3D printing of HEK 293FT cell-laden hydrogel
into macroporous constructs with high cell viability and normal biological functions. Biofab-
rication 7(1):015010
20. Ouyang L, Yao R, Mao S, Chen X, Na J, Sun W (2015) Three-dimensional bioprinting of
embryonic stem cells directs high-throughput and highly uniform embryoid body formation.
Biofabrication 7(4):044101
Chapter 2
3D Bioprinting and Bioink: Background
This chapter generally introduces the field of 3D bioprinting, starting with the related
concepts and their history. The definitions of bioprinting and bioink are clarified
based on the most recent literature. The brief history of this field is reviewed, and the
technology trends are presented from different angles, which all indicate a booming
development. Then a comprehensive overview of state of the art is presented in terms
of bioprinting technologies, bioinks, and application areas. Given the inconsistent
classification method in literature, here we classify the bioprinting techniques accord-
ing to the dimensions of the building blocks, which meet the essential characteristics
of this bottom-to-up methodology. This chapter ends with the general challenges and
some perspectives from the author.
2.1 Concepts and History
As an emerging technology, 3D bioprinting has been called numerous names, includ-

ing biofabrication, cell printing, tissue/organ printing, and bioplottering [1–3]. Cur-
rently, the word “bioprinting” is more frequently used regarding the 3D printing
with living cells, which is one of the most important contents of biofabrication [4].
In 2015, the Oxford Dictionary defined bioprinting as “the use of 3D printing tech-
nology with materials that incorporate viable living cells, e.g., to produce tissue
for reconstructive surgery.” Compared to other well-known 3D printing fields that
deal with plastic, elastomer, ceramics, and metals, bioprinting is recognized by the
highlight of manipulating living cells.
The concept of bioink comes along with that of bioprinting, referring to the inks
used in the 3D bioprinting process. Researchers have recently suggested clarifying
this concept as a cell-laden ink, to distinguish from the work of printing acellular scaf-
folds seeded with cells afterward [4, 5]. To enable living cell encapsulation, the bioink
formulation is usually a hydrated solution with suitable physiological conditions
(e.g., suitable osmotic pressure and pH). This is also correlated to the nature of the
extracellular matrix (ECM), which is a 3D network of extracellular macromolecules
© Tsinghua University Press, Beijing and Springer Nature Singapore Pte Ltd. 2019 7
L. Ouyang, Study on Microextrusion-based 3D Bioprinting and Bioink Crosslinking
Mechanisms, Springer Theses, https://doi.org/10.1007/978-981-13-9455-3_2
8 2 3D Bioprinting and Bioink: Background
providing structural and biochemical support of surrounding cells [6]. These consid-
erations have driven the use of hydrogel materials as the bioink network as they could
create similar 3D microenvironment to ECM [7]. Apart from cells, different com-
ponents are possible to be incorporated in bioink, including nano/microparticles,
growth factors, and other biomacromolecules [7–9]. It should be noted that some
novel types of bioinks have also been explored, including cell aggregates and cell-
laden microgels [10, 11].
As a fabrication technology, 3D bioprinting is believed to enjoy great poten-
tial over some conventional approaches for tissue engineering (Table 2.1). Casting
method is widely used to replicate a solid model with fine features, which, however,
fails to engineer materials heterogeneity and complex geometry with 3D voids. The
porous scaffold is treated as one of the most important elements in tissue engineer-
ing, where it provides structural support for cell growth. However, such conditions
on a substrate (either flat or asperous) might not represent the microenvironment
in vivo, where cells are surrounded by ECM. Moreover, the challenges in hetero-
geneity and cell seeding efficiency also hinder the use of porous scaffolds [14]. Based
on the principle of additive manufacturing, 3D bioprinting provides a new strategy
for engineering 3D tissue model by manipulating biological components spatially
and temporarily. It allows for direct locating of multiple cells into a 3D matrix envi-
ronment replicating a customized model.
3D bioprinting have attracted massive attention since 2003 when researchers
printed living cells using an ink-jet printer for the first time [15, 18], followed by the
use of other techniques such as microextrusion approach (Fig. 2.1). In 2009, the listed
company, Organovo, launched the first commercial bioprinter, and IOP Publishing
released a high-impact peer-review journal Biofabrication, focusing on bioprinting
field. With the foundation of the International Society for Biofabrication (ISBF) in
2010, the community has been well organized via the ISBF annual conference and
other activities (Fig. 2.1). 3D bioprinting is regarded as one of the most promising
biotechnologies that would bring revolutions to human health. In the last decade, it
Table 2.1 Comparison of different biofabrication technologies. Reproduced, with permission from
[12, 13]
Casting Porous scaffold 3D bioprinting
Materials Natural/synthetic Natural/synthetic Natural/synthetic
polymer, cell polymer, mental, polymer, cell
suspension ceramic suspension
Resolution >500 nm 100 nm–1000 μm 10–1000 μm
Advantages Reusable mold, high Controllable porosity, Directly cell
resolution, cell-friendly mechanical properties, manipulation, complex
materials versatility and heterogeneous
architecture
Disadvantages Lack of porosity, Low cell seeding Possible cell damage,
heterogeneity efficiency, poor limitation to materials
heterogeneity
2.1 Concepts and History 9
Fig. 2.1 Brief timeline of bioprinting history. Reproduced, with permission from [15–17]
has been seen with dramatic growth in both academia and industry: The numbers
of bioprinting publications and newly established companies have been growing
exponentially since 2003 (Fig. 2.2a, b). According to the recent Gartner curve hype
cycle on 3D printing technologies, it will still take 5–10 years for 3D bioprinting to
reach mainstream adoption in life science R&D (Fig. 2.2c), which also indicates the
necessity of further investigation into this technology toward the translational stage.
2.2 State of the Art
2.2.1 Bioprinting Technologies
Numerous bioprinting technologies have emerged since the ink-jet printing was firstly
used in cell printing. People have tried to classify them based on different criteria, such
as driven force (e.g., thermal, piezoelectric, pneumatic, piston, and screw driving)
and depositing approach (e.g., nozzle-based jetting or extruding, nozzle-free printing
such as laser-direct-write and stereolithography) [12, 19–21]. Taking the nature of
additive manufacturing, here we sort all the technologies into three categories based
on the geometry of building blocks (Fig. 2.3).
2.2.1.1 Drop-Based Bioprinting (Zero-Dimensional Building Block)
Various techniques have been applied to generate cell-laden drop as a building block,
including ink-jet, valve-based, acoustic, and laser-assisted approaches (Fig. 2.3a).
Similar to the commercial ink-jet printer, ink-jet bioprinting patterns ink drops onto
a surface according to the computer-aided design (CAD). The deposited drops are
usually generated by the burst of microbubbles induced by thermal or piezoelectric
effects. Valve-based bioprinting applies a mechanical or solenoid valve in the nozzle
to control the generation of drops. Both ink-jet and valve-based approaches allow
for the creation of submicron drops with the volume low to 1 pl per drop, which
Fig. 2.2 Growth of bioprinting. Numbers of a publications and b new companies each year since
2003. c Gartner curve hype cycle for 3D printing technologies as of July 2017. Data source a Web of
Science searched with “bioprinting” as keyword; b IDTechEX report “3D Bioprinting 2014–2024”;
c Gartner, Inc
also means that only low-viscosity bioinks are applicable (<20 mPa·s) [22, 23]. In
acoustic bioprinting, bioink drops are generated due to the acoustic force in the local
area, where a microhole is designed to allow for drop jetting [24]. In a laser-based
approach, the laser is used to either directly guide cell movement or induce a cell-
laden drop generation on a reversed platform coated with cell-laden bioinks. This
is possible because of the exposure of a microbubble generated by the laser energy
[25]. In this case, viscous bioinks are usually used to maintain the coating layer.
2.2 State of the Art 11
Fig. 2.3 Schematic of different types of bioprinting technologies based on the building blocks of
a drop, b filament, and c plane. Reproduced, with permission from [6, 12, 21]
These drop-based approaches rely on the specific driving mechanisms for drop
generation and deposition. Cell damages would come from the thermal effects, sur-
face tension when bubbles break, impulse force when drops hit the bed, and water
evaporation. Generally, 85% or higher cell viability could be maintained after process
optimization [26, 27].
2.2.1.2 Filament-Based Bioprinting (One-Dimensional Building Block)
Microextrusion bioprinting is the main approach that uses cell-laden filaments as the
building blocks. Similar to the commonly used fused deposition modeling (FDM)
Fig. 2.4 Schematic of crosslinking strategies used in microextrusion bioprinting: a pre-

crosslinking, b thermally pre- plus post-crosslinking, and c various post-crosslinking performed
by (i) injecting crosslinker in situ, (ii) applying a core–shell needle, (iii) printing into crosslinker
batch, and (iv) spraying crosslinkers. Reproduced, with permission from [28]
3D printing, raw materials are extruded into microfilaments, driven by pneumatic,

piston, or screw force (Fig. 2.3b). Pneumatic system can generate a consistently
stable force on the bioinks, which also means that the extrusion flux might vary with
different bioinks. Thus, air pressure should be optimized whenever new bioinks are
applied. Piston-driving system can preciously control the volume of bioinks being
extruded and thus the printing flux. One of the disadvantages of this approach is
that the extrusion force might be delayed and accumulated when printing viscous
materials, which might induce unstable extrusion. Screw-driving system is suitable
for dealing with highly viscous materials as it mechanically forces the inks to flow
through the thread until the nozzle end. Though high force and precious control can
be achieved, the screw system adds to the risk of cell contamination and damage.
Currently, pneumatic and piston systems are more used in cell printing with hydrogel-
based inks.
Usually, bioinks are required to maintain structural integrity after deposition to
achieve 3D construction. Apart from the extrusion driving force, other printing con-
figurations would also affect the printing outcome, such as the bioink crosslink-
ing strategy. Corresponding to the bioink rheological properties, varied crosslinking
strategies have been applied. For example, pre-crosslinking is supposed to enhance
the bioink viscosity and thus to contribute to the printing fidelity (Fig. 2.4a). For
some thermo-sensitive bioink formulations, comprehensive temperature control is
performed pre- and post-printing to achieve optimal printing results (Fig. 2.4b) [16].
In more cases, post-crosslinking is applied to stabilize the printed filaments immedi-
ately (Fig. 2.4c). For instance, a dual-needle or core–shell needle is used to simulta-
neously introduce crosslinker along with bioinks (Fig. 2.4c (i, ii)) [29, 30]. Printing
bioinks into crosslinker batch or spraying crosslinker on the printing area would also
contribute to the fast gelation of bioinks (Fig. 2.4c (iii, iv)).
2.2.1.3 Plane-Based Bioprinting (Two-Dimensional Building Block)
Recently, digital light processing (DLP) has been introduced to fabricate 3D cell-
laden constructs for tissue engineering [31]. Based on a nozzle-free optical system,
DLP allows for simultaneous gelation of a 2D plane and could possibly achieve
2.2 State of the Art 13
3D construction by combining multiple planes. Basically, the curing light source is

projected with a designed pattern into photo-crosslinkable bioinks to obtain a desired
2D plane. By refreshing the bioinks, several planes could be successively generated
and combined. Using a 2D pattern as the building block, DLP is likely to achieve
higher printing speed. However, the continuous light irradiation might induce high
cell damage, and it remains a challenge for an easy switch between layers and thus
for thick 3D construct fabrication.
Comprehensive comparison of three types of technologies is presented in
Table 2.2. Drop-based approaches are suitable for precise 2D patterning, but fail
to build up 3D constructs effectively due to the use of normal low-viscosity inks.
The plane-based type represented by DLP seems to enjoy higher printing speed as
the fast generation of a two-dimensional building block. Coming out very recently,
this technology is not seen with much literature (8.4% fraction) and still needs to be
promoted further, such as in terms of bioinks development and effective multilayer
construction. Generally, the microextrusion approach is more straightforward in the
applications of tissue engineering and other biomedicine fields. Moreover, most of
the commercial bioprinters in the market are based on a filament building block
strategy.
Table 2.2 Comparison of bioprinting technologies based on different building blocks. Reproduced,
with permission from [12, 20, 28]
Drop-based Filament-based Plane-based
Cost Low (ink-jet) or high Low High
(laser-based)
Cell viability (%) >85 40–95 >85
Cell density Low Flexible Medium
Ink materials Low viscosity Various hydrogels Photo-crosslinkable
and cell aggregates
Resolution High Medium High
Fidelity Poor Good Good
Structural complexity Suitable for 2D Suitable for thick 3D Suitable for 2D
pattern constructs pattern and thin 3D
constructs
Applications Tissue engineering Tissue engineering Tissue engineering
(bone, cartilage, skin, (vascular, bone, (bone, cartilage, etc.)
etc.) cartilage, neural,
muscle, cancer, etc.),
drug delivery,
organ-on-a-chip
Literatures’ fractiona 55.4 36.2 8.4
(%)
a Data from Web of Science, searched with “bioprinting” and specific technology names as keywords,
from 2003 to 2016

2.2.2 Bioinks
As discussed previously, bioinks usually apply hydrogel formulations as cell carriers

and matrix networks. Various hydrogel materials have been used in 3D bioprinting
(Table 2.3), which could be presented as follows based on the source.
Table 2.3 Comparison of different hydrogel materials used in bioprinting. Reproduced, with per-
mission from [7, 57–59]
Materials Type Crosslinking Gelation time Applicable Literatures’
technologies fractiona
(%)
Collagen I Natural pH, >10 min Drop-, 24.9
protein temperature filament-
based
Fibrin Natural Fibrinogen Minutes Drop-, 8.1
protein + thrombin filament-
based
Hyaluronic Natural gly- Lightb , 1–5 min Filament- 6.3
acid cosamino- enzyme based
glycan
MatrigelTM Natural Temperature >10 min Drop-, 1.6
ECM filament-
based
Gelatin Protein Temperature, Minutes Drop-, 18.9
hydrolysis lightb filament-,
from plane-based
collagen
Alginate Natural Ionic Seconds Drop-, 23.7
polysaccha- filament-
ride based
Chitosan Natural pH >10 min Filament- 6.1
polysaccha- based
ride
Agarose Natural Temperature Minutes Drop-, 4.0
polysaccha- filament-
ride based
PEG Synthetic Lightb Minutes Drop-, 5.6
polymer filament-,
plane-based
Pluronic Synthetic Temperature Minutes Filament- 0.9
F-127 polymer based
a Web of Science, searched with “bioprinting” and name of individual materials as keywords, from
2003 to 2016
b With chemical modification, e.g., methacrylation
Another random document with
no related content on Scribd:
“invictus,” and “felix” as freely as his Pagan predecessors. From this
period the notion of the “divinity that doth hedge a king” descended
to comparatively modern times, and “Sacred Majesty” was an epithet
of our own kings down to the reign of the last Stuart. Probably, too, it
was the custom of releasing an eagle at a royal funeral which so
impressed the popular imagination that the metaphor became
transferred, as such things generally are sooner or later, to the lower
ranks of the community, and the figure of the soul being borne aloft
on wings took the place that it still occupies in popular Christian
literature.
The share that Mithraism had in diffusing the practices of magic and
astrology is by no means so clear. That the Mithraists, like other
pagans of the early centuries, were addicted to magic is one of the
most frequent accusations brought against them by Christian writers,
and the word magic itself, as has been said above, is derived from
those Magi from whom the Mithraists were said to have derived their
doctrine. In support of this, it can certainly be said that the
worshippers of Mithras by rendering a modified cult to Ahriman,
whom the Christians identified with Satan, laid themselves open to
the suspicion of trafficking with devils, and it is quite possible that
they, like the followers of many other religions at the time, looked
with favour upon the compulsion rather than the propitiation of the
lower powers. Yet the strict monotheism of the faith which practically
looked to Mithras for the ultimate control and regulation of all
sublunary things, is certainly against this conclusion; and it should be
noticed that the laws against the practices of magic and astrology,
then so intertwined that it is difficult to separate them[986], were quite
as severe under emperors like Commodus and Diocletian who
worshipped Mithras, as under those of their successors who
professed the faith of Christ. The rites of Hecate, however, were, as
we have seen, closely connected with those of Mithras and were
generally in the hands of Mithraists. These Hecatean rites seem to
have been almost entirely magical in their character, and it is the
name of Hecate that was handed down as that of the patroness of
sorcerers through the Middle Ages and the Renaissance[987]. One of
the priests of Mithras also goes out of his way to declare on his
epitaph that he is studiosus astrologiae, and on the whole the
Christian accusation was probably not without foundation.
CHAPTER XIII
MANES AND THE MANICHAEANS
It is generally said that the religion of Mithras ended and was

absorbed in Manichaeism, which may thus be supposed to have
inherited some, at least, of its doctrines[988]. This is one of those
statements which are copied by one author from another until they
acquire by mere repetition the force of an axiom; but its truth is not
obvious, nor does it appear to rest upon any sound foundation.
Except in the fact that both Mithraism and Manichaeism came in the
first instance from Persia, there is little likeness between the two
faiths, which are in all essential respects diametrically opposed to
each other. A strict dualism, or the eternal antagonism of two equal
principles, is the distinguishing feature of the religion of Manes, while
the worship of Mithras rested, as has been said in the last chapter,
on an equally uncompromising monotheism, which made the
Supreme Being, whether known as Jupiter or Ormuzd, at once the
creator and the governor of the universe. In this respect, it drew near
to Judaism, which it may have aimed at incorporating with itself, and
was not ashamed to place on its monuments scenes which can be
referred to the Old Testament[989]. Manichaeism, on the other hand,
looked on Judaism with horror, rejected the Old Testament entirely,
and was not improbably born in an outbreak of anti-Semitic fury[990].
But the discrepancy of doctrine is as nothing compared to the wide
difference in those external matters which in a new religion most
strike the imagination of the crowd, and have therefore much to do
with its success or failure. The Mithraist was accustomed, as we
have seen, to an allegorical and symbolical ritual in which the
material image of his god was for ever before him; but the
Manichaean, as we shall see later, forbade the use of images and
his worship consisted merely of prayers and hymns. The Mithraists
made frequent use in their ceremonies of the sacrifice of animals;
but the Manichaeans looked with displeasure on the taking of the life
even of plants. The worshipper of Mithras not only gloried in the
outward profession of his religion, but by his avoidance of the
wearing of garlands forced the notice of it on those of his fellows who
were not of the faith. The follower of Manes, on the contrary,
concealed his religion as carefully as Basilides wished his followers
to conceal theirs, and even went to the length of outwardly adopting
a creed different from his own. It is not therefore to be wondered at
that the rulers of the Roman Empire, whose acquaintance with the
worship of Mithras was a thousand times more profound than our
own, should have favoured Mithraism and have made every effort to
suppress Manichaeism. The very emperors who placed their
reformed State under the protection of Mithras imposed the penalty
of death upon those of their subjects who should venture to teach
the religion of Manes[991].
Not less different were the sanctions with which Mithraism and
Manichaeism appeared in the West. The worship of Mithras came
into the Roman world unobtrusively and without any claim to an
exclusive revelation or special means of propaganda. But
Manichaeism had at its back the personality of one of those
wonderful men who appear at rare intervals in the world’s history, to
leave behind them a memorial of their empire over the minds of their
fellows in the shape of a new creed. Manes was indeed, as the
discoveries of the last decade have taught us, an innovator in
religion entirely worthy to rank with Zoroaster, Buddha, and
Muhammad, and when the difficulties in the way of his missionary
activity are considered, his influence upon the religious ideas of
those who came after him was at least as marked as that of any of
them. Manes or Mânî—the first being the Greek form of the name—
was born, according to his own deliberate statement, about the year
216 A.D., in a village of Babylonia called Mardînû situate on the Kutha
canal to the south of Ctesiphon[992]. According to Christian tradition,
his real name was Corbicius or Kubrik and he was a slave of
unknown birth[993]; according to the Mahommedan writers his father
was one Patecius or Fatak, while his mother is sometimes described
as the “Lady Mary,” sometimes as a Parthian princess, and is
sometimes named Karossa[994]. Such legends grow up naturally
round the birth of all founders of religions, and we should believe
them the less in this case that they have been handed down to us by
the professors of religions bitterly opposed to that of Manes. Yet the
story about the Parthian princess seems confirmed by the free
access that he seems to have always possessed to the court of the
Persian monarchs of his time. Manes himself says, according to Al-
Bîrûnî, that illumination came to him in his thirteenth year[995]; but this
is contradicted by the Fihrist, which puts the age at which he
received revelation as twenty-four[996]. The Acta Archelai, a Christian
source obviously suspect in the state it has come down to us, would
make him a priest of Mithras[997], a tradition which may have
originated at a date when the Catholic Church recognized the danger
to itself involved in the spread of the Mithraic religion. Another story
would make him a Magus or one of the priestly caste entrusted by
Ardeshîr with the propagation of the reformed religion of
Zoroaster[998], which is discredited by the fact that it was the Magi
who were from the outset his bitterest enemies[999]. A late Oriental
writer says that he was a Christian priest having a cure of souls at
Ahvâz[1000], the capital city of the province of Huzitis, which again is
negatived by the fact that he seems from his writings to have had
little more than a hearsay knowledge of Catholic Christianity,
although they show some acquaintance with the heresies of
Bardesanes and Marcion[1001]. He is said to have acquired great skill
in painting which he used to illustrate his teaching[1002], and to have
been a learned mathematician and astronomer. This is likely enough;
but the only events of his life which seem well attested, are that he
began at an early age to propagate his doctrine and that he
succeeded in converting to it Peroz or Fîrûz the son of Ardeshîr,
through whose means he obtained a formal hearing from Sapor or
Shâpûr, the conqueror of Valerian and Ardeshîr’s successor, shortly
after this king’s accession to the throne[1003]. Sapor seems to have
listened to Manes with respect and, according to an Oriental writer,
to have even favoured his propaganda, until the Magi, to whom the
revival of the Zoroastrian religion had been committed, convinced
him of his error[1004]. On this, Manes was exiled from Persia and
retired, says Al-Bîrûnî, to India, China, and Thibet preaching his
gospel[1005]. On Sapor’s death, he returned to Persia under
Hormisdas or Ormuz, and again, it is said, succeeded in converting
to his tenets the reigning monarch[1006]. On Varanes’ or Bahram’s
accession to the throne the following year, however, he was seized
and put to death as a heretic after a disputation with the Chief of the
Magi, in which he failed to support the test of an ordeal by molten
metal proposed to him[1007]. The most likely account of his death
narrates that he was decapitated, and that his skin stuffed with straw
was suspended at the gate of the town where the execution took
place[1008]. This was followed by a great persecution of the
Manichaeans throughout Persia, and it is fairly evident that this, like
his own fate, was due to the hostility he had aroused in the Magi[1009].
The date of his death is fixed with some accuracy at 275 A.D., so that
he would then have reached the age of sixty years[1010].
The causes underlying this sudden appearance of a new religion are
doubtless to be looked for in the political and religious history of
Persia at the time. Ardeshîr, as has been said above, gave new life
to the feeling of Persian nationality which the Parthian Kings had
kept alive during Greek supremacy in Asia, and succeeded in again
founding a Persian Empire. Like Alexander, Antiochus Epiphanes,
and again, Diocletian, he seems to have been thoroughly alive to the
great effect that a faith common to the whole empire would have in
uniting the peoples under his sway.
“Never forget,” he says in the supposed testament that he is said

to have left for the guidance of his son Sapor, “that as a king you
are at once the protector of religion and of your country. Consider
the altar and the throne as inseparable and that they must
always sustain each other. A sovereign without religion is a
tyrant, and a people which have no religion may be deemed the
most monstrous of all societies. Religion may exist without a
State, but a State cannot exist without religion; and it is by holy
laws that a political association can alone be bound[1011].”
Yet in spite of these sentiments, more pithily expressed perhaps in

the “No bishop, no king” of our own James I, the task of founding a
common religion for the whole of the new Persian empire must have
presented some uncommon difficulties. Apart from the strong Semitic
element dominant in their Babylonian province, the Parthians had
always been eclectic in matters of faith, and Vonones, one of the last
kings of Parthia, had shown himself to be a Philhellene of a type
which must have been peculiarly offensive to a sovereign who was
trying to revive the old Persian nationality[1012]. The worship of
Mithras, the god most favoured by the legions with whom Ardeshîr
was soon to be at death-grips, must have been equally out of the
question; and the knowledge of this is probably to be seen in the low
place in the celestial hierarchy assigned to the old Vedic god in the
Avesta of Ardeshîr’s day[1013]. The Jewish religion in Central Asia had
lately given signs of proselytizing fervour, and it was the going-over
of a Parthian kinglet against the will of his people to the Jewish faith
which first, according to one account, gave the excuse for the
intervention of Vologeses or Valkhash and the subsequent
reformation or revival of the Zoroastrian religion[1014]. At the same
time, Christianity had already begun to share with Mithraism the
devotion of the legions stationed on the Roman frontier, and in the
Gnostic form favoured by the teaching of Marcion and Bardesanes
was pushing into Persia from Armenia and Edessa[1015]. Nor can we
doubt that Buddhism, already perhaps struck with decay in its native
country of India[1016], but flourishing exceedingly further East, was
trying to obtain a foothold in that very Bactria which was afterwards
said to have been the historic scene of Zoroaster’s activity. Other
small, but, as the event was to show, highly vitalized faiths, were
current in Western Asia, and the power of the Magi when Ardeshîr
overthrew the Parthian power had declined so greatly that the
statues of the Parthian kings were placed in the temples of the gods
and adored equally with those of the divinities[1017]. The Persians of
Herodotus’ time, who did not believe in deities who had the same
nature as men, would have blushed at such a profanation.
From this unpromising welter of creeds and cults, Ardeshîr delivered
the State by restoring the worship of Ahura Mazda as the State
religion. One of his first cares was to collect the fragments of the
books which we now know as the Zend Avesta, in which the
revelations of the national prophet Zoroaster were set down in a
language not then understanded of the people. It was afterwards
said that the MSS. of these books had purposely been destroyed or
scattered by Alexander; but the fact seems to be that they had fallen
into discredit through the turning-away of the Persians towards
Hellenic and Semitic gods; and that a previous attempt to restore
their authority by Valkhash or Vologeses I, the Parthian king who
reigned from 50 to 75 A.D., had met with little encouragement from
his subjects[1018]. Most modern scholars are now agreed that the
Avesta and the literature that grew up round it contain many
doctrines not to be found in the Persian religion current in
Achaemenian times, and evidently brought into it from foreign
sources under the Hellenistic and Parthian kings. Such as it is,
however, the Avesta formed the Sacred Book of Ardeshîr’s
reformation; while, in the order of the Magi, by him restored to more
than their former power, the reformed Zoroastrian faith possessed an
active, established, and persecuting Church, which reigned in Persia
without a serious rival until the Mahommedan invasion.
Yet the first struggles of the reformation must have been sharp, and
Darmesteter was doubtless justified when he saw in Manichaeism
the first and possibly the strongest expression of the revulsion of
Ardeshîr’s subjects against the rigid orthodoxy which he sought to
impose upon them[1019]. That such a feeling persisted for some time
is plain from the fact that Manes’ “heresy” is said by Al-Bîrûnî to have
been followed by that of Mazdak, who seems to have preached, like
the Antinomian sects of Cromwell’s time, a kind of Socialism
including the community of women and of property[1020]. There arose
also about the same time or a little later the sect of Zervanists
referred to in the chapter on Mithras, who taught that Boundless
Time was the origin of all things and was superior to Ormuzd and
Ahriman, to both of whom he was said to have given birth. They
seemed to have gained great power in the reign of Yezdegerd II;
and, if we may trust the Armenian authors, a proclamation
commanding adherence to their doctrines was put forth by
Yezdegerd’s general Mihr Nerses on his invasion of Armenia in 450
A.D.[1021] But the earliest and most enduring of these heresies or
rebellions against the purified and restored religion of Ahura Mazda
appears to have been that of Manes.
Were now the doctrines that Manes preached to his own undoing his
invention, or did he draw them from some pre-existent source? It is
said, in a Christian account which has come down to us, that they
were the work of one Scythianus[1022], a native, as his name implies,
of “Scythia” (which here probably means Turkestan) and a
contemporary of the Apostles, who married an Egyptian slave and
learned from her all the wisdom of the Egyptians[1023]. With the help
of this and the tincture of dualism which he extracted from “the works
of Pythagoras,” the story goes on to say, Scythianus constructed a
system which he taught to a disciple named Terebinthus, otherwise
called Buddas or Buddha, before his own death in Judaea[1024]. This
Terebinthus gave out that he was born of a virgin and had been
nursed by an angel on a mountain; and he also wrote four books in
which the doctrines of Scythianus were set down[1025]. These books
he entrusted to an aged widow with whom he lived, and he was
afterwards struck dead while performing a magical ceremony. On his
death, she bought a boy of seven years old named Corbicius, whom
she enfranchised, and to whom she left her property and
Terebinthus’ books some five years later. Thus equipped, Corbicius
took the name of Manes, which may signify “Cup” or “Vessel[1026],”
and began to preach. This history has evidently been much
corrupted and by no means agrees with the account before quoted
from Oriental sources which bears greater marks of authenticity; but
it is thought by some to be, like the 14th chapter of Genesis, a sort of
allegory in which the names of peoples and systems are given as
those of individual men[1027]. If this be so, we should perhaps see in
Scythianus the representative of those non-Aryan tribes of Medes of
whom the Magi formed part, while in the name of Buddha we might
find that of one of those Judaean communities holding a mixture of
Magian and Buddhist tenets who according to one tradition were for
long encamped near the Dead Sea[1028]. Yet there is nothing
specifically Buddhist or Egyptian about the doctrines of Manes as we
know them[1029], and if there were any likeness between the
mythology and observances of the cult and those of its
predecessors, it was probably introduced by Manes’ followers rather
than by himself[1030]. As to the doctrines of the Magi, Manes certainly
had no occasion to go to Judaea to find them; for in the Persia of
Ardeshîr and Sapor he must have heard quite as much of them as
he wished.
Probably, therefore, the Christian account of Manes’ sources is
untrue, or rather, as M. Rochat suggests, it was composed at a time
and place in which Manichaeism had become a heresy or alternative
creed attached, so to speak, not to Zoroastrianism but to Christianity,
and had picked up from this and other faiths many accretions[1031].
The doctrine of Manes which has come down to us from other
sources is extremely simple, and seems to accord better with the
Puritanical simplicity of life and ritual afterwards practised by his
followers. Both the Christian and the Mahommedan traditions agree
that he believed that there were two gods, uncreated and eternal,
and everlastingly opposed to each other[1032]. One of these is the
God of Light and the other the God of Darkness; but he does not
seem to have given any specific or proper name to either[1033]. It is
possible that this last-named being may have been identified by him
with Matter[1034], although this would seem to be a remnant of the
Platonic philosophy of which there is no other trace in his teaching.
But it is certain that he regarded the God of Darkness as entirely evil,
that is to say, malevolent, and as a power to propitiate whom man
should make no attempt. “I have considered it needful to despatch
this letter to you” says an epistle which there is much reason to
consider expresses the opinions, if not the actual words, of Manes
himself[1035]:
“first for the salvation of your soul and then to secure you against
dubious opinions, and especially against notions such as those
teach who lead astray the more simple (ἁπλούστεροι), alleging
that both good and evil come from the same Power, and
introducing but one principle, and neither distinguishing nor
separating the darkness from the light, and the good from the
bad and the evil (φαῦλον), and that which is without man from
that which is within him, as we have said formerly, so that they
cease not to confuse and mingle one thing with another. But do
not thou, O my son, like most men, unreasonably and foolishly
join the two together nor ascribe them both to the God of
Goodness. For these teachers attribute to God the beginning
and the end, and make him the father of these ills the end of
which is near a curse[1036].”
Although this epistle bears evident marks of having been worked

over and amplified by some writer of a later age than that of the
founder of Manichaeism, there cannot be much doubt that it contains
his teaching on the Two Principles of all things. In the Christian
account of Manes’ doctrine which M. Rochat thinks earlier than the
epistle quoted above, Manes’ quondam follower Turbo says after
recantation that his master reverences two gods “unbegotten, self-
existing (αὐτοφυεῖς), eternal and set over against each other,” and
that “he represents one as good, the other as wicked, giving to the
one the name of Light and to the other that of Darkness[1037].” So,
too, the Mahommedan writers who give what seems to be an
independent account of Manes’ opinions are agreed that he deduced
the origin of the world from “two Original Principles, one of which is
Light and the other Darkness, and which are separated one from the
other[1038].” The absolute opposition from the outset of good and evil
therefore formed the pivot of Manes’ whole system, and was
opposed quite as much to the Christian and Jewish creeds as to the
Mithraic and other modifications of Persian religious ideas then or
later in vogue, which held that evil like good was the creation of the
Supreme Being, and that Ahriman or Pluto was a god having
subordinate authority to, but of the same nature as, Ormuzd or Zeus.
This uncompromisingly dualistic theory gives an origin to evil
independent of that of good, and can only lead logically to the
assertion of its eternity. Whether Manes gave utterance to it for the
first time, or derived it from a theology then current in Persia, there is
little evidence to show[1039]. The Zend Avesta itself in its Sassanian
recension does not seem to pronounce clearly on this point, and has
been thought by some high authorities to teach the subordinate
origin and ultimate extinction of evil[1040], and by others exactly the
reverse. It does, however, seem to be clear that unless Manes
invented de novo the doctrine above quoted, it must have been from
Persia that he obtained it. No other country with which he can have
become acquainted has yet been shown to possess it[1041].
Exclusively Oriental, too, in its origin must be the history of the
conflict between these two Principles which follows. Each of them
apparently dwelt in his own domain for countless ages untroubled by
the existence of the other. The Light is the uppermost and is,
according to the Mahommedan version of Manes’ doctrine, without
bounds in height and on each side. The Darkness lies below it, and
is in like manner boundless in depth and in lateral extent[1042]. Hence
there is a long frontier at which they touch, and this spot was filled
from the beginning by the celestial air and the celestial earth. If we
may read into the tradition something which is not expressed there,
but which seems to follow logically from it, this atmosphere and this
earth were the heavier parts of the Divine substance, which sinking
down formed a kind of sediment or deposit[1043]. Each of these Two
Principles has five “members” or components, and this partition into
five seems in the Manichaean teaching to run through all things.
Thus, the Mahommedan tradition tells us that the “members” of the
God of Light are Gentleness, Knowledge, Intelligence, Discretion,
and Discernment, those of the Air the same five, of the (celestial)
earth, the Breeze or Ether, Wind, Light, Water, and Fire, and of the
Darkness Smoke, Flame, Hot Wind, Poison or Pestilence, and
Gloom or Fog[1044]. In this, and especially in its deification of abstract
principles, we may see a reflection of Gnostic teaching which may
easily have reached Manes from Valentinus by way of Bardesanes
and the Oriental or Edessan School. On the other hand, the
borrowing may have been the other way, and Simon Magus may
have obtained these notions from the Persian Magi and have
handed them on to Valentinus and his successors. This does not
seem so likely as the other, but the point can hardly be settled until
we know more than we do at present of the state of the Persian
religion from the time of the Achaemenian kings to the Sassanian
reform.
However that may be, both the Christian and Mahommedan
traditions are agreed that the aggressor in the struggle between the
good God and the bad was the Evil One. The Mahommedan source,
here fuller than the Christian, tells us that the Darkness remained in
an unorganized condition for ages, although consisting of the five
members enumerated above. These parts, however, seem to have
sunk down and produced another Earth called the Darker Earth,
from which in course of time came forth Satan. Satan was not, like
the King of the Paradise of Light, without beginning, but came into
being from the union of these five members of Darkness, having the
head of a lion, the body of a serpent, the wings of a bird, the tail of a
fish, and four feet like those of crawling animals[1045], in which figure
we may see a kind of reflection of the Mithraic Ahriman[1046]. Satan,
on his emergence on the Darker Earth, perceived the rays of light
from the upper world, piercing as we may suppose through the
gloomy atmosphere of his own world, and conceived a hatred for
them. Seeing, too, that these rays gained much in strength by their
combination and mutual support, he withdrew within himself so as to
unite himself more closely with his members[1047]. Then again
springing upwards, he invaded the realms of Light with the intention
of there spreading calamity and destruction. The aeon—or world as
the Fihrist calls it—of Discernment was the first to be aware of this
invasion[1048], and reported it to the aeon Knowledge, from whom it
passed to the others in turn until it at last reached the ear of the
Good God, here, as elsewhere in the Fihrist, called the King of the
Paradise of Light. With the aid of the Spirit of his Right Hand, of his
five worlds or members before mentioned, and of his twelve
elements, of which we have before heard nothing[1049], he made the
First Man, clothing him by way of armour with the five “species” or
powers of the celestial earth, the Breeze, Wind, Light, Water and Fire
as before enumerated[1050]. With these He despatched him to fight
Satan, who in his turn did on his armour in the shape of his five
“species,” Smoke, Flame, Poison, Hot Wind, and Gloom[1051]. The
fight lasted long, but in the end Satan triumphed, and dragged the
First Man down into the Realm of Darkness, where he took from him
his light[1052]. During the fight, too, the elements had become mingled,
so that the Ether henceforth was mixed with the Smoke, the Fire with
the Flame, the Light with the Darkness, the Wind with the Hot Wind,
and the Cloud with the Water. This it is which brings about the
confusion or mixture seen in the present world, wherein everything
which is beautiful, pure, or useful, such as gold and silver, comes
from the armour of the First Man, and everything foul, impure, and
gross, from that of his infernal opponent[1053]. After the fight, the King
of the Paradise of Light descended with another Power called the
Friend of the Lights, who overthrew Satan, and the Spirit of the Right
Hand or Mother of Life recalled, either by her voice or by another
power called the Living Spirit, the First Man from his prison in the
lowest Darkness. The First Man, on his deliverance, in this account
mounts again to the Realms of Light, but before doing so “cuts the
roots” of the Five Infernal Elements so that they can no more
increase[1054]. Then the King of the Paradise of Light orders an angel
to draw the Confusion or Mixture of the Elements to that part of the
Realm of Darkness which touches the Realm of Light, and to create
out of it the present world, so as to deliver the imprisoned elements
of Light from the Darkness with which they are contaminated. This is
done, and a Universe having six heavens and eight earths is formed,
each heaven having twelve gates, together with terraces, corridors,
and places in such profusion as to point to some confusion in the
translation into the Syriac which has come down to us. The only
thing that concerns us in this, perhaps, is that the visible world,
presumably the lowest of the eight, has a ditch dug round it in which
is thrown the Matter of Darkness as it is separated from the Light,
and outside this a wall so that it cannot escape. This is in view of the
End of the World[1055].
So far there is no great difference—at all events, no irreconcilable
difference—between the Christian and the Mahommedan accounts
of Manes’ doctrines. The machinery set up for the process of the
redemption of the light, however, differs somewhat conspicuously in
the two traditions. The Mahommedan writers declare that in Manes’
teaching the Sun and Moon were created for the purification of the
Light, the Sun drawing to itself those light-elements which had
become contaminated by the demons of heat and flame and the
Moon exercising a like attraction on those which had suffered from
the embrace of Satan’s other powers. Both luminaries bear these
elements into the Column of Praises or Glory which is perpetually
mounting from the Sun to the World of Light, bearing with it the
praises of men, their hymns of gratitude, and their pure words and
good works[1056]. This will continue until none but a feeble fragment of
the Light remains in this world, when the angels charged with its
maintenance will abandon their task, and return to the World of Light.
A fire will then break out, which will burn for 1468 years and will set
free the remainder of the Light imprisoned in matter by consuming its
envelope. Satan or Hummâma, the Spirit of Darkness, will then
acknowledge his defeat, and will be driven into the tomb prepared for
him, the entrance to which will be closed with a stone the size of the
world[1057]. In the Christian tradition these matters are more
complicated, and Manes is said to have taught that there exists a
great wheel bearing twelve vases or buckets after the fashion of an
Egyptian sakiyeh, which raise the redeemed portions of Light to the
Sun, who gives them to the Moon, who in her turn delivers them to
the Aeons of the Light, who place them in the Column of Glory here
called the Perfect Air[1058]. The Christian account is also more
detailed with regard to the functions of the angels charged with the
conduct of the world, making out that one of them supports this earth
on his shoulders and is therefore called Omophorus, great
earthquakes and commotions taking place when from weariness he
shifts his burthen from one shoulder to the other, while another,
called Splenditenens, holds the heavens by their backs[1059]. The
stars are also in the Christian tradition fashioned out of the purer part
of the Light which was not captured by the Satanic powers, whereas
the Mahommedan tradition says nothing about their origin[1060]. The
Christian writers also make the Manichaeans tell a story about the
appearance of a beautiful virgin who appears to the male and female
devils who were crucified or fixed in this world on the deliverance of
the First Man. She appears to the male fiends as a beautiful woman
and to the female as a desirable young man; and when they covet
and pursue her, she flies from them and disappears. The anger of
the Great Archon or Satan on this causes the appearance of clouds
in this world and thereby obscures the Sun’s light, whilst his sweat
becomes rain[1061].
On the origin of terrestrial man, there is also considerable
discrepancy between the two streams of tradition. The
Mahommedan tells us that Adam was born from the conjunction of
one of “these Archons” or Princes, and a star. Nothing is said to tell
us what is meant by “these” princes, but as the phrase is used in
other passages by the same writer to denote the Satanic hierarchy
one can but suppose that it is one of the rulers of darkness who is
here indicated[1062]. The same writer goes on to say that the
conjunction was “beheld” [or aided?] by a pair of Archons, one male
and the other female, and that a second similar conjunction resulted
in the birth of Eve. There is evidently a reference here to some
legend of which we have lost the trace[1063], and the Christian
tradition assigns to Adam an entirely different origin and declares
that he was made by all the “princes” or archons on the advice of
one of their number, who persuaded the others to give up some of
the light they had received which they knew would otherwise be
taken from them and to make from it man in their own image and
after the form of the “First Man” against whom they had fought with
temporary success[1064]. This story is clearly the same as that which
we have already seen current among the Ophites, and it now seems
most probable that it here appears not—as was once thought—as an
interpolation foisted into the teaching of Manes by the Christian
writer, but because both Ophite and Manichaean derived the story
independently of each other from legends current in Western
Asia[1065].
The Mahommedan writer then plunges into a long and elaborate
account of how the “Five Angels,” meaning thereby apparently the
“members” Gentleness, Knowledge, Intelligence, Discretion and
Discernment, on beholding Adam and Eve, prayed to certain powers
which seem to be those which descended with the King of the
Paradise of Light after the defeat of the First Man properly so called.
These Powers include the First Man himself and the Mother of
Life[1066], and the Living Spirit[1067], and were besought by the Five to
send to earth a Saviour who should give Adam and Eve Knowledge
and Goodness and deliver them from the devils. Their prayer was
heard, and Jesus was sent upon earth “accompanied by a god,” with
whose aid the Archons were again overthrown and imprisoned, while
Adam and Eve were set free[1068]. Jesus then addressed Adam and
revealed to him the whole secret of the cosmogony, enlightening him
upon the origin and functions of the different heavenly worlds or
paradises, of the gods, of hell, of the devils, of the earth and sky, and
of the sun and moon. He then showed him, continues the
Mahommedan tradition, the seductive power of Eve, put him on his
guard against it, and breathed into him the fear of yielding to it.
Adam, it is said, listened to these commands obediently.
The result of this abstinence on Adam’s part—we are still pursuing
the Mahommedan account of the Manichaean teaching—was seen
in the sequel. The Archon or Demon who was practically the father
of the present race of mankind became enamoured of Eve, and
engendering with her begot a son “ugly and of a reddish colour,” who
was named Cain. Cain in turn had relations with his mother Eve, and
from this incest was born a son of white colour who was named
Abel. From the further intercourse of Cain and Eve were born two
daughters, one called “the Wisdom of the World,” and the other “the
Daughter of Pleasure.” Cain took the last-named to wife and gave
the other in marriage to Abel; but he did not know that the Wisdom of
the World was filled with Light and divine wisdom, while the
Daughter of Pleasure possessed nothing of the kind. In the sequel,
one of the Angels had relations with the Wisdom of the World and
begot two daughters, called Help (Farjâd) and Bringer of Help
(Barfarjâd). Abel accused Cain of being the father of these girls,
whereupon Cain killed him and took the “Wisdom of the World” as
his own second wife. The Rulers of Darkness were annoyed at this,
and the “Great Devil,” here called Sindîd, taught Eve magical
formulas by the aid of which she again enticed Adam to intercourse.
The result was a son “beautiful and of an agreeable countenance,”
whom Eve wished to kill as having nothing of the Archons in him.
Adam arranged to have the child fed exclusively on milk and fruits,
and drew three magic circles round him bearing the names of the
King of the Paradise of Light, the First Man, and the Spirit of Life
respectively, to protect him against the devils. He then went to a high
place and entreated God for him, whereupon one of the Three
Powers last named appeared and gave him a Crown of Glory, at the
sight of which Sindîd and the Archons fled away. Then a tree
appeared to Adam called the Lotus, from which he drew milk with
which to nourish his son whom he called first after the tree, and then
Seth (Schâthîl). Eve, on the instigation of Sindîd, again persuaded
Adam to intercourse, which so disgusted Seth that he took with him
the Wisdom of the World, her two daughters Help and Bringer of
Help, and “Siddikût,” which seems to be the community of the elect
or Perfect Manichaeans, and journeyed to the East in search of the
Divine Light and Wisdom. At their death all these entered into
Paradise, while Eve, Cain, and the daughters of Desire went to
hell[1069].
The story about the protoplasts of the Book of Genesis has been
given in more detail than it perhaps deserves because of its manifest
connection with the doctrines of the extant sect of Mandaites,
Hemerobaptists, or Disciples of St John still to be found in certain
villages near the Shât-el-Arab and even in considerable towns like
Bussora. These sectaries declare themselves to have inherited the
faith of John the Baptist, and have a sacred book called the Sidra
Rabba, which has been known to Europeans since the XVIIth century,
and contains, among other things, many stories like those given
above. The Mandaites are a violently anti-Christian sect, and say
that the historical Jesus was a fiend who obtained baptism from St
John the Baptist by means of a trick, and they display a similar
hatred of the religions of both the Jews and the Mahommedans.
Nevertheless, most modern writers consider them related to, and
perhaps the modern representatives of, the Mughtasilah or
“Washers[1070].” This last sect is certainly very ancient, and its history
can in fact be traced as far back as the beginning of the reign of
Trajan[1071], while the Mahommedan author, from whom the
traditional account of Manes’ doctrines has been quoted above, says
that Manes was in his youth one of the Mughtasilah. From this Prof.
Kessler, who perhaps devoted more attention to the Manichaean
religion than any living scholar, built up the theory that the doctrines
of the Mughtasilah were one of the principal sources from which
Manes formed his system. He even says that the Fatak or Patecius
whom tradition gives as a father to Manes must be identified with
that Scythianus or Terebinthus whom the Christian tradition makes
Fatak’s predecessor, was one of the Mughtasilah, and helped Manes
both in the construction of his system and in its propagation[1072]. This
may be so, but very little evidence is available in support of the
theory; and the points which the Mandaites and the Manichaeans
undoubtedly possess in common do not seem to be more than can
be explained by the contact which must necessarily have taken
place between two neighbouring sects both persecuted successively
by Persian Shahs, Christian Emperors, and Mahommedan Caliphs.
The Christian tradition of Manes’ teaching concerning the protoplasts
says merely that “he who said ‘Let us make man in our own image’”
was the same Prince of Darkness who thereby counselled the other
Archons to give up their light in order to make man in the likeness
“of the form that we have seen, that is to say, of the First Man.
And in that manner,” it continues, “he created the man. They
created Eve also after the like fashion, imparting to her of their
own lust, with a view to the deceiving of Adam. And by these
means the construction of the world proceeded from the
operations of the Prince[1073].”
The teaching of Manes with regard to Jesus is not very clear in the
Christian tradition, no doubt because the writers who recorded it
were careful to remove from it as much as possible everything which
in their view savoured of blasphemy. Yet the Christian author before
quoted makes Manes say that the God of Light whom he calls “the
Good Father” sent his well-beloved son upon earth for the salvation
of man’s soul and “because of Omophorus” or the world-sustaining
angel. This son, by whom he can hardly mean any other than the
historical Jesus, “came and transformed himself into the semblance
of a man and showed himself to men as a man, although he was not
a man, and men imagined that he had been begotten[1074].” It is also
to Him that is attributed the construction of the wonderful wheel
before alluded to as equipped with twelve vases which the sphere
causes to revolve, and which thus scoops up, as it were, the souls of
the dying[1075]. The Christian account also narrates that in
“the Paradise which is called the Cosmos [Qy the ‘heavenly’

earth or the Sun?], there are trees such as Desire and other
deceits, whereby the minds of those men [those who reach it?]
are corrupted. But the tree in Paradise, whereby they know the
good, is Jesus and the knowledge of Him which is in the
Cosmos. And whoso receives it, distinguishes between good and
evil. Yet the Cosmos itself is not of God, but it was made from
portions of matter, and therefore all things in it will
disappear[1076].”
There is not really any very great difference between this and the
Mahommedan tradition quoted above which makes Jesus the
messenger sent from above to give knowledge to Adam, especially if
we consider that Manes probably, like most of the Gnostics, placed
Paradise not upon the earth but in one of the heavens intermediate
between us and the abode of the Supreme Being[1077]. That Manes
supposed Jesus to have descended to this earth also is plain from
his own words quoted by Al Bîrûnî from the Shapurakan or book
written by Manes for King Sapor:
“Wisdom and deeds have always from time to time been brought
to mankind by the messenger of God. So in one age they have
been brought by the messenger called Buddha to India, in
another by Zaradusht [i.e. Zoroaster] to Persia, in another by
Jesus to the West. Thereupon this revelation has come down,
this prophecy in this last age through me, Mânî, the messenger
of the God of Truth to Babylonia[1078].”
Manes’ ideas as to the salvation of man’s soul again differ little in the
two streams of tradition. The Christian, here perhaps the fuller of the
two, describes him as teaching that the soul of man, as also that of
beasts, birds, other animals, and plants, is part of the light which was
won by the demons from the First Man, while all bodies are of that
matter which is the same as darkness. Man’s body, we are told, is
called a cosmos by parallelism with the great Cosmos, and all men
have roots here below bound to things which are above[1079]. It is the
cutting of these roots by the demons which causes death. On the
death of a man who has attained the knowledge of the truth during
this life, his soul is taken up in the wheel to the Sun, by whom after it
has been purified it is passed over to the Moon, the two luminaries
being represented as ships or ferry-boats sailing to-and-fro in the
upper air. When the Moon is full, she ferries the souls with which she
is filled towards the East, and then delivers them to the Aeons of
Light who place them in the Pillar of Glory before described. She

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Study on Microextrusion based 3D

Bioprinting and Bioink Crosslinking

3D Bioprinting in Medicine Technologies Bioinks and

3D Bioprinting in Regenerative Engineering Principles

3D Bioprinting Modeling In Vitro Tissues and Organs

3D Rotations Parameter Computation and Lie Algebra

Design and Test Strategies for 2D 3D Integration for

How to Be a Successful Student 20 Study Habits Based on

Explorations in the History and Heritage of Machines

Microarray Bioprinting Technology Fundamentals and

Recognizing Outstanding Ph.D. Research

Theses are accepted into the series by invited nomination only

More information about this series at http://www.springer.com/series/8790

Prof. Jason A. Burdick

ISSN 2190-5053 ISSN 2190-5061 (electronic)

It is my great pleasure to introduce Dr. Ouyang’s thesis work, which is recognized

Beijing, China/Philadelphia, USA Prof. Wei Sun, Ph.D.

3.4 Research Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

6.1.3 Printing Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

3DP Three-dimensional Printing

Nanog Homeodomain-bearing Transcriptional Factor

3D printing (3DP), also known as additive manufacturing, is believed to be a rev-

As an interdisciplinary area, tissue engineering combines the fundamentals of

1.2 Scope and Contents

The topic of this thesis is 3D bioprinting, a multidisciplinary field involving man-

ation, structure fabrication, post-stabilization, cell damage control, and fundamental

1.3 Chapter Outline

Fig. 1.1 Flow diagram of

is developed to allow for versatile bioprinting of different bioinks at a low viscosity.

2.1 Concepts and History

As an emerging technology, 3D bioprinting has been called numerous names, includ-

2.2 State of the Art

2.2.1 Bioprinting Technologies

2.2.1.1 Drop-Based Bioprinting (Zero-Dimensional Building Block)

2.2.1.2 Filament-Based Bioprinting (One-Dimensional Building Block)

Fig. 2.4 Schematic of crosslinking strategies used in microextrusion bioprinting: a pre-

3D printing, raw materials are extruded into microfilaments, driven by pneumatic,

2.2.1.3 Plane-Based Bioprinting (Two-Dimensional Building Block)

3D construction by combining multiple planes. Basically, the curing light source is

from 2003 to 2016

As discussed previously, bioinks usually apply hydrogel formulations as cell carriers

It is generally said that the religion of Mithras ended and was

“Never forget,” he says in the supposed testament that he is said

Yet in spite of these sentiments, more pithily expressed perhaps in

Although this epistle bears evident marks of having been worked

“the Paradise which is called the Cosmos [Qy the ‘heavenly’

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