TISSUE ENGINEERING

Soft Tissue Biomaterials

Alyssa Panitch
Harrington Department of Bioengineering
Arizona State University

Soft Tissue Engineering

Biology and materials
Historical perspective
Proteins, polysaccharides, cells and
tissues
Examples of biologically interactive
biomaterials

What Issues Need to Be

Considered?
How does the body respond to the
material?
Molecular level
Cellular level

Surface features/chemistry matter

Protein
Adsorption

Free From
Fouling

Free From
Fouling

Cell
Adhesion

What Issues Need to Be

Considered?

How does the material respond to the

body?
Surface rearrangement
Erosion
Degradation
Chemical and mechanical failure
Body
Fluid

Body
Fluid

Body
Fluid

Body
Fluid

Historical Perspective
Currently Used Biomaterials

Silicone Rubber
Dacron
Teflon
PMMA

Polyurethanes
Carbon
Stainless Steel
Titanium
Hydroxy Apatite
Collagen

Catheters, tubing
Vascular Grafts
Catherters, Vascular Grafts
Intraoccular Lenses, Bone
Cement
Catheters, Pace Makers
Heart Valves
Orthopedic Devices
Orthopedic Devices, Dental
Orthopedic Devices
Burns, Sponges
Ratner, JBMR, 27, 1993

Wheres the Engineering?

Traditionally, the body responds to all
materials the same way
Recognized as non-self and walled off
No longer able to interact with the body to
induce tissue regeneration
May act as mechanical support or structural
replacement

Protein Adsorption
Plasma Contains over 200 different
proteins
Vroman effect different proteins
adsorbed to surface over time

t = 10s

t = 5 min

t = 30 min

Proteins and Interfaces

Vroman and Adams looked at protein
adsorption from plasma on Ge, Pt, Si, Ta
Within 10s of exposure ~6 nm thick layer
of fibrinogen formed
Within 60s layer was less uniform, ~12.5
nm mostly fibrinogen
Fibrinogen-340kDa plasma glycoprotein
Major protein component of clotting
Promotes platelet adhesion
Vroman and Adams, J. Biomed. Mat. Res. 3(1969): 669

Clotting and Biomaterials

Two pathways lead to clot formation
Intrinsic pathway is activated by damage to or
change in vascular endothelium or exposure
of blood to collagen
7-12 minutes to form a soft clot

Extrinsic pathway is activated by Tissue

Thrombospondin or a foreign body
5-12 s to form a soft clot

Inhibiting Protein Adsorption

Coat with Hydrogel

Precoat with albuman

End-graft PEG

Attach heparin

Complement - The Major Defense

Clearance System
Can be activated through immunoglobulin
Or if a particle provides a site for amplified
self-activation of the early activating
components

Complement and Materials

Complement activating factor C3b is an
opsonin
Opsonins, when bound to a surface
promote adhesion of and activation of
neutrophils and macrophages
Lead to frustrated phagocytosis
Frustrated
Phagocytosis

Phagocytosis

Foreign Body Response

All materials elicit some level of foreign
body response.
Fibroblasts secrete collagen
Wall off the object from the body
The thickness of the capsule depends on
material properties.
Can we ensure that the desired response is
faster than the undesired one?

Can We Engineer the Biological

Response?
Not all materials are created equal
Clearly, Biology has found a way to
develop materials, which support healing
or regeneration
Can we tap into biology to deliver the
appropriate clues for tissue regeneration?
Adhesion, migration, proliferation,
differentiation, appropriate scaffold
synthesis

What is it that we are trying to

engineer?

Skin
Vasculature
Liver
Nervous tissue
Muscle
Cartilage
Ocular lenses
Others

Skin

Bone

Blood Vessel

Peripheral Nervous Tissue

What is a tissue?
Tissue is a blend or
composite of
materials

Cells
Proteins
Polysaccharides
Small molecules
Water (~90%)

What is a cell?
How does the cell interact with its environment?
Soluble factors
Extracellular matrix
Receptors

Cell

Extracellular Matrix (ECM)

Structural proteins:
Collagen
Elastin

Specialized proteins:
Fibronectin
Laminin

Proteoglycans

Glycosaminoglycans
Hyaluronic Acid
Chondroitin Sulfate
Heparin/Heparan
Sulfate
Dermatan Sulfate

Basal Lamina

Amino Acid Structures

H

OH

H3N C C
R

General Structure

H 3N

O
O

Serine
+

NH3

H3 N

O
O

Phenylalanine

H 3N

O
O

Lysine

Amino Acids
Amino Acid

R Group

Amino Acid

R Group

Aspartic acid

Acidic

Alanine

Nonpolar

Glutamic acid

Acidic

Glycine

Nonpolar

Arginine

Basic

Valine

Nonpolar

Lysine

Basic

Isoleucine

Nonpolar

Histidine

Basic

Leucine

Nonpolar

Asparigine

Polar

Proline

Nonpolar

Glutamine

Polar

Phenylalanine

Nonpolar

Serine

Polar

Methionine

Nonpolar

Threonine

Polar

Tryptophan

Nonpolar

Tyrosine

Polar

Cysteine

Nonpolar

Denatured Protein
Polar Groups

Nonpolar Groups

Folded Protein

Nonstructural ECM Proteins

Contain several biological domains
Bind collagen and/or cells
Many bind to GAGs such as heparin or heparan
sulfate

Fibrin,
Collagen
heparan sulfate

Integrin

Heparan
sulfate

Fibrin
COOH

H2N

Schematic of Domains within Fibronectin

Polysaccharides
Many cell surface proteins are
glycosylated
Affects protein function
Influence recognition by other proteins/cells

Most cells present heparan sulfate

Binds to many ECM proteins (e.g. fibronectin,
collagen, growth factors)

Polysaccharides
ECM often is rich in polysaccharides
Sulfated/charged polysaccharides interact
with water which provides beneficial
mechanical properties
Provides compressive strength of collagen

Sequestering of growth factors/creation of

chemical gradients

Polysaccharides
O
O

S
O O
H 2C
O

O
O
OH

O
O S O
O

OH

OH

O
OH

HN
O S O
O

NH
O

Heparin

Chondroitin Sulfate
CH2

Dextran
Sulfate

O
O S O OH
CH2
O
O

CH2

O
O
O
O
O S O
O S O
O
O
O
O
O S O
O S O
O
O
O
O
O S O
O S O
O
O

What does a Cell see?

Difficult question to answer
Depends on tissue type

I spy

Maybe highly hydrated polysaccharide rich scaffold

cartilage
Maybe dense, hard composite, bone
Collagen and hydroxyapatite

Certainly a complex milieu of both covalently

linked and physically linked macromolecules

How do we design a material for

tissue engineering ?
Keep in mind that: Dacron vascular grafts
(>0.6 mm in diameter) work well
And PLGA has been used to create an
acceptable skin substitute and as a
controlled release vehicle
PGA is used for degradable sutures
Polyanhydrides are used for release of
chemotherapeutic agents

How do we design a material for

tissue engineering?
With that said:
Do we attempt to incorporate more biology?
Well see excellent examples of continued use of
PLGA in the following talks

Do we design a scaffold that mimics that of a

healthy form of the tissue to be replaced?
Or do we look to development?

Incorporation of biological signals

Ligand / Origin

Activity

SIKVAV / Laminin

Angiogenesis, Nerve regeneration

YIGSR / Laminin

Angiogenesis, Nerve Regeneration

RGDS / Ubiquitous Cell Adhesion

REDV / Fibronectin Cell Adhesion
FHRRIKA / BSP*
*

BSP = Bone Sialoprotein

Heparin binding

Biology is Selective and Precise

Orientation of ligand is critical for cell
adhesion and biological function

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

Y
S
D
G
R
G

Y
S
D
G
R
G

Y
S
D
G
R
G

Massia and Hubbell, JBMR, 1991, 25:223-42

Y
S
D
G
R
G

Y
S
D
G
R
G

Biology is Selective and Precise

Density of signal is important for function

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

G
R
G
D
S
Y

Massia and Hubbell, J. Cell Biol, 1991, 114:1089-1100

G
R
G
D
S
Y

Degradable Materials
Polylactide, polyglycolide, etc. are
hydrolytically degradable
Copolymers of varying monomer ratios
degrade at different rates

Polyanhydrides also degrade hydrolytically

Hydrogels
Materials that are composed of hydrophilic, crosslinked polymer chains
Have extremely high water content (often >90%)
Physicochemical properties can be tailored

Closely mimic mechanical properties of soft tissue

Can be modulated for specific tissue or application

May be polymerized into any desired geometry

Can even be gelled in situ

May be composed of degradable or nondegradable polymers

Some Unique Attributes of

Hydrogels
High water content permits free diffusion of
cellular nutrients and waste products
In situ polymerization possible
Facilitates localized delivery of the material
Gel conforms to the geometry of the wound or defect

Mechanisms of polymerization allow

incorporation of bioactive signals or
bioresponsive domains
Cellular growth or guidance cues
Enzymatic degradation domains

Hydrogels in Tissue Engineering

Interfacial barrier systems
Material provides physical barrier between
target tissue and other tissue or external
environment
Wound healing applications (dermal sealants, etc.)

Mitigates post-operative adhesion wounds

Can prevent thrombosis and restenosis
subsequent to a vascular injury
Materials can be highly resistant to protein
deposition and platelet adhesion
K.T. Nguyen, J.L. West, B
iomaterials 23(2002): 430

Hydrogels in Tissue Engineering

Drug delivery systems
Act as localized drug sequestration depots
Release kinetics can be controlled via
physicochemical properties of the polymer
Cross-link density (pore size)
Degradation rate of matrix
Density of degradable domains/chains

In situ polymerization provides directed

therapy precisely to target area
K.T. Nguyen, J.L. West, B
iomaterials 23(2002): 430

Hydrogels in Tissue Engineering

Cell encapsulation systems
Cells are included in pre-polymerization
solution and the material is gelled around
them
Provides immunoisolation
Gels are permeable to nutrients and waste
products
Thus, cells are allowed to function normally
while protected from host immune system
K.T. Nguyen, J.L. West, B
iomaterials 23(2002): 430

Hydrogels in Tissue Engineering

Tissue scaffold systems
Material acts a physical framework for cell
attachment and proliferation
Mechanical properties may be customized for
the native values for a particular tissue
Can be formed into any geometry
Scaffolds can be seeded with cells and precultured prior to implantation
Degradable systems allow integration of
newly formed native tissue
K.T. Nguyen, J.L. West, B
iomaterials 23(2002): 430

Bioactive Hydrogel Example:

Overview
Photoinitiated poly(vinyl alcohol) gels were
used to encapsulate fibroblasts
Modified with RGD peptide to facilitate cell
adhesion

Cell viability >80% after two weeks

Youngs modulus for 15% and 30% gels
15 wt% gels: 125 +/- 13 kPa
30 wt% gels: 838 +/- 194 kPa
R.H. Schmedlen et al., Bi
omaterials 23(2002): 4325

Biologically Degradable Materials

PEG hydrogels were
designed to degrade
in response to
biological events
VRN plasmin
degradation
APGL collagenase
degradation

West and Hubbell, Macromolecules, 1999, 32(1): 241-244

Protein Sequences
100% Collagenase activity
ECSAVG
where

ECSAVG

ECSAVG

ECS

is PQGIAGQRGDSSIKVAVG

30% Collagenase activity

ECSAVG
where

ECSAVG

ECSAVG

is PDGIAGQRGDSSIKVAVG

ECS

Hydrogel Formation
C

SH

40% hydrogels
Proteins are dissolved in
phosphate buffered saline with
EDTA ( pH 8.0)
Molar ratios of cysteine groups
to vinyl sulfone groups
Cross-linked with PEG-vinyl
sulfone (8-arm) at 37 for 15
min-2 hours via Michael addition

SH
C

+ VS - PEG - VS

C
S

VS
VS

PE
PE
G
G

VS
VS

S
S
C
C

Mechanical Data

100% Collagenase activity

ECSAVG

ECSAVG

ECSAVG

ECS

Degradation Data

Artificial ECM
Making use of heparin affinity

= PBD1
= PEG-8VS
= Polysaccharide
= PBDx to be released or
sequestered

Heparin-Binding Peptides
Name

Sequence

KD (M)

HBD1*

dansyl-GKAFAKLAARLYRKAGC

5.10.2 x 10-8

HBD2*

dansyl-GAAFAKLAARLYRKAGC

3.70.6 x 10-7

HBD3*

dansyl-GKAFAALAARLYRKAGC

6.60.1 x 10-7

TAT #

dansyl-GYGRKKRRQRRRGC

3.01.4 x 10-8

Tyler-Cross et al. (1994). Prot. Sci. 3: 620 # Rusnati, M. et al. (1999). J. Biol. Chem. 274: 28198.

KD varies based on number of TAT bound per heparin

*

Rheological Evaluation

Modulus (Pa)

10000

1650 Pa at 100 rad/s

* *
* * *

1000
100

830 Pa at 25 rad/s

10

* p<0.001
1
0.1

10

Angular Frequency (rad/s)

100

G'
G''

Release
1

% Released

0.9
0.8

p<0.005

0.7

* p<0.0001

0.6

PBD2
PBD3
TAT
PTD2
RGDS
Control

0.5

0.4

0.3

0.2
0.1
0
0

5
Time (days)

10

Examples of Materials Used

in Nerve Regeneration
Grafts
Allografts
Xenografts

Low water content

polymers
Poly(L-lactic acid) poly(glycolic acid) copolymers
Poly(pyrrole)
Silicone tubes

Hydrogels
Synthetic
Methyl cellulose
Acrylamide

Biological

Calcium-alginate
Agarose
Collagen
Fibrin
Laminin
Hyaluronic acid

Fibrin Gels for Nerve

Regeneration
Fibrin is a natural wound healing matrix
Gel structure can be controlled based on Ca+2 and
fibrinogen concentrations
Amenable to inclusion of neurotrophic factors via Fa
XIIIa sites
Degraded by plasmin, which is released by extending
neurites

Fibrin Gel NGF Delivery

System

Fibrin-based hydrogel
Incorporates NGF
Human recombinant
Factor XIIIa crosslink site
Plasmin degradation
site
Releases on-demand

Other Polymer Systems in

Neural Tissue Engineering
Hyaluronic acid (HyA)
Ubiquitous native ECM component
Found at high levels in CNS
Neuronal pericellular matrices
Myelin-rich fiber tracts

Cell surface receptors are expressed by many

neuronal cell types
Thiolation permits addition of other polymers or factors

PEG modification of other systems

Fibrin
HyA

Hyaluronic Acid
Found in many Tissue Types
Prominent during development
S

SH SH SH SH SH

S
S
S

S
S

Shu, et al, Biomacromolecules, 2002, 3(6):1304

Neural Cell Adhesion Peptides

in Polymer Matrices
Peptide sequences
from N-cadherin
Conjugated to
functionalized PEG
Crosslinked into fibrin
and HyA
Along with CRGDS
Chick DRGs cultured
within for ~48 hours
Will be compared to
CRGDS alone and
unmodified polymer

Legend
Cell Adhesion
Peptide
Sequences
Multi-arm Star
PEG
Factor XIIIa (Fa
XIIIa) Crosslink Substrate
(Artificial
Peptide
Sequence)
Factor XIIIa (Fa
XIIIa) Crosslink Substrate
(Intrinsic on
Native Protein)
Fibrinogen
Molecule

Cells and ECM Talk to One Another

Cells play a role in organizing the ECM
while the ECM sends signals to the cells
deHart, et al studied the effect of 31 on
the organization of laminin-5
Keratinocytes reorganized extracellular
laminin-5 into structures near the cell surface
Similar reorganization is seen with other ECM
molecules and integrins.
G. W. deHart, et al. Exp. Cell Res, 283 (2003) 67-79

In Conclusion
Multiple parameters play a role in material tissue interactions
Material design needs to take into account initial
contact with the biological system

Complement
Coagulation
Foreign body response
Immunology

Biology holds secrets for specific relevant

interactions between materials and cells