TISSUE ENGINEERING

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TISSUE ENGINEERING Soft Tissue Biomaterials
Alyssa Panitch Harrington Department of
Bioengineering Arizona State University
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Soft Tissue Engineering

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

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What Issues Need to Be Considered?

• How does the body respond to the material?
• Molecular level
• Cellular level
• Surface features/chemistry matter

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What Issues Need to Be Considered?

• How does the material respond to the body?
• Surface rearrangement
• Erosion
• Degradation
• Chemical and mechanical failure

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Historical PerspectiveCurrently Used Biomaterials

• Silicone Rubber Catheters, tubing
• Dacron Vascular Grafts
• Teflon Catherters, Vascular Grafts
• PMMA Intraoccular Lenses, Bone Cement
• Polyurethanes Catheters, Pace Makers
• Carbon Heart Valves
• Stainless Steel Orthopedic Devices
• Titanium Orthopedic Devices, Dental
• Hydroxy Apatite Orthopedic Devices
• Collagen Burns, Sponges

Ratner, JBMR, 27, 1993
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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

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Protein Adsorption

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

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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
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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

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Inhibiting Protein Adsorption
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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

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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
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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?

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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

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What is it that we are trying to engineer?

• Skin
• Vasculature
• Liver
• Nervous tissue
• Muscle
• Cartilage
• Ocular lenses
• Others

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Skin
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Bone
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Blood Vessel
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Peripheral Nervous Tissue
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What is a tissue?

• Tissue is a blend or composite of materials
• Cells
• Proteins
• Polysaccharides
• Small molecules
• Water (90)

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What is a cell?

• How does the cell interact with its environment?
• Soluble factors
• Extracellular matrix
• Receptors

Cell
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Extracellular Matrix (ECM)

• Structural proteins
• Collagen
• Elastin
• Specialized proteins
• Fibronectin
• Laminin
• Proteoglycans

• Glycosaminoglycans
• Hyaluronic Acid
• Chondroitin Sulfate
• Heparin/Heparan Sulfate
• Dermatan Sulfate

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Basal Lamina
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Amino Acid Structures
General Structure
Serine
Phenylalanine
Lysine
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Amino Acids
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Denatured Protein
Folded Protein
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Nonstructural ECM Proteins

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

Schematic of Domains within Fibronectin
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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)

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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

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Polysaccharides
Heparin
Chondroitin Sulfate
Dextran Sulfate
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What does a Cell see?
I spy

• Difficult question to answer
• Depends on tissue type
• 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

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How do we design a material for tissue
engineering ?

• Keep in mind that Dacron vascular grafts (gt0.6
  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

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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?

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Incorporation of biological signals
BSP Bone Sialoprotein
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Biology is Selective and Precise
Orientation of ligand is critical for cell
adhesion and biological function
Massia and Hubbell, JBMR, 1991, 25223-42
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Biology is Selective and Precise

• Density of signal is important for function

Massia and Hubbell, J. Cell Biol, 1991,
1141089-1100
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Degradable Materials

• Polylactide, polyglycolide, etc. are
  hydrolytically degradable
• Copolymers of varying monomer ratios degrade at
  different rates
• Polyanhydrides also degrade hydrolytically

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Degradable Polyanhydrides

• Mugli, et al. synthesized polymers with varying
  ratios of sebasic acid and 1,6-bis(carboxyphenoxy)
  hexane
• Degradation rates 2 days to 1 year
• Mechanical properties 1.4 GPa to 0.14 GPa
• Polyanhydrides are surface eroding
• retained 70 of their mechanical integrity when
  50 of the materials has eroded

Mugli, BurKoth, and Anseth, JBMR, 1999, 46
271-278
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Hydrogels

• Materials that are composed of hydrophilic,
  cross-linked polymer chains
• Have extremely high water content (often gt90)
• 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 non-degradable
  polymers

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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

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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

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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

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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

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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
  pre-cultured prior to implantation
• Degradable systems allow integration of newly
  formed native tissue

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Bioactive Hydrogel Example Overview

• Photoinitiated poly(vinyl alcohol) gels were used
  to encapsulate fibroblasts
• Modified with RGD peptide to facilitate cell
  adhesion
• Cell viability gt80 after two weeks
• Youngs modulus for 15 and 30 gels
• 15 wt gels 125 /- 13 kPa
• 30 wt gels 838 /- 194 kPa

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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
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Protein Sequences
100 Collagenase activity
ECSAVG
ECSAVG
ECSAVG
ECS
where
is PQGIAGQRGDSSIKVAVG
30 Collagenase activity
ECSAVG
ECSAVG
ECSAVG
ECS
where
is PDGIAGQRGDSSIKVAVG
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Hydrogel Formation

• 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

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Mechanical Data
100 Collagenase activity
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Degradation Data
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Artificial ECM
Making use of heparin affinity
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Heparin-Binding Peptides
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
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Rheological Evaluation
1650 Pa at 100 rad/s





830 Pa at 25 rad/s
plt0.001
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Release
plt0.005 plt0.0001





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Examples of Materials Used in Nerve Regeneration

• Grafts
• Allografts
• Xenografts
• Low water content polymers
• Poly(L-lactic acid) - poly(glycolic acid)
  co-polymers
• Poly(pyrrole)
• Silicone tubes

• Hydrogels
• Synthetic
• Methyl cellulose
• Acrylamide
• Biological
• Calcium-alginate
• Agarose
• Collagen
• Fibrin
• Laminin
• Hyaluronic acid

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Fibrin Gels for Nerve Regeneration

• Fibrin is a natural wound healing matrix
• Gel structure can be controlled based on Ca2 and
  fibrinogen concentrations
• Amenable to inclusion of neurotrophic factors via
  Fa XIIIa sites
• Degraded by plasmin, which is released by
  extending neurites

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Fibrin Gel NGF Delivery System

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

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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

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Hyaluronic Acid

• Found in many Tissue Types
• Prominent during development

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

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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 a3b1 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
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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