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TISSUE ENGINEERING Soft Tissue Biomaterials

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

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TISSUE ENGINEERING Soft Tissue Biomaterials

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

  2. Soft Tissue Engineering • Biology and materials • Historical perspective • Proteins, polysaccharides, cells and tissues • Examples of biologically interactive biomaterials

  3. Protein Adsorption Free From Fouling Free From Fouling Cell Adhesion What Issues Need to Be Considered? • How does the body respond to the material? • Molecular level • Cellular level • Surface features/chemistry matter

  4. Body Fluid Body Fluid Body Fluid Body Fluid What Issues Need to Be Considered? • How does the material respond to the body? • Surface rearrangement • Erosion • Degradation • Chemical and mechanical failure

  5. 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

  6. Where’s 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

  7. t = 30 min t = 10s t = 5 min Protein Adsorption • Plasma Contains over 200 different proteins • Vroman effect – different proteins adsorbed to surface over time

  8. 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

  9. 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

  10. End-graft PEG Coat with Hydrogel Precoat with albuman Attach heparin Inhibiting Protein Adsorption

  11. 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

  12. 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

  13. 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?

  14. 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…

  15. What is it that we are trying to engineer? • Skin • Vasculature • Liver • Nervous tissue • Muscle • Cartilage • Ocular lenses • Others…

  16. Skin

  17. Bone

  18. Blood Vessel

  19. Peripheral Nervous Tissue

  20. What is a tissue? • Tissue is a blend or composite of materials • Cells • Proteins • Polysaccharides • Small molecules • Water (~90%)

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

  22. Structural proteins: Collagen Elastin Specialized proteins: Fibronectin Laminin Proteoglycans Glycosaminoglycans Hyaluronic Acid Chondroitin Sulfate Heparin/Heparan Sulfate Dermatan Sulfate Extracellular Matrix (ECM)

  23. Basal Lamina

  24. Amino Acid Structures General Structure Serine Phenylalanine Lysine

  25. Amino Acids

  26. Polar Groups Nonpolar Groups Denatured Protein Folded Protein

  27. Fibrin, heparan sulfate Heparan sulfate Collagen Integrin Fibrin COOH H2N 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

  28. 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)

  29. 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

  30. Polysaccharides Heparin Chondroitin Sulfate Dextran Sulfate

  31. 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

  32. 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

  33. How do we design a material for tissue engineering? • With that said: • Do we attempt to incorporate more biology? • We’ll 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?

  34. Incorporation of biological signals *BSP = Bone Sialoprotein

  35. GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY YSDGRG YSDGRG YSDGRG YSDGRG YSDGRG Biology is Selective and Precise Orientation of ligand is critical for cell adhesion and biological function Massia and Hubbell, JBMR, 1991, 25:223-42

  36. GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY GRGDSY Biology is Selective and Precise • Density of signal is important for function Massia and Hubbell, J. Cell Biol, 1991, 114:1089-1100

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

  38. Hydrogels • Materials that are composed of hydrophilic, cross-linked 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 non-degradable polymers

  39. 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

  40. 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, Biomaterials 23(2002): 4307-4314

  41. 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, Biomaterials 23(2002): 4307-4314

  42. 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, Biomaterials 23(2002): 4307-4314

  43. 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 K.T. Nguyen, J.L. West, Biomaterials 23(2002): 4307-4314

  44. 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 • Young’s modulus for 15% and 30% gels • 15 wt% gels: 125 +/- 13 kPa • 30 wt% gels: 838 +/- 194 kPa R.H. Schmedlen et al., Biomaterials 23(2002): 4325-4332

  45. 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

  46. Protein Sequences 100% Collagenase activity ECSAVG ECSAVG ECSAVG ECS where is PQGIAGQRGDSSIKVAVG 30% Collagenase activity ECSAVG ECSAVG ECSAVG ECS where is PDGIAGQRGDSSIKVAVG

  47. C + VS - PEG - VS C  SH  SH C C  S  VS  PEG  VS  S   S  VS  PEG  VS  S  C C 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

  48. ECSAVG ECSAVG ECS ECSAVG Mechanical Data 100% Collagenase activity

  49. Degradation Data

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