Lecture 3: Degradable Materials with Biological Recognition Last time: Theory of hydrolytic polymer erosion Enzymatic degradation of polymers Designing Biodegradable Macromolecules Today: Biological recognition in vivo Engineering biological recognition of biomaterials: cell adhesion/migration Reading: S.E. Sakiyama-Elbert and J.A. Hubbell, ‘Functional Biomaterials: Design of Novel biomaterials,’ Annu. Rev. Mater. Sci. 31, 183-201 (2001) J.C. Schense et al., ‘Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension,’ Nat. Biotech. 18, 415-419 (2000) Supplementary Reading: ‘The Extracellular Matrix,’ pp. 1124-1150, Molecular Biology of the Cell, Lodish et al. Biological Recognition in vivo Interactions of cells with their environment at the molecular level ECM = extracellular matrix Motivation: ? Cell interactions with simple synthetic materials are governed by nonspecific interactions: o e.g. surface energies; hydrophobic interactions, charge-charge interactions o DRAW OXIDE SURFACE, POLYMER SURFACE ? …but this is not how cells interact with ECM ? Cells use receptor-receptor/receptor-ligand interactions to guide their functions, including: o Adhesion, migration o Growth o Differentiation Secretion of molecules Binding of molecules Specialized functions Functions of ECM: ? Mechanical Support ? cues for cell survival/function o anchorage-dependent cell growth o differentiation cues ? organization of tissue o control of tissue morphology, localization of cell types Lecture 3 – Biological Recognition 1 of 15 Structure of native ECM scaffold Prototypical soft tissue ECM (varies from tissue to tissue): ? structural fibers o collagen o other fibers? (SLIDE) collagen gel reticular network of lymph node TEM of collagen fibril ? generalized matrix assembly: o fibrils assemble into fibers null fibers may be organized or isotropic, and form tight (~10 nm separation between fibers) or open (20-30 μm between fibers) meshes o adhesion proteins ‘decorate’ fibers o other signals (cytokines, etc.) may also be sequestered on fiber surfaces Collagen fiber hydrogel P. Friedl et al.: Eur. J. Immunol. 28, 2331 (1998). Collagen triple helices Collagen fiber Adhesion motifs Sequestered Cytokines/ Chemokines/ Etc. Lecture 3 – Biological Recognition 2 of 15 ? Other major matrix-structure proteins: o Elastin ? Key length scales: o diameter of collagen fibrils: 50-200 nm o diameter of collagen fibers: 0.5-5 μm o diameter of collagen triple helices: o diameter of of collagen chain: o length of collagen triple helix: 300 nm ? Adhesion proteins o Complexity of adhesion proteins (SLIDE) laminin structureCollagen I fibril ligand binding site map 1 (from Lodish) o Adhesion proteins designed to bind to structural ECM components, and present binding sites to receptors o Adhesion proteins can present multiple binding sites for different receptors that work in synergy Lecture 3 – Biological Recognition 3 of 15 Interactions of cells with native ECM ? Signals from the extracellular environment: DRAW ON BOARD: ￥Growth factors ￥Cytokines ￥Extracellular matrix ￥Chemokines Cell adhesion: integrin-mediated cell-ECM interactions ? Cells interact with specific adhesion motifs in adhesion proteins via cell surface receptors; the microsctructure of ECM protein arrangement and its composition can tune cell adhesion o Adhesion in turn regulates growth, differentiation, and migration o Major family of cell-ECM receptors: integrins null Composed of noncovalently-associated α and β chains Actin filaments (cytoskeleton) α β Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ Ca ++ integrin ECM fiber Adhesion protein Cell membrane (Extracellular space) ? Integrins and signaling o ‘inside-out’ signaling: biochemical signal triggers affinity change in integrins o focal contacts and signaling null integrin clustering drives actin filament assembly and can signal through multiple biochemical pathways, some of which synergize with growth factors to tell the cell ‘where it is’ Lecture 3 – Biological Recognition 4 of 15 Ca++ Ca++ Ca ++ Ca++ Ca++ Ca ++ Ca + Ca++ ON BOARD: o Length scales in cell adhesion: o Size of integrins, focal contacts, relationship to cell size, fiber spacing DRAW ON BOARD Integrin structure (Lodish) focal contacts 2 (SLIDE) Actin stress fibers Interference reflection microscopy: dark spots indicate cell-substrate separations < 50 nm (stress fibers pics from Maheshwari et al.) ? 20 different integrins known, many different pairs possible with different ligand specificities, thus cell-specific adhesion can be modulated by ECM Lecture 3 – Biological Recognition 5 of 15 Engineering Biological Recognition of Synthetic Degradable Polymers: Incorporation of peptides in synthetic polymers ? Lit on peptides in polymers 3-7 ? So far, we’ve focused on making degradable synthetic materials, and ignored biological recognition o How do we make materials that can interact in an engineered way with their biological environment? o ANSWER: incorporation of ECM cues ? Peptides have been introduced in synthetic polymers to provide scaffolds that appear less foreign and have some engineered response from cells ? Why use peptides instead of full proteins? 1. Proteins fragile 2. Proteins not soluble in organic solvents, but peptides often are 3. Cost 4. Immunogeneicity of peptides is less than complete protein sequences (reduce likelihood of provoking inflammatory response to devices) ? Peptide sequences conjugated to synthetic polymers have been used to provide signals for: (SLIDE) o Adhesion null Fragments from ECM adhesion proteins o Remodeling null Short sequences recognized by remodeling enzymes null Support transformation of synthetic scaffolds into de novo natural matrix null Support cell migration through solid scaffolds Lecture 3 – Biological Recognition 7 of 15 Lecture 3 – Biological Recognition 8 of 15 o Growth/differentiation null Peptide cytokines o Other potential functions null Chemotaxis?: gradients of peptide attractants ? What sizes are we talking about when we discuss peptides vs. proteins? o Ideal case are peptides of ~30 amino acids or less that can be prepared on a solid-phase synthesizer o This is usually more than adequate for adhesion peptides, enzyme-recognized peptides o Cytokines used on biomaterials may be slightly larger (30-60 amino acids total MW~5K g/mole), but may also be produced efficiently in mass quantities null These peptides sometimes have some folding or intra-chain bonding that is not reproducible with shorter peptide sequences ? We’ll discuss approaches to incorporating peptides in biomaterials as we go through the representative applications: 1. adhesion 2. remodeling 3. ytokine signaling Recognition of Biomaterials by Adhesion Receptors: Controlling Cell Adhesion on Degradable Polymers Paradigm of Cellular responses to synthetic biomaterials ? Proteins adsorb (SLIDE) ? Cells respond to adsorbed protein layer o Why is this an issue? 1. Adsorbed layer often unstable and reconstructs as environment changes ? Vroman effect -> protein exchange 2. Cells may adhere poorly to this surface 3. Proteins denatured/presented in non-native conformations DRAW ON BOARD ? Non-native signals transmitted that are not readily controlled ? Immunogenic epitopes may be generated cell matrix c Protein-resistant surfaces 8,9 ? ONLY LANGMUIR ADSORPTION COVERED IN 3.051J ? 1 st order: present peptide fragments from adhesion proteins ? more control, perhaps better: present peptide fragments from adhesion proteins on ‘inert’ background o modify material to resist protein adsorption o present peptides on this ‘blank slate’ background ? resisting protein adsorption o current hypotheses: achieve a surface structure with some or all of these characteristics: null water-rich surface layer – reduce protein-surface interactions null dynamics chains at surface – steric interference with adsorption o SCHEMATIC VIEW OF HOW PROTEIN RESISTANCE IS ACHIEVED o molecules known to provide protein resistance: (essentially, the most hydrophilic nonionic polymers) null poly(ethylene glycol) ? most water-soluble non-ionic synthetic polymer ? fastest chain dynamics of any polymer in water ? can be synthesized with good control to many molecular weights and incorporate end- functional groups ? reminder: PEG = PEO; nomenclature MW < 20K is called PEG null dextran o note that resisting protein adsorption may not always be necessary for eliminating nonspecific cell adhesion ? mechanically soft surfaces – work of Rubner lab 10 (SLIDE) Polyelectrolyte multilayers (Rubner lab): dehydrated Highly hydrated null poly(acrylic acid)/poly(allyl amine) multilayers with varying charge density- thus loopiness and swelling Lecture 3 – Biological Recognition 9 of 15 ? Achieving controlled cell adhesion in practice: 1 method o Incorporation of poly(ethylene glycol) at surface with adhesion peptides Creating a ‘blank slate’: 11-14 (SLIDE) PMMA O O OH O O O O O x y z Fr a c t ion of c e lls a dhe r e d re la tiv e to TCPS O O O O OH n ~ 6 O O O OH + O O O n ~ 6 (1) (1) O m n M = 20K ?100K g/mol O OH 0.2 9-unit side chains comb (1) + O N O O n ~ 6 O O O O N O0.15 (2) 0.1 O O O O O O O O n ~ 6 (3) N GRGDSP OH 0.05 (2) + H2N GRGDSP OH H 0 20 30 45 weight fraction PEO units now add adhesion peptides: (Here, Arg-Gly-Asp-based peptide) TCPS 0.5 0.4 Unmodified PLA 0.3 0.2 0.1 0 GRGDSP GRGESP Fraction seeded cells adhered Fr acti on S eeded Cel l s Adhered + soluble RGD Tethered RGD null concept of soluble peptide control: integrin-mediated attachment is low-affinity but high avidity: many receptors binding on and off relatively quickly Lecture 3 – Biological Recognition 10 of 15 Biophysical Effects of Cell Adhesion Peptides null controlling the physical distribution of cells (work of Shakesheff) 15 Lecture 3 – Biological Recognition 11 of 15 null Cell adhesion ligand density effects on cell migration 16 Cell migration on fibronectin coated substrates: Cells too strongly adherent to release and extend filipodia Cells too weakly adherent; no traction for mechanical forces Lauffenburger lab null Clustering effects DRAW ON BOARD: Lecture 3 – Biological Recognition 12 of 15 o PEO surface modified with PEO star polymers: CONTROL OF ARCHITECTURE FOR CONTROL OF LIGAND PRESENTATION (SLIDE) null Utility of a branched polymer architecture ? self-assembled monolayers – K.L Prime and G.M. Whitesides , J. Am. Chem . Soc ., 115 , 10714 (1993) ? end-grafted polymers – C.G. Golander et al., in Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications ? adsorbed polymers – D. Gingell and N. Owens, J. Biomed . Mat. Res ., 28 , 491 (1994) ? plasma discharge coating – G.P. Lopez et al., J. Biomed . Mat. Res., 26(4), 415 (1992) ? hydrogels – J.P. Bearinger , D.G. Castner , S.L. Golledge , A. Rezanio , S. Hubchak , and K.E. Healy, Langmuir , 13, 5175 (1997) Lecture 3 – Biological Recognition 13 of 15 Example peptides used to modulate cell adhesion on biomaterials Peptide sequence Derived from Conjugate receptor Role References IKVAV Laminin α-chain LBP110 (110 KDa laminin binding protein) Cell-ECM adhesion J. Biol. Chem. 264, 16174 (1989) RGD Laminin α-chain, fibronectin, collagen Multiple integrins Cell-ECM adhesion J. Cell Physiol. 146, 451 (1991) YIGSR Laminin β1-chain α 1 β 1 and α 3 β 1 integrins Cell-ECM adhesion Cell 48,989 (1987); Arch. Biochem. Biophys. 272, 39 (1989); J. Biol. Chem. 268, 8053 (1993) RNIAEIIKDI Laminin γ-chain unknown CM adhesion FEBS Lett. 244, 141 (1989) HAV N-cadherin herin adhesion Dev. Biol. 139, 227(1990) DGEA Type I collagen α 2 β 1 integrin Cell-ECM adhesion VAPG tase Elastase receptor Cell-ECM adhesion KQAGDV Fibrinogen γ-chain β 3 integrins Cell-ECM adhesion Cell-E N-cad Cell-cell Elas null Applying the ‘comb polymer’ approach on biodegradable materials: QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. Comb polymer thin film coating Hydrolytically-degradable polymer QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. QuickTime? and a Graphics decompressor are needed to see this picture. TISSUE ENGINEERING scaffold o This general approach has also been applied to fully biodegradable polymers null use peptide or side chain-bearing ring monomers to create biodegradable backbones with PEG side chains that can be functionalized (not trivial) Lecture 3 – Biological Recognition 14 of 15 References 1. Di Lullo, G. A., Sweeney, S. M., Korkko, J., Ala-Kokko, L. & San Antonio, J. D. Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem 277, 4223-31 (2002). 2. Lemire, J. M., Merrilees, M. J., Braun, K. R. & Wight, T. N. Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro. J Cell Physiol 190, 38-45 (2002). 3. Hubbell, J. A., Massia, S. P. & Drumheller, P. D. Surface-grafted cell-binding peptides in tissue engineering of the vascular graft. Ann N Y Acad Sci 665, 253-8 (1992). 4. Drumheller, P. D. & Hubbell, J. A. Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates. Anal Biochem 222, 380-8 (1994). 5. Kuhl, P. R. & Griffith-Cima, L. G. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat Med 2, 1022-7 (1996). 6. Cook, A. D. et al. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, resorbable biomaterial. J Biomed Mater Res 35, 513-23 (1997). 7. Mann, B. K., Schmedlen, R. H. & West, J. L. Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells. Biomaterials 22, 439-44 (2001). 8. de Gennes, P. G. Conformations of polymers attached to an interface. Macromolecules 13, 1069-1075 (1980). 9. Milner, S. T. Polymer brushes. Science 251, 905-914 (1991). 10. Mendelsohn, J. D., Yang, S. Y., Hiller, J., Hochbaum, A. I. & Rubner, M. F. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 4, 96-106 (2003). 11. Banerjee, P., Irvine, D. J., Mayes, A. M. & Griffith, L. G. Polymer latexes for cell-resistant and cell-interactive surfaces. J Biomed Mater Res 50, 331-9. (2000). 12. Irvine, D. J., Mayes, A. M. & Griffith, L. G. Nanoscale Clustering of RGD Peptides at Surfaces Using Comb Polymers. 1. Synthesis and Characterization of Comb Thin Films. Biomacromol. 2, 85-94 (2001). 13. Irvine, D. J. et al. Comparison of tethered star and linear poly(ethylene oxide) for control of biomaterials surface properties. J Biomed Mater Res 40, 498-509. (1998). 14. Irvine, D. J., Ruzette, A. V., Mayes, A. M. & Griffith, L. G. Nanoscale clustering of RGD peptides at surfaces using comb polymers. 2. Surface segregation of comb polymers in polylactide. Biomacromolecules 2, 545-56 (2001). 15. Patel, N. et al. Spatially controlled cell engineering on biodegradable polymer surfaces. Faseb Journal 12, 1447- 1454 (1998). 16. Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A. & Horwitz, A. F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385, 537-40 (1997). Lecture 3 – Biological Recognition 15 of 15