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 Readin 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 Cel, 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 DRAW OXIDE SURFACE. POL YMER 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 Differentiation Secretion of molecules Binding of molecules 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 1of15
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 Prototy pical soft tissue ECM(varies from tissue to tissue) o collagen o other fibers SLIDE) reticular network collagen gel TEM of collagen fibril generalized matrix assembly o fibrils assemble into fibers fibers may be organized or isotropic, and form tight(-10 nm separation between fibers)or open (20-30 um between fibers)meshes o adhesion proteins decorate'fibers o other signals(cytokines, etc )may also be sequestered on fiber surfaces Sequestered Cytokines/ Chemokines/ Collagen fiber hydrogel Adhesion motifs P Friedl et al. Eur J Immuno. 28, 2331(1998) collagen triple helices Lecture 3- Biological Recognition 2of15
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 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 k-structure proteins · Key length scales ter of collagen fibrils 50-200nm o diameter of collagen fibers 05-5pm o diameter of collagen triple helices: o diameter ofof collagen chain o length of collagen triple helix 300nm Adhesion proteins Collagen I fibril ligand binding site map H!! lal Binds to surface recep:Drs on 400000 Binds to Heparan sulfate prote Laminin 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 Lecture 3- Biological Recognition 3of15
• 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) Collagen I fibril ligand binding site map laminin structure 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 ￥ Extracellular matrix DRAW ON BOARD 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 Composed of noncovalently-associated a and B chains Actin filaments(cytoskeleton integrin (Extracellular space) Adhesion protein ECM fiber o inside-out signaling: biochemical signal triggers affinity change in integrins o focal contacts and signaling 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 4of15
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 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 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
ON BOARD o Length scales in cell adhesion o Size of integrins, focal contacts, relationship to cell size, fiber spacing DRAW ON BOARD focal contacts"(SLIDEl 12-15nm 12-15nm Actin stress fibers LXSN LV3SN Interference reflection microscopy lark spots indicate cell-substrate eparations 50 nm 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 5of15
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 contacts2 (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 tides in 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 to devices) Peptide sequences conjugated to synthetic polymers have been used to provide signals for: (SLIDE) o Adhesion Fragments from ECM adhesion protein Short sequences recognized by remodeling enzymes Support transformation of synthetic scaffolds into de novo natural matrix Support cell migration through solid scaffolds Lecture 3- Biological Recognition 7of15
Engineering Biological Recognition of Synthetic Degradable Polymers: Incorporation of peptides in synthetic polymers • Lit on peptides in polymers3-7 • 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 Fragments from ECM adhesion proteins o Remodeling Short sequences recognized by remodeling enzymes Support transformation of synthetic scaffolds into de novo natural matrix Support cell migration through solid scaffolds Lecture 3 – Biological Recognition 7 of 15
o Growth/differentiation Peptide cytokines o Other potential functions 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 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 1. cell adhesion 2. matrix remodeling 3. cytokine 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 onment changes Vroman effect-> protein exchang 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 Lecture 3- Biological Recognition 8of15
Lecture 3 – Biological Recognition 8 of 15 o Growth/differentiation Peptide cytokines o Other potential functions 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 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, 9 ONLY LANGMUIR ADSORPTION COVERED IN 3.051J 1 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 res/stin present peptides on this blank slate background o current hypotheses: achieve a surface structure with some or all of these characteristics water-rich surface layer-reduce protein-surface interactions 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) 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 dextran o note that resisting protein adsorption may not always be necessary for eliminating nonspecific cell adhesion mechanically soft surfaces-work of Rubner lab (SLIDE) Polyelectrolyte multilayers(Rubner lab) COOH COOH COOH COOH COOHCOOH dehydrated Highly hydrated b poly(acrylic acid)/poly(allyl amine)multilayers with varying charge density-thus loopiness and swelling Lecture 3- Biological Recognition 9of15
Protein-resistant surfaces8,9 • ONLY LANGMUIR ADSORPTION COVERED IN 3.051J • 1st 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: water-rich surface layer – reduce protein-surface interactions 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) 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 endfunctional groups • reminder: PEG = PEO; nomenclature MW < 20K is called PEG dextran o note that resisting protein adsorption may not always be necessary for eliminating nonspecific cell adhesion • mechanically soft surfaces – work of Rubner lab10 (SLIDE) Polyelectrolyte multilayers (Rubner lab): dehydrated Highly hydrated 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 slat (SLIDE PMMA M=20K G100K a/mol comb 里 2) now add adhesion peptides:(Here, Arg-Gly-Asp-based peptide) TCPS Unmodified Pla Tethered rgd 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
• 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 Fraction of cells adhered relative to TCPS O O O O OH n ~ 6 O O O OH + O O O n ~ 6 (1)(1) m O n M = 20K Ğ100K g/mol O OH 0.2 9-unit side chains comb (1) + O N O O n ~ 6 O O O O N 0.15 O (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 Fraction Seeded Cells Adhered + soluble RGD Tethered RGD 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 controlling the physical distribution of cells(work of Shakesheff5 FONs t.ld t写m Incubation in ag solution Aaps m2a er lague 2. a esre I she mat e representa on of the surLace engineering m和肿 higands All steps in the surface hme, B scheEle engineering are peformed m aqueous environmen reprentation of the microfluidic puttering technique. ission images Panel C B a pha 200 Lecture 3- Biological Recognition 11 of 15
Biophysical Effects of Cell Adhesion Peptides controlling the physical distribution of cells (work of Shakesheff)15 Lecture 3 – Biological Recognition 11 of 15