C. Synthesis of biologically active scaffolds (regeneration templates) 1. History of biologically active scaffolds (regeneration templates). 2. Physical chemistry of collagen: Melting of collagen quaternary structure (thromboresistance). Melting of collagen tertiary structure (gelatinization). 3. Synthesis of ECM analogs: Ionic complexation collagen/GAG, formation of pore structure, crosslinking. 4. Biological activity of ECM analogs depends critically on structure. 1. History of biologically active scaffolds (regeneration templates). ? 1974-75 Synthesis and characterization of the first biologically active scaffolds. Scaffolds defined as very highly porous polymeric constructs that are commonly used, either unseeded or seeded with cells, to synthesize tissues and organs in vitro or in vivo (Yannas et al., 1975a,b,c; 1979; 1980a,b,c). ? 1979-80 First clinical use of a biologically active scaffold to regenerate the dermis (treatment of massively burned children) (Burke et al., 1981). 1. History of biologically active scaffolds (regeneration templates). (continued) ? 1981-82 Implantation (grafting) of a cell-seeded scaffold. Keratinocyte-seeded template regenerates simultaneously dermis and epidermis in animals (Yannas et al., 1982). ? 1985 Regeneration of peripheral nerves across unprecedented distances in animals using a biologically active scaffold (Yannas et al., 1985). ? 1989 Identification of structural features that account for template regenerative activity (Yannas et al., 1989). ? 1996 FDA approves first scaffold (Integra) for treatment of burned patients and, later, for plastic and reconstructive surgery of skin (2001). Analogs of extracellular matrix 2. Physical chemistry of collagen: --- Melting of collagen tertiary structure: acceleration of biodegradation rate. --- Melting of collagen quaternary structure: thromboresistance. COLLAGEN STRUCTURE Images removed due to copyright considerations I r d due t c right c iderations Primary structure (amino acid sequence) of Type I collagen Image removed due to copyright considerations. Image removed due to copyright consi erations. Mechanical (viscoelastic) behavior of colagen and gelatin. Proteins were Progressively diluted with glycerol to elicit the entire spectrum of their viscoelastic behavior. Gelatin shows a rubberlike state. Collagen does not. Image removed due to copyright considerations. Image removed due to copyright consi erations. Degradation of collagen fibers by collagenase Image removed due to copyright considerations. Image removed due to copyright consi erations. Degradation of collagen molecule by collagenase to gelatin. Gelatin itself degrades much faster than collagen. Image removed due to copyright considerations. Image removed due to copyright consi erations. Melting of quaternary structure of collagen fibers occurs below pH 4.5. Melting confers thromboresistance to the scaffold. Platelets Do not aggregate unless the quaternary structure Is intact. Blocking of platelet aggregation leads to downregulation of the inflammatory response at the site of grafting or implantation. Image removed due to copyright considerations. Image removed due to copyright consi erations. 3. Synthesis of active ECM analogs: --- Ionic complexation of collagen/GAG. --- Formation of pore structure. --- Crosslinking. Collagen-GAG membrane formation process Collagen Dispersion (or solution) Coprecipitation and homogenization Freeze-drying Crosslinking GAG solution Γλυκοσαμινογλυκανε? Glycosamino- glycans disaccharide repeat unit Image removed due to copyright considerations. Diagram of Chondroitin 4-Sulfate Image removed due to copyright considerations. Diagram of Dermatan Sulfate. Image removed due to copyright considerations. Diagram of Heparan Sulfate. Biologically active collagen/GAG scaffold (dermis regeneration template) Image removed due to copyright considerations. Image removed due to copyright consi erations. Procedures used to study the pore structure of scaffolds. Unlike collagen sponges (used as hemostatic agents), regeneration templates have very high pore volume fraction. typically >95%. Image removed due to copyright considerations. Image removed due to copyright consi erations. Crosslinking binds GAG covalently to collagen to produce a graft copolymer. Solvents with high ionic strength fail to separate the two polymers from each other. Image removed due to copyright considerations. Image removed due to copyright consi erati ns. 4. Biological activity of ECM analogs depends critically on structure. Images removed due to copyright considerations I r d due t c right c iderations Which ECM analogs are biologically active as regeneration templates? Critical Structural Feature Role in regeneration A. SKIN Chem. Composition >2% GAG Deleted collagen quaternary structure Ligand identity Downregulation of inflammatory response Pore diameter 20—120 μm Ligand density Degradation half-life 10-15 d Duration of ligands B. NERVE Chem. Composition Deleted collagen quaternary structure [not studied] [not studied] Pore diameter ～ 5 μm Ligand density Degradation half-life ～ 1-10 wk Duration of ligands Conclusions 1. Certain ECM analogs are biologically active scaffolds (regeneration templates) that induce regeneration of tissues and organs: skin, peripheral nerve and the conjunctiva (eye) in humans and experimental animals. 2. Regeneration templates lose their activity if the following structural features fall outside a narrow range: chemical composition, collagen quaternary structure, pore diameter, degradation rate. 3. The data suggest that templates induce regeneration in a defect by blocking selectively the contraction process that leads to closure of the defect in adults. 4. Templates block contraction by two basic mechanisms. First, by downregulating differentiation of fibroblasts to myofibroblasts. Second, by binding most of the contractile cells in the defect over a period corresponding to the duration of contraction in that defect. Binding requires the presence of appropriate ligands (chem. composition) at a minimal density (pore diameter) over a critical duration (degradation rate).