2.79J/2.79J/3.96J/BE.441J/HST.522J A theory of induced regeneration in adults. Note: [C, S, R] data only cited; no kinetics Outline 1. Irreversible injury 2. Regenerative and nonregenerative tissues 3. Antagonistic relation between contraction and regeneration 4. Present theory: Selective inhibition of contraction necessary but not sufficient for regeneration 5. Mechanism 1. Irreversible injury Reversible injury Image removed due to copyright considerations. See Figure 1.1 in Yannas, I. V. Tissue and Organ Regeneration in Adults. New York: Springer-Verlag, 2001. Image removed due to copyright consi erations. See Figure 1.1 in Yannas, I. V. Tissue and Organ Regeneration i Adults. New York: Springer-Verlag, 2001. Spontaneous regeneration of amputated limb in the newt occurs independently of severity of injury Goss, 1992 Irreversible injury Image removed due to copyright considerations. Image removed due to copyright consi erations. Burn victim suffering from severe contraction and scar formation Tomasek et al., 2000 2. Regenerative and nonregenerative tissues in adult mammals The tissue triad in skin and nerves Skin The epidermis is a regenerative tissue. After excision, it regenerates spontaneously. Reversible injury. No contraction. The dermis is a nonregenerative tissue in the adult. After excision, it does not regenerate spontaneously. Irreversible injury. Contraction occurs with scar formation. Skin Peripheral nerve The myelin sheath is a regenerative tissue. Following nerve crushing with myelin disruption, the myelin regenerates spontaneously. Reversible injury. No contraction. crushed nerve heals spontaneously by regeneration Peripheral nerve The endoneurial stroma is a nonregenerative tissue.Following transection, it forms neural scar (neuroma). Irreversible injury. Contraction occurs. transected nerve heals spontaneously by contraction and neuroma (neural scar) formation SUMMARY SO FAR Regenerative tissues. Reversible injury. No contraction. Nonregenerative tissues. Irrever- sible injury. Contraction +scar. SKIN epidermis dermis BM NERVE myelin endoneurial stroma BM Conclusion: Inverse relation between contraction and regeneration ? During adult healing, no contraction is observed following injury to epithelia or basement membrane. ? Contraction is only observed following injury to stroma. ? Contraction only observed following irreversible injury. 3. Antagonistic relation between contraction and regeneration a. Data from spontaneously healing wounds b. Blocking of contraction using scaffolds c. Isolation of contraction during “island” grafting d. Scar formation vs inhibition of contraction e. Contraction during impaired healing Quantitative description of healing processes ? Initial wound area is A o ? Wound eventually closes up spontaneously. Final area is A f . ? Final wound area is distributed among fractions that closed by contraction (%C), scar formation (%S) or regeneration (%R). ? This is the configuration of the final state. ? Wound closure rule: C + S + R = 100 Spontaneously healing defect Configuration of final state general case [C, S, R] ideal fetal healing [0, 0, 100] dermis-free skin-- adult rodents [96, 4, 0] dermis-free skin-- adult human [37, 63, 0] peripheral nerve– adult rat [96, 4, 0] conjunctiva-- adult rabbit [45, 55, 0] a. Data from spontaneously healing wounds Tadpole → Frog Developmental changes in configuration of final state [C, S, R] following healing: (early stages) → (late stages) [41, 0, 59] → [62, 0, 38] → [66, 0, 34] → [90, 10, 0] tadpole → frog b. Blocking of contraction using scaffolds Organ/ species Treatment used Spontan- eous Treated skin-guinea pig scaffold A [91, 9, 0][89, 0, 11] skin-guinea pig scaffold DRT+ KC [92, 8, 0][28, 0, 72] nerve-rat silicone tube+scaf- fold DRT [95, 5, 0][53, 0, 47] nerve-rat collagen tube+scaf- fold NRT [95, 5, 0][0, 0, 100] conjunctiva -rabbit scaffold DRT [45, 55, 0][13, 0, 87] Skin Skin Burn patient has closed severe skin wounds in neck partly by contraction Image removed due to copyright considerations. Image removed due to copyright consi erati ns. Wound area closure using three protocols [89, 0, 11] [28, 0, 72] [91, 9, 0] Skin Image removed due to copyright considerations. See Figure 8.1 in [Yannas]. Image removed due to copyright consi erations. See Figure 8.1 in [Yannas]. Dermis regeneration template (DRT) Image removed due to copyright considerations. Image removed due to copyright consi erations. Structural features of biologically ECM analogs 3. pore structure (ligand density) 2. macromolecular structure (ligand duration) 1. chemical composition (ligand identity) 4. orientation of pore channels (ligand orientation) Diagrams removed due to copyright considerations. Contraction inhibited maximally in pore diameter range 20 μm — 120 μm. Scaffolds with pores in that range induced dermis regeneration. Scar formed outside that range. DRT Image removed due to copyright considerations. See Figure 8.5 - top in [Yannas]. ue to copyright consi erations. See Figure 8.5 - top i [Yannas]. rete ridges with capillary loops and vascular plexus underneath Normal skin Image removed due to copyright considerations. See Figure 5.2 (top left) in [Yannas]. Image removed due to copyright consi erations. See Figure 5.2 (top left) in [Yannas]. Burkitt et al., 1992 Verify induced regeneration of skin basement membrane. I: Immunostaining: Factor VIII for capillary loops Image removed due to copyright considerations. Image removed due to copyright consi erations. 75 μm Compton et al., 2000 Verify induced regeneration of skin basement membrane. II. Immunostaining: α 6 β 4 Integrin for hemidesmosomes Image removed due to copyright considerations. Image removed due to copyright consi erations. 100 μm Compton et al., 2000 Verify induced regeneration of skin basement membrane. III. Immunostaining: Collagen VII for anchoring fibrils Image removed due to copyright considerations. Image removed due to copyright consi erations. 150 μm Compton et al.,2000 Conjunctiva Anatomy of the conjunctiva Fornix Eyelid Cornea Sclera Epithelium Substantia Propria Conjunctival stroma low mag high mag Conjunctiva wound model Conjunctiva Tenon’s capsule Sclera Sclera Scaffold DRT Sutures Effect of DRT on contraction kinetics of conjunctival defect. It is experimentally convenient to study contraction of the fornix, a tissue attached to the conjunctiva. ungrafted grafted with scaffold DRT 30 % Fornix Shor tening 15 0 15 300 Days Hsu et al., 2000 Test of synthesis of conjunctival stroma (use microscope polarizing stage to study orientation of collagen fibers) Image removed due to copyright considerations. Image removed due to copyright consi erations. ungrafted conjunctival scar grafted with scaffold DRT normal conjunctiva Hsu et al., 2000 Peripheral nerve Rat sciatic nerve model Image removed due to copyright considerations. Image removed due to copyright consi erations. Experimental model used to study PNS regeneration ? Transect rat sciatic nerve ? Insert nerve stumps into tube ? “Nerve chamber” model is standard proximal stump tube, empty or filled distal stump gap Nerve regenera- ted across 8- mm gap Image removed due to copyright considerations. See Figure 10.7 (lower right) in [Yannas]. Image removed due to copyright consi erations. See Figure 10.7 (lower right) in [Yannas]. Jenq and Coggeshall, 1985 Contractile cell zone surrounds regenerating nerve contractile cells original stump surface regenerated nerve Image removed due to copyright considerations. Image removed due to copyright consi erations. Spilker and Seog, 2000 Nerve regeneration template (NRT) Image removed due to copyright considerations. Image removed due to copyright consi erations. silicone tube scaffold tube Images removed due to copyright considerations. I r d due t c right c iderations. 15-20 contractile cell layers poor quality of regeneration 0-1 contractile cell layer superior quality Chamberlain et al., 2000 c. Scar formation vs inhibition of contraction Data in table will show: Inhibition of contraction, even modest, leads to virtual abolition of scar Scar appears to be a by-product of contraction Organ/ species Treatment used Spontan- eous Treated skin-guinea pig scaffold A [91, 9, 0][89, 0, 11] skin-guinea pig scaffold DRT+ KC [92, 8, 0][28, 0, 72] nerve-rat silicone tube+scaf- fold DRT [95, 5, 0][53, 0, 47] nerve-rat collagen tube+scaf- fold NRT [95, 5, 0][0, 0, 100] conjunctiva -rabbit scaffold DRT [45, 55, 0][13, 0, 87] d. Impaired healing of skin wounds Dermis-free wounds in: ? genetically diabetic mouse ? genetically obese mouse ? infected wounds ? mechanically splinted ? treated with steroids all impaired-healing wounds showed strong delay in contraction but not regeneration 4. Theory: Selective inhibition of contraction Conclusions 1. During development, contraction increases in importance, while regeneration corespondingly decreases (rana catesbeiana). 2. Certain scaffolds block contraction “selectively” and induce partial regeneration in adult mammals (rodents, swine, human). 3. Scar is abolished when contraction is blocked. 4. Impaired healing blocks contraction but does not induce regeneration. Theory refers to [C, S, R] ? Inhibition of contraction is necessary but does not suffice to induce regeneration ?R > 0 and S → 0 if ?C < 0 Mechanism of contraction inhibition by DRT scaffold in skin wound ? Scaffold does not aggregate platelets (during its preparation, abolish collagen banding but not triple helix). Hypothesis: Downregulate release of TGF-β. ? Scaffold binds TGF- β1 avidly (but nonspecifically). Hypothesis: Downregulate soluble cytokine concentration. ? Scaffold binds myofibroblasts extensively. Hypothesis: myofibroblast contractile axes disoriented, lose vectorial character. ? Scaffold competitively inhibits natural ECM Myofibroblast Image removed due to copyright considerations. Image removed due to copyright consi erations. Tomasek et al., 2000 100 μm 100 μm A. No scaffold. Contracting wound B. Grafted with scaffold. No contraction Myofibroblasts stain brown-red. Scaffold unstained. surface of wound surface of wound Image removed due to copyright considerations. Image removed due to copyright consi erations. Image removed due to copyright considerations. Image removed due to copyright consi erations. Kidney Test applicability of theory to a new organ Rat kidney fibrotic tissue stains blue untreated scar formation and contraction of perimeter significantly smaller scar and less contraction of perimeter treated with scaffold DRT Image removed due to copyright considerations. Image removed due to copyright consi erations. Image removed due to copyright considerations. Image removed due to copyright consi erations.