MIT 20 441J - Skin synthesis in vivo (induced regeneration)

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Skin synthesis in vivo (induced regeneration)*1. Experimental parameters (skin)1. Experimental parameters (skin) [Cont.]2. Synthesis of an epidermis2. Synthesis of an epidermis [Cont.]2B. Synthesis of BM2B. Synthesis of BM [Cont.]2B. Synthesis of BM [Cont.]2B. Synthesis of BM [Cont.]3. Synthesis of dermis3. Synthesis of dermis (Cont.)3. Synthesis of dermis (Cont.)4. Partial synthesis of skin4. Partial synthesis of skin [Cont.]Verify basement membrane. I: Immunostaining: Factor VIII for capillary loopsVerify basement membrane. II. Immunostaining: 64 Integrin for hemidesmosomesVerify basement membrane. III. Immunostaining: Collagen VII for anchoring fibrils4. Partial synthesis of skin [Cont.]In vitro or in vivo? Skin synthesis5. Comparative regenerative ability of reactants6. Overall mechanism of skin regenerationContraction-blocking vs regenerationIsolation of contraction from tissue synthesis during “island” graftingSimilarity of configuration + synchronization of template degradation rate.Isomorphous and synchronized tissue replacement ruleOVERALL MECHANISM OF SKIN SYNTHESIS Fact: Contraction blocking is required but does not suffice to induce regeneration. ThisSkin synthesis in vivo (induced regeneration)*1. Experimental parameters2. Synthesis of epidermis and BM3. Synthesis of dermis4. Partial synthesis of skin5. Comparative regenerative activity of reactants6. Overall mechanism of skin regeneration________________________*Tissue and Organ Regeneration in Adults, Yannas, Springer, 2001, Ch. 5.1. Experimental parameters (skin)A. Anatomically well-defined defect–Designate experimental volume–Delete nonregenerative tissue(s)–Anatomical bounds–Containment of exudateB. Timescale of observations–Initial state: defect generated–Final state: defect closedNote: Remodeling continues after closureFigure removed due to copyright restrictions. See Figure 3.1 in [TORA].Diagram with four types of anatomically well-defects for study of induced skin regeneration.[TORA] = Yannas, I. V. Tissue and Organ Regeneration in Adults. New York, NY: Springer-Verlag, 2001. ISBN: 9780387952147. [Preview in Google Books]1. Experimental parameters (skin) [Cont.]C. Assays of final state (product of synthesis)-Literature describes several assays, unrelated to nature of product synthesized (e.g., time of closure by epithelialization, % take of graft, ability to cross histocompatibility barriers).-Required assays are both qualitative (which tissue was synthesized?) and quantitative (How much?)2. Synthesis of an epidermisStructure. Five cell layers (strata); 100 μm thick. Basal layer is closest to BM…stratum corneum is farthest out. Cell maturation gradient (increasing keratin content away from BM). Tissue turns over every 25-50 days.Function. Protection against dehydration and microorganisms (primarily stratum corneum). Also protection against mechanical, thermal, chemical, UV insults.Schematic view ofepidermisFigure by MIT OpenCourseWare.2. Synthesis of an epidermis [Cont.]Synthesis in vitro. Epidermis ⇒ trypsinization ⇒ dissociated keratinocytes (KC). Condensation of KC to epidermis requires nondiffusible substrate (e.g., plastic surface) but not growth factors or dermal substrate.In vivo. Epidermis synthesized spontaneously by KC, originally at the defect edge. KC dissociate spontaneously, migrate over residual dermis toward “center” of defect, synthesize BM and reform epidermis.2B. Synthesis of BMStructure. BM structure similar in all organs. 100 nm thick. Egg-carton topology in skin. Layer closest to epidermis is 20-40 nm thick (lamina lucida; mostly laminin). Intermediate layer 40-50 nm thick (lamina densa; type IV collagen). Next to stroma is fibroreticular layer (anchoring fibrils based on type VII collagen) that connects with type I collagen fibers in dermis via anchoring plaques. Hemidesmosomes connect basal cells to BM (tonofilaments).injury mode(blister)through dermis:irreversible healingthrough epidermis: reversible healingbetween epidermis and dermis:reversible healingFigure removed due to copyright restrictions. See Figure 2.6 in [TORA].Diagram showing regeneration of the basement membrane after blistering.Skin basement membraneLL, lamina lucidaLD, lamina densad, dermisFigure removed due to copyright restrictions. See Figure 5.1 (top) in [TORA].Electron micrograph showing the two major layers of the basement membrane.Skin BMFigure removed due to copyright restrictions. See Figure 5.1 (bottom) in [TORA].Diagram showing the structure of the intact basement membrane in skin.2B. Synthesis of BM [Cont.]Function. Boundary restricting transfer of cells and molecules; anchorage matrix for epithelial cells; mechanically competent “adhesive” layer binding epithelia to stroma; possibly “scaffold” facilitating repair after injury.2B. Synthesis of BM [Cont.]Synthesis in vitro. KC cultures in serum-free medium are transferred to solid surface. BM minus anchoring fibrils is synthesized.In vivo. KC sheets are grafted on dermis-freedefect; however, synthesized BM minus anchoring fibrils does not adhere to underlying tissue. Complete BM formed when cultured KC sheets are grafted on dermis.2B. Synthesis of BM [Cont.]Mechanical failure of dermal-epidermal junction.-- 1952-56 Billingham et al. Epidermal sheets or KC suspensions grafted on dermis-free surface failed to adhere (“avulsion”).-- 1977 Rheinwald and Green (RG) achieved KC culture expansion to KC sheets by 10,000X in 3 weeks.-- 1980-95 Clinical studies of KC sheets prepared by RG method were terminated after completing 105 of them. Problem: avulsion of KC sheets from muscle substrate.-- 1988-95 Woodley, Grinnell, Carver, Cooper et al. identified source of failure: lack of integration of BM to muscle substrate.3. Synthesis of dermisStructure. Consists of two layers: Papillarydermis just below epidermis, comprising loosely packed, thin, type I collagen fibers, as well as dermal papillae with vascular loops and nerve endings. Reticulardermis comprises closely packed, thicker, type I collagen fibers; also elastin fibers. Mechanically robust tissue comprises two interpenetrating networks of stiff, crystalline collagen fibers and extensible, amorphous, elastin fibers.3. Synthesis of dermis (Cont.)Function. Supports epidermis. -- tough base absorbs mechanical forces.-- rich vascular network supports metabolically the avascular dermis. -- thermoregulatory control for organism (sweat glands). -- tactile, pain, hot/cold


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