Simplest synthetic pathways*---outlineA. Symbolism of Organ SynthesisInformation stored in a chemical equationTransition to biology I. Reactants Transition to biology II. ReactorsSkin: In vitro or in vivo synthesis? Nerves: In vitro or in vivo?Standardized in vivo reactors for study fo skin synthesis and peripheral nerve synthesis Transition to biology. III. ProductsHistomorphometry-cross sections of peripheral nerves regenerated using scaffolds with variable degradation rateProblems and advantages of chemical symbolismB. The central question in organ synthesis Which tissues in the triad do not regenerate spontaneously?SKIN: The epidermis regenerates spontaneouslySKIN: Scar formation. The dermis does not regenerate.NERVE: The injured myelin sheath regenerates spontaneouslyNeuroma formation. The endoneurium does not regenerate.The central question is…C. What is required to synthesize an organ?Required vs. redundant reactantsMethod used to identify required reactantsResults. Use color code for reactantsConventions used in reaction diagramsSynthesis of an Epidermis (E)Synthesis of a basement membrane (E·BM)Synthesis of a dermis (D)Synthesis of skin (partial skin = PS = EBMD )Select simplest routes for skin synthesisSequential vs. simultaneous synthesis of skin tissuesSimplest routes for nerve synthesisD. Trans-organ reaction diagrams Select only the simplestSummary of trans-organ rules for organ synthesisVarious synthetic routesSummary of synthetic rules for tissues and organsSimplest synthetic pathways*---outlineA. Symbolism of organ synthesis.B. The central question of organ synthesis.C. What is requiredto synthesize an organ?D. Trans-organ rules of synthesis.*Tissue and Organ Regeneration in Adults, Yannas IV, New York, Springer, 2001A. Symbolism of Organ SynthesisInformation stored in a chemical equationAmmonia synthesis (F. Haber)T, P3H2+ N2 → 2NH3reactorreactants → productsNOTE: The stoichiometry (masses on both sides) of a chemical equation expresses conservation of mass (Lavoisier)Transition to biologyI. Reactants• Cells migrate, proliferate, synthesize matrices and cytokines, degrade matrices, etc.• Cytokinesare soluble molecules that diffuse. They serve as “language”between cells.• Matricesare insoluble macromolecular networks and do not diffuse. They control cell behavior (phenotype) via integrin-ligand binding. Usually porous (“scaffolds”).100 μm 100 μm A biologically active ECM analogScaffold by scanning electron microscopyNerve regeneration template100 μm Scaffold by scanning electron microscopy100 μmScaffold by optical microscopySource: Freyman, T. M., I. V. Yannas, R. Yokoo, and L. J. Gibson. "Fibroblast contraction of a collagen-GAG matrix."Biomaterials 22 (2001): 2883-2891. Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.Scaffold by optical microscopyAnother sequence showing a cell (A) elongating and deforming matrix struts (B)Scaffold by optical microscopyCourtesy Elsevier, Inc.,http://www.sciencedirect.com.Used with permission.Transition to biologyII. Reactors• In vitro reactors are dishes or flasks for cell culture. • In vivo reactors are anatomical sites of organ loss in the living organism. • Experimental in vivo reactors are generated by surgical excision (scalpel, laser, etc.). • When organ synthesis takes place in vivo at the correct anatomical site of living organism it is referred to as “induced regeneration”.Skin: In vitro or in vivo synthesis?Figure by MIT OpenCourseWare.Nerves: In vitro or in vivo?Figure by MIT OpenCourseWare.Standardized in vivo reactorsfor study fo skin synthesis and peripheral nerve synthesis• T• T• Transected nerveransected nerveransected nervenerve stumps nerve stumpsnerve stumpsdermis epidermis dermisepidermisdermisepidermisSKIN PERIPHERAL NERVE Figures by MIT OpenCourseWare.Rat sciatic nerve modelproximal stumpscaffold/chamberdistal stumpLandstrom, Aria. “Nerve Regeneration Induced by Collagen-GAG Matrix in Collagen Tubes.” MS Thesis, MIT, 1994.Transition to biology. III. Products• Organs are made up of tissues.• Products of the synthesis can be tissues or organs.• Almost all organs are essentially made up of three types of tissues: epithelial, basement membrane and stroma (connective tissue).• Describe degree of completion of product of synthesis using the triad.Scaffold slowlydegradingScaffold seeded with epithelial cellsKINETICS OF SKINSYNTHESISI.Epithelial tissue being synthesizedtogether with stromaButler et al., 1998Three histology images removed due to copyright restrictions.See Butler, CE, et al. “Effect of Keratinocyte Seeding of Collagen-Glycosaminoglycan Membranes on the Regeneration of Skin in a Porcine Model.” Plast. Reconstr. Surg. 101, no. 6 (May 1998): 1572-1579.Scaffold degraded; diffuses awayKINETICS OF SKINSYNTHESISII.Epithelial tissue separating out from stromaButler et al., 1998Three histology images removed due to copyright restrictions.See Butler, CE, et al. “Effect of Keratinocyte Seeding of Collagen-Glycosaminoglycan Membranes on the Regeneration of Skin in a Porcine Model.” Plast. Reconstr. Surg. 101, no. 6 (May 1998): 1572-1579.Normal skin has capillary loops and a wavelike border separating epidermis from dermis. Burkitt et al., 1992Partially regenerated skin in the swine. Compton et al., 1998Partially regenerated skin is not scar.Scar does not have capillary loops. Nor does scar have a wavelike border separating epidermis from dermis↑capillary loopscapillary loops75 μmPartially regenerated skin is not scar.Diagram removed due to copyright restrictions. See Figure 5.2a in [TORA].[TORA] = Yannas, I. V. Tissue and Organ Regeneration in Adults. New York, NY: Springer-Verlag, 2001. ISBN: 9780387952147. Histology photo removed due to copyright restrictions. See Compton, C.C., et al. J. Invest. Dermatol. 110 (1998): 908-916.normal skin(guinea pig)scarregenerated skinv, blood vessels(absent in scar)d, dermisStudy blood vessels at interface of epidermis-dermis.Scar has no blood vessels at interface. Regenerated skin is not scar.Three histology photos removed due to copyright restrictions. See Figure 5.4 in [TORA].Normal Regenerated-polarized lightScar-polarized light Regenerated-natural lightComparison of stroma (dermis) in regenerated skin, normal skin and scar(guinea pig)Orgill, D. P. MIT PhD Thesis, 1983.Identify scar using laser light scattering assay1(a)cos2S2−=ScarNormal DermisDermis