Cell-scaffold interactions during regeneration Two major questionsThree scales of understandingThe tissue triad in skin and nervesNormal skin wound healingWhy does our adult body fail to regenerate when injured? What does the scaffold do? Examples from five organs. Blocking of adult healing response following grafting with active scaffold Organ #1. Skin RegenerationHypothetical Stress Fields in Skin WoundOrgan #2. Peripheral Nerve RegenerationThe pressure cuff theoryNerve RegenerationOrgan #3. Conjunctiva RegenerationAnatomy of the conjunctivaSummarize evidence from healing in five adult organs Back to the old question: Does scar block regeneration?Does the scaffold act as a mechanical splint?Model of wound contraction by myofibroblasts (MFB)In the absence of the scaffold, the macroscopic force that contracts wound cannot be easily explained by hypothesizing indepenMechanism of scaffold activity # 1. Scaffold reduces the number of contractile cells Mechanism of scaffold activity # 2. Scaffold disorients MFB thereby reducing sum of forces generated by MFBHypothesis of loss of cell-cell cooperativityTwo examples of clinical application of the dermis regeneration template, DRT Bilayer medical device approved by FDAPhotos by Dr. Andrew Byrd, M.D., Bristol, UKCASE 2. Care of Chronic Skin WoundsPhenotype change: fibroblast α2β1 integrin binds to collagen ligand GFOGER (hexapeptide)Conclusions from transcriptional analysis and proteomics analysis in poorly vs successfully regenerating modelsHow does wound healing induced in the early fetal stage (regeneration) differ from wound healing in late fetal stage (no regenCell-scaffold interactions during regeneration1. Two questions.2. Evidence supporting antagonistic relation between contraction and regeneration.3. Similarity between early fetal regeneration and induced regeneration in adults.conception birthRegeneration Adult RepairMammals: Early fetal healing → Late fetal healingA remarkable transition from early fetal to late fetal healingGestation time →Longaker et al.; Ferguson et al.Two major questions1. What is the mechanism of induced organ regeneration in adultsat the cell-biological and the molecular-biological levels? 2. Is the process of induced regeneration of organs in adultssimilar to spontaneous regeneration at the early fetal stage?Three scales of understanding• Macroscopic---what we see with our eyes alone. Scale > 1 mm• Cell biological----what microscopy shows. Scale > 1 μm• Molecular biological---what biochemical assays or gene expression assays tell us is happening. Scale > 1 nmSpontaneous Regeneration in an AmphibianUnlike adult mammals, certain adult amphibians can regenerate arms and legs that have been amputated Figure removed due to copyright restrictions. See Figure 1.1 in [TORA].[TORA] = Yannas, I. V. Tissue and Organ Regeneration in Adults. New York, NY: Springer-Verlag, 2001. ISBN: 9780387952147. [Preview in Google Books]Tomasek et al., 2002Adult repair. Healing by contraction and scar formation in burn victim.Adult Repair of Injured ArmPhoto removed due to copyright restrictions. See Figure 6 in Tomasek, J. J. et al. “Myofibroblasts and mechano-regulation of connective tissue remodelling.” Nature Reviews Molecular Cell Biology 3 (May 2002): 349-363.http://dx.doi.org/10.1038/nrm809Spontaneous contraction and scar formation in burn victim.Mass Gen Hospital, 1986Adult Repair of Burned SkinPhoto removed due to copyright restrictions.The tissue triad in skin and nervesFigure by MIT OpenCourseWare.Figure by MIT OpenCourseWare.Skin: Adult RepairThe dermis does not regenerate spontaneously. Wound closes with contraction and scar formation. Yannas, 2001E: epidermisD: dermisS: scarEDEDhealingscarSFigure by MIT OpenCourseWare.Peripheral nerve: Adult Repair of InjuryTransected (cut) nerve heals by contraction and scar formation (paralysis)Figure by MIT OpenCourseWare.Ramirez et al., 1969: Rudolph, 1979; Yannas, 1981Speed of contraction of skin wounds Skin: Adult Repair of Injury (followup of original studies by Medawar and Billingham in early 1950s)10 20 30 40 50 60120100806040200Time, d% Initial defect areaHumanSwineGuinea pigFigure by MIT OpenCourseWare.Skin: Adult Repair with Wound Contraction and Scar Formation (guinea pig)ScarTroxel, MIT Thesis, 1994Dermis has contractedMicroscopy of a healed skin woundDermis has contractedNormal skin wound healingA short film by D. TzeranisWhy does our adult body fail to regenerate when injured? Prevalent explanation:“Regeneration is blocked by scar formation”Yannas et al., 1981An unexpected result in 1979( “artificial skin”)Contraction dramatically delayed when full-thickness skin wounds grafted with a particular scaffold. Scar formation blocked. First observation of induced organ “regeneration” (partial) in adult mammal. Grafted% Wound area remainingUngrafted0 10 20 30 40 50 60 120Days020406080100Figure by MIT OpenCourseWare. After Yannas, 1981.The scaffold was an analog of the Extracellular Matrix (ECM) based on collagen and a glycosaminoglycan (GAG).Harley, 2006Image by MIT OpenCourseWare. After Ricci.Collagen/GAG scaffold was synthesized as graft copolymer from purified macromolecules in highly porous state. DRT, dermis regeneration template.100 μmDRTYannas et al., 1989Only scaffolds within narrow pore size range are active. DRT, dermis regeneration template, blocks contraction maximally.Wound contraction half-life, dBiological Activity Courtesy of National Academy of Sciences,U.S.A. Used with permission. © 1989 NationalAcademy of Sciences, U.S.A. Source: Yannas,I., et al. "Synthesis and Characterization ofa Model Extracellular Matrix that Induces PartialRegeneration of Adult Mammalian Skin." PNAS 86 (1989): 933-937.4. macromolecular structure (duration of ligands on surface)Structural determinants of scaffold biological activity.1. chemical composition (ligand identity) 2. pore structure---pore volume fraction, pore size (ligand density)3. orientation of pore channels (ligand spatial coordinates)Yannas et al., 1989What makes a scaffold active biologically ? Active scaffold reduces number of contractile cells in the wound---and binds these cells on “ligands” on the scaffold surfaceDiagram removed due to copyright restrictions.What does the scaffold do?It blocks contraction and abolishes scar formation during wound healing. If used without cell seeding, it leads to regeneration of dermis.Eventually,