Mammals fail to regenerate organs when wound contraction drives scar formation

Abstract

To understand why mammals generally do not regenerate injured organs, we considered the exceptional case of spontaneous skin regeneration in the early lamb fetus. Whereas during the early fetal stage skin wounds heal by regeneration, in the late fetal stage, and after birth, skin wounds close instead by scar formation. We review independent evidence that this switch in wound healing response coincides with the onset of wound contraction, which is also enabled during late fetal gestation. The crucial role of wound contraction in determining the wound healing outcome in adults has been demonstrated in three mammalian models of severe injury (excised guinea pig skin, transected rat sciatic nerve, excised rabbit conjunctival stroma) where grafting the injury with DRT, a contraction-blocking scaffold of highly-specific structure, altered significantly the wound healing outcome. While spontaneous healing resulted in scar formation in these animal models, DRT grafting significantly reduced the extent of wound contraction, prevented scar synthesis, and resulted in partial regeneration. These findings, as well as independent data from species that heal spontaneously via regeneration, point to a striking hypothesis: The process of regeneration lies dormant in mammals until appropriately activated by injury. In spontaneous wound healing of the late fetus and in adult mammals, wound contraction impedes such endogenous regeneration mechanisms. However, engineered treatments, such as DRT, that block wound contraction can cancel its effects and favor wound healing by regeneration instead of scar formation.

Fig. 1. Wound contraction and associated wound healing response in three adult injury models in the absence (spontaneous healing) and presence of a DRT graft.

a–e guinea pig full-thickness excised skin wound. f–j fully transected rat sciatic nerve. k–o excised rabbit conjunctival stroma. a, f, and k Schematic of the corresponding injury site model. b, d, g, i, l, and n Immunohistochemical localization of αSMA⁺ myofibroblasts (red brown) 10 days after skin injury (b, d), 7 days after peripheral nerve transection (g, i), or 14 days after conjunctiva injury (l, n). b, g, and l Lack of DRT grafting or grafting with control grafts led to large, dense, highly-aligned MFB configurations. d, i, and n DRT grafting led to significantly fewer dispersed, and almost randomly aligned MFBs. Scale bars: skin and nerves, 100 μm; conjunctiva, 10 μm. c, e, h, j, m, and o Evaluating the structure of the resulting tissue. c, e In full-thickness skin wounds birefringence microscopy of collagen fibers demonstrates the formation of scar in ungrafted wounds (c) and the synthesis of the nearly-physiological dermis in DRT-grafted wounds (e)^,,. h In DRT-ungrafted peripheral nerve wounds electron microscopy reveals that 26 months following transection, the original nerve fibers have been replaced by a dense sheaf of collagen fibrils that enclose groups of Schwann cells (Büngner bands, Bb). j In contrast, histological micrographs of cross-sections from DRT-grafted peripheral nerve wounds demonstrate the formation of neural tissue whose histomorphometric (equivalent diameter, number of myelinated fibers, number of A-fibers) and electrophysiological assays were similar to those for the autograft. m, o In conjunctiva wounds, immunohistochemical analysis and birefringence microcopy demonstrate scar formation in ungrafted wounds (m, note marked orientation of birefringent collagen fibers) and synthesis of near-normal conjunctival stroma collagen fibers (absence of orientation) in DRT-grafted wounds (o). Scale bars: skin, 50 μm; peripheral nerves, neural scar (top), 1 μm; peripheral nerve, regenerated nerve (bottom), 25 μm; conjunctiva, 50 μm. Figure 1a, f was reproduced with permission from Springer Nature. Fig. 1b, d, g, i, and j were reproduced with permission from Biomaterials, 33, 4783–91, ^©Elsevier (2012). Figure 1c, e were reproduced with permission from MIT. Figure 1h was reproduced with permission from J. Anat., 192, 529–39, ^©Wiley (1998). Figure 1l–o was reproduced with permission from Invest. Ophthalmol. Vis. Sci, 41, 2404–11, ^©Association for Research in Vision and Ophthalmology (2000).

Fig. 2. An inverse relationship between wound contraction and induced regeneration was demonstrated in transected rat sciatic nerves grafted by a library of five different porous collagen scaffolds, either DRT or analogs of DRT, differing only in degradation half-life.

a OsO₄ staining of tissue sections reveals the formation of a contractile MFB capsule (thickness shown between red arrows) around the newly-formed nerve tissue (stars) 9 weeks post-injury. b Quantification of the inverse relationship between the intensity of wound contraction (assayed by the radial thickness of the MFB capsule) and quality of induced regeneration, assayed both by the equivalent tissue diameter (left) and the number of myelinated fibers (right). Data (mean ± se) were obtained at the midpoint of the gap distance, 15 mm, initially separating the two nerve stumps, measured 9 weeks post-injury. Scale bars 50 μm. Figure 2a, b were reproduced with permission from Biomaterials, 33, 4783–91, ^©Elsevier (2012).

Fig. 3. The absence of wound contraction has been reported in several cases of spontaneous scarless healing in various species.

a No αSMA⁺ fibroblasts (red) were detected 12 days after full-thickness ear injury in A. kempi, which are able to spontaneously regenerate severe skin injuries. b In contrast, a significant number of αSMA⁺ cells (red) were detected in ear injuries in Mus musculus mice, which spontaneously heal such injuries by forming a scar. c, d The axolotl can regenerate spontaneously injuries in several organs, including its limbs and tail. c αSMA staining was not detected in skin injuries, 12 days after injury. d In the same animal, αSMA (brown) was detected in control tissue (small intestine)^–. Figure 3a, b were reproduced with permission from Nature 489, 561–66, ^©Springer Nature (2012). Figure 3c, d was reproduced with permission from J. Exp. Zool. B. Mol. Dev. Evol., 314B, 684–97, ^©Wiley (2010).