Seifert AW et al. (2012), Skin regeneration in adult axolotls: a blueprin...

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PLoS One 2012 Jan 01;74:e32875. doi: 10.1371/journal.pone.0032875.

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Skin regeneration in adult axolotls: a blueprint for scar-free healing in vertebrates.

Seifert AW , Monaghan JR , Voss SR , Maden M .

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While considerable progress has been made towards understanding the complex processes and pathways that regulate human wound healing, regenerative medicine has been unable to develop therapies that coax the natural wound environment to heal scar-free. The inability to induce perfect skin regeneration stems partly from our limited understanding of how scar-free healing occurs in a natural setting. Here we have investigated the wound repair process in adult axolotls and demonstrate that they are capable of perfectly repairing full thickness excisional wounds made on the flank. In the context of mammalian wound repair, our findings reveal a substantial reduction in hemostasis, reduced neutrophil infiltration and a relatively long delay in production of new extracellular matrix (ECM) during scar-free healing. Additionally, we test the hypothesis that metamorphosis leads to scarring and instead show that terrestrial axolotls also heal scar-free, albeit at a slower rate. Analysis of newly forming dermal ECM suggests that low levels of fibronectin and high levels of tenascin-C promote regeneration in lieu of scarring. Lastly, a genetic analysis during wound healing comparing epidermis between aquatic and terrestrial axolotls suggests that matrix metalloproteinases may regulate the fibrotic response. Our findings outline a blueprint to understand the cellular and molecular mechanisms coordinating scar-free healing that will be useful towards elucidating new regenerative therapies targeting fibrosis and wound repair.

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Species referenced: Xenopus
Genes referenced: acta2 acta4 actc1 actl6a dio3 fn1 gtsf2 krt12.4 lcp1 mmp2 mmp28 pnma2 tnc

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	Figure 1. Scar-free healing of full thickness excisional wounds in adult axolotls.A) Massonâs trichrome staining of uninjured dorsal flank skin in axolotls showing epidermis (E), dermis (D), hypodermis (H) and underlying muscle (M). B) Magnified image of epidermis and dermis. The epidermis is pseudo-stratified and contains epithelial cells and leydig cells (yellow arrows) while the dermis is divided into the stratum spongiosum (containing glands and dermal fibroblasts) atop the densely compacted ECM of the stratum compactum. C-H) Scar-free healing over 80 day period following full thickness excisional wounding. C) One day post injury (dpi) the wound bed is completely re-epithelialized. Some blood plasma has accumulated beneath the neoepidermis (green arrows) (wound marginâ=âWM). D) Seven dpi there is little evidence of a fibrin clot between the epidermis and underlying muscle and no new ECM has been deposited. Green arrows depict residual blood plasma. E) Fourteen dpi fibroblasts are visible beneath the epidermis where new ECM is deposited (blue staining) and muscle cells are fragmenting into individual myoblasts. F) Twenty-one dpi a thick band of transitional matrix is visible beneath the epidermis. Collagen is visible within the regenerating muscle. G) Forty-seven dpi the underlying muscle has completely regenerated and is devoid of collagen. Skin glands (yellow arrow) have regenerated and descended from the epidermis. The dermal stratum spongiosum has reformed and the stratum compactum is coalescing. H) Eighty dpi all skin layers have regenerated. Scale barsâ=â100 Âµm.
	Figure 2. Scar-free healing in adult axolotls.Regeneration of dorsal flank skin following 4 mm full thickness biopsy punch wounding. White circles depict area of original injury. Individual pigment cells are visible at D14 in the overlying epithelium. Regeneration and scar-free healing at D89. Contraction is evident at the wound edges after D14. Scale barsâ=â2.0 mm.
	Figure 3. Lamina lucida and lamina densa regenerate before new ECM deposition.A) Histological examination of basement membrane (BM) regeneration in axolotls. The uninjured BM is visible as a thick blue-stained fibrous band (yellow arrows). An immature BM has begun to reform (yellow arrow D1) after re-epithelialization and is visible at the wound margin (WM) in contrast to the uninjured BM. The regenerated BM is visible at D47. Yellow arrows at D7 and D21 indicate reforming BM. B) Examination of lamina lucida (laminin) and lamina densa (collagen type IV) during basement membrane regeneration. The uninjured BM is positive for laminin and collagen type IV (yellow arrows) as are the basement membranes surrounding glands and muscle fibers. Following re-epithelialization the basal lamina of the epidermis is negative for laminin and collagen type IV (white arrows) and this is clearly evident at the wound margin (WM). Seven days post injury the BM stains strongly for laminin indicating reformation of the lamina lucida, while staining for collagen type IV is punctuated. The lamina densa is regenerated by D14 based on continuous collagen type IV staining and persists during dermal regeneration.
	Figure 4. Metamorphic axolotl skin heals scar-free but slower compared to paedomorphs.AâB) Morphology of uninjured axolotl skin after metamorphosis. A) Massonâs trichrome staining showing epidermis (E), dermis (D) containing enlarged granular glands in stratum spongiosum atop stratum compactum, hypodermis (H) and muscle (M). B) High magnification of epidermis showing stratified epithelium. Leydig cells have disappeared and the epidermis now contains a well-defined stratum spinosum, granulosum and corneum. C-H) Wound healing following FTE wounds over 82-day period. C) One day post injury (dpi) epithelial cells have begun to migrate but the wound is not re-epithelialized. Erythrocytes are visible in the wound bed and between muscle fibers undergoing histolysis. D) Re-epithelialization is complete 3 dpi and at 7 dpi coagulated plasma (green arrows), erythrocytes and leukocytes are visible in the wound bed. E) Fourteen dpi fibroblasts are visible in the wound bed and new ECM is deposited (blue staining). The wound margin (WM) is still clearly visible. F) The wound bed 21 dpi is rich in ECM. This ECM extends deep into the underlying muscle fibers which are fragmenting into myoblasts. G). Regenerating glands (yellow arrows) are present in the dermis 42 dpi and the stratum spongiosum is beginning to develop. The underlying muscle continues to remain damaged with deep pockets of collagen persisting beneath the wound. H) Eighty dpi the dermis is partially regenerated but the stratum compactum has not coalesced. Some collagen still persists deep in the muscle and both mucous and granular glands have regenerated (yellow arrows). Scale barsâ=â100 Âµm.
	Figure 5. The rate of re-epithelialization is slower in metamorphs compared to paedomorphs.Epidermis was labeled with a pan-cytokeratin antibody. A) The wound bed is completely re-epithelialized 24 hrs after injury in paedomorphs. The wound margins (WM) are visible where the stratum compactum is disrupted. B, C) Wound edge keratinocytes have just begun migrating 24 hrs dpi and re-epithelialization is complete by 72 hrs dpi in metamorphs. D) Leydig cells are present in the paedomorph neoepidermis. E) The leading edge of migrating metamorph keratinocytes. The epidermal cells appear to move as a group of cells with one cell at the leading edge. F) After re-epithelialization is complete in metamorphs the epidermis becomes re-stratified.
	Figure 6. Matrix metalloproteinase (MMP) expression during re-epithelialization and new tissue formation.Expression values (y-axis) reflect absolute expression values from Affymetrix axolotl genechips (see Table S1 for exact values). Error bars represent standard error. Blue lines represent paedomorphs and red lines represent metamorphs. Expression kinetics for selected MMPs generally followed two patterns; (1) a strong upregulation at D1 followed by an equally strong decrease at D3 and a continued decrease or leveling off at D7 [MMP3/10a, MMP9] and (2) a strong upregulation at D1 which remained high at D3 and then downregulated at D7 [MMP3/10b, MMP19]. For MMP28 the kinetics followed pattern 1 for paedomorphs and pattern 2 for metamorphs suggesting that MMP28 expression was highly connected to re-epithelialization. The expression profile for MMP2 was unique in that is was downregulated following injury and was upregulated after re-epithelialization was complete.
	Figure 7. Higher initial leukocyte levels are present in terrestrial axolotls coincident with slower re-epithelialization, but neutrophil levels are not different between morphs.L-plastin was used as a pan-leukocytic marker to quantify total leukocyte levels following injury. A) Total leukocytes counted per mm2 in the wound bed at 1, 3, 7 and 14 dpi (nâ=â4 for each morph). L-plastin positive cells are red (yellow arrows), nuclei are stained blue (Hoescht) and green fluorescence was used to account for autofluorescencing erythrocytes that were excluded as leukocytes. An influx of leukocytes was observed 24 hrs dpi, with higher numbers present in terrestrial axolotls. Leukocyte levels dropped in metamorphs at D7 and converged with paedomorph levels. Levels for paedomorphs were not significantly changed after D1. B) All neutrophils present in the wound bed (yellow arrows) were counted on 5 Âµm sections above the muscle using myloperoxidase (nâ=â8 for each morph; see Figure S6 for positive control staining in liver). Neutrophil levels were generally low and were not significantly different between paedomorphs and metamorphs, although they did drop significantly at D7. C) Modified Wright-Giemsa stain used to indentify individual leukocytes in circulating axolotl blood. Sudan black was used to stain neutrophils. Yellow arrows indicate the specific leukocyte type.
	Figure 8. Contraction during scar-free healing in axolotls is similar to tight-skinned mammals.A) Percent wound closure in paedomorphs and metamorphs over 21 days (when contraction is complete). Metamorph wounds expanded by 10% following wounding and wounds contracted at about the same rate in both morphs. B) Paedomorph wounds contracted â¼27% more than metamorph wounds with contraction accounting for 37.9% of wound closure in metamorphs. C) Alpha-smooth muscle actin (alpha-SMA) localization in unwounded skin and during wound repair in Mus musculus and axolotls (A. mexicanum) to identify myofibroblasts. Alpha-SMA localized to blood vessels and a few cells in mouse skin and around glands and the stratum compactum in axolotl skin. Ten days post injury (dpi), when contraction rates are highest in mouse wounds, alpha-SMA positive cells are detected at high levels at the wound margins (white dotted circle). In contrast, we detected very few alpha-SMA positive cells ten days after wounding in axolotl tissue. We did, however, detect alpha-SMA in the regenerating basement membrane (yellow arrows). Twenty-one dpi we detected a high number of myofibroblasts in mouse tissue (red arrows). We detected a few myofibroblasts in axolotl tissue at D21 (red arrows) near the underlying muscle.
	Figure 9. Regenerative ECM in axolotl wounds is characterized by high levels of tenascin-C.A-B) Fibronectin (FN) and tenascin-C (TN-C) levels were detected during scar-free healing in paedomorphs and metamorphs using an antibody to axolotl fibronectin and a polyclonal antibody to chick tenascin-C. We detected low levels of FN in the basement membrane at D7, and at the wound margins in both morphs. FN was present during ECM deposition at D14 in the center of the wound bed, but in relatively small amounts. By D21 little FN persisted in the regenerating dermis. B) TN-C was detected at the wound margins, in the basement membrane and surrounding some cells at D7. Fourteen days post injury we detected high levels of TN-C throughout the wound bed and in regenerating muscle. A sharp boundary formed between intact muscle and regenerating muscle. These high levels of TN-C persisted during dermis regeneration. Green fluorescence was used to detect autofluorescing erythrocytes. Epidermis (E), dermis (D), muscle (M), wound margin (WM).
	Figure 10. Collagen type III predominates early during new tissue formation and is slowly replaced by collagen type I during scar-free healing.Picrosirius red staining was used to detect collagen type III and collagen type I during scar-free healing in the wound bed. Using polarized light to detect bifringence, collagen type III (green fibers) was deposited first during new ECM deposition in both morphs. As the dermis regenerated, collagen type III was slowly replaced by collagen type I (red fibers) in both morphs.
	Figure 11. Summary of wound healing processes comparing axolotls and mammals.The x-axis represents time and the y-axis represents percent maximal response for each process. Information for mammals has been approximated from the literature and from our own experiments with 4mm FTE wounds in mice. Colors represent individual processes overlapping across the three phases of wound healing; inflammation, new tissue formation and tissue remodeling. Comparing paedomorphic and metamorphic axolotls, metamorphs exhibited an increased hemostatic response, slower re-epithelialization, increased early inflammatory response, increased and prolonged deposition of extracellular matrix (ECM) and an almost doubling in the time required for complete skin regeneration. Comparing scar-free healing in terrestrial axolotls to scar formation in mammals, terrestrial axolotls exhibited a reduced hemostatic response, lower neutrophil levels, faster re-epithelialization rate, delay in ECM production, differences in the relative composition of the new ECM, regeneration of glands and dermis regeneration instead of scarring. Schematic for mammals is adapted from Mikael HÃ¤ggstrÃ¶m.

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