Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

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

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.     

Epidermal downgrowths in regenerating rabbit ear holes.

Rabbits are unique among mammals in that their ears can regenerate tissues from the margins of full thickness holes which grow in and completely fill the opening in about two months. The circular blastema that forms around the edges of the hole differentiates a new sheet of cartilage as it regenerates in a centripetal direction. Similar holes in other mammals fail to regenerate and form scar tissue instead of a blastema. Histological studies of the healing around the edges of rabbit ear holes reveal that during the second week, when the epidermis is completing its migration across the wound from the opposite sides of the ear, conspicuous tongues of epidermal cells grow down into the underlying tissues at the edges of the wound. These epidermal downgrowths are situated between the original intact dermis of the skin and the more central tissues which give rise to the blastema. Such downgrowths are of a transient nature, and are no longer found once the blastema rounds up toward the end of the second week. Since they are not found in the healing of similar wounds in rabbit ears prevented from regenerating by prior removal of their cartilaginous sheets, nor in the naturally nonregenerating ears of sheep and dogs, it is considered that these downgrowths of healing epidermis may play a role in the unusual regenerative response of ear tissues in the rabbit.     

Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells

Skin wound healing involves Notch/Jagged1 signaling. However, little is known how Jag1 expression level in epidermal stem cells (ESCs) contributes to wound healing and scar formation. We applied multiple cellular and molecular techniques to examine how Jag1 expression in ESCs modulates ESCs differentiation to myofibroblasts (MFB) in vitro, interpret how Jag1 expression in ESCs is involved in wound healing and scar formation in mice, and evaluate the effects of porcine acellular dermal matrix (ADM) treatment on wound healing and scar formation. We found that Jag1, Notch1 and Hes1 expression was up-regulated in the wound tissue during the period of wound healing. Furthermore, Jag1 expression level in the ESCs was positively associated with the level of differentiation to MFB. ESC-specific knockout of Jag1 delayed wound healing and promoted scar formation in vivo. In addition, we reported that porcine ADM treatment after skin incision could accelerate wound closure and reduce scar formation in vivo. This effect was associated with decreased expression of MFB markers, including α-SMA Col-1 and Col-III in wound tissues. Finally, we confirmed that porcine ADM treatment could increase Jag1, Notch1 and Hesl expression in wound tissues. Taken together, our results suggested that ESC-specific Jag1 expression levels are critical for wound healing and scar formation, and porcine ADM treatment would be beneficial in promoting wound healing and preventing scar formation by enhancing Notch/Jagged1 signaling pathway in ESCs.     

The blastema and epimorphic regeneration in mammals

Studying regeneration in animals where and when it occurs is inherently interesting and a challenging research topic within developmental biology. Historically, vertebrate regeneration has been investigated in animals that display enhanced regenerative abilities and we have learned much from studying organ regeneration in amphibians and fish. From an applied perspective, while regeneration biologists will undoubtedly continue to study poikilothermic animals (i.e., amphibians and fish), studies focused on homeotherms (i.e., mammals and birds) are also necessary to advance regeneration biology. Emerging mammalian models of epimorphic regeneration are poised to help link regenerative biology and regenerative medicine. The regenerating rodent digit tip, which parallels human fingertip regeneration, and the regeneration of large circular defects through the ear pinna in spiny mice and rabbits, provide tractable, experimental systems where complex tissue structures are regrown through blastema formation and morphogenesis. Using these models as examples, we detail similarities and differences between the mammalian blastema and its classical counterpart to arrive at a broad working definition of a vertebrate regeneration blastema. This comparison leads us to conclude that regenerative failure is not related to the availability of regeneration-competent progenitor cells, but is most likely a function of the cellular response to the microenvironment that forms following traumatic injury. Recent studies demonstrating that targeted modification of this microenvironment can restrict or enhance regenerative capabilities in mammals helps provide a roadmap for eventually pushing the limits of human regeneration.     

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2 mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.     

Notch与相关调控信号的相互作用在伤口愈合中的调控

皮肤是哺乳动物最重要的组织之一.当皮肤受损时,受损组织通过系列伤口愈合反应的生理和心理作用被修复,实现组织再生.再生反应主要发生在胚胎发育早期,伤口自愈能力随着机体的成熟而减弱;并且哺乳动物的组织重塑过程较为复杂,在不正确的信号引导下,可能引起并发症而导致创面愈合异常.研究表明,伤口微环境的稳态和信号分子的辅助作用是愈...     

Regeneration of articular cartilage in healer and non-healer mice

Mammals rarely regenerate their lost or injured tissues into adulthood. MRL/MpJ mouse strain initially identified to heal full-thickness ear wounds now represents a classical example of mammalian wound regeneration since it can heal a spectrum of injuries such as skin and cardiac wounds, nerve injuries and knee articular cartilage lesions. In addition to MRL/MpJ, a few other mouse strains such as LG/J (a parent of MRL/MpJ) and LGXSM-6 (arising from an intercross between LG/J and SM/J mouse strains) have now been recognized to possess regenerative/healing abilities for articular cartilage and ear wound injuries that are similar, if not superior, to MRL/MpJ mice. While some mechanisms underlying regenerative potential have been begun to emerge, a complete set of biological processes and pathways still needs to be elucidated. Using a panel of healer and non-healer mouse strains, our recent work has provided some insights into the genes that could potentially be associated with healing potential. Future mechanistic studies can help seek the Holy Grail of regenerative medicine. This review highlights the regenerative capacity of selected mouse strains for articular cartilage, in particular, and lessons from other body tissues, in general.     

Isolation of wound healing/regeneration genes using restrictive fragment differential display-PCR in MRL/MPJ and C57BL/6 mice

Wound healing in mammals can take several weeks to months and the process is always accompanied by scar formation. Wound healing mechanisms that mimic regeneration are not found in most mature mammalian tissues. However, the MRL/MPJ (MRL) mouse has the unique capacity to regenerate ear hole wound completely in less than a month. To identify genes involved in wound healing without a scar, we chose to use restriction fragment differential display-PCR to isolate genes differentially expressed in the MRL (good healer) mouse and the C57BL/6 (poor healer) mouse at different stages of wound healing. We identified 36 genes that were differentially expressed in the regenerating tissue of good and poor healer strains of which several genes are also genetically linked to wound healing and thus are potential candidate genes for scarless wound healing.     

Learning from regeneration research organisms: The circuitous road to scar free wound healing

The skin is the largest organ in the body and plays multiple essential roles ranging from regulating temperature, preventing infection and ultimately defining who we are physically. It is a highly dynamic organ that constantly replaces the outermost cells throughout life. However, when faced with a major injury, human skin cannot restore a significant lesion to its original functionality, instead a reparative scar is formed. In contrast to this, many other species have the unique ability to regenerate full thickness skin without formation of scar tissue. Here we review recent advances in the field that shed light on how the skin cells in regenerative species react to injury to prevent scar formation versus scar forming humans.     

Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells

Damage to neonatal and adult tissues always incites an influx of inflammatory neutrophils and macrophages. Besides clearing the wound of invading microbes, these cells are believed to be crucial coordinators of the repair process, acting both as professional phagocytes to clear wound debris and as a major source of wound growth factor signals. Here we report wound healing studies in the PU.1 null mouse, which is genetically incapable of raising the standard inflammatory response because it lacks macrophages and functioning neutrophils. Contrary to dogma, we show that these "macrophageless" mice are able to repair skin wounds with similar time course to wild-type siblings, and that repair appears scar-free as in the embryo, which also heals wounds without raising an inflammatory response. The growth factor and cytokine profile at the wound site is changed, cell death is reduced, and dying cells are instead engulfed by stand-in phagocytic fibroblasts. We also show that hyperinnervation of the wound site, previously believed to be a consequence of inflammation, is present in the PU.1 null wound, too.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

c-Met is essential for wound healing in the skin

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.     

Extracellular matrix considerations for scar-free repair and regeneration: Insights from regenerative diversity among vertebrates

The extracellular matrix (ECM) is an essential feature of development, tissue homeostasis and recovery from injury. How the ECM responds dynamically to cellular and soluble components to support the faithful repair of damaged tissues in some animals but leads to the formation of acellular fibrotic scar tissue in others has important clinical implications. Studies in highly regenerative organisms such as the zebrafish and the salamander have revealed a specialist formulation of ECM components that support repair and regeneration, while avoiding scar tissue formation. By comparing a range of different contexts that feature scar-less healing and full regeneration vs. scarring through fibrotic repair, regenerative therapies that incorporate ECM components could be significantly enhanced to improve both regenerative potential and functional outcomes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Therapeutic effect of human mesenchymal stem cells in skin after radiation damage

Over 50% of all cancer patients presently receive radiotherapy at one stage in their treatment course. Inevitably skin is one of the most frequently damaged tissue due to its localization and constant turn-over. Our present goal is to reduce radiation-induced complications in human skin through stem cell therapy, particulary in human epidermis. Mesenchymal Stem Cells (MSCs) have been shown to be multipotent cells able to engraft in many tissues after injury. Herein, we isolated human MSCs and tested their capability to improve skin wound healing after irradiation. This potential was assessed in NOD/SCID mice which received 30 Gy locally on the thigh. This dose caused within 3 weeks local epidermis necrosis which was repaired within 13 weeks. MSCs were intravenously injected in irradiated mice 24 hours after exposure. Clinical scoring throughout 6 weeks gave indications that human MSCs reduced the extent of damage and accelerated the wound healing process. We show by quantitative qPCR and histological studies the presence of human MSCs derived cells into the scar. Human MSCs homed to the damaged skin and participated to the wound healing process. These results open prospects for cellular therapy by MSCs in irradiated epithelial tissues and could be extended to the whole general field of cutaneous cicatrization, particularly after burns.