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.     

Smad4 disruption accelerates keratinocyte reepithelialization in murine cutaneous wound repair

Keratinocyte reepithelialization is a rate-limiting event in cutaneous wound repair, which involves the migration and proliferation of keratinocytes to cover the denuded dermal surface. Transforming growth factor-β1 (TGF-β1) has the ability to induce epithelial cell migration while inhibiting proliferation, and controversial results have been generated regarding the effect of TGF-β signaling on reepithelialization. In this study, full-thickness skin wounds were made in keratinocyte-specific Smad4 knockout and the control mice. The wound closure, reepithelialization, keratinocyte proliferation, myofibroblast numbers and collagen deposition of were assessed. The results showed that the proliferation of keratinocytes increased, which accelerated the reepithelialization, and led to faster wound repair in the epidermis of Smad4 mutant mice. Upregulation of keratin 17, 14-3-3 sigma and phosphorylated AKT in the hyperproliferative epidermis may be correlated with the accelerated reepithelialization. We conclude that Smad4 plays an inhibitory role in the keratinocyte-mediated reepithelialization of wound healing.     

Inhibition by the soluble syndecan-1 ectodomains delays wound repair in mice overexpressing syndecan-1

Wound repair is a tightly regulated process stimulated by proteases, growth factors, and chemokines, which are modulated by heparan sulfate. To characterize further the role of the heparan sulfate proteoglycan syndecan-1 in wound repair, we generated mice overexpressing syndecan-1 (Snd/Snd) and studied dermal wound repair. Wound closure, reepithelialization, granulation tissue formation, and remodeling were delayed in Snd/Snd mice. Soluble syndecan-1 was increased, and shedding was prolonged in wounds from Snd/Snd mice. Excess syndecan-1 increased the elastolytic activity of wound fluids. Additionally, cells in the granulation tissue and keratinocytes at wound edges showed markedly reduced proliferation rates in Snd/Snd mice. Skin grafting experiments between Snd/Snd and control mice indicated that the slower growth rate was mainly due to a soluble factor in the Snd/Snd mouse skin. Syndecan-1 immunodepletion and further degradation experiments identified syndecan-1 ectodomain as a dominant negative inhibitor of cell proliferation. These studies indicate that shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair.     

EGF and TGF-alpha in wound healing and repair

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

Accelerated healing of full-thickness skin wounds in a wet environment

Full-thickness skin wounds are preferably allowed to heal under controlled hydration dressings such as hydrocolloids. It was hypothesized that a wet (liquid) environment rather than a dry or moist one would accelerate the wound healing process. We compared skin repair by secondary intention in full-thickness skin wounds in wet (saline), moist (hydrocolloid), and dry (gauze) conditions in an established porcine wound healing model. The study included three animals with a total of 70 wounds layered in a standardized fashion on the back of young Yorkshire pigs. Twelve days after wounding, 0 percent of dry, 20 percent of moist, and 86 percent of saline-treated wounds were completely reepithelialized (p values = 0.0046 and 0.027 for saline wounds compared with dry and moist wounds, respectively). The accelerated healing was caused at least in part by faster contraction in wet wounds (p value < 0.005 compared with that of other groups 9 and 12 days after wounding). Development of granulation tissue was faster in moist conditions than it was for dry and wet wounds. The thickness and number of cell layers of the newly formed epidermis were greater in dry and wet wounds than in moist ones. It was concluded that these full-thickness porcine skin wounds healed faster in a wet environment than in a moist one. Dry wounds healed more slowly than moist wounds. The basic mechanisms of skin wound repair were influenced by the treatment modality as demonstrated by the observed differences in granulation tissue formation, reepithelialization, and rate of wound contraction.     

Trends in wound repair: cellular and molecular basis of regenerative therapy using electromagnetic fields

Chronic ulceration of the leg represents a major, underestimated problem of modern health care, involving physical and cosmetic impairment and social stigma along with high community costs for patients' treatment. The increasing prevalence of chronic ulcers, currently reported to be as much as 0.3% in the general population, should stimulate identification of more efficacious therapeutic approaches to achieve complete healing. The strategies of regenerative medicine based on small molecules, biomimetic scaffolds, gene or cell therapy, and electromagnetic field manipulation represent some of the modern therapeutic alternatives for wound healing. Here we review in an integrated, interdisciplinary approach the modern cellular and molecular mechanistic concepts regarding the involvement of extremely low frequency electromagnetic fields (ELF-EMF) in the complex process of tissue repair, with particular focus on chronic wounds. The data analysis supports three main effects of electromagnetic fields on the wound healing pathways: 1) an antiinflammatory effect, by modulation of cytokine profile that induces the transition of the healing process from a chronic pro-inflammatory to an anti-inflammatory state; 2) a neo-angiogenic effect, by increased endothelial cells proliferation and tubulization and production of fibroblast growth factor (FGF)-2; and 3) a reepithelialization effect, by stimulation of collagen formation. We believe that utilization of ELF-EMF in larger clinical trials designed to optimize these functional parameters would facilitate a better understanding of ELFEMF- induced healing mechanisms and lead to improved therapeutic outcomes for this disabling condition which is often totally resistant to treatment.     

Stimulation of growth factor synthesis in skin wounds using tissue extract (G-90) from the earthworm Eissenia foetida

Growth factors are biologically-active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. In the case of wound healing, a proper wound dressing is needed to cover the wound area, protect the damaged tissue, and if possible to activate cell proliferation and stimulate the healing process. In this study we examined the efficacy of a glycolipoprotein tissue homogenate extract from Eisenia foetida (G-90) to activate signal transduction pathways, leading to wound healing. We measured the activation of EGF and FGF in healthy skin, in wounds with physiological healing and in wounds treated with G-90. The activation of EGF and FGF was measured during the first 24 h of wound healing under both physiological conditions and treatment with G-90. In both cases an increased concentration of EGF and FGF was observed 6 h after wounding. In comparison with healthy skin, the concentration of EGF increased 10-fold and FGF five-fold in wounds treated with G-90 (10 ng ml(-1)). Healing in physiological conditions resulted in a two-fold increase of EGF and 1.5-fold of FGF.     

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.     

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.     

Rapid preparation of a noncultured skin cell suspension that promotes wound healing

Autologous skin cell suspensions have been used for wound healing in patients with burns and against normal pigmentation in vitiligo. To separate cells and the extracellular matrix from skin tissue, most researchers use enzymatic digestion. Therefore, this process is difficult to perform during a routine surgical procedure. We aimed to prepare a suspension of noncultured autologous skin cells (NCSCs) using a tissue homogenizer as a new method instead of harsh biochemical reagents. The potential clinical applicability of NCSCs was analyzed using a nude-rat model of burn healing. After optimization of the homogenizer settings, cell viability ranged from 52 to 89%. Scanning electron microscopy showed evidence of keratinocyte-like cell morphology, and several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the NCSCs. The rat model revealed that NCSCs accelerated skin regeneration. NCSCs could be generated using a tissue homogenizer for enhancement of wound healing in vivo. In the NCSC group of wounds, on day 7 of epithelialization, granulation was observed, whereas on day 14, there was a significant increase in skin adnexa regeneration as compared to the control group (PBS treatment; p < 0.05). This study suggests that the proposed process is rapid and does not require the use of biochemical agents. Thus, we recommend a combination of surgical treatment with the new therapy for a burn as an effective method.     

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.     

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ?mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.     

Cellular mechanisms of skin repair in humans and other mammals

The increased incidence of non-healing skin wounds in developed societies has prompted tremendous research efforts on the complex process known as “wound healing”. Unfortunately, the weak relevance of modern wound healing research to human health continues to be a matter of concern. This review summarizes the current knowledge of the cellular mechanisms that mediate wound closure in the skin of humans and laboratory animals. The author highlights the anatomical singularities of human skin vs. the skin of other mammals commonly used for wound healing research (i.e. as mice, rats, rabbits, and pigs), and discusses the roles of stem cells, myofibroblasts, and the matrix environment in the repair process. The majority of this review focuses on reepithelialization and wound closure. Other aspects of wound healing (e.g. inflammation, fibrous healing) are referred to when relevant to the main topic. This review aims at providing the reader with a clear understanding of the similarities and differences that have been reported over the past 100 years between the healing of human wounds and that of other mammals.     

Soft tissue fibroblasts from well healing and chronic human wounds show different rates of myofibroblasts in vitro

Due to an increasing life expectancy in western countries, chronic wound treatment will be an emerging challenge in the next decades. Because therapies are improving slowly appropriate diagnostic tools enabling the early prediction of the healing success remain to be developed. We used a well-established in vitro assay in combination with the analysis of 27 cytokines to discriminate between fibroblasts from chronic (n = 6) and well healing (n = 8) human wounds. Proliferation and migration of the cells as well as their response to hypoxia and their behaviour in co-culture with microvascular endothelial cells were analyzed. Myofibroblast differentiation, a time-limited essential process of regular wound healing, was also quantified. Besides weaker proliferation and migration significantly higher rates of myofibroblasts were detected in chronic wounds. With respect to the cytokine release, there was a clear trend within the group of chronic wound fibroblasts, which were releasing interferon-γ, monocyte chemotactic protein-1, granulocyte–macrophage colony stimulating factor and basic fibroblast growth factor in higher amounts than fibroblasts from healing wounds. Although the overall response of both groups of fibroblasts to hypoxia and to the contact with endothelial cells was similar, especially chronic wound fibroblasts seemed to benefit from the endothelial interaction during hypoxia and displayed better migration characteristics. The study shows (1) that the assay can identify specific features of fibroblasts derived from different human wounds and (2) that wound fibroblasts are varying in their response to the chosen parameters. Thus, current therapeutic approaches and individual healing prediction might benefit from this assay.     

创伤合并海水浸泡后愈合过程的病理学观察

目的观察创伤合并海水浸泡后对伤口愈合时间的影响及愈合过程中的病理学改变。方法以大鼠为实验动物,建立背部双侧圆形创伤模型,创后随机分为对照组和实验组,每组50只,实验组创后置海水中浸泡30min。观察2组创面的愈合生长状况。观察伤口局部愈合过程中的病理学改变,应用免疫组化方法观察愈合过程中伤口修复细胞增殖指数及微血管密度变化。结果对照组平均愈合时间为（12.0±1.0）d,而实验组的平均愈合时间为（14.3±0.8）d;两组在肉芽组织形成时间上以及修复细胞增殖能力上存在差异。结论创伤合并海水浸泡可导致伤口愈合时间延迟,加重创伤局部的炎症反应、延缓肉芽组织形成时间。     

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.