Models of epidermal wound healing

The spreading of cells across the surface of an epidermal wound enables epidermal migration to be studied independently of the wound contraction that occurs in deeper wounds. In particular, the stimulus for the increase in epidermal mitosis during would healing is uncertain. Our modelling suggests that biochemical regulation of mitosis is fundamental to the process, and that a single chemical with a simple regulatory effect can account for the healing of circular epidermal wounds. The model results compare well with experimental data.     

Changes in cytokeratin expression in epidermal keratinocytes during wound healing

In order to investigate the re-epithelialization process during wound healing, the hair on the back of guinea pigs was shaved and then excisional wounds were made through the entire thickness of the skin. Histological changes were observed and changes in the expression of different cytokeratin polypeptides were examined using an immunohistochemical technique. Immunohisto chemical study revealed that the proliferating and migrating keratinocytes expressed the same cytokeratins as the basal cells of normal epidermis. In addition, the entire epidermis of fairly remote areas from the edges of the wound where no thickening was observed showed a temporarily abnormal staining pattern. The suprabasal cells in the regenerating epidermis temporarily expressed cytokeratins not only specific for suprabasal cells but also specific for basal cells. The cytokeratins expressed in normal basal keratinocytes were also present in the thickened granular layers. These data indicate that the expression of cytokeratins in the epidermal keratinocytes (even in fairly remote areas from the wound edges) changes during wound healing, that the origin of the migrating keratinocytes from the remaining epidermis seems to be the basal cells in the epidermis, and that the appearance of keratohyalin granules is not related to changes in cytokeratin expression.     

Cyclophilin C-associated protein is up-regulated during wound healing

Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.     

Actin aggregation and embryonic epidermal wound healing

Recent experiments on the response of embryonic epidermis to wounding have revealed a cable of filamentous actin at the wound edge, which may be responsible for healing (Martin and Lewis 1991, 1992). We investigate the important question of how the cable forms as a response to wounding. We modify the mechanical model of Murray and Oster (1984) to investigate the post-wounding equilibrium in the epidermal sheet. We analyse the model in both one-dimensional and radially symmetric two-dimensional geometries, to determine the parameter domain in which a solution exists. We show that in both geometries the model solutions reflect the phenomenon of the actin cable for parameter values close to one edge of this domain. We interpret these results in terms of the relative rates of intracellular reorganization of actin and myosin, and thus suggest a possible mechanism for the formation of the actin cable.     

A secreted MMP is required for reepithelialization during wound healing

Matrix metalloproteinases (MMPs) are extracellular proteases highly expressed at wound sites. However, the precise function of MMPs during reepithelialization in vivo has been elusive in mammalian models because of the high level of redundancy among the 24 mammalian MMPs. For this reason we used Drosophila melanogaster, whose genome encodes only two MMPs-one secreted type (Mmp1) and one membrane-anchored type (Mmp2)-to study the function and regulation of the secreted class of MMPs in vivo. In the absence of redundancy, we found that the Drosophila secreted MMP, Mmp1, is required in the epidermis to facilitate reepithelialization by remodeling the basement membrane, promoting cell elongation and actin cytoskeletal reorganization, and activating extracellular signal-regulated kinase signaling. In addition, we report that the jun N-terminal kinase (JNK) pathway upregulates Mmp1 expression after wounding, but that Mmp1 is expressed independent of the JNK pathway in unwounded epidermis. When the JNK pathway is ectopically activated to overexpress Mmp1, the rate of healing is accelerated in an Mmp1-dependent manner. A primary function of Mmp1, under the control of the JNK pathway, is to promote basement membrane repair, which in turn may permit cell migration and the restoration of a continuous tissue.     

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.     

Elimination of foreign material by epidermal malpighian cells during wound healing in fish skin

Cultured epidermal malpighian cells and experimental wounds of Atlantic salmon Salmo salar were challenged with a variety of particulate materials. Latex beads and the bacteria Carnobacterium piscicola, Pseudomonas fluorescens and Aeromonas salmonicida salmonicida were engulfed by cultured cells, whereas Staphylococcus intermedius were not. The cells engulfed bacteria that proliferated in culture medium devoid of antibiotics and melanin granules and other cellular debris. Cells at wound margins engulfed latex beads and C. piscicola, P. fluorescens and A. s. salmonicida , but not S. intermedius. Malpighian cells thus appear to be both phagocytic and discriminatory. The results support the hypothesis that malpighian cells remove foreign material from fish skin by sloughing after becoming laden with engulfed material.     

Fibronectin is overproduced by keloid fibroblasts during abnormal wound healing.

Wound healing in certain individuals leads to the development of keloid tumors which exhibit abnormal collagen metabolism and an increased abundance of extracellular matrix components. Comparison of fibronectin levels in fibroblasts derived from keloids and normal dermis revealed a relative increase in intracellular and extracellular fibronectin in the keloid-derived cells. While fibronectin was similarly processed, compartmentalized, and degraded by both cell types, fibronectin biosynthesis was found to be accelerated as much as fourfold in keloid fibroblasts due to a corresponding increase in the amount of accumulated fibronectin mRNA. These changes account for the elevated steady-state level of the molecule in keloid fibroblasts and suggest that increased fibronectin in keloid lesions is due to overproduction by the wound-healing fibroblasts. Glucocorticoid treatment stimulated fibronectin biosynthesis in both normal and keloid fibroblasts. However, the amount of stimulation was less for the keloid-derived cells, indicating a limitation on maximal rates of fibronectin biosynthesis. These observations suggest that separate mechanisms act to control basal and maximal rates of fibronectin production. Biosynthesis of the 140-kilodalton fibronectin receptor was also found to be increased in keloid fibroblasts, suggesting some level of coordinate regulation for fibronectin and fibronectin receptor expression.     

Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection

The basement membrane of myofibers plays an important role during orderly regeneration of skeletal muscle after injury. In this report, changes in various basement membrane components were analyzed in skeletal muscle grafts undergoing regeneration (autografts) or immune rejection (allografts). The immunofluorescence technique using specific antibodies against laminin, types IV and V collagen, heparan sulfate proteoglycan, fibronectin, in combination with binding of concanavalin A (ConA) was used to monitor basement membranes. In normal muscle, these components were localized in the pericellular region of myofiber corresponding to its basement membrane. After transplantation, the majority of myofibers underwent degeneration as a result of ischemic injury, followed by regeneration from precursor myosatellite cells. Various components of basement membrane zone disappeared from the degenerating myofibers, leaving behind some unidentifiable component that still bound ConA. A new basement membrane appeared around the regenerated myotubes which persisted during maturation of the regenerating muscle. In rejected skeletal muscles, the immunoreactivity of various components persisted even after the disappearance of myotubes and myofiber cytoplasm. In addition, an accumulation of fibronectin was seen throughout the rejected muscle with the onset of immune rejection. These results demonstrate that the major basement membrane components disappear and reappear sequentially during myofiber degeneration and regeneration. Such a turnover is not seen in rejected skeletal muscles. Thus, the myofiber basement membrane is not a static structure as previously thought but one which changes chemically during degeneration and regeneration. This feature of basement membrane may be important in the orderly regeneration of skeletal muscle after injury.     

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.     

Scanning electron microscopical observations on epidermal wound healing in the PlanarianDugesia tigrina

Summary Epidermal wound healing in regeneratingDugesia tigrina (Planaria) has been studied using scanning electron microscopy (SEM). The normal epidermal surface and its differentiations have been descrebed. Observations on living material reveal the highly dynamic state of the wound in invididual animals and its more or less continously changing size due to the state of activity of the animals. These observations show good agreement with the SEM studies, which allow a clear delineation of cellular details of the wound, the wound margins and the apposing epidermal regions. These details are described. The over-all picture of planarian wound healing that emerges is briefly as follows: Epithelization is characterized by absence of proliferation from the old intact epidermis. Variable contraction of smooth muscle cells reduces the wound size to a certain extent. Simultaneously with this and also during a longer period epidermal cells adjacent to the wound are extending and some become highly attenuated. These two processes together are only to a certain degree effective in wound closure because of a definite epidermal cell deficit which is reflected in the emergence of an epidermal wound edge reflecting the maximal contribution of these two processes to an attempt to close the wound. Complete epithelization is effected by the operation of a third mechanism: Recruitment of cell through flow of subjacent blastemal cells (including rhabdite-forming cells) along the wound border; these cells subsequently occupy a peripheral position in the wound. This process is supplemented by cell immigration and insertion into the adjacent old epidermis and in the wound cell sheet. Rhabdite-forming cells contribute predominantly to this process. Eventually integration between old epidermal cells and the newly recruited cells which differentiate into epidermal cells results in final epithelization. Complete wound healing is based on interactions between the epidermal cell system and the regenerating subepidermal membrane-connective tissue filament-muscle cell system.     

Mathematical analysis of a basic model for epidermal wound healing

The stimuli for the increase in epidermal mitosis during wound healing are not fully known. We construct a mathematical model which suggests that biochemical regulation of mitosis is fundamental to the process, and that a single chemical with a simple regulatory effect can account for the healing of circular epidermal wounds. The numerical results of the model compare well with experimental data. We investigate the model analytically by making biologically relevant approximations. We then obtain travelling wave solutions which provide information about the accuracy of these approximations and clarify the roles of the various model parameters.     

Chemokine changes during oral wound healing

The oral mucosa is susceptible to tissue injury from many causes, including infection, autoimmune disorders, surgical and accidental trauma, and gingival and periodontal inflammation; however, little is known about the events that influence wound healing in the mouth. Recent studies in non-oral tissues have implicated immune system-derived factors, in particular chemokines, in the wound healing process. Tissues from mice with experimental gingival wounds were studied for expression of genes for four chemokine ligands or receptors (CCL19, CCL20, CCL25, and CCR5) that are important in leukocyte trafficking or inflammation. Notably, during the peak phase of wound healing, chemokine gene expression was up-regulated for CCL19, CCL20, and CCL25, and down-regulation of CCR5, suggesting an orchestrated process of chemokine-mediated recruitment or retention of lymphocytes and macrophages into wound areas, while simultaneously suppressing a potentially adverse inflammatory response. These findings have implications for developing therapeutic strategies aimed at promoting more effective tissue healing at oral surfaces.     

Collagen fibril formation in a wound healing model

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.     

Rac activation upon cell-cell contact formation is dependent on signaling from the epidermal growth factor receptor

Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829-1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305-3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling.     

Basement membrane formation and lung cell differentiation in vitro

In high density cultures of mouse fetal lung cells, so-called "mass cultures", development of organoid structures, formation of a basement membrane (BM), and differentiation of pneumocytes type II occur accompanied by synthesis and secretion of lamellar bodies. The relationship between the formation of a BM, on the one hand, and morphogenesis as well as differentiation of pneumocytes type II, on the other hand, has been investigated by use of antibodies against BM components in the lung mass culture. It is shown here that anti-laminin antibodies prevented BM formation, but morphogenesis and pneumocyte differentiation occurred as in untreated cultures. Short-term treatment with the antibody revealed that the BM is formed only during the first 2 to 3 days in vitro. Already formed BM could not be removed by anti-laminin. Anti-collagen type IV antibodies showed no effect in the lung mass culture except for a stronger staining of the BM. Anti-BM-1 antibodies caused no changes in morphogenesis, cell differentiation and BM formation either, but the mesenchymal intercellular space exhibited a dark staining, which is probably due to antigen-antibody complexes. The results obtained with anti-laminin antibodies indicate that a BM is not necessary for lung cell differentiation in vitro.     

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.     

Dimethyl sulfoxide (DMSO) does not affect epidermal wound healing

We studied the effect of 95% DMSO on dermal/epidermal healing and microbiol flora in partial-thickness wounds. Wounds of 0.3 mm were made in the skin of Yorkshire pigs with a keratome and treated daily with either 95% DMSO, water, or they were left untreated. Wounds were excised on Days 2-7 and the dermis was separated from the epidermis. The dermis was assayed for collagen biosynthesis (by measuring the production of [14C]hydroxyproline (HP) and amount of radioactive peptides released after collagenase digestion) and absolute HP (by spectrophotometric analysis). The epidermis was evaluated macroscopically for resurfacing. Aerobic bacteria from unwounded and wounded skin were identified and quantitated. There were no significant differences between treatment groups in HP incorporation or absolute collagen content from Days 2-6 after wounding. HP incorporation in the total protein fractions and in the collagenase digestible fractions were analogous. Collagen biosynthesis was similar in both unwounded, untreated, and unwounded DMSO-treated skin. Epidermal healing did not differ between treatment groups. There were no differences in the number or types of bacteria in wounds between treatment groups. These results indicate that topical DMSO is neither beneficial nor harmful in the healing of superficial wounds.