Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.     

Inhibition of wound closure by transforming growth factor-β and dexamethasone in a fetal mouse limb organ culture model

Tissue repair comprises several physiologic processes including the deposition of a newly synthesized connective tissue matrix and the regeneration of the epidermis by reepithelialization. A fetal mouse limb organ culture system facilitates the investigation of both reepithelialization and connective tissue deposition in repair within a controlled environment. A sutured closed wound in an intact 18.5-day old fetal mouse limb completely heals by 7 days. Including dexamethasone in the media inhibited both reepithelialization and connective tissue deposition. Concurrent administration of transforming growth factor-beta with dexamethasone restored the deposition of connective tissue but did not restore reepithelialization. When transforming growth factor-beta was given alone, connective tissue deposition was enhanced at both the wound site and in contiguous dermis, but reepithelialization did not proceed. Transforming growth factor-beta inhibited wound closure by blocking the migration of epidermal cells. The organ-cultured, wounded fetal mouse limb system is sufficiently sensitive to show both mesenchymal cell-enhancing activity as well as epithelial migration inhibiting activity by transforming growth factor-beta. The in vitro repair of fetal mouse limbs may serve as an in vitro system to test the influence of soluble agents on the repair process.     

Gastrin-Releasing Peptide, a Bombesin-like Neuropeptide, Promotes Cutaneous Wound Healing

BACKGROUND: Little is known about the effects of neuropeptides on wound healing. OBJECTIVE: To investigate the effect of gastrin-releasing peptide (GRP), one of the bombesin-like neuropeptides, on wound healing. METHODS: The effects of GRP on cultured keratinocyte proliferation and migration were measured by BrdU uptake and in vitro scratch assay, respectively. Various concentrations of GRP ointments (0, 10(-9), 10(-8), 10(-7), 10(-6) M) were topically applied to 1.0 mm wounds on porcine flanks. RESULTS: GRP stimulated keratinocyte growth and locomotion in a dose-dependent manner. Topical administration of GRP accelerated macroscopic epidermal regeneration in a dose-dependent manner, as measured by planimetry. Histologic studies also showed that GRP promoted reepithelialization, including epidermal thickness as well as superficial skin coverage. CONCLUSION: Topical use of GRP may clinically accelerate wound healing of burns, injuries, chronic ulcers, and skin graft donor sites through the enhancement of keratinocyte growth and spreading.     

Lesional skin in vitiligo exhibits delayed in vivo reepithelialization compared to the nonlesional skin

Vitiligo, a common skin disorder, is characterized by the loss of functional melanocytes resulting in the depigmentation of skin. Previous studies have demonstrated molecular and architectural alterations in the epidermal keratinocytes upon loss of melanocytes. The physiological implications of these “altered” keratinocytes are yet not known. We investigated the wound healing efficiency of lesional vs nonlesional skin in 12 subjects with stable nonsegmental vitiligo using histological and ultrastructural evaluation of partial‐thickness wounds. The wounds were examined 12 days postinjury, coinciding with the reepithelialization phase of healing marked primarily by keratinocyte migration and proliferation. This study demonstrated a significant difference in the reepithelialization potential between the lesional and nonlesional skin. While all 12 nonlesional wounds demonstrated considerable neoepidermis formation on the 12th day post wound, only four of the corresponding lesional samples showed comparable reepithelialization; the rest remaining in the inflammatory phase. Ultrastructural studies using transmission electron microscopy as well as immunohistochemical staining revealed a reduced number of desmosomes, shorter keratin tonofilaments and an increase in myofibroblast population in the dermis of lesional reepithelialized tissue compared to the nonlesional reepithelialized samples. This study implicates gross functional perturbations in the lesional skin during physiological wound healing in vitiligo, suggesting that the breakdown of keratinocyte‐melanocyte network results in delayed wound repair kinetics in the lesional skin when compared to patient‐matched nonlesional skin.     

Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation

Various growth factors such as epidermal growth factor and keratinocyte growth factor have been reported to promote wound closure and epidermal regeneration. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of epidermal growth factor and keratinocyte growth factor on keratinocyte proliferation, migration and differentiation. Our results show that epidermal growth factor supplemented cultures share many of the features which are observed during regeneration of wounded epidermis: a thickening of the entire epidermis, an enhanced rate of proliferation and migration, and an increase in keratin 6, keratin 16, skin-derived antileukoproteinase, involucrin and transglutaminase 1 expression. The increase in transglutaminase 1 protein is accompanied by an increase in the amount of active transglutaminase 1 enzyme. Surprisingly no increase in keratin 17 is observed. Prolonging the culture period for more than two weeks results in rapid senescence and aging of the cultures. In contrast, keratinocyte growth factor supplemented cultures have a tissue architecture that is similar to healthy native epidermis and remains unchanged for at least 4 weeks of air-exposure. The rate of proliferation and the expression of keratins 6, 16 and 17, skin-derived antileukoproteinase, involucrin and transglutaminase 1 is similar to that found in healthy epidermis and furthermore keratinocyte migration does not occur. When the culture medium is supplemented with a combination of keratinocyte growth factor and a low concentration of epidermal growth factor, skin-derived antileukoproteinase, involucrin and keratins 6, 16 and 17 expression is similar to that found in cultures supplemented with keratinocyte growth factor alone and in healthy epidermis. Only high transglutaminase 1 expression remains similar to that observed in cultures supplemented with epidermal growth factor alone. Our results show that the regulation of keratinocyte growth, migration and differentiation depends on the availability of these growth factors. Epidermal growth factor may play a dominant early role in wound healing by stimulating keratinocyte proliferation and migration while keratinocyte growth factor may play a role later in the repair process by stabilizing epidermal turnover and barrier function.     

FAK induction in keratinocytes in an in vitro model of reepithelialization

During reepithelialization, keratinocytes must become activated in order to migrate over the provisional extracellular matrix of the wound. Previously we have shown that focal adhesion kinase (FAK) is induced in activated keratinocytes. The mechanisms responsible for keratinocyte activation are unknown. Here we use an organ culture system to investigate FAK up-regulation and regulation of keratinocyte activation. Normal human skin was cultured on type I collagen. Keratinocytes migrated out of the explant onto the supporting collagen. Immunostaining for FAK showed induction in the migrating epithelium and also in the center of the explant some distance from the cut edge. Cells from the center of the explant expressed FAK and showed the activated phenotype as defined by their ability to spread on collagen. Since FAK is a tyrosine kinase, the tyrosine kinase inhibitors genistein or herbimycin A were added to the explant medium for 24 h. Inhibition of tyrosine kinase activity delayed epithelial migration, but keratinocytes were able to begin migrating after removal of the inhibitors. We conclude that FAK is up-regulated in keratinocytes in this whole skin explant model. Furthermore FAK up-regulation and keratinocyte activation are not confined to the migrating cells but are found in cells some distance from the skin margin. These data suggest that (1) cell migration, contact with wound matrix molecules, loss of cell-cell contact, or loss of basement membrane contact is not necessary for FAK up-regulation or keratinocyte activation; and (2) tyrosine kinase signaling pathways are important for reepithelialization.     

Effects of keratinocyte conditioned medium,amniotic fluid and EGF in reepithelialization of human skin wounds in vitro

Growth factors and combinations of growth promoting substances in amniotic fluid, platelet extracts or factors secreted by cultured human keratinocytes have been shown to stimulate cell proliferation and wound healing. In this report, the possibility of studying reepithelialization of wounds in human skin, using small biopsies maintained in vitro, was examined. The effects of fetal calf serum (FCS), keratinocyte conditioned medium (KCM), amniotic fluid and epidermal growth factor (EGF) on the reepithelialization process was investigated after seven days. KCM alone did not affect healing, but when added to a suboptimal concentration of FCS (2%), KCM induced reepithelialization of the wounds. Amniotic fluid (25%) alone stimulated the formation of a new epidermis, whereas EGF (10 ng/ml) alone or added to 2% FCS did not induce reepithelialization. The model used in this study includes an autologous matrix comprising living fibroblasts and endothelial cells and may thus be used to study aspects of wound healing in human skin.     

Inhibition of keratinocyte migration by lipopolysaccharide

A critical process in cutaneous wound healing is reepithelialization by keratinocytes that closes the breach in the epidermis. Chronic wounds fail to reepithelialize despite the presence of activated and proliferative keratinocytes around the wound perimeter. This type of wound is generally colonized to a greater or lesser extent by bacteria. This study examines the possibility that bacterial products might directly inhibit keratinocyte migration. Using conventional scratch assays, we observed a dose-dependent inhibition of keratinocyte migration by lipopolysaccharide (LPS) derived from either Pseudomonas aeruginosa or Escherichia coli . Although the P. aeruginosa preparation appeared to be slightly more inhibitory, both gave half-maximal inhibition at 0.5–0.6 ng/mL. Migration of fibroblasts was not inhibited. The result could not be attributed to a cytotoxic effect of the LPS. LPS inhibition of migration was relieved by neutralizing antibodies to toll-like receptors (TLR), 40% by anti-TLR2 and 75% by anti-TLR4. We conclude that keratinocyte migration is inhibited by bacterial products, detected through TLR4 and also through TLR2. Because chronic wounds always show some presence of bacteria, these findings provide a possible explanation for the lack of healing found in ulcers.     

Development of a multilayered in vitro model for studying events associated with wound healing

Simplified in vitro models such as cellular monolayer cultures have only limited usefulness in the study of cutaneous wound repair processes. This has stimulated the investigation of three-dimensional tissue equivalent systems such as the dermal and skin equivalent models. With the use of a wound system constructed of rat tail type I collagen and human dermal fibroblasts, experimental wounding was accompanied by problems with mechanical scoring of the plastic substratum which prevented cell migration. These problems were overcome with the use of a multilayered model in which a punch biopsy-wounded dermal equivalent (bilayered model) or skin equivalent (tri-layered model) was placed onto an acellular collagen lattice and fixed in place with polymerizing collagen. This model permitted observation of the process of cellular repopulation of the "wound space," into which both fibroblast and keratinocyte migration commenced within 1 day. The number of fibroblasts in this space increased dramatically over a period of 9 days, the cells appearing to migrate both over and through the acellular lower collagen layer. Keratinocyte reepithelialization of the "wound space" was completed after 5 days. With the model it was shown that platelet-derived growth factor--AB and epidermal growth factor had positive effects in increasing fibroblast number within the wound space. In conclusion, the model described here should facilitate the study of fibroblast and keratinocyte responses to a wound stimulus in vitro and be a plausible in vitro system for evaluating agents which may have a potential stimulatory or inhibitory effect on numerous cellular responses associated with wound healing.     

Transforming growth factor-β and its effect on reepithelialization of partial-thickness ear wounds in transgenic mice

Transforming growth factor-beta (TGF-beta) is known to affect nearly every aspect of wound repair. Many of the effects have been extensively investigated; however, the primary effect of endogenously derived TGF-beta on wound reepithelialization is still not completely understood. To examine this, two types of wounds were made on a transgenic mouse over-expressing TGF-beta1. Full-thickness back wounds were made to compare the wound healing process in the presence of compensatory healing mechanisms. Superficial partial-thickness ear wounds involving only the epidermis were made to determine the effect of TGF-beta on reepithelialization. In the partial-thickness ear wounds, at later time points, the transgenic group had smaller epithelial gaps than the wild-type mice. A greater number of actively proliferating cells, as determined by bromodeoxyuridine incorporation, was also found in the transgenic mice at post-injury day 8. These results show that TGF-beta1 stimulates the rate of reepithelialization at later time points in partial-thickness wounds. However, in the full-thickness back wounds, the transgenic animals exhibited a slower reepithelialization rate at all time points and the number of bromodeoxyuridine-positive cells was fewer. Our findings would suggest that the overexpression of TGF-beta1 speeds the rate of wound closure in partial-thickness wounds by promoting keratinocyte migration. In full-thickness wounds, however, the overexpression of TGF-beta1 slows the rate of wound reepithelialization.     

Role of wound healing myofibroblasts on re-epithelialization of human skin

In human skin, large burned surfaces heal using two concomitant phenomena: re-epithelialization and dermal neoformation. Numerous studies report the role of interactions between keratinocytes and fibroblasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. We investigated the influence of myofibroblasts on keratinocyte growth and differentiation using an in vitro skin model. A histological study was performed to determine the speed and quality of epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7-10 days. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10-day period studied. After 7 further days of epidermal differentiation, histology showed an epidermis more disorganized and expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. These results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGFbeta1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change re-epithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus, TGFbeta1 action is not sufficient to explain all the different keratinocyte reactions towards fibroblasts and wound healing myofibroblasts. Our conclusion is that myofibroblasts seem to have a limited role in the re-epithelialization process and might be more associated with the increased extracellular matrix secretion.     

An in vitro wound healing model for evaluation of dermal substitutes

Reepithelialization of skin wounds is essential to restore barrier function and prevent infection. This process requires coordination of keratinocyte proliferation, migration, and differentiation, which may be impeded by various extrinsic and host‐dependent factors. Deep, full‐thickness wounds, e.g., burns, are often grafted with dermal matrices before transplantation of split‐skin grafts. These dermal matrices need to be integrated in the host skin and serve as a substrate for neoepidermis formation. Systematic preclinical analysis of keratinocyte migration on established and experimental matrices has been hampered by the lack of suitable in vitro model systems. Here, we developed an in vitro full‐thickness wound healing model in tissue‐engineered human skin that allowed analysis of the reepithelialization process across different grafted dermal substitutes. We observed strong differences between porous and nonporous matrices, the latter being superior for reepithelialization. This finding was corroborated in rodent wound healing models. The model was optimized using lentivirus‐transduced keratinocytes expressing enhanced green fluorescent protein and by the addition of human blood, which accelerated keratinocyte migration underneath the clot. Our model shows great potential for preclinical evaluation of tissue‐engineered dermal substitutes in a medium‐throughput format, thereby obviating the use of large numbers of experimental animals.     

Gallium nitrate accelerates partial thickness wound repair and alters keratinocyte integrin expression to favor a motile phenotype

The nitrate form of the Group III transitional element gallium (GN) increases expression of specific structural components of the provisional wound matrix (i.e., collagen type I, fibronectin) in human dermal fibroblasts. To evaluate the potential of GN as a therapeutic option in management of cutaneous trauma, GN-treated partial thickness porcine wounds and experimentally "injured" human keratinocyte (NHK) monolayer cultures were compared with mirror image control (i.e., saline-treated) sites. GN suppressed cell proliferation in both models, as determined by reduced Ki-67 reactivity and significant lengthening of keratinocyte cell cycle transit times, while effectively promoting reepithelialization. The primary effect of GN was apparently to promote cell migration, as neither epidermal thickness nor epidermal differentiation was altered as a result of GN exposure in vivo or in vitro. Significantly enhanced epidermal reepithelialization was associated with alterations in expression of several keratinocyte integrin subunits. GN induced a significant increase in alpha5 expression. alpha5beta1 switching is a characteristic of the motile phenotype in the setting of cutaneous injury. Concomitantly, GN treatment also induced a dramatic (70%) decrease in the expression of the alpha3 subunit; alpha3beta1 binds laminin 5 and is associated with hemidesmosome formation and reestablishment of a nonmotile phenotype. Taken together, the GN-induced changes in integrin expression favor acellular migration. While the molecular mechanism of GN action on resident cells of the skin remains to be defined, these data suggest that GN administration which represses MMP activity in the wound and increases matrix synthesis also accelerates NHK motility and, thereby, may be a useful therapeutic agent for wound repair.     

Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization

To investigate the mechanisms behind the antifibrotic effect associated with epidermal regeneration, the expression of 12 fibroblast genes important for the modulation of the extracellular matrix (ECM), as well as α‐smooth muscle actin, was studied in a keratinocyte‐fibroblast organotypic skin culture model. The study was performed over time during epidermal generation and in the presence or absence of the profibrotic factor transforming growth factor‐β. the Presence of epidermal differentiation markers in the model was essentially coherent with that of native skin. Fibroblast gene expression was analyzed with real‐time polymerase chain reaction after removal of the epidermal layer. After 2 days of air‐exposed culture, 11 out of the 13 genes studied were significantly regulated by keratinocytes in the absence or presence of transforming growth factor‐β. The regulation of connective tissue growth factor, collagen I and III, fibronectin, plasmin system regulators, matrix metalloproteinases and their inhibitors as well as α‐smooth muscle actin was consistent with a suppression of ECM formation or contraction. Overall, the results support a view that keratinocytes regulate fibroblasts to act catabolically on the ECM in epithelialization processes. This provides possible mechanisms for the clinical observations that reepithelialization and epidermal wound coverage counteract excessive scar formation.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Development of an in vitro burn wound model

Healing of a deeper burn wound is a complex process that often leads to scar formation. Skin wound model systems are important for the development of treatments preventing scarring. The aim of this study is to develop a standardized in vitro burn wound model that resembles the in vivo situation. A burn wound (10 × 2 mm) was made in ex vivo skin and the skin samples were cultured at the air–liquid interface for 7, 14, and 21 days. Cells in the skin biopsies maintained their viability during the 21-day culture period. During culture, reepithelialization of the wound took place from the surrounding tissue and fibroblasts migrated into the wound area. Cells of the epithelial tongue and fibroblasts near the wound margin were proliferating. During culture, skin-derived antileukoproteinase and keratin 17 were expressed only in the epithelial tongue. Both collagen type IV and laminin were present underneath the newly formed epidermis, indicating that the basement membrane was restored. These results show that the burn wound model has many similarities to in vivo wound healing. This burn wound model may be useful to study different aspects of wound healing and testing pharmaceuticals and cosmetics on, e.g., migration and reepithelialization.     

Autologous keratinocyte suspensions accelerate epidermal wound healing in pigs

BACKGROUND: Tissue culture techniques enable in vitro expansion of keratinocytes that can be used to treat burns and chronic wounds. These keratinocytes are commonly grafted onto the wounds as differentiated sheets of mature epithelium. Less is however known about the effects of transplanting the cells as suspensions. This study evaluated epidermal regeneration in fluid-treated skin wounds treated with suspensions of cultured and noncultured autologous keratinocytes. MATERIALS AND METHODS: Eighty-seven full-thickness excisional skin wounds were created on the back of 6 pigs and then transplanted with either cultured or noncultured autologous keratinocytes. The wounds were enclosed with liquid-tight chambers containing saline to provide a hydrated and standardized environment. RESULTS: Keratinocyte transplantation resulted in several cell colonies within the granulation tissue of the wound. These colonies progressively coalesced and contributed to a new epithelium. The origin of the transplanted keratinocytes was confirmed by histochemical staining of wounds transplanted with transfected keratinocytes expressing beta-galactosidase. Transplantation of 0.125 x 10(6), 0.5 x 10(6), and 2.0 x 10(6) cultured keratinocytes, and 0.5 x 10(6) and 5.0 x 10(6) noncultured keratinocytes, increased reepithelialization dose dependently over saline-treated controls. The epithelial barrier function recovered faster in transplanted wounds as demonstrated by less protein leakage over the wound surface on Days 7-10 as compared to control wounds. Wound reepithelialization and the number of keratinocyte colonies observed in granulation tissue were significantly less in wounds transplanted with noncultured keratinocytes compared to wounds seeded with cultured keratinocytes. CONCLUSION: Our study demonstrates successful transplantation of keratinocyte suspensions and their dose-dependent acceleration of wound repair. Selection of proliferative cells during culture and higher colony-forming efficiency may explain the greater effects observed with cultured keratinocytes.     

Use of an in vitro model of tissue-engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization

To produce a stable epidermis, keratinocytes need to be firmly attached to the basement membrane. However, following wounding, keratinocytes are required to develop a migratory phenotype in order to reepithelialize the wound. To investigate some of the issues underlying reepithelialization, we have developed a three-dimensional in vitro model of tissue-engineered skin, comprising sterilized human dermis seeded with human keratinocytes and dermal fibroblasts. Using this model, we have shown that the inclusion of fibroblasts within the model increases the stability of keratinocyte attachment. We have also demonstrated that keratinocyte migration occurs most effectively in the absence of a basement membrane and following the inclusion of fibroblasts in the model. In addition, subjecting the keratinocyte layer to mechanical trauma induces a migratory phenotype. We conclude that this three-dimensional in vitro wound model can be used to increase our understanding of the factors that enhance keratinocyte migration and hence wound healing in vivo.