Keratinocyte outgrowth from human skin explant cultures is dependent upon p38 signaling

Organ culture of skin is known to recapitulate several early events in the process of wound healing. Here we investigate the function of p38 kinase signaling as a regulator of keratinocyte behavior in human skin organ culture. We first show that skin organ culture recapitulates the transition from migration to proliferation that is known to characterize the reepithelialization process. We next show that inhibition of p38 markedly impairs the formation of keratinocyte outgrowth in human skin explant cultures, as well as the migration of keratinocytes in an in vitro wound assay. In contrast, the marked induction of mRNA encoding the ErbB ligand heparin-binding epidermal growth factor-like growth factor, known to occur after skin wounding, was not blocked by inhibition of p38. As assessed by immunoblotting, phosphorylation of p38 was limited and was not increased between 0 and 7 days of organ culture. Our results show the sensitivity of reepithelialization to inhibition by p38 and suggest that p38 acts primarily during the migration phase of this process. These data also indicate that autocrine heparin-binding epidermal growth factor expression is not regulated by p38.     

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.     

Up‐regulation of fibroblast growth factor (FGF) 9 expression and FGF‐WNT/β‐catenin signaling in laser‐induced wound healing

Fibroblast growth factor (FGF) 9 is secreted by both mesothelial and epithelial cells, and plays important roles in organ development and wound healing via WNT/β‐catenin signaling. The aim of this study was to evaluate FGF9 expression and FGF‐WNT/β‐catenin signaling during wound healing of the skin. We investigated FGF9 expression and FGF‐WNT/β‐catenin signaling after laser ablation of mouse skin and adult human skin, as well as in cultured normal human epidermal keratinocytes (NHEKs) upon stimulation with recombinant human (rh) FGF9 and rh‐transforming growth factor (TGF)‐β1. Our results showed that laser ablation of both mouse skin and human skin leads to marked overexpression of FGF9 and FGF9 mRNA. Control NHEKs constitutively expressed FGF9, WNT7b, WNT2, and β‐catenin, but did not show Snail or FGF receptor (FGFR) 2 expression. We also found that FGFR2 was significantly induced in NHEKs by rhFGF9 stimulation, and observed that FGFR2 expression was slightly up‐regulated on particular days during the wound healing process after ablative laser therapy. Both WNT7b and WNT2 showed up‐regulated protein expression during the laser‐induced wound healing process in mouse skin; moreover, we discerned that the stimulatory effect of rhFGF9 and rhTGF‐β1 activates WNT/β‐catenin signaling via WNT7b in cultured NHEKs. Our data indicated that rhFGF9 and/or rhTGF‐β1 up‐regulate FGFR2, WNT7b, and β‐catenin, but not FGF9 and Snail; pretreatment with rh dickkopf‐1 significantly inhibited the up‐regulation of FGFR2, WNT7b, and β‐catenin. Our results suggested that FGF9 and FGF‐WNT/β‐catenin signaling may play important roles in ablative laser‐induced wound healing processes.     

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.     

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.     

Optical coherence tomography for assessment of epithelialization in a human ex vivo wound model

The ex vivo human skin wound model is a widely accepted model to study wound epithelialization. Due to a lack of animal models that fully replicate human conditions, the ex vivo model is a valuable tool to study mechanisms of wound reepithelialization, as well as for preclinical testing of novel therapeutics. The current standard for assessment of wound healing in this model is histomorphometric analysis, which is labor intensive, time consuming, and requires multiple biological and technical replicates in addition to assessment of different time points. Optical coherence tomography (OCT) is an emerging noninvasive imaging technology originally developed for noninvasive retinal scans that avoids the deleterious effects of tissue processing. This study investigated OCT as a novel method for assessing reepithelialization in the human ex vivo wound model. Excisional ex vivo wounds were created, maintained at air‐liquid interface, and healing progression was assessed at days 4 and 7 with OCT and histology. OCT provided adequate resolution to identify the epidermis, the papillary and reticular dermis, and importantly, migrating epithelium in the wound bed. We have deployed OCT as a noninvasive tool to produce, longitudinal “optical biopsies” of ex vivo human wound healing process, and we established an optimal quantification method of re‐epithelialization based on en face OCT images of the total wound area. Pairwise statistical analysis of OCT and histology based quantifications for the rate of epithelialization have shown the feasibility and superiority of OCT technology for noninvasive monitoring of human wound epithelialization. Furthermore, we have utilized OCT to evaluate therapeutic potential of allogeneic adipose stem cells revealing their ability to promote reepithelialization in human ex vivo wounds. OCT technology is promising for its applications in wound healing and evaluation of novel therapeutics in both the laboratory and the clinical settings.     

Akermanite bioceramic enhances wound healing with accelerated reepithelialization by promoting proliferation,migration, and stemness of epidermal cells

Reepithelialization is an important step of wound healing, which is mainly completed by proliferation and migration of epidermal cells. Akermanite is a Ca‐, Mg‐, and Si‐containing bioceramic. This study evaluated the effects of Akermanite on wound healing and investigated the mechanisms. Using scald burn mice models, we demonstrated that local Akermanite treatment significantly accelerated wound healing by increasing reepithelialization and the stemness of epidermal cells. Epidermal cells were cultured in medium containing Akermanite extracts to explore the cellular mechanism of reepithelialization. Akermanite promoted the cell proliferation and migration, maintaining more cells in the S and G₂/M phases of the cell cycle. An additional study showed that Akermanite enhanced the expressions of integrinβ1, Lgr4, Lgr5, and Lgr6, which are specific molecular markers of epidermal stem cells, accompanied by the activation of the Wnt/β‐catenin pathway. These results suggested that Akermanite accelerated reepithelialization by increasing the proliferation, migration, and stemness of epidermal cells in a manner related to the Wnt/β‐catenin pathway, which might contribute, at least partially, to accelerated wound healing by Akermanite therapy.     

Identification of a transcriptional signature for the wound healing continuum

There is a spectrum/continuum of adult human wound healing outcomes ranging from the enhanced (nearly scarless) healing observed in oral mucosa to scarring within skin and the nonhealing of chronic skin wounds. Central to these outcomes is the role of the fibroblast. Global gene expression profiling utilizing microarrays is starting to give insight into the role of such cells during the healing process, but no studies to date have produced a gene signature for this wound healing continuum. Microarray analysis of adult oral mucosal fibroblast (OMF), normal skin fibroblast (NF), and chronic wound fibroblast (CWF) at 0 and 6 hours post‐serum stimulation was performed. Genes whose expression increases following serum exposure in the order OMF < NF < CWF are candidates for a negative/impaired healing phenotype (the dysfunctional healing group), whereas genes with the converse pattern are potentially associated with a positive/preferential healing phenotype (the enhanced healing group). Sixty‐six genes in the enhanced healing group and 38 genes in the dysfunctional healing group were identified. Overrepresentation analysis revealed pathways directly and indirectly associated with wound healing and aging and additional categories associated with differentiation, development, and morphogenesis. Knowledge of this wound healing continuum gene signature may in turn assist in the therapeutic assessment/treatment of a patient's wounds.     

Gene expression profiling of negative-pressure-treated skin graft donor site wounds

Negative-pressure wound therapy (NPWT) is widely used to improve skin wound healing. Although NPWT has been studied as a treatment for wound closure and healing, the molecular mechanisms explaining its therapeutic effects remain unclear. To investigate the effect of NPWT on gene expression, and to discover the genes most dominantly responding to this treatment during skin wound healing, we applied negative pressure on split-thickness skin graft donor sites from the first postoperative day (POD) to the seventh POD. Biopsies were collected from 4 NPWT-treated and 2 control patients. Two biopsy samples were taken from each patient: one from intact skin before graft harvesting, and one on the seventh POD from the donor site wound. Genome-wide microarrays were performed on all samples. Gene expression changes on the seventh POD were compared between NPWT and control patients, and were analyzed for statistical significance. In addition, we analyzed wound exudates for volume, and for concentrations of leukocytes, erythrocytes, and haemoglobin. NPWT induced major changes in gene expression during healing. These changes ranged from 10-fold induction to 27-fold suppression. The genes most induced were associated with cell proliferation and inflammation, and the most down-regulated genes were linked to epidermal differentiation. Our results provide the first insight into the molecular mechanisms behind NPWT, and suggest that NPWT enhances specific inflammatory gene expression at the acute phase associated with epithelial migration and wound healing. However, its continued use may inhibit epithelial differentiation.     

Accelerated healing in NONcNZO10/LtJ type 2 diabetic mice by FGF‐1

The development of novel therapies to treat chronic diabetic ulcers depends upon appropriate animal models for early stage investigation. The NONcNZO10/LtJ mouse is a new polygenic strain developed to more realistically model human metabolic syndrome and obesity‐induced type 2 diabetes; however, detailed wound healing properties have not been reported. Herein, we describe a quantitative wound healing study in the NONcNZO10/LtJ mouse using a splinted excisional wound. The rate of wound healing is compared to various controls, and is also quantified in response to topical administration of normal and mutant fibroblast growth factor‐1 (FGF‐1). Quantitation of reepithelialization shows that the diabetic condition in the NONcNZO10/LtJ mouse is concomitant with a decreased rate of dermal healing. Furthermore, topical administration of a FGF‐1/heparin formulation effectively accelerates reepithelialization. A similar acceleration can also be achieved by a stabilized mutant form of FGF‐1 formulated in the absence of heparin. Such accelerated rates of healing are not associated with any abnormal histology in the healed wounds. The results identify the NONcNZO10/LtJ mouse as a useful model of impaired wound healing in type 2 diabetes, and further, identify engineered forms of FGF‐1 as a potential “second‐generation” therapeutic to promote diabetic dermal wound healing.     

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.     

Cutaneous wound healing in aging small mammals: a systematic review

As the elderly population grows, so do the clinical and socioeconomic burdens of nonhealing cutaneous wounds, the majority of which are seen among persons over 60 years of age. Human studies on how aging effects wound healing will always be the gold standard, but studies have ethical and practical hurdles. Choosing an animal model is dictated by costs and animal lifespan that preclude large animal use. Here, we review the current literature on how aging effects cutaneous wound healing in small animal models and, when possible, compare healing across studies. Using a literature search of MEDLINE/PubMed databases, studies were limited to those that utilized full‐thickness wounds and compared the wound‐healing parameters of wound closure, reepithelialization, granulation tissue fill, and tensile strength between young and aged cohorts. Overall, wound closure, reepithelialization, and granulation tissue fill were delayed or decreased with aging across different strains of mice and rats. Aging in mice was associated with lower tensile strength early in the wound healing process, but greater tensile strength later in the wound healing process. Similarly, aging in rats was associated with lower tensile strength early in the wound healing process, but no significant tensile strength difference between young and old rats later in healing wounds. From studies in New Zealand White rabbits, we found that reepithelialization and granulation tissue fill were delayed or decreased overall with aging. While similarities and differences in key wound healing parameters were noted between different strains and species, the comparability across the studies was highly questionable, highlighted by wide variability in experimental design and reporting. In future studies, standardized experimental design and reporting would help to establish comparable study groups, and advance the overall knowledge base, facilitating the translatability of animal data to the human clinical condition.     

Localization of small leucine‐rich proteoglycans and transforming growth factor‐β in human oral mucosal wound healing

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine‐rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor‐β (TGF‐β) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF‐β during non‐scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3–60 days postwounding. Localization of SLRPs, TGF‐β1 and TGF‐β3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF‐β in distinct cells. Decorin, biglycan, fibromodulin, and TGF‐β isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF‐β isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.     

Complement activation at the interface of wound dressings and blood does not influence keratinocyte migration/proliferation in vitro

Recently, we reported that some wound dressings caused complement activation at the interface of wound dressing and blood. Since complement activation is associated with impaired wound healing, we investigated whether this activation of the complement cascade at the interface of wound dressings and blood does impair reepithelialization in a scratch wound healing assay. Although some samples showed higher levels of the complement activation marker SC5b‐9 in our study, reepithelialization of the samples did not significantly differ from the control group. Further studies have to clarify if complement activation at the interface of wound dressings and blood plays a relevant role in the healing process especially in long‐time experiments.     

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.     

Exogenous calreticulin improves diabetic wound healing

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full‐thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild‐type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRT's potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.     

Gene expression of tenascin is altered in normal scars and keloids

Tenascin is an extracellular matrix molecule with structural similarity to fibronectin. An increase in extracellular matrix content of both tenascin and fibronectin is associated with early wound healing and with various skin fibroses. However, the relationship of tenascin and fibronectin expression during scar remodeling and the formation of pathologic scars such as keloids is unknown. Expression of tenascin in normal and abnormal human scars was examined and compared with that of fibronectin by immunohistochemistry and in situ hybridization. Tenascin and fibronectin protein and messenger RNA contents were elevated in normal, mature scars relative to quiescent skin, similar to the situation during earlier stages of healing. Tenascin and fibronectin expression was further enhanced in keloids relative to normal skin and scar, and, as has been shown for fibronectin, tenascin expression in uninjured skin adjacent to keloids was indistinguishable from that in quiescent skin from unaffected individuals. These data suggest that tenascin and fibronectin gene expression are coordinated during later stages of normal wound healing and that a defect involving common regulatory elements for these genes is associated with the formation of keloids.     

Cytokeratin, filaggrin, and p63 expression in reepithelialization during human cutaneous wound healing

Cytokeratin (CK), filaggrin (filament aggregating protein), and p63 expression and cellular distribution during reepithelialization has not been systemically studied in the healing stage of human cutaneous wounds. We examined these proteins by immunohistochemical methods in 12 cases of skin ulcer, using seven anti-keratin antibodies, anti-filaggrin, and anti-p63 antibody. At the edge of the wound in skin ulcers, CK1 and 10 expression was reduced, while CK14, 16, and 17 expression was raised. Beneath the wound bed, all layers of the epidermal tongue, deriving from sweat ducts, were positive for CK14 and 17. Both cytokeratins were also found in basal and luminal cells of the dermal duct. CK expression by epithelia continuous with hair follicles showed that, CK14, 16, and 17 were present, and CK1 and 10 were absent. Filaggrin expression was elevated in reepithelialized epithelium. Expression of p63 expression was verified in the suprabasal layer in reepithelialized epithelia. CK, filaggrin, and p63 expression in the reepithelialization stage at the wound edge and at epidermal appendages remaining in the wound bed is undifferentiated and hyperproliferative. The presence of CK14 and 17 in the remaining epidermal appendages in the pathological wound may be important in epidermal replacement.