Epidermal Re-epithelialization Research Articles

Single-cell profiling and functional screening reveal crucial roles for lncRNAs in the epidermal re-epithelialization of human acute wounds.

Re-epithelialization is an important physiological process for repairing skin barrier function during wound healing. It is primarily mediated by coordinated migration, proliferation, and differentiation of keratinocytes. Long noncoding RNAs (lncRNAs) are essential components of the noncoding genome and participate in various biological processes; however, their expression profiles and function in re-epithelialization during wound healing have not been established. We investigated the distribution of lncRNAs during wound re-epithelialization by comparing the genomic profiles of uninjured skin and acute wound (AW) from healthy donors. We performed functional screening of differentially expressed lncRNAs to identify the important lncRNAs for re-epithelialization. The expression of multiple lncRNAs is changed during human wound re-epithelialization process. We identified VIM-AS1, SMAD5-AS1, and LINC02581 as critical regulators involved in keratinocyte migration, proliferation, and differentiation, respectively. LncRNAs play crucial regulatory roles in wound re-epithelialization. We established lncRNA expression profile in human acute wounds compared with intact skin, offering valuable insights into the physiological mechanisms underlying wound healing and potential therapeutic targets.

Nanoapatite-Loaded κ-Carrageenan/Poly(vinyl alcohol)-Based Injectable Cryogel for Hemostasis and Wound Healing.

Immediate control of excessive bleeding and prevention of infections are of utmost importance in the management of wounds. Cryogels have emerged as promising materials for the rapid release of medication and achieving hemostasis. However, their quick release properties pose the challenge of exposing patients to high concentrations of drugs. In this study, hybrid nanocomposites were developed to address this issue by combining poly(vinyl alcohol) and κ-carrageenan with whitlockite nanoapatite (WNA) particles and ciprofloxacin, aiming to achieve rapid hemostasis and sustained antibacterial effects. A physically cross-linked cryogel was obtained by subjecting a blend of poly(vinyl alcohol) and κ-carrageenan to successive freezing-thawing cycles, followed by the addition of WNA. Furthermore, ciprofloxacin was introduced into the cryogel matrix for subsequent evaluation of its wound healing properties. The resulting gel system exhibited a 3D microporous structure and demonstrated excellent swelling, low cytotoxicity, and outstanding mechanical properties. These characteristics were evaluated through analytical and rheological experiments. The nanocomposite cryogel with 4% whitlockite showed extended drug release of 71.21 ± 3.5% over 21 days and antibacterial activity with a considerable growth inhibition zone (4.19 ± 3.55 cm). Experiments on a rat model demonstrated a rapid hemostasis property of cryogels within an average of 83 ± 4 s and accelerated the process of wound healing with 96.34% contraction compared to the standard, which exhibited only ∼78% after 14 days. The histopathological analysis revealed that the process of epidermal re-epithelialization took around 14 days following the skin incision. The cryogel loaded with WNAs and ciprofloxacin holds great potential for strategic utilization in wound management applications as an effective material for hemostasis and anti-infection purposes.

Eco-Friendly Synthesis of Silver Nanoparticles by Nitrosalsola vermiculata to Promote Skin Wound Healing

Eco-friendly synthesis of silver nanoparticles (SN) by using a naturally occurring plant, such as Nitrosalsola (Salsola) vermiculata (SV), could be a novel way for appropriate wound healing. AgNO3 was reduced by SV to produce safe SN (SN-SV) extract and hasten the wound healing process. The obtained SN-SV were characterized by size, charge, wavelength, and surface morphology. The optimized formulation was dispersed in O/W cosmetic cream. Then, it was characterized in terms of pH, viscosity, homogeneity, and permeability. The ex vivo and in vivo studies have been conducted in a rat animal model to assess the potential of SN-SV cream on skin tissue regeneration. A skin punch biopsy was obtained to investigate the histopathological (HP) changes in the skin lesions of all rats by the H&E staining and PCNA immunostaining methods. The skin wounds in all subgroups were examined on days 5, 11, and 15 to analyze the effectiveness of SN-SV cream for treating surgical skin wounds. The prepared SN-SV had a particle size of 37.32 ± 1.686 nm, a charge of −1.4 ± 0.7 mV, non-aggregated SN-SV, and a λmax of 396.46 nm. The formed SN-SV cream showed a pH near the skin’s pH, with suitable viscosity and homogeneity and an apparent permeability of 0.009 ± 0.001. The HP changes in the SN-SV subgroups revealed a substantial reduction in wound size and improvement in wound granulation tissue formation and epidermal re-epithelialization (proliferation) compared to the healing in the SN subgroups. The current work revealed that SN-SV could be a novel skin-wound-healing agent with a practical application as a wound-healing platform.

Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway

Deep second-degree burns heal slowly, and promoting the healing process is a focus of clinical research. Sestrin2 is a stress-inducible protein with antioxidant and metabolic regulatory effects. However, its role during acute dermal and epidermal re-epithelialization in deep second-degree burns is unknown. In this study, we aimed to explore the role and molecular mechanism of sestrin2 in deep second-degree burns as a potential treatment target for burn wounds. To explore the effects of sestrin2 on burn wound healing, we established a deep second-degree burn mouse model. Then we detected the expression of sestrin2 by western blot and immunohistochemistry after obtaining the wound margin of full-thickness burned skin. The effects of sestrin2 on burn wound healing were explored in vivo and in vitro through interfering sestrin2 expression using siRNAs or the small molecule agonist of sestrin2, eupatilin. We also investigated the molecular mechanism of sestrin2 in promoting burn wound healing by western blot and CCK-8 assay. Our in vivo and in vitro deep second-degree burn wound healing model demonstrated that sestrin2 was promptly induced at murine skin wound edges. The small molecule agonist of sestrin2 accelerated the proliferation and migration of keratinocytes, as well as burn wound healing. Conversely, the healing of burn wounds was delayed in sestrin2-deficient mice and was accompanied by the secretion of inflammatory cytokines as well as the suppression of keratinocyte proliferation and migration. Mechanistically, sestrin2 promoted the phosphorylation of the PI3K/AKT pathway, and inhibition of PI3K/AKT pathway abrogated the promoting role of sestrin2 in keratinocyte proliferation and migration. Therefore, sestrin2 plays a critical role in activation of the PI3K/AKT pathway to promote keratinocyte proliferation and migration, as well as re-epithelialization in the process of deep second-degree burn wound repair.

Comparison of Propolis Extracts and Bioplacenton at Epidermal Re-epithelialization Process in Burn Wound of Mice (Mus musculus)

Effective treatment is needed in accelerating the healing process of burn. Propolis extract is a bee product that has a high flavonoid content. Propolis extract is thought to stimulate the process of re-epithelialization of the epidermis. This research was an experimental study with a post-test only control group design. This study used experimental animal, male mice (Mus musculus) Swiss webster strain. Mice were grouped randomly into a control group and a treatment group. Each mouse was given treatment in the form of burn on the back. The control group was the group that was not given propolis or bioplacenton extract. The treatment group was given 5%, 10%, and bioplacenton propolis extract, respectively. Skin histopathological tissue collection was carried out on day 1, day 4, and day 21 to observe the process of re-epithelialization of the epidermis. The results showed that the means of epithelial thickness in the control group and the 5% and 10% propolis extract treatment were significantly thicker on the 21st day of observation when compared to the control group. The administration of bioplacenton showed an increase in the re-epithelialization process of the epidermis on the 4th day. The administration of 10% propolis extract was better in increasing the mean epithelial thickness on the 4th day than the group given 5% propolis extract and bioplacenton.

Potential Wound Healing Effect of Gel Based on Chicha Gum, Chitosan, and Mauritia flexuosa Oil.

Wounds are considered a clinically critical issue, and effective treatment will decrease complications, prevent chronic wound formation, and allow rapid healing. The development of products based on naturally occurring materials is an efficient approach to wound healing. Natural polysaccharides can mimic the extracellular matrix and promote cell growth, thus making them attractive for wound healing. In this context, the aim of this work was to produce a gel based on chicha gum, chitosan, and Mauritia flexuosa oil (CGCHO) for wound treatment. TG and DTG analyzed the thermal behavior of the materials, and SEM investigated the surface roughness. The percentages of total phenolic compounds, flavonoids, and antioxidants were determined, presenting a value of 81.811 ± 7.257 µmol gallic acid/g Mauritia flexuosa oil, 57.915 ± 0.305 µmol quercetin/g Mauritia flexuosa oil, and 0.379 mg/mL, respectively. The anti-inflammatory was determined, presenting a value of 10.35 ± 1.46% chicha gum, 16.86 ± 1.00% Mauritia flexuosa oil, 10.17 ± 1.05% CGCHO, and 15.53 ± 0.65% chitosan, respectively. The materials were tested against Gram-negative (Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus) bacteria and a fungus (Candida albicans). The CGCHO formulation showed better antimicrobial activity against Gram-positive bacteria. In addition, an in vivo wound healing study was also performed. After 21 days of treatment, the epidermal re-epithelialization process was observed. CGCHO showed good thermal stability and roughness that can help in cell growth and promote the tissue healing process. In addition to the good results observed for the antimicrobial, antioxidant, anti-inflammatory activities and providing wound healing, they provided the necessary support for the healing process, thus representing a new approach to the wound healing process.

MSC-derived exosomes attenuate cell death through suppressing AIF nucleus translocation and enhance cutaneous wound healing

BackgroundSkin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing.MethodsIn vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton’s jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant.ResultsThe hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis.ConclusionsThese findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings.

Investigation of chemogenic and biogenic derived nano ZnO activity on excision wound in rat model: a comparative study

Wound care management and wound treatment is one of the common and challenging tasks in medicinal field. Development of cost-effective and novel nano material based drugs with wound healing properties is a promising research arena. In the present study, we report systematic comparison of the physical properties and biological activity of biogenic (ZnO-B) and chemogenic (ZnO-C) zinc oxide nanoparticles (nano ZnO). Pomegranate peel is used as a reducing agent (otherwise a waste) for the synthesis of biogenic nano ZnO. Synthesized nano ZnO were characterized using PXRD, FESEM and TEM. Wound healing potential was assessed using excision wound model for different concentrations of ZnO-B, ZnO-C and are compared with standard drug. Excision wound treated with 2% ZnO-B (95.57 ± 1.031%) exhibited highest wound closure rate compared to 2% ZnO-C (93.95 ± 1.143%) and standard drug povidone–iodine ointment (5 w v−1%) (93.29 ± 0.391%). Sections of animals treated with 2% ZnO-B showed epidermal re-epithelialization with proliferation of fibroblast and mononuclear inflammatory infiltrates, new blood vessels and significant increase in collagen formation in the dermis. When compared with the standard drug, both ZnO-B and ZnO-C showed higher MIC against all the tested strains (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli). ZnO-B showed higher antibacterial activity compared to ZnO-C nanoparticles. In addition to this, in silico molecular docking studies were also carried out for GSK-3β synthase and GlcN-6-P synthase. The in silico studies proves the interactions of GSK-3β synthase and GlcN-6-P synthase with ZnO. Based on the results of antibacterial activity and docking studies a probable wound healing mechanism is proposed.

Cytokeratin Expression at Different Stages in Sweat Gland Development of C57BL/6J Mice.

Sweat glands exhibit a documented role in epidermal reepithelialization after wounding. However, the regenerative potential of sweat glands has remained underappreciated due to the absence of useful markers for the analysis of determination and differentiation processes in the developing eccrine sweat gland from epithelium. Although the current knowledge of keratin expression in most of the different origins has been described, it remains widely shared and not unified in eccrine sweat glands of C57BL/6J mice that are commonly used as animal models for sweat gland and wound healing studies, both at the molecular and cellular levels. Aiming to answer this question, we have investigated the changes in cytokeratin expression patterns during the embryonic, neonatal, juvenile, and young adult stages (E12.5, E17.5, P0.5, P5, and P28). In this article, we demonstrate that the morphology of murine sweat gland progenitor cells are similar to epidermal stem cells before birth (E12.5 and E17.5); at postnatal stages, the duct formed gradually and curled to glob. K8 and K19 were expressed in the eccrine sweat gland cells at all times and highly expressed after birth at both gene and protein levels. Also, histological results revealed K8 and K19 positive cells localized in the secretary portion of glands. Meanwhile, K14 strongly expressed both in vivo and in vitro at E12.5, while it weakly expressed at other stages. Moreover, K10 was rarely detected before birth, but it expressed positively in vivo and in vitro only at the protein level after birth. These data indicate the pattern of main cytokeratin expression at different stages during murine sweat gland development and might provide an efficient tool for sweat gland research and exciting potential for developing targeted therapies for wound healing.

Platelet-activating factor induces proliferation in differentiated keratinocytes

Increased levels of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) are found in several inflammatory dermatoses, but PAF's exact role in epidermis is uncertain. In order to better understand the physiological consequences of excess PAF production in epidermis, we examined the gene regulatory effects of PAF short-term stimulation in differentiated HaCaT keratinocytes by transcriptional profiling. Even though PAF induces COX2 expression, we found that PAF regulates only few genes associated with inflammation in differentiated keratinocytes. Rather, we show that natural PAF rapidly regulates genes involved in proliferation, (anti)-apoptosis and migration, all sub-processes of re-epithelialization and wound healing. Moreover, profiling of phosphorylated kinases, cellular wound-scratch experiments, resazurin assay and flow cytometry cell cycle phase analysis all support a role for PAF in keratinocyte proliferation and epidermal re-epithelialization. In conclusion, these results suggest that PAF acts as an activator of proliferation and may, therefore, function as a connector between inflammation and proliferation in differentiated keratinocytes.

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.

Doxycycline hydrogels with reversible disulfide crosslinks for dermal wound healing of mustard injuries

Doxycycline hydrogels containing reversible disulfide crosslinks were investigated for a dermal wound healing application. Nitrogen mustard (NM) was used as a surrogate to mimic the vesicant effects of the chemical warfare agent sulfur mustard. An 8-arm-poly(ethylene glycol) (PEG) polymer containing multiple thiol (-SH) groups was crosslinked using hydrogen peroxide (H 2O 2 hydrogel) or 8-arm-S-thiopyridyl (S-TP hydrogel) to form a hydrogel in situ. Formulation additives (glycerin, PVP and PEG 600) were found to promote dermal hydrogel retention for up to 24 h. Hydrogels demonstrated high mechanical strength and a low degree of swelling (< 1.5%). Doxycycline release from the hydrogels was biphasic and sustained for up to 10-days in vitro. Doxycycline (8.5 mg/cm 3) permeability through NM-exposed skin was elevated as compared to non vesicant-treated controls at 24, 72 and 168 h post-exposure with peak permeability at 72 h. The decrease in doxycycline permeability at 168 h correlates to epidermal re-epithelialization and wound healing. Histology studies of skin showed that doxycycline loaded (0.25% w/v) hydrogels provided improved wound healing response on NM-exposed skin as compared to untreated skin and skin treated with placebo hydrogels in an SKH-1 mouse model. In conclusion, PEG-based doxycycline hydrogels are promising for dermal wound healing application of mustard injuries.

Silver Nanoparticles Mediate Differential Responses in Keratinocytes and Fibroblasts during Skin Wound Healing

With advances in nanotechnology, pure silver has been recently engineered into nanometer-sized particles (diameter <100 nm) for use in the treatment of wounds. In conjunction with other studies, we previously demonstrated that the topical application of silver nanoparticles (AgNPs) can promote wound healing through the modulation of cytokines. Nonetheless, the question as to whether AgNPs can affect various skin cell types--keratinocytes and fibroblasts--during the wound-healing process still remains. Therefore, the aim of this study was to focus on the cellular response and events of dermal contraction and epidermal re-epithelialization during wound healing under the influence of AgNPs; for this we used a full-thickness excisional wound model in mice. The wounds were treated with either AgNPs or control with silver sulfadiazine, and the proliferation and biological events of keratinocytes and fibroblasts during healing were studied. Our results confirm that AgNPs can increase the rate of wound closure. On one hand, this was achieved through the promotion of proliferation and migration of keratinocytes. On the other hand, AgNPs can drive the differentiation of fibroblasts into myofibroblasts, thereby promoting wound contraction. These findings further extend our current knowledge of AgNPs in biological and cellular events and also have significant implications for the treatment of wounds in the clinical setting.

In vivo confocal imaging of epidermal cell migration and dermal changes post nonablative fractional resurfacing: study of the wound healing process with corroborated histopathologic evidence

In vivo wound healing response post nonablative fractional laser treatment is evaluated. Seven healthy subjects receive treatments with a Fraxel re:store laser system on the forearm with pulse energies ranging from 10 to 70 mJ. The treatment sites are imaged at 1-h increments up to 40 h using confocal microscope z-stacks using 10-mum-depth spacing. At least five individual microscopic treatment zones are imaged per subject, time point, and treatment energy. Images are analyzed for tissue structure and morphology to classify each lesion as healed or not healed, depending on epidermal re-epithelialization at each time point and treatment energy. Probit analysis is used to statistically determine the ED(50) and ED(84) probabilities for a positive dose response (healed lesion) as a function of treatment energy. Confocal observations reveal epidermal keratinocyte migration patterns confirmed with histological analysis using hematoxylin and eosin (HE) and lactate dehydrogenase (LDH) staining at 10 mJ at 0, 7, 16, and 24-h post-treatment. Results indicate that more time is required to conclude re-epithelialization with larger lesion sizes (all less than 500 mum) corresponding to higher treatment energies. For the entire pulse energy range tested, epidermal re-epithelialization concludes between 10 to 22-h post-treatment for ED(50) and 13 to 28 h for ED(84).

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.

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 α5 expression. α5β1 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 α3 subunit; α3β1 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.

The use of the erbium:YAG laser for the treatment of class III rhytids.

The erbium:YAG (Er:YAG) laser can be used for precise tissue ablation with minimal thermally induced damage. Because of its ability to superficially ablate tissue, the Er:YAG laser can be used to improve fine rhytids with rapid epidermal reepithelialization. It has not been shown that the Er:YAG laser can successfully treat deeper rhytids where some thermal damage may lead to more optimal collagen remodeling. We chose to evaluate the efficacy of multiple superficial Er:TAG laser sessions in the treatment of deeper class III rhytids. Twenty subjects were selected for this study. All treated subjects had class III rhytids and were Fitzpatrick I-III skin phenotypes. All subjects were treated with four Er:YAG laser passes at 5 J/cm2. Three months after the initial treatment, a second treatment with similar parameters was repeated. Six months after the initial treatment, a third laser session with identical parameters was undertaken. Subjects were evaluated for laser efficacy and postlaser complications. Although no subjects showed improvement after the first laser session, mild to excellent improvement was noted 6 months after the third Er:YAG laser treatment. The Er:YAG may be used successfully in the treatment of class III rhytids. Multiple sessions may be required.

Characterization of Epidermal Wound Healing in a Human Skin Organ Culture Model: Acceleration by Transplanted Keratinocytes1

Few data are available on early regeneration of human epidermis in vivo. We have established a supravital skin organ culture model for epidermal wound healing by setting a central defect (3 mm diameter) in freshly excised skin specimens and culturing under air exposure. Re-epithelialization was followed for up to 7 d by histology and immunohistologic analysis of various markers for differentiation and proliferation. In 12 of 19 cases (63%; 5% fetal calf serum) or six of 21 cases (29%; 2% fetal calf serum), the wounds were re-epithelialized spontaneously after 7 d. After transplantation to the wounds of 1-2 x 10(6) dissociated allogenic cultured epidermal or about 1 x 10(6) autologous outer root sheath keratinocytes, 18 of 21 cases (86%; 5% fetal calf serum) or 17 of 21 cases (81%; 2% fetal calf serum) were healed within the same period. Histologically, early neoepithelium (3 d) was disordered after keratinocyte transplantation, whereas later (7 d) it had gained a more ordered stratification, exhibiting a thin discontinuous granular and a compact horny layer. At this stage, not only hyperproliferative (CK 6) but also, abundantly, maturation-associated cytokeratins (CK 1, CK 10) were detected immunohistochemically. Analyses of regenerated epidermis after transplantation of (i) keratinocytes labeled in vitro with BrdU and (ii) heterosexual keratinocytes by immunohistochemistry and fluorescence in situ hybridization for the Y chromosome, respectively, clearly showed that external keratinocytes are physically integrated into the regenerated epidermis and extendedly contribute to its formation. The data presented here demonstrate improvement and acceleration of epidermal re-epithelialization by transplantation of keratinocytes.