Autologous skin transplantation: comparison of minced skin to other techniques

BACKGROUND: Skin grafting may be necessary to close nonhealing skin wounds. This report describes a fast and minimally invasive method to produce minced skin suitable for transplantation to skin wounds. The technique was evaluated in an established porcine skin wound healing model and was compared to split-thickness skin grafts and suspensions of cultured and noncultured keratinocytes. MATERIALS AND METHODS: The study included 90 wounds on 3 pigs. Fluid-treated full-thickness skin wounds were grafted with minced skin, split-thickness skin grafts, noncultured keratinocytes, or cultured keratinocytes. Controls received either fluid or dry treatment. The wound healing process was analyzed in histologies collected at Days 8 to 43 postwounding. Wound contraction was quantified by photoplanimetry. RESULTS: Wounds transplanted with minced skin and keratinocyte suspension contained several colonies of keratinocytes in the newly formed granulation tissue. During the healing phase, the colonies progressed upward and reepithelialization was accelerated. Minced skin and split-thickness skin grafts reduced contraction as compared to keratinocyte suspensions and saline controls. Granulation tissue formation was also reduced in split-thickness skin-grafted wounds. CONCLUSIONS: Minced skin grafting accelerates reepithelialization of fluid-treated skin wounds. The technique is faster and less expensive than split-thickness skin grafting and keratinocyte suspension transplantation. Minced skin grafting may have implications for the treatment of chronic wounds.     

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.     

Cell suspension cultures of allogenic keratinocytes are efficient carriers for ex vivo gene transfer and accelerate the healing of full-thickness skin wounds by overexpression of human epidermal growth factor

The concept of using growth factor therapy to induce wound repair has been endorsed in studies that show reduced growth factors in wound fluid from chronic and aged wounds. In this study, we used cell suspensions of allogenic keratinocytes as gene-delivery vehicles for human epidermal growth factor (hEGF) and analyzed their impact on wound repair in a porcine wound-healing model. Full-thickness wounds were created on the backs of six Yorkshire pigs and covered with a wound chamber to create a wet wound-healing environment. First, 5 x 10(5) allogenic, autogenic, or mixed keratinocytes were transplanted into wounds and healing parameters were analyzed. Second, we measured long-term reepithelialization and contraction rates from day 8 until day 35. In the third experiment, allogenic keratinocytes were transfected with an hEGF-expressing plasmid pCEP-hEGF and transplanted in full-thickness wounds to improve repair. Wounds treated with autogenic, allogenic, or mixed keratinocytes showed a significantly higher rate of reepithelialization relative to saline-treated control wounds. Repetitive biopsies indicated that the use of allogenic keratinocytes did not lead to long-term wound breakdown. Wounds treated with hEGF-expressing allogenic keratinocytes reepithelialized faster than wounds treated with allogenic keratinocytes or control wounds. With a peak hEGF expression of 920.8 pg/mL, hEGF was detectable until day 5 after transplantation compared with minimal hEGF expression in control wounds. This study shows that allogenic keratinocytes can serve as efficient gene transfer vehicles for ex vivo growth factor delivery to full-thickness wounds and overexpression of hEGF further improves reepithelialization rates.     

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.     

Lysophosphatidic acid enhances healing of acute cutaneous wounds in the mouse.

Lysophosphatidic acid is a phospholipid growth factor and intercellular signaling molecule released by activated platelets and injured fibroblasts that affects keratinocytes, fibroblasts, neutrophils,and monocytes. We therefore hypothesized that lysophosphatidic acid could influence the inflammation and reepithelialization phases of wound healing. Lysophosphatidic acid (100 microM) was applied daily for 5 days to 2 mm-diameter excisional mouse ear skin wounds and re-epithelialization was measured. We also evaluated whether the bioactivity of lysophosphatidic acid could be increased by zinc (Zn2+, 1 mM). Inflammatory cells were counted in wound sections after 1, 3, or 5 days of healing. Reepithelialization was accelerated significantly by either lysophosphatidic acid or lysophosphatidic acid + Zn2+ (p < 0.01 and p < 0.0001, respectively). Both lysophosphatidic acid solutions significantly increased the amount of new epithelium in the wounds on days 1, 2, and 3 (p < 0.05). Little wound area remained on day 4, and all wounds were fully reepithelialized by day 5. Lysophosphatidic acid did not affect the number of neutrophils or macrophages present in the wound area. Our findings show that lysophosphatidic acid increased the amount of reepithelialization in the early stages of cutaneous wound healing in excisional ear wounds, without affecting inflammatory function.     

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.     

Fetal wound healing: an in vitro explant model

The ability of fetal skin wounds to heal without scar formation is remarkable. The mechanisms that endow the fetus with this unique healing ability remain unknown. We have developed an in vitro explant model using fetal sheep skin to investigate fetal wound healing. This model eliminates the complex systemic mechanisms that modulate in vivo wound healing. We demonstrated that using an enriched medium, midgestation fetal sheep skin explants following wounding reepithelialized within 4 days. By 7 days after wounding the confluent epidermis was thicker, but the dermal wound remained open. This model demonstrates that it is possible to achieve conditions in culture that maintain tissue viability and support reepithelialization. This model may allow us to resolve some of the individual components that participate in the process of scarless fetal skin healing.     

Interferon‐gamma inhibits healing post scald burn injury

Impaired healing after severe burns remains a reason for prolonged hospitalization, opportunistic infections, and debilitating scarring. Interferon‐gamma (IFN‐γ) is an important immune regulator that has been shown to inhibit collagen synthesis by fibroblasts, resulting in delayed healing in incision wounds. To determine whether IFN‐γ plays similar roles in the healing process after severe burn, we induced scald injury in mice deficient or sufficient in IFN‐γ and examined local responses. In the absence of IFN‐γ, scalded areas healed faster. This was associated with attenuated local inflammatory responses, enhanced reepithelialization, increased proliferation of keratinocytes in reepithelialized leading edges, and up‐regulation of growth factors in burned skin areas. Furthermore, angiogenesis and myofibroblast formation commenced and terminated earlier in IFN‐γ^–/– mice compared with wild type (WT) controls. Our observations demonstrate that inhibition of IFN‐γ results in accelerated healing after burn injury by dampening excessive inflammation and facilitating reepithelialization, collagen deposition, and wound contraction.     

Wound healing in a fetal,adult, and scar tissue model: A comparative study

Early gestation fetal wounds heal without scar formation. Understanding the mechanism of this scarless healing may lead to new therapeutic strategies for improving adult wound healing. The aims of this study were to develop a human fetal wound model in which fetal healing can be studied and to compare this model with a human adult and scar tissue model. A burn wound (10 × 2 mm) was made in human ex vivo fetal, adult, and scar tissue under controlled and standardized conditions. Subsequently, the skin samples were cultured for 7, 14, and 21 days. Cells in the skin samples maintained their viability during the 21‐day culture period. Already after 7 days, a significantly higher median percentage of wound closure was achieved in the fetal skin model vs. the adult and scar tissue model (74% vs. 28 and 29%, respectively, p<0.05). After 21 days of culture, only fetal wounds were completely reepithelialized. Fibroblasts migrated into the wounded dermis of all three wound models during culture, but more fibroblasts were present earlier in the wound area of the fetal skin model. The fast reepithelialization and prompt presence of many fibroblasts in the fetal model suggest that rapid healing might play a role in scarless healing.     

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.     

Cultured autologous fibroblasts augment epidermal repair

BACKGROUND: Autologous dermal fibroblasts may be useful in the treatment of skin wounds and for the enhancement of keratinocyte proliferation. This paper addressed the following questions: (1) can cultured fibroblasts (CF) be transplanted as suspensions to full-thickness skin wounds and do they influence wound healing; (2) will the transplanted CF be integrated into the new dermis; (3) can a transgene that encodes a secretable marker, human epidermal growth factor (hEGF), be expressed in the wound fluid by the transplanted CF; and (4) do CF cotransplanted with cultured keratinocytes (CK) influence the rate of wound healing? METHODS: Suspensions of CF were transplanted alone or together with CK to full-thickness wounds covered with liquid-containing chambers in an established porcine model. RESULTS: Transplantation of CF accelerated reepithelialization as determined from wound histologies and sequential measurements of protein efflux over the wound surface. CF transfected with a marker gene, beta-galactosidase, resulted in in vivo gene expression and demonstrated that transplanted CF integrated into the developing dermis. Transplantation of hEGF gene-transfected CF resulted in significant hEGF expression in wound fluid. The hEGF levels peaked at day 1 (2450 pg/ml) and then sharply decreased to low levels on day 6. CF cotransplanted with CK led to greater number of keratinocyte colonies in the wound and accelerated reepithelialization as compared with CK alone. CONCLUSIONS: Transplanted CF integrated into the dermis, accelerated reepithelialization, and improved the outcome of CK transplantation. CF may also be used for the expression of transgenes in wound and wound fluid.     

Amniotic membrane induces epithelialization in massive posttraumatic wounds

Large‐surface or deep wounds often become senescent in the inflammatory or proliferation stages and cannot progress to reepithelialization. This failure makes intervention necessary to provide the final sealing epithelial layer. The best current treatment is autologous skin graft, although there are other choices such as allogenic or autologous skin substitutes and synthetic dressings. Amniotic membrane (AM) is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial reepithelialization effects, with antiinflammatory, antifibrotic, antimicrobial, and nontumorigenic properties. These properties are related to its capacity to synthesize and release cytokines and growth factors. We report the use of AM as a wound dressing in two patients with large and deep traumatic wounds. Negative pressure wound therapy followed by AM application was capable of restoring skin integrity avoiding the need for skin graft reconstruction. AM induced the formation of a well‐structured epidermis. To understand this effect, we designed some assays on human keratinocyte‐derived HaCaT cells. AM treatment of HaCaT induced ERK1/2 and SAP/JNK kinases phosphorylation and c‐jun expression, a gene critical for keratinocytes migration; however, it did not affect cell cycle distribution. These data suggest that AM substantially modifies the behavior of keratinocytes in chronic wounds, thereby allowing effective reepithelialization.     

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.     

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.     

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.     

Effect of hyperbaric oxygen on human skin cells in culture and in human dermal and skin equivalents

A critical stage of cutaneous wound healing is the development and maturation of the epidermis. In the aged, and in certain pathologies, this repair process is compromised due to a variety of deficiencies, one of which is tissue oxygenation. Several phases of wound healing are dependent on adequate tissue oxygen levels, and hyperbaric oxygenation has been shown to transiently elevate these levels. The use of human cell monolayers, dermal equivalents and human skin equivalents provide excellent opportunities for studying wound healing using in vivo relevant models. The goal of this study was to examine the effect of hyperbaric oxygen on cell proliferation, differentiation, and matrix biosynthesis in monolayer cultures and epidermopoiesis in the developing skin equivalent. Normal human dermal fibroblasts, keratinocytes and melanocytes, dermal equivalents and skin equivalents were exposed to hyperbaric oxygen at pressures up to three atmospheres, for up to 10 consecutive daily treatments lasting 90 minutes each. Increase in fibroblast proliferation (cf., 30% at 1 atmosphere after 10 days treatment), was observed without a significant effect on proliferation of normal human melanocytes and glycosaminoglycan synthesis. Stimulation of collagen synthesis after two days of treatment was only significant at 1 atmosphere (about 20% increase) but this differential was not observed after 5 days of treatment. Hyperbaric oxygenation above 2 atmospheres, inhibited proliferation of fibroblasts and keratinocytes in cell monolayer cultures (e.g., a 10 day treatment at 3 atmospheres appeared cytostatic to keratinocytes). In contrast, hyperbaric treatment up to 3 atmospheres dramatically enhanced keratinocyte differentiation, and epidermopoiesis in the complete human skin equivalent. These results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue. The results also show the utility of the human skin equivalent as a model for evaluation of parameters involved in wound healing.     

Locomotion of human skin keratinocytes on polystyrene, fibrin, and collagen substrata and its modification by cell-to-cell contacts

Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.