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.     

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.     

A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study

A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.     

Comparison of cultured and uncultured keratinocytes seeded into a collagen-GAG matrix for skin replacements.

A well-characterised collagen-glycosaminoglycan (CG) matrix functions as an extracellular matrix analogue (ECMA) of dermis on full-thickness wounds. The epidermis can be reconstituted by seeding autologous uncultured keratinocytes into the matrix prior to grafting. We hypothesised that seeding the CG matrix with keratinocytes cultured to sub-confluence may provide the ECMA with more proliferating keratinocytes than with uncultured keratinocytes. Autologous cells were isolated from split-thickness skin grafts and cultured to sub-confluence. ECMAs were seeded by centrifuging cultured (n = 8) or uncultured (n = 8) autologous keratinocytes into a CG matrix at a density of 100,000 cells/cm2, then applied onto full-thickness wounds on Yorkshire pigs. Gross and histologic observations were made up to 21 days post-grafting. At 14 days, a fully differentiated epidermis was present on all graft sites, but the epidermis of the cultured-cell-seeded matrices was thicker, 180 (19) microns, than the uncultured-cell-seeded matrices, 110 (18) microns. The epidermis of cultured-cell-seeded matrices was acanthotic, containing 14 (4) cell layers, as compared to uncultured-cell-seeded matrices, 9 (1) cell layers. The number of subepithelial keratinocyte cysts/cm cross-section present in the neodermis was also greater in cultured-, 1.35 (0.37), than in uncultured-cell-seeded matrices, 0.47 (0.35). Epidermal confluence on day 14 was 96 (3)% on cultured-cell-seeded grafts and 50 (17)% on uncultured-cell-seeded grafts. These results are consistent with the hypothesis that the process of in vitro cell cultivation increases the proportion of dividing cells in preference to differentiated cells. This technology may be useful in reconstruction of specialised bilayer tissues with minimal donor sites.     

Tissue expression of transforming growth factor-β1 and transforming growth factor-α during wound healing in human skin explants

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.     

Composite skin graft: frozen dermal allografts support the engraftment and expansion of autologous epidermis

Rapid closure of burn wounds significantly reduces the complications associated with thermal injury. Successful wound coverage, however, is often limited by the lack of suitable autografts. To circumvent this limitation a composite graft was developed which combines the utility and availability of allogeneic skin with the permanence of an autograft. Composite grafts were first employed in a rat wound model and subsequently to treat six patients with thermal injuries. In experiments with rats, full-thickness excised (1") wounds were prepared on thoracic walls, covered with previously frozen allograft skin, dressed, and secured. Five days later, the dead epidermis was removed and trypsin-disaggregated syngeneic epidermal cells applied to the exposed dermal surface. Successful engraftment with complete epidermal coverage could be observed within 7 to 10 days. In eight patients, split-thickness skin bank allografts were placed on full-thickness burn wounds. Four days later the dead epidermis was removed and vacuum blister-prepared sheets of autologous epidermis grafted to the exposed dermal surface. In all eight patients successful engraftment ensued. Increased pigmentation at the site of each original epidermal graft confirmed the stability of underlying allograft dermis. Epidermal expansion ranged from 1:20 to 1:100. All patients were followed from 10 to 12 months with no demonstrated graft loss or significant wound contracture. Composite skin grafts which combine allogeneic dermis and an expanded autologous epidermis can effect rapid wound closure and will remain stable without evidence of rejection or graft breakdown for at least 12 months.     

Healing of full-thickness wounds treated with lyophilized cultured keratinocyte cell lysate in genetically diabetic mice

The effect of a lyophilized cell lysate prepared from cultured human keratinocytes on the healing of full-thickness wounds was evaluated in an impaired healing model. Full-thickness wounds (8 mm in diameter) were made on the dorsal areas of female genetically diabetic mice C57 BL/KsJ (db/db) and their normal (db/+) littermates. Wounds were covered with an occlusive polyurethane film dressing and were treated for 5 days either with the lyophilized cell lysate from cultured human keratinocytes prepared in phosphate-buffered saline solution or with phosphate-buffered saline solution. In normal (db/+) mice, all wounds were closed 16 days after wounding, and more than 90% of the wound closure was due to wound contraction. Wound contraction accounted for a similar extent of wound closure in both lyophilized cell lysate-treated and phosphate-buffered saline solution-treated wounds. In contrast, in the diabetic (db/db) mice, after histologic examination of the wounds 32 days after wounding, four of ten lyophilized cell lysate-treated wounds and four of seven phosphate-buffered saline-treated wounds were found to be closed. Moreover, applications of lyophilized cell lysate from cultured human keratinocytes to full-thickness wounds in diabetic db/db mice significantly decreased the contribution of contraction to wound closure. Day 32 after wounding, contraction contribution to wound closure amounted to 57.7%+/- 4.7% and 80.4%+/- 3.2% (mean +/- standard error of the mean, p < 0.005) of the initial wound areas, respectively, for lyophilized cell lysate-treated and phosphate-buffered saline solution-treated wounds. At this time of wound healing, the thickness of the dermis was increased 1.7-fold by the keratinocyte cell lysate treatment, but neither epithelial migration from the wound edges nor the thickness of the regenerated epithelium were significantly affected. In conclusion, in diabetic (db/db) mice the application of lyophilized cell lysate from cultured human keratinocytes influenced the healing of the dermis and wound contraction, but had no effect on reepithelialization.     

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.     

Porcine wound healing in full-thickness skin defects using Integra? with and without fibrin glue with keratinocytes

BACKGROUND:

An artificial dermal matrix such as Integra (Integra Life Sciences Corporation, USA) provides a wound bed template for vascular and fibrocyte ingrowth as well as collagen remodelling. Dermal repair leads to epidermal and basement membrane regeneration. Burn wounds in particular have been shown to benefit from Integra by enhanced wound healing.

OBJECTIVE:

To evaluate the effect of fibrin glue to modify the integration of Integra in large excised cutaneous wounds. It was hypothesized that applying fibrin glue on a wound bed would reduce the time needed for matrix vascularization and incorporation of Integra and take of the cultured keratinocytes.

METHODS:

Four separate full-thickness wounds were created on the dorsum of two swine. Wound beds were randomly assigned to either application of fibrin glue or no application of fibrin glue before application of Integra. Full-thickness biopsies were performed at days 7, 14, 21, 29 and 35. On day 21, keratinocytes were applied either as sheets or aerosolized fibrin glue suspension.

RESULTS:

Histological analysis revealed a wave of inflammatory cells and early granulation tissue ingrowth into the Integra from the fascia below on day 7. Only this initial phase was augmented by application of fibrin glue to the wound bed. By day 14, most and by day 21, all of the Integra thickness was incorporated. Accelerated dermal repair proceeded from the base with new collagen deposition in Integra spaces. There was no evidence of keratinocyte engraftment, although re-epithelialization occurred at wound edges extending onto the incorporated Integra.

CONCLUSIONS:

It appears there is an acceleration of early phase (day 7 to day 21) dermal incorporation with fibrin glue application to the wound bed, perhaps secondary to increased cellular migration. Day 21 appears to be too early to apply cultured keratinocytes either as sheets or aerosolized suspension.     

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.     

Wound healing by transplantation of mesenchymal stromal cells loaded on polyethylene terephthalate scaffold: Implications for skin injury treatment

BackgroundSeveral clinical studies have shown that cellular therapy based on mesenchymal stromal cells (MSCs) transplantation may accelerate wound healing. One major challenge is the delivery system used for MSCs transplantation. In this work, we evaluated the capacity of a scaffold based on polyethylene terephthalate (PET) to maintain the viability and biological functions of MSCs, in vitro. We examined the capacity of MSCs loaded on PET (MSCs/PET) to induce wound healing in an experimental model of full-thickness wound.MethodsHuman MSCs were seeded and cultured on PET membranes at 37 °C for 48 h. Adhesion, viability, proliferation, migration, multipotential differentiation and chemokine production were evaluated in cultures of MSCs/PET. The possible therapeutic effect of MSCs/PET on the re-epithelialization of full thickness wounds was examined at day 3 post-wounding in C57BL/6 mice. Histological and immunohistochemical (IH) studies were performed to evaluate wound re-epithelialization and the presence of epithelial progenitor cells (EPC). As controls, wounds without treatment or treated with PET were established.ResultsWe observed MSCs adhered to PET membranes and maintained their viability, proliferation and migration. They preserved their multipotential capacity of differentiation and ability of chemokine production. MSCs/PET implants promoted an accelerated wound re-epithelialization, after three days post-wounding. It was associated with the presence of EPC Lgr6⁺ and K6⁺.DiscussionOur results show that MSCs/PET implants induce a rapid re-epithelialization of deep- and full-thickness wounds. MSCs/PET implants constitute a potential clinical therapy for treating cutaneous wounds.     

Bioactive peptide amphiphile nanofiber gels enhance burn wound healing

BackgroundBurns are physically debilitating and potentially fatal injuries. The standard-of-care for burn wounds is the coverage with gauze dressings designed to minimize trauma to the regenerating epidermis and dermis during dressing changes. However, deep partial- and full-thickness burns always heal slowly when standard wound care alone is performed. We have previously reported that peptide amphiphile (PA) gels, pH-induced self-assembling nanostructured fibrous scaffolds, promote cell proliferation and have great potential in regenerative medicine for rapid repair of tissues. In this study, we hypothesized that the PA gels are capable of accelerating wound healing in burn injury.MethodsArtificially generated thermally damaged fibroblasts and human umbilical vein endothelial cells were seeded onto the various PA nanofiber gels including bioactive and nonbioactive peptide sequences. Cell proliferation was assessed at different time points, and thermally damaged fibroblasts and HUVECs manifested increased proliferation with time when cultured with various PA gels. To determine in vivo effects, burn wounds of rats were treated with the bioactive Arg-Gly-Asp-Ser (RGDS)-modified gel that showed greater cell proliferation in vitro. The wound closure was observed, and skin samples were harvested for histologic evaluation.ResultsCell proliferation using the RGDS-PA gel was significantly higher than that observed in other gels. The RGDS-PA gel significantly enhanced re-epithelialization during the burn wound healing process between days 7 and 28. Application of PA gels accelerates the recovery of deep partial-thickness burn wounds by stimulation of fibroblasts and the creation of an environment conducive to epithelial cell proliferation and wound closure.ConclusionsThis biomaterial represents a new therapeutic strategy to overcome current clinical challenges in the treatment of injuries resulting from burns.     

CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway

Background

CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing.

Materials and methods

Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied.

Results

The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration.

Conclusions

CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.     

The co-application of sprayed cultured autologous keratinocytes and autologous fibrin sealant in a porcine wound model.

We have explored the potential for cultured autologous keratinocytes to form an epidermis when delivered as a spray intermixed with autologous fibrin sealant. Twelve full-thickness wounds in Large White pigs (six wounds in each of two pigs) were isolated from the surrounding skin by 4 cm diameter polytetrafluoroethylene chambers, and grafted with Integra artificial skin (Ethicon). Autologous fibrin sealant was produced 10 days later, using an automated processor unit (Vivostat System, ConvaTec, Bristol Myers Squibb), from 120 ml of autologous citrated blood taken 30 min before keratinocyte application. Nine wounds were sprayed, using a Vivostat System automated applicator unit, with a mixture of the sealant preparation and freshly trypsinised cultured autologous keratinocytes in growth medium, at a density of 1-3 x 10(5) cm(-2). Three control wounds were sprayed with the same mixture without cells. The sealant-cell mixture polymerised and adhered to the wound surface immediately. Histological analysis of biopsies taken following sealant-cell application showed that isolated spherical keratinocytes were distributed throughout the sealant at between 3.1 x 10(4) cm(-2) and 7.6 x 10(4) cm(-2). After 4 days discreet colonies of keratinocytes were observed on the wound bed. At 14 days a multi-layered undulating epidermis was formed, punctuated by sporadic epidermal cysts; the mean area of epithelium was 50.1% (s.d. = 19.7%, n = 9). There was no epithelium in the controls (s.d. = 0, n = 3). The difference was statistically significant (P=0.016). This study suggests that co-sprayed cultured keratinocytes and autologous fibrin sealant may be an effective means of delivering epithelial cells to assist wound 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.     

bFGF对移植术后自体全厚皮片色素代谢的影响

探索bFGF对移植术后自体全厚皮片色素代谢的影响及其影响机制。方法：在自体全厚皮片移植术中应用bFGF,于术后一月内,应用组化和电镜的方法,对皮片内黑色素代谢进行动态观察。结果：实验浓度的bFGF对黑色素细胞的数量和形态无明显影响,但促进了角肮细胞的增值,使术后14天～28天皮片表皮层黑色素含量降低。结论：全厚皮片移植中应用bFGF在术后一定时期内有助于减轻皮片黑色素沉着。     

Transient hyperproliferation of a transgenic human epidermis expressing hepatocyte growth factor

Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial cells including epidermal keratinocytes. To investigate the role of HGF in cutaneous biology and to explore the possibility of using it in a tissue engineering approach, we used retroviral-mediated gene transfer to introduce the gene encoding human HGF into diploid human keratinocytes. Modified cells synthesized and secreted significant levels of HGF in vitro and the proliferation of keratinocytes expressing HGF was enhanced compared with control unmodified cells. To investigate the effects of HGF in vivo, we grafted modified keratinocytes expressing HGF onto athymic mice using acellular dermis as a substrate. When compared with controls, HGF-expressing keratinocytes formed a hyperproliferative epidermis. The epidermis was thicker, had more cells per length of basement membrane, and had increased numbers of Ki-67-positive proliferating cells, many of which were suprabasal in location. Hyperproliferation subsided and the epidermis was equivalent to controls by 2 weeks, a time frame that coincides with healing of the graft. Transient hyperproliferation may be linked to the loss of factors present in the wound that activate HGF. These data suggest that genetically modified skin substitutes secreting HGF may have applications in wound closure and the promotion of wound healing.     

Transplantation of autologous cells and porous gelatin microcarriers to promote wound healing

Definitive treatment to achieve wound healing in major burns frequently include skin transplantation, where split-thickness skin grafts is considered gold standard. This method is associated with several drawbacks. To overcome these hurdles, efforts have been made to develop tissue engineered skin substitutes, often comprised of a combination of cells and biomaterials. In the present study, we aimed to investigate transplantation of autologous keratinocytes and fibroblasts seeded on porous gelatin microcarriers using a porcine wound model. Pre-seeded microcarriers were transplanted to a total of 168 surgical full-thickness wounds (2 cm diameter) on eight adult female pigs and covered with occlusive dressings. The experimental groups included wounds transplanted with microcarriers seeded with the combination of keratinocytes and fibroblasts, microcarriers seeded with each cell type individually, microcarriers without cells, each cell type in suspension, and NaCl control. Wounds were allowed to heal for one, two, four or eight weeks before being excised and fixated for subsequent histological and immunohistochemical analysis. In vitro, we confirmed that viable cells populate the surface and the pores of the microcarriers. In vivo, the microcarriers were to a large extent degraded after two weeks. After one week, all treatment groups, with the exception of microcarriers alone, displayed significantly thicker neo-epidermis compared to controls. After two weeks, wounds transplanted with microcarriers seeded with cells displayed significantly thicker neo-epidermis compared to controls. After four weeks there was no difference in the thickness of neo-epidermis. In conclusion, the experiments performed illustrate that autologous cells seeded on porous gelatin microcarriers stimulates the re-epithelialization of wounds. This method could be a promising candidate for skin transplantation. Future studies will focus on additional outcome parameters to evaluate long-term quality of healing following transplantation.     

Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model

Background

Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing.

Methods

Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air–liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing.

Results

Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human IL-10 increased vascular endothelial growth factor expression and neovascularization compared with controls.

Conclusions

The human ex vivo wound model provides a simple and viable design to study dermal wound healing. Both IL-10 homologs demonstrate vulnerary effects. The viral homolog demonstrates enhanced effects on wound closure compared with human IL-10. These data represent a novel tool that can be used to screen therapeutics, such as CMV IL-10, before preclinical studies.     

Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds.

The purpose of this study was to establish a method for transplantation of autologous keratinocytes on an allogenic cell-free dermis. From four healthy volunteers two full thickness skin grafts, 1 x 1 cm, were taken. The epidermis was separated from the dermis enzymatically and the cells of the dermal part were removed by incubation in Triton X-100. Keratinocytes were seeded on a cell-free dermis and the combined graft transplanted back to one of the wounds of the donor of the keratinocytes. The other wound was covered with cell-free dermis without keratinocytes. After 2, 3, 4 and 6 weeks, respectively, the grafted wounds were removed from the subjects and investigated histologically and immunohistochemically regarding re-epithelialisation, fibroblast ingrowth and angiogenesis. The wounds covered with cell-free dermis and keratinocytes showed a complete epidermal coverage 2 weeks after transplantation, in contrast to the wounds covered with un-seeded dermis which only showed epidermal coverage at the wound edges. There was also a marked difference concerning fibroblast ingrowth and angiogenesis. In this study we have shown that autologous keratinocytes can be seeded on a cell-free dermis and transplanted as a kerato-dermal graft which stimulate re-epithelialisation as well as fibroblast ingrowth and angiogenesis in the wound.