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 human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds. The technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (KFGS) has been recently developed to overcome common disadvantages of standard cultured epidermal sheet grafts. The combination of this method with glycerolized (nonvital) xenograft overlays in standardized nude mice full-thickness wounds, as compared to KFGS alone or controls with no grafts, showed enhancement of epithelial regeneration in terms of epithelial thickness and diminished wound contraction during the 6-week follow-up. Total scar thickness was increased after the combined KFGS/xenograft technique. The time taken to complete wound reepithelialization was similar in the two groups. Reconstitution of the dermo-epidermal junction zone as shown by electron microscopy and immunohistochemistry was enhanced by the KFGS+xenograft technique, showing structures resembling rete ridges 6 weeks postoperatively. The combined KFGS/xenograft technique is able to transfer proliferative single keratinocytes. The method simplifies the application when compared to conventional epithelial sheet grafting and reduces wound contraction when compared to pure keratinocyte grafting. Received: 15 October 1998 / / Accepted: 10 March 1999     

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.     

Employing human keratinocytes cultured on macroporous gelatin spheres to treat full thickness-wounds: an in vivo study on athymic rats

Providing cutaneous wounds with sufficient epidermis to prevent infections and fluid loss is one of the most challenging tasks associated with surgical treatment of burns. Recently, application of cultured keratinocytes in this context has allowed this challenge to be met without several of the limitations connected with the use of split-thickness skin grafts. The continuous development of this novel approach has now revealed that transplantation of cultured autologous keratinocytes as single-cell suspensions exhibits several advantages over the use of cultured epidermal grafts. However, a number of methodological problems remain to be solved, primarily with regards to the complexity of culturing these cells; loss of viability and other negative effects during their preparation and transportation; the relatively long period of time required following transplantation to obtain a sufficiently protective epidermis. In the present investigation we attempted to eliminate these limitations by culturing the keratinocytes on macroporous gelatin spheres. Accordingly, the efficacies of normal human keratinocytes in single-cell suspension or growing on macroporous gelatin spheres, as well as of split-thickness skin grafts in healing wounds on athymic rats were compared. Human keratinocytes were found to adhere and proliferate efficiently both on the surface and within the pores of such spheres. Transplantation of such cells adherent to the spheres resulted in significantly more rapid formation of a stratified epidermis than did transplantation of single-cell suspensions or spheres alone. Twenty-three days after transplantation, the epidermis formed from the cells bound to the spheres was not as thick as the epidermis on wounds covered with split-thickness skin grafts, but significantly thicker than on wounds to which single-cell suspensions, spheres alone or no transplant at all was applied. Furthermore, fluorescence in situ hybridisation revealed that the transplanted keratinocytes, both those adherent to gelatin spheres and those in single-cell suspension, were components of the newly formed epidermis. These findings indicate that application of biodegradable macroporous spheres may prove to be of considerable value in designing cell-based therapies for the treatment of acute and persistent wounds.     

Reconstructed human skin produced in vitro and grafted on athymic mice

BACKGROUND: The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. METHODS: The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. RESULTS: After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. CONCLUSIONS: This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.     

Skin substitutes in chronic wounds

Skin substitutes in chronic wounds. There is a large demand for skin substitutes for the coverage of chronic wounds. Due to a intensive and local wound treatment the impaired dermal and epidermal repair still remains a major problem. Known treatmentes as split-skin-, reverdin- and pinch grafts make a sufficient and solid wound closure possible. However in such procedures the take rate of grafting often failes. The graft healing is increased with combined mesh-graft and vacuum-sealing technique. Temporary and permanent skin substitutes extend the spectrum in closure of chronic defects. Sheets and single cell suspensions of keratinocytes are available for clincial treatments. First clinical results with autologous keratinocyte transplantation are described. In the future approaches in gen therapy becomes more and more important for skin substitutes.     

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.     

Allogeneic cultured keratinocytes vs. cadaveric skin to cover wide-mesh autogenous split-thickness skin grafts.

Improved shock therapy has extended the limits of survival in patients with massive burns, and nowadays skin coverage has become the major problem in burn management. The use of mesh skin grafts is still the simplest technique to expand the amount of available donor skin. However, very wide-mesh skin grafts take a very long time to heal, often resulting in unaesthetic scar formation. On the other hand, allogeneic cultured keratinocytes have been reported as a natural source of growth factors and thus could be useful to improve wound healing of these wide-mesh grafts. A clinical study was performed to compare the use of cryopreserved allogeneic cultured keratinocytes vs. the traditional cadaveric skin as a double layer over widely expanded autogenous skin grafts. This procedure was performed in 18 pairs of full-thickness burn wounds (with similar depth and location) in 11 severely burned patients. Early clinical evaluation was made at 2, 3, and 4 to 5 weeks. Parameters such as epithelialization, granulation tissue formation, infection, and scar formation were evaluated. Biopsies were taken to compare the histological characteristics of the epidermis, the epidermal-dermal junction, and the dermis. Late evaluations were performed at 6 and 12 months regarding color, softness, thickness, and subjective feeling of the scar tissue. Aside from a faster (p < 0.05) epithelialization in the keratinocyte group at 2 weeks, there were no statistically different results in any of the early evaluated parameters, neither clinically nor histologically. At long-term follow-up, clinical results and scar characteristics were not significantly different in the two compared groups. It is concluded from the results of this study that, during the early phase, epithelialization was faster with allogeneic cultured keratinocytes compared with cadaveric skin. However, taking into account the substantial difference in costs, the described use of cryopreserved allogeneic cultured keratinocytes as a double layer on meshed autogenous split-thickness skin grafts can hardly be advocated.     

Preparation of laser micropore porcine acellular dermal matrix for skin graft: an experimental study

In our previous study, we used composite grafts consisting of meshed porcine acellular dermal matrix (PADM) and thin split-thickness autologous epidermis to cover full thickness burn wounds in clinical practice. However, a certain degree of contraction might occur because the distribution of dermal matrix was not uniform in burn wound. In this study, we prepare a composite skin graft consisting of PADM with the aid of laser to improve the quality of healing of burn wound. METHODS: PADM was prepared by the trypsin/Triton X-100 method. Micropores were produced on the PADM with a laser punch. The distance between micropores varied from 0.8, 1.0, 1.2 to 1.5mm. Full thickness defect wounds were created on the back of 144 SD rats. The rats were randomly divided into six groups: micropore groups I-IV in which the wound were grafted with PADM with micropores, in four different distances, respectively and split-thickness autograft; mesh group rats received meshed PADM graft and split-thickness autograft; control group received simple split-thickness autografting. The status of wound healing was histologically observed at regular time points after surgery. The wound healing rate and contraction rate were calculated. RESULTS: The wound healing rate in micropore groups I and II was not statistically different from that in control group, but was significantly higher than that in mesh group 6 weeks after grafting. The wound healing rate in micropore groups III and IV was lower than that in mesh and control groups 4 and 6 weeks after grafting. The wound contraction rate in micropore groups I and II was remarkably lower than that in control group 4 and 6 weeks after surgery and it was significantly much lower than that in mesh group 6 weeks after surgery. Histological examination revealed good epithelization, regularly arranged collagenous fibers and integral structure of basement membrane. CONCLUSION: Laser micropore PADM (0.8 or 1.0mm in distance) grafting in combination with split-thickness autografting can improve wound healing. The PADM with laser micropores in 1.0mm distance is the better choice.     

Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A

Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK- graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.     

A novel recombinant human collagen hydrogel as minced split-thickness skin graft overlay to promote full-thickness skin defect reconstruction

To overcome limited donor-site availability in patients with extensive burns, split-thickness skin grafts (STSGs) are sometimes minced into micrografts (MGs) to improve the expansion ratio of the grafts, but this may reduce wound healing. We aimed to produce a novel hydrogel as an overlay of minced STSGs to improve wound healing. The new hydrogel was produced using recombinant human collagen type III powder as a raw material. Morphological and physical characteristics (degradation and swelling rate), cytotoxicity, and cell viability of the hydrogel were evaluated in vitro. A full-thickness in vivo skin defect model was constructed with male Sprague-Dawley rats. The animals were randomly assigned to experimental and control groups in which the new hydrogel and Vaseline gauze, respectively, were overlaid on minced STSGs to repair and regenerate skin wound. The healing rates and recovery status were compared between the two groups. The hydrogels exhibited good water retention properties and a suitable degradation rate, which can promote the proliferation and migration of wound healing-related cells in vitro. Further, using the hydrogel as an overlay accelerated wound closure and angiogenesis, increased dermal tissue and basement membrane formation, enhanced collagen synthesis and wound healing-related growth factor expression, while reducing scar formation compared to the Vaseline gauze group. In conclusion, the novel, low-cost recombinant human collagen hydrogel can accelerate wound closure and improve wound healing when used as an overlay of minced STSGs. The new hydrogel could become a new treatment option for traumatic skin wounds caused by burns or injuries.     

Rejection of cultured keratinocyte allografts in the rat. Clinical implications and a possible clue to the enigma of skin graft rejection

We have made a detailed analysis of the fate of Langerhans cell-free cultured keratinocyte allografts in two rat strain combinations, DA-to-PVG and DA-to-LEW, and compared the results with the rejection of conventional skin allografts in these strain combinations. The cultured keratinocyte layers were grafted both to the body surface using a technique to prevent wound contraction, and to the renal subcapsular site. Histological examination of grafts was made on days 2, 7, 10, 14, and 28 after transplantation. Donor-specific anti-class I MHC monoclonal antibodies were used to verify the donor origin of the keratinocytes. We report that the keratinocyte allografts are acutely rejected but, in contrast to the conventional allografts, do not evoke alloantibody responses. Rejection of the keratinocytes at the renal subcapsular site was as rapid as that of conventional skin grafts. However, rejection of keratinocyte grafts on the body surface was delayed by a few days when compared with conventional skin grafts. Immunosuppression with cyclosporine prevented the rejection of DA keratinocyte layers placed at the renal subcapsular site of PVG rats, but rejection followed soon after cessation of cyclosporine therapy. These data suggest that rejection is a major constraint for the clinical application of cultured keratinocytes, and that autografts must be used if permanent cover is required. Moreover, the findings have interesting theoretical implications relating to the much greater vulnerability to rejection of skin grafts compared with organ grafts. Our current and previous data also suggest that class II positive dendritic cells are the major stimulus to alloantibody production after tissue transplantation.     

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.     

皮肤混合移植的现状和未来

1 皮肤混合移植的演变过程 皮肤移植是外科修复皮肤缺失的主要方法之一.大面积深度烧伤患者可供移植的自体皮有限,探索相应的皮肤移植技术成为烧伤医学的重要研究课题之一[1].     

Treatment of burns and chronic wounds using a new cell transfer dressing for delivery of autologous keratinocytes

This study describes a new methodology for delivering cultured autologous keratinocytes to wounds on a sterile medical grade polymer coated with a chemically defined plasma polymerised functional surface containing 20% carboxylic acid (referred to as PPS). Seven patients (two acute major burns and five chronic non-healing wounds) were treated with applications of autologous keratinocytes delivered on a 6 cm diameter medical grade polymer disc whose surface was functionalised by PPS. For initial keratinocyte expansion a split-thickness skin biopsy was taken from each patient followed by keratinocyte isolation and expansion and, where required for repeated applications, freezing down of keratinocytes. After expansion, cells were cultured on the PPS for 2 days then the PPS with cells was inverted onto the patients wound bed to allow cell transfer to wound beds. For two burns patients transfer of cells from PPS onto donor sites was seen for both patients and it appeared to facilitate healing of grafted burns wounds. For five patients with intractable chronic wounds (with nine ulcers in total) repeated applications of cells resulted in complete healing in 5/9 ulcers with a major reduction in ulcer size for all other (4/9) ulcers. This reduction in ulcer size improved the wound conditions for two of these patients such that they were then considered suitable for conventional grafting and orthopaedic surgery respectively. In conclusion, PPS delivery of autologous cells is a promising approach for acute burns injuries and chronic wounds.This paper is dedicated to the memory of Mr Archibald Newman (Patient 3) who sadly died in January 2005.     

Allogeneic skin substitutes applied to burns patients

Early re-surfacing of burn wounds remains the ideal but is limited by the availability of skin graft donor sites. Cultured grafts overcome these problems and autologous keratinocytes can be grown in culture and placed on a dermal substitute, but this results in delay and requires two operations. We developed an organotypic skin substitute, which achieves cover in one procedure, and have previously found allogeneic cell survival up to 2.5 years after grafting onto clean elective wounds (tattoo removal). Here, we report a short series using the same model applied to burns patients with less than 20% total body surface area affected. The skin substitutes consisted of allogeneic dermal fibroblasts embedded in a collagen gel overlain with allogeneic epidermal keratinocytes, and were grafted to patients with tangentially excised burns. A side-by-side comparison with meshed split-thickness autografts was performed. No grafts became infected. The allogeneic skin substitute showed little effective take at 1 week, and by 2 weeks only small islands of keratinocytes survived. These sites were subsequently covered with meshed split-thickness autograft, which took well. It is concluded that further development of this model is needed to overcome the hostile wound bed seen in burns patients.     

The application of new biosynthetic artificial skin for long-term temporary wound coverage

Temporary dressings protect wounds from desiccation and infection. In our previous study, we used meshed acellular porcine dermis (APD) to enhance wound healing and decrease wound contraction; however, the wounds showed meshed scar [Wang HJ, Chen TM, Cheng TY. Use of a porcine dermis template to enhance widely expanded mesh autologous split-thickness skin graft growth: preliminary report. J Trauma 1997;42(2):177–82]. In this study, we produced an artificial skin composed of a cross-linked silicon sheet on the surface of APD which we have called silicone acellular porcine dermis (SAPD). This new artificial skin can protect the wound long enough to promote wound healing either by second intention or covered long enough until cultured epithelium autograft (CEA) or autologous skin graft can be harvested for permanent coverage.

We delivered 4 cm × 5 cm full-thickness wound on the back of 350 g Sprague–Dawley rats. Thirty-six rats were divided into two groups. Eighteen rats had SAPD and the other 18 were covered with Biobrane. The wounds were first examined 2 weeks after grafting and followed weekly for an additional 4 weeks to evaluate the wound and study pathological changes by using H.E. and Masson's stains. Wound size was calculated by ruler and analyzed by Student's t-test.

At the 2-week inspection, both SAPD and Biobrane showed tight adherence to the wound with no change of wound size. Both the SAPD and Biobrane dermal templates were pink. In the Biobrane-covered group, the wounds contracted soon after the tie-over dressing was removed. Its dermal layer is a layer of thin porcine dermal substance, which was promptly digested by tissue hyaluronidase and provides no real dermal template. In the SAPD-covered group however, the wound size was maintained significantly from third to sixth week after grafting (p < 0.001). SAPD was designed with thick epidermal silicone and a well-organized porcine dermis so that it incorporates into the recipient wound. Clinically the silicone layer of SAPD dislodged from APD about 6–7 weeks after grafting and was followed by dermal matrix exposure and infection. In pathological examination, much like a human skin graft, new vessels were found in APD about 1 week after grafting with minimal inflammatory cells infiltrated in the graft and wound. Six weeks after grafting, the collagen of APD incorporated into the wound, showing palisade arrangement and no sign of rejection. In the Biobrane group however, the wounds showed severe inflammation, the porcine dermal matrix was digested and disappeared 3 weeks after coverage.

In conclusion, SAPD is a thick biosynthetic artificial skin, which protects the rat wound significantly longer than Biobrane and prevents contraction. We expect that using of SAPD for temporary wound coverage will provide enough time to grow autologous-cultured epithelium or to reharvest skin grafts.     

血小板源性生长因子基因修饰的人工复合皮移植大鼠创面效果观察

目的观察血小板源性生长因子B(PDGFB)基因修饰的人工复合皮移植大鼠创面后的效果。方法构建PDGFB真核表达质粒,在脂质体介导下转染大鼠成纤维细胞。分别构建复合皮1(角质形成细胞 猪脱细胞真皮基质 PDGFB基因转染的成纤维细胞)和复合皮2(角质形成细胞 猪脱细胞真皮基质 未转染的成纤维细胞),移植于大鼠背部全层皮肤缺损创面,相应设为转染组、未转染组(各18只)。以不作皮肤移植的8只大鼠全层皮肤缺损创面为对照组。术后2周观察大鼠创面移植皮片存活情况。术后2、4、6周观察大鼠创面大体情况,计算创面收缩率,并取创面组织标本进行组织学观察。结果(1)术后2周,转染组大鼠中皮片完全存活者14只、部分存活者3只、未存活者1只;未转染组大鼠中皮片完全存活者10只、部分存活者4只、未存活者4只。(2)术后2周,对照组创面结痂。术后6周转染组移植皮片表面光滑,有弹性,抗磨擦性强,愈合效果优于其他两组。(3)术后2、4、6周,对照组大鼠创面收缩率均高于其他两组,转染组创面收缩率低于未转染组(P<0.05)。(4)术后2周转染组移植皮片内可见较多毛细血管分布;6周时表皮细胞分化达7～10层,纤维排列致密整齐,毛细血管分布均匀。结论用含PDGFB基因的人工复合皮移植修复创面,可明显提高创面愈合质量。     

Effect of autologous epidermal cell suspension transplantation in chronic nonhealing wounds: a pilot study

Background

Chronic nonhealing wounds are difficult to manage. Various substances are being used to heal these wounds. We sought to observe the effects of autologous epidermal cell suspension dressings on chronic nonhealing ulcers.

Methods

We enrolled patients of the wound clinic at University Hospital, Varanasi, India, with nonhealing wounds of more than 6 weeks’ duration. We treated the wound beds with sterile dressings and antibiotics until the swab cultures became sterile. We prepared autologous epidermal cell suspensions from skin grafts and used them on the ulcer beds along with Vaseline gauze dressings. Follow-up visits with patients occurred weekly for assessment of wound healing and other changes.

Results

Fifteen patients enrolled in our study. Of these, 6 patients had completely healed at 12 weeks, 1 patient at 16 weeks and 2 patients at 20 weeks after treatment. We observed a slow healing response in 6 patients, of whom 1 patient had healed completely at 32 weeks and another at 48 weeks. One patient needed skin grafting, and 3 patients were lost to follow-up.

Conclusion

Autologous noncultured epidermal cell suspension transplantation seems to be an effective, simple and time-saving method to treat chronic nonhealing wounds.     

The use of cultured autologous epidermis in the treatment of extensive burn wounds

Seventeen patients with deep second- and third-degree burn wounds have been grafted with cultured autologous epidermis. These epidermal cell sheets were cultivated according to the feeder layer technique as described by Rheinwald and Green. After dispase treatment and detachment from the culture vessel, the cell sheets, mounted on a polyamide mesh, were ready for grafting. Patients with wounds excised at an early stage, prepared with human cadaver allografts or synthetic dressings, showed a significantly better graft take than nonexcised, chronic granulating wounds which were grafted at a later stage (47% versus 15%; p less than 0.002). Sandwich treatment of expanded mesh autografts and cultured autograft overlay did not improve the graft take, although in some cases wound healing was accelerated. The graft take was inversely correlated with the age of the patient (p = 0.01), and showed a weak inverse correlation with the day of first (and subsequent) culture grafting (p = 0.07). Wound infection was the main cause of graft failure. Up to 4 years after grafting, the grafted areas showed continued stability and the regenerated skin became supple, smooth, and pliable. Hypertrophic scar formation was less than observed in comparable areas treated with meshed grafts. Wound contraction occurred approximately to the same extent as in split-thickness skin grafts. We emphasize that by a better control of wound infection the graft take, also in secondary-stage procedures, can significantly improve.