[Current place of cultured epithelial autografts in the management of massive burns and future prospects: Literature review]. / Place actuelle des cultures d'épidermes autologues dans la prise en charge des brûlures étendues et perspective d'avenir : revue de littérature.

Cultured Epithelial Autografts (CEAs), developed at the end of the 1970s from in vitro culture amplification of keratinocytes, have led to a therapeutic revolution in the treatment of major burns. The areas of improvement of the cultures initially involved the manufacturing processes (culture media, support matrices, etc.) and then clinical applications (use of a largely expanded allogeneic or autologous dermal bed). These advances have enabled burn centers (BC) using CEAs to obtain very satisfactory percentages of graft integration and survival of major burns patients. However, since CEAs are not without major drawbacks (fragility, high rate of infection, high cost, unstable scars), these pitfalls have restricted their use worldwide. As of 2014, CEAs produced by Genyzme Tissue Repair are no longer available in Europe, which has considerably reduced an indispensable therapeutic arsenal for severe and extensive burns. To overcome these therapeutic limitations, current research is focusing on techniques combining surgery, tissue engineering and cell therapy. The advent of regenerative medicine, based on the use of stem cells, in particular mesenchymal stem cells (MSC), can contribute to an improvement in the management of these massively burned patients (optimization of the environmental medium, attenuation of the systemic inflammatory response and the immunosuppressive effects of the burn, acceleration of tissue regeneration, etc.). Cell therapy, therefore, offers alternatives to CEAs, which must imperatively retain their place in the therapeutic arsenal, namely an effective emergency coverage technique that can be improved.

A case report of the first application of culture epithelial autograft (JACE®) for giant congenital melanocytic nevus after its approval in Japan.

In Japan, the JACE® cultured epidermal autograft (CEA) was approved and covered by public healthcare insurance for use in the treatment of giant congenital melanocytic nevus (GCMN) in 2016. We herein report the results of the application of JACE® after curettage and Q-switched ruby laser therapy. The current patient was the first patient with GCMN to be treated with JACE® since its approval. A 3-month-old girl had a hairy GCMN of 9.5 cm in diameter from her cheek to her temple on the left side of her face. We first performed curettage of the nevus on the temple and applied irradiation using a Q-switched ruby laser; however, erosion relapsed at 2 months after first surgery. After preparing JACE®, we performed curettage a second time at 7 months with irradiation of a Q-switched ruby laser and the application of the CEA. The CEA took successfully and the wound was completely epithelized at 1 week after grafting. Re-pigmentation is an important issue that remains to be solved; however, overcoming this would allow for a deeper abrasion or more intense laser irradiation to be performed in cases in which CEA will be subsequently applied.

The application of cultured epithelial autografts improves survival in burns.

This prospective observational study was performed to analyze the clinical outcomes of patients with massive burns treated using cultured epithelial autografts (CEAs) and to determine the association of this treatment with survival outcomes. During 2006-2013, total 177 massive-burns subjects treated with (96 subjects) or without (81 subjects) CEAs. Data were analyzed using the independent t test or chi-square test. Multivariate logistic regression, Kaplan-Meier survival, and Cox regression analyses were performed to evaluate the factors that influenced mortality. Age, percentage of total body surface area burned, incidence of inhalation injury, allograft-application rate, Abbreviated Burn Severity Index score, length of hospital stay, and mortality significantly differed between the CEA and noncultured epithelial autograft groups. Mortality and other clinical parameters did not differ between the sheet-type and spray-type CEA groups. Allograft application (odds ratio, 4.44; p < 0.01) significantly influenced CEA application. The CEA group showed significantly higher survival rates (p = 0.05). Cultured epithelial autografting had a hazard ratio of 0.55 (p = 0.02) and 0.59 (p = 0.05) according to the uni- and multivariate Cox regression analysis, respectively. In conclusion, early and aggressive allograft application is required to facilitate CEA application. Furthermore, the use of CEAs was associated with a lower mortality, but this result should be interpreted with caution as the groups were not randomized.

Additional treatments after curettage of congenital melanocytic nevi in the craniofacial region: A report from a single center in Japan.

BACKGROUND: Congenital melanocytic nevus (CMN) is a benign skin lesion present from birth, which may present with a risk of malignant transformation if extensive. Curettage, a treatment method involving the removal of the superficial layer of the nevus, is often used in the early stages of life. However, recurrence of the nevus and postoperative scarring may present as problems. Additional treatments, such as resection and/or laser treatment, are regularly required after curettage, particularly in the craniofacial region. However, no systematic treatment strategy has been reported. This study investigated additional treatments used after curettage to treat CMN in the craniofacial region and compared the frequency of treatments with respect to specific sites. METHODS: CMN cases involving curettage as an initial treatment were retrospectively reviewed at Kyoto University Hospital between May 2019 and April 2022. RESULTS: This study comprised 23 cases. Curettage was performed at a mean of 3.8 (1-10) months of age. No additional treatments were provided for 80% of head CMN. Additional treatments were performed in all cases, including the forehead and cheek. Laser treatment was performed in 86% of eyelid CMN and 75% of nasal CMN. Tissue expansion and flap closure were used in 33% of forehead CMN and 33% of cheek CMN. CONCLUSIONS: Additional treatments used for CMN in the craniofacial region varied in accordance with the lesion site.

Advances in spray products for skin regeneration.

To date, skin wounds are still an issue for healthcare professionals. Although numerous approaches have been developed over the years for skin regeneration, recent advances in regenerative medicine offer very promising strategies for the fabrication of artificial skin substitutes, including 3D bioprinting, electrospinning or spraying, among others. In particular, skin sprays are an innovative technique still under clinical evaluation that show great potential for the delivery of cells and hydrogels to treat acute and chronic wounds. Skin sprays present significant advantages compared to conventional treatments for wound healing, such as the facility of application, the possibility to treat large wound areas, or the homogeneous distribution of the sprayed material. In this article, we review the latest advances in this technology, giving a detailed description of investigational and currently commercially available acellular and cellular skin spray products, used for a variety of diseases and applying different experimental materials. Moreover, as skin sprays products are subjected to different classifications, we also explain the regulatory pathways for their commercialization and include the main clinical trials for different skin diseases and their treatment conditions. Finally, we argue and suggest possible future trends for the biotechnology of skin sprays for a better use in clinical dermatology.

Cultured epithelial autografts for the treatment of large-to-giant congenital melanocytic nevus in 31 patients.

INTRODUCTION: Giant congenital melanocytic nevus (GCMN) is a large melanocytic nevus, and its full-thickness removal is usually difficult due to the lack of skin available for reconstruction. Curettage is an alternative approach in cases of GCMN to remove the superficial dermis above the cleavage plane with a curette in the neonatal period, and its major complications include repigmentation, retarded epithelization, and hypertrophic scar formation. In Japan, the JACE® cultured epidermal autograft (CEA) was approved and covered by public healthcare insurance for the treatment of congenital melanocytic nevus (CMN) that is difficult to treat with conventional methods in 2016. We have used CEA for wounds after curettage in the neonatal period or following ablation after the neonatal period in combination with laser therapies to reduce the above-mentioned complications. METHODS: In this study, we summarized all consecutive CMN patients treated using CEA from December 2016 to April 2019 and evaluated the duration required for epithelialization, incidence of hypertrophic scar, and color change in the target nevus by comparing the L∗ values one year later between the Curettage group, the non-Curettage group with initial treatment or the subsequent group. RESULTS: No significant differences were seen in the epithelization period or incidence of hypertrophic scars among the groups, but the color of the target nevus was improved significantly in the Curettage group (p < 0.01) and non-Curettage group with initial treatment (p < 0.01). CONCLUSIONS: In conclusion, CEA seems to accelerate epithelization after curettage or ablation of CMN, and this treatment could improve the color of CMN when applied initially.

Treatment of giant congenital melanocytic nevi with cultured epithelial autografts: Clinical and histopathological analysis.

INTRODUCTION: Curettage and dermabrasion are effective in the treatment of giant congenital melanocytic nevi (GCMN); however, local infection and hypertrophic scar formation are major issues. Thus, we applied cultured epithelial autografts (CEA) on skin defects after curettage or abrasion of GCMN and assessed the postoperative outcomes. METHODS: Seven nevi lesions of five patients (aged 3 months to 24 years) were treated with CEA after curettage or abrasion with a dermatome or a surgical bar, respectively. We assessed the postoperative outcomes, including CEA take ratio, erosion and/or ulcer formation in the acute phase, hospitalization days, Vancouver scar scale, and color improvement one year after the operation. In addition, a histological evaluation of a skin biopsy was performed over one year after the operation. RESULTS: The CEAs took well on the wound, and the wound surface was mostly epithelized by postoperative day 7 in all cases. While hypertrophic scar formation and slight pigmentation were observed in some lesions, the color was improved in all of the treated lesions. Histopathological examination revealed that the regenerated epidermis had stratified keratinocytes with rete ridges, and the dermal layer without nevus cells regenerated above the remaining dermis layer. CONCLUSIONS: In this study, we found that early epithelialization and regeneration of the dermal layer was achieved after the application of CEA, suggesting that CEA could be an effective option after curettage or abrasion of GCMN.

[Research status and prospect on autologous tissue-engineered skin as permanent graft].

To date, skin substitute that can provide permanent wound closure is still autologous tissue-engineered skin using autologous skin cells as seed cells. The development of cultured epithelial autograft has experienced a long and tortuous process. Autologous tissue-engineered composite skin is closer to autologous split-thickness skin graft in terms of structure, function, and efficacy, which has become a research focus in recent years. Based on the actual status of research on tissue-engineered skin application, this paper reviewed the main research progresses and existing problems, and the prospect of research and development and clinical application of autologous tissue-engineered skin as permanent graft in order to provide a reference for the improvement and application of autologous tissue-engineered skin.

Surgical Management Evolution Between 2 Massive Burn Cases at 17-Year Interval: Contribution of Cell Therapies in Improving the Surgical Care.

We report the cases of 2 patients admitted to our hospital at a 17-year interval, both with 90% total body surface area (TBSA) burns. These two young patients were in good health before their accident, but major differences in time of intensive care and hospitalization were observed: 162 versus 76 days in intensive care unit and 18 versus 9.5 months for hospitalization, respectively. We have analyzed the different parameters side-by-side during their medical care and we have identified that the overall improved outcomes are mainly due to a better adapted fluid reanimation in combination with the evolution of the surgical management to encompass allogenic cellular therapy (Biological Bandages). Indeed, autologous cell therapy using keratinocytes has been used for over 30 years in our hospital with the same technical specifications; however, we have integrated the Biological Bandages and routinely used them for burn patients to replace cadaver skin since the past 15 years. Thus, patient 1 versus patient 2 had, respectively, 83% versus 80% TBSA for autologous cells, and 0% versus 189% for allogenic cells. Notably, it was possible that patient 2 was able to recover ∼6% TBSA with the use of Biological Bandages, by stimulating intermediate burn zones toward a spontaneous healing without requiring further skin grafting (on abdomen and thighs). The body zones where Biological Bandages were not applied, such as the buttocks, progressed to deeper-stage burns. Despite inherent differences to patients at their admission and the complexity of severe burn care, the results of these two case reports suggest that integration of innovative allogenic cell therapies in the surgical care of burn patients could have major implications in the final outcome.

Wound healing after cultured epithelial autografting in patients with massive burn injury: A cohort study.

BACKGROUND/AIM: Last century, our laboratory produced Cultured Epithelial Autograft (CEA) for clinical use by the affiliated adult burn service and other burn units across the country. Production of CEA for clinical use was discontinued after several years because of a low success rate and subsequent low demand. Recently, at our burns unit, a cell culture program was reintroduced as a direct response to the need for improvement in ongoing deficiencies and clinical requirements in burn wound closure. The aim of this study was to validate the laboratory processes and clinical algorithms established and share our recent clinical experiences involving CEA. METHODS: This observational cohort study recruited patients with burns exceeding 35% TBSA admitted to the Victorian Adult Burns Service at The Alfred (December 2013-December 2016). Autologous keratinocytes were expanded and delivered through sheets of fibrin carrier. RESULTS: Twelve patients were recruited to participate in the study. Thirty-two sites were treated with CEA. CEA applied in combination with widely meshed SSG led to the highest take rate (90.1%) at 7-10 days. Further, debridement and grafting were necessary in sixteen of thirty-two sites treated, all involving wound beds prepared with Cuono method or sites treated with CEA only. CONCLUSION: It is important to address the problem of wound bed contamination, either through increased resistance on the part of the construct or wound bed sterilization. Improved understanding of the relative importance of vascularization, control of cell behavior, the extracellular matrix, immune function, and intrinsic antimicrobial capacity for graft take would then inform a more targeted approach to skin tissue engineering for wound closure in severe burns.

Isolation and Culture of Human Keratinocytes.

Culturing keratinocytes to form coherent epithelial tissue sheets has improved the treatment of extensively burned patients. Keratinocyte culture is also used to investigate various cellular and molecular mechanisms involved in different skin pathologies. To preserve stem cells during epithelial cell culture, reliable methods and conditions are of the utmost importance. Properly cultured keratinocytes will exhibit a consistent cuboid morphology and can proliferate for many passages. This chapter details materials needed and methods for all aspects of efficient keratinocyte culture for clinical applications, namely tissue sampling and transportation, isolation, routine culture, subculture, and cryopreservation.

Successful application of keratinocyte suspension using autologous fibrin spray.

OBJECTIVE: The back is a challenging anatomical area to resurface in acute burns due to its large surface area, its dependent position with the patient lying down and the shearing forces applied to any method of resurfacing employed. This case study presents the use of Vivostat® (Vivostat A/S, Lillerød, Denmark) in resurfacing the back in conjunction with Recell® regenerative epithelial suspension. Vivostat® (Vivostat A/S, Lillerød, Denmark) is a "novel patented biotechnological process that enables reproducible preparation of autologous fibrin sealant or platelet rich fibrin without cryoprecipitation or a separate thrombin component" [1]. METHODS: A 29-year-old female sustained 27% total body surface area (TBSA) flame burns, including the whole back. This area was initially grafted with the sandwich autograft/allograft technique on day four after injury, with approximately 80% graft take on day eight. Unfortunately, there was subsequent significant graft loss on the back proving to be a stubborn area to treat despite further grafting sessions. This challenge led to the decision to use Vivostat® (Vivostat A/S, Lillerød, Denmark) and Recell® to resurface the back. RESULTS: The patient underwent one session of resurfacing with Vivostat® (Vivostat A/S, Lillerød, Denmark) and Recell® and went on to have full healing on the back. CONCLUSIONS: NICE (The National Institute for Health and Care Excellence) states that Recell® shows potential to improve healing in acute burns and we believe that its co-delivery with fibrin via Vivostat® (Vivostat A/S, Lillerød, Denmark) allows for precise delivery of the fibrin suspended cells while minimising loss in the "run off" encountered when Recell® is just simply sprayed on, assisting the anchoring of keratinocytes to the wound surface and thus aiding in the treatment of challenging areas.

Harvesting epithelial keratinocyte sheets from temperature-responsive dishes preserves basement membrane proteins and improves cell survival in a skin defect model.

Cultured epithelial autograft (CEA) therapy has been used in clinical applications since the 1980s. However, there are some issues related to this treatment that still remain unsolved. Enzymatic treatment is typically used in the collection of epithelial keratinocyte sheets, but it tends to break the adhesion and basement membrane proteins. It is thought that the loss of proteins after enzymatic treatment is responsible for the poor survival of transplanted cell sheets. Our laboratory has developed a temperature-responsive culture dish that does not require enzymatic treatment to harvest the cells. In this study, we compare morphological and survival results from rat epithelial keratinocyte cell sheets harvested by temperature-reducing treatment (TT sheets) against cell sheets harvested by enzymatic (dispase) treatment (DT sheets). TT sheets preserve keratin structure in better conditions and express higher levels of collagen IV and laminin 5 than DT sheets. In order to evaluate cell sheet survival after transplantation, we created an in vivo transplant model. Keratinocyte sheets obtained from GFP-positive animals were transplanted into athymic rats. The survival rate 7 days after transplantation of TT sheet was higher than that of DT sheets. Collagen IV and Laminin 5 expression was observed in the TT sheet transplantation group. These results indicate that the remaining basement membrane proteins are important for initial attachment and cell survival. We believe that the cell sheet harvesting method using temperature-responsive culture dishes provides superior cell survival and can solve one of the roadblocks in CEA therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Keratinization induced by air exposure in the reconstructed human epidermal model: an in vitro model of a cultured epithelial autograft.

A reconstructed human epidermis, an in vitro model of a cultured epithelial autograft, was used to examine the formation of a stratum corneum induced by exposure to air. A prolonged wet condition and excess application of petrolatum on the dressing reduced efficient production of the stratum corneum.

Spray-on-skin cells in burns: a common practice with no agreed protocol.

OBJECTIVES: Cultured epithelial autograft (CEA) has been used for skin coverage after burn wound excision since 1981. It is used in burn units and centres throughout the U.K.; however, there appears to be no agreed standards of practice. We aimed to investigate the experience and current practice with its usage in the management of acute burn injury. METHODS: An online survey was sent to twenty-five burns consultants in the U.K., who are members of the British Burn Association. RESULTS: We received 14 responses. Rarely have the responders agreed to the same practice in most of the questions. Different choices were given by responders with regards the indications for cell culture, techniques used, primary and secondary dressings used, first wound review timing, and measures used to evaluate outcomes. CONCLUSION: In the current economic environment, the NHS needs to rationalize services on the basis of cost effectiveness. CEA is an expensive procedure that requires an adequately sterile laboratory, special equipments and highly experienced dedicated staff. When dealing with expensive management options, it is important to have an agreed protocol that can form the standard that can be referred to when auditing practices and results to improve burn management and patients' care.