Human cadaveric allografts are the gold standard for temporary coverage of severe burns, but the supply is limited due to a paucity of clinically acceptable donors and onerous processing requirements. Porcine skin shares fundamental properties with human skin; thus live-cell porcine xenografts represent a potential alternative to achieve temporary coverage of severe burns. Previous research demonstrated that genetically-engineered, knock-out porcine grafts, which lack α-1,3-galactose sugar responsible for hyperacute rejection observed in wild-type xenografts, provide an equivalent barrier and adherence function as allografts in primate models. However, little data exists on the impact of extended cryopreservation on such grafts. In this study, we examined the impact of long-term cryopreservation on graft viability, graft take, and barrier function in a preclinical model. Over a 7-year period, over sixty porcine split thickness skin grafts were cryopreserved and stored at -80oC. Cellular viability of stored grafts was assessed using formazan-MTT and phenyl acetate assays to quantify mitochondrial activity. Stored grafts were compared to fresh skin grafts (100% viability) and heat denatured controls. To test for biological properties, grafts cryopreserved for varying lengths were grafted on swine recipients. Each swine (n=4) received 4 grafts: one autograft and three allografts of identical MHC-profiles. Grafts were clinically assessed every other day postoperatively for overall graft-take, adherence to wound bed, and time until complete graft rejection. The rejection sequelae was observed and assessed histologically via a blinded-pathologist based on the BANFF grading scale. There were no technical failures; all grafts initially adhered to their respective wound beds and re-vascularized. On POD 8, fresh and frozen autografts were indistinguishable on gross examination. Allogeneic grafts appeared comparable to autografts through POD 4. However, by POD 8, all allogeneic grafts, fresh and frozen, demonstrated mild erythema, consistent with rejection and were considered fully rejected by POD 10. In contrast, autografts, remained warm, viable, and appeared healthy. The authors found no statistically significant difference in the duration or quality of adherence among the comparator groups. Similarly, cellular viability (mitochondrial activity) was comparable. Relative quantitative viability and mitochondrial activity of fresh, recently preserved and long-term preserved skin grafts were comparable. The results of this study offer the potential for tissue banking for longer periods. This would be beneficial both in preparation for mass casualty events and to address global shortages.
PURPOSE: Fasciocutaneous free flaps (FFF) have become one of the most advanced techniques for various defect reconstructions not treatable by a local flap. Workable vessels close to the defect are needed to allow the transfer, and failure occurs mainly by vessel thrombosis. Machine perfusion (MP) could help bypass some therapeutic impasses and provide solutions for complex cases. We studied ex-vivo acellular MP on FFF to allow their preservation for several days. The objectives are to provide a reliable protocol for these techniques in swine, for short and long-term perfusion. We compared the outcomes for continuous and intermittent perfusion. METHODS: We harvested fourteen saphenous flaps on 35kgs Yorkshire pigs under general anesthesia. We dissected the flap vessels until the femoral vessels, allowing easier cannulation. We optimized an MP setup, and the flaps were perfused with oxygenated custom-made acellular Steen+ solution at 20-22°C. Monitoring included vascular resistances, edema, angiography, and biochemical and gas measurements in the perfusate. Continuous perfusion was compared to intermittent perfusion (30 to 45 min of perfusion every 90 min of ischemia). Perfusion was performed until failure (Increasing resistances, edema >50%). Histology at the end of the perfusion was compared to non-perfused and fresh controls. RESULTS: We performed eight continuous perfusions and six intermittent perfusions. Both allowed successful 12h perfusion with minimal weight gain related to preserving the vascular tree. After 12h, continuous perfusion showed higher vascular resistance and significant edema. At 24h, the weight of the flaps was higher in the continuous perfusion group (167.97% of initial weight versus 91.61% in the intermittent perfusion group, p=0.04). Intermittent perfusion showed slightly higher lactate values, but no differences were found for other measurements. Flow values were comparable between groups, and histology showed no difference between both perfused groups and controls. Intermittent perfusion allowed successful perfusion until 48h, but increased resistances and edema were found at 72h. CONCLUSIONS: Continuous and intermittent acellular perfusion can lead to short-duration perfusions (<12h), eventually allowing flap salvage (ex-vivo thrombolysis, revision). Longer perfusions seem to present better outcomes with intermittent perfusion. Further optimization is needed to allow extracorporeal perfusion of FFF for several days, eventually leading to reconstructions without micro anastomosis as suggested in the recent literature.
Background: The thumb plays a key role in hand function for precise dexterity. The surgical treatment of full avulsions of the digital skin sheath by tearing (ring syndrome) is complex and the management of these lesions is challenging for the reconstructive surgeon. When replantation is not possible, other alternatives are available if the covering tissue is thin and flexible. Case presentation: A 30-year-old male, tobacco addict, had a thumbsequelae similar to a stage III ring finger. Revascularization attempt failed and the skin envelope necrosed secondarily. A groin pocket flapgraft allowed a conservation of his thumb with a satisfactory aesthetic and functional result. Conclusions: We described a thumb coverage by a groin flapgraft using an original pocket design. This simple and safe procedure, in the carefully selected patient can successfully address functional and aesthetic concerns simultaneously, with minimal sequelae at the donor site. This technique can be used to treat a cutaneous tearing of the other fingers.
Vascularized composite allotransplantation (VCA) refers to the transplantation of multiple tissues as a functional unit from a deceased donor to a recipient with a severe injury. These grafts serve as potential replacements for traumatic tissue losses. The main problems are the consequences of the long immunosuppressive drugs and the lack of compatible donor. To avoid these limitations, decellularization/recellularization constitutes an attractive approach. The aim of decellularization/recellularization technology is to develop immunogenic free biological substitutes that will restore, maintain, or improve tissue and organ's function. A PubMed search was performed for articles on decellularization and recellularization of composite tissue allografts between February and March 2021, with no restrictions in publication year. The selected reports were evaluated in terms of decellularization protocols, assessment of decellularized grafts, and evaluation of their biocompatibility and repopulation with cells both in vitro and in vivo. The search resulted in a total of 88 articles. Each article was reviewed, 77 were excluded, and the remaining 11 articles reported decellularization of 12 different vascular composite allografts in humans (4), large animals (3), and small animals (rodents; 5). The decellularization protocol for VCA varies slightly between studies, but majority of the reports employ 1% sodium dodecyl sulfate as the main reagent for decellularization. The immunological response of the decellularized scaffolds remain poorly evaluated. Few authors have been able to attempt the recellularization and transplantation of these scaffolds. Successful transplantation seems to require prior recellularization. Decellularization/recellularization is a promising, growing, and emerging developing research field in vascular composite allotransplantation. Impact statement Tissue engineering for vascular composite allotransplantation using decellularization and recellularization approach is a fast-growing area of interest in the reconstructive surgery field. This review will be a very useful tool to get a clear overview for researchers interested in this field.
INTRODUCTION: The incidence of acute rejection (AR) of the skin within the first year after hand or face transplantation is approximately 85% and up to 56% of patients experience multiple episodes1. Significant immunosuppression is required to prevent allograft loss, and recent studies suggest that repeated AR episodes can lead to VCA dysfunction and loss2. The mechanisms underlying variability in AR presentation remain poorly defined however. MATERIALS AND METHODS: 8 cynomolgus monkeys received either an orthotopic hand (n=2) or heterotopic face VCA (n=6) from MHC-mismatched donors following induction with anti-thymocyte globulin. Post-operatively, triple immunosuppression – tacrolimus, mycophenolate mofetil, methylprednisolone – was maintained for up to 120 days before bone marrow transplantation (BMT) was performed. Protocol biopsies of VCA skin were performed at 30-day intervals for histopathology and flow cytometric analysis of resident skin leukocyte populations; VCA-resident cells were differentiated by H38 status (mouse antihuman HLA class I monoclonal antibody that cross reacts with cynomolgus monkeys) for donor or recipient derivation. Clinical AR was treated with steroids and further biopsies were taken for histologic confirmation; corresponding anti-donor responses were evaluated by mixed lymphocyte reaction (MLR) and allo-antibody formation. RESULTS: Up to three episodes of AR (from POD 14, Banff I to II) developed while recipient animals were maintained on triple immunosuppression. Corresponding flow cytometric analyses demonstrate > 80% of skin-resident T lymphocytes (CD4+, CD8+) within VCA dermis were of recipient origin, suggesting rapid immigration of various lineages into the VCA. These observations coincided with the first episode of AR in fully mismatched recipients but haplomatched animals remained rejection-free. All but one episode of AR were successfully treated. No allo-antibodies were detected and anti-donor responses by MLR were comparable to that against third-party. Following BMT, mixed chimerism was detected and enabled immunosuppression withdrawal. However, this was transient and once lost, clinical AR developed and nearly 100% of both dermal and epidermal lymphocytes were recipient-derived. CONCLUSION: We report a clinically-relevant model for studying AR in VCA. Our results suggest that further understanding of the relative importance of MHC differences in transplant pairs may lead to differences in outcomes for VCA recipients maintained under standard immunosuppression. Immunosuppression-free tolerance of non-hematopoietic antigens in composite tissues can be achieved, but require additional strategies to achieve stable, rather than transient mixed chimerism following BMT. DISCLOSURE/FINANCIAL SUPPORT:Supported by the Reconstructive Transplantation Research Consortium grant W81XWH-13-2-0062. None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript. REFERENCES: 1. Petruzzo P, Lanzetta M, Dubernard JM, Landin L, Cavadas P, Margreiter R, et al. The International Registry on Hand and Composite Tissue Transplantation. Transplantation. 2010;90:1590–1594 2. Unadkat JV, Bourbeau D, Afrooz PN, Solari MG, Washington KM, Pulikkottil BJ, et al. Functional outcomes following multiple acute rejections in experimental vascularized composite allotransplantation. Plast Reconstr Surg. 2013;131:720e-30e
