Allogeneic Skin Research Articles

CD8+ T Cells Differentiate into a Potent CD43+ Effector Population During Acute Allograft Rejection

Abstract Although acute rejection threatens allograft function, little is known about the functional properties of graft-specific effector CD8 +T cells. Using the Balb/c H-2L(d) into C57Bl/6 H-2(b) fully allogeneic skin graft model, we identified host CD8 +T cells specific for the L(d) QL9 epitope using MHC tetramers. L(d) QL9 +T cells increased in number and differentiated into CD44 +effector populations by day 10 post-graft with Balb/c skin, but not third-party C3H skin. Using dimensionality reduction analysis, we found that a population of CD62L loeffector CD8 +T cells expressing the surface receptor CD43 formed during graft rejection in the draining lymph nodes, spleen, and blood. The frequency of CD43 +effectors peaked from day 10–14 post-graft, indicative of a short-lived population. CD43 +CD8 +T cells displayed potent effector functions relative to CD43 −cells, including Granzyme B expression, IFN-γ/TNF-α co-production, and accelerated Balb/c graft rejection (p<0.01 for each). Adoptive transfer experiments revealed that only 31% of the L(d) QL9 +T cells divided on day 14 post-graft, versus 95.6% of OT-I T cells after mOVA skin grafting. CD43 expression was only expressed on cells in their final division. Finally, we rechallenged flow cytometry purified CD45.1 CD43 +and CD43 −effector CD8 +T cells with a Balb/c skin graft, and found that approximately half of the CD43 −population became CD43 +, while all of the CD43 +population remained CD43 +on day 21 (49% vs 96%, p<0.001). Together, these data demonstrate that CD8 +T cells differentiate into a CD43 +population after grafting that undergoes weak proliferation and yet is functionally potent.

The Effect of Fetal Bovine Acellular Dermal Matrix Seeded with Wharton's Jelly Mesenchymal Stem Cells for Healing Full-Thickness Skin Wounds.

The treatment of full-thickness skin wounds is a problem in the clinical setting, as they do not heal spontaneously. Extensive pain at the donor site and a lack of skin grafts limit autogenic and allogeneic skin graft availability. We evaluated fetal bovine acellular dermal matrix (FADM) in combination with human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) to heal full-thickness skin wounds. FADM was prepared from a 6-month-old trauma-aborted fetus. WJ-MSCs were derived from a human umbilical cord and seeded on the FADM. Rat models of full-thickness wounds were created and divided into three groups: control (no treatment), FADM, and FADM-WJMSCs groups. Wound treatment was evaluated microscopically and histologically on days 7, 14, and 21 post-surgery. The prepared FADM was porous and decellularized with a normal range of residual DNA. WJ-MSCs were seeded and proliferated on FADM effectively. The highest wound closure rate was observed in the FADM-WJMSC group on days 7 and 14 post-surgery. Furthermore, this group had fewer inflammatory cells than other groups. Finally, in this study, we observed that, without using the differential cell culture media of fibroblasts, the xenogeneic hWJSCs in combination with FADM could promote an increased rate of full-thickness skin wound closure with less inflammation.

Bone marrow mesenchymal stem cell-derived exosomes attenuate the maturation of dendritic cells and reduce the rejection of allogeneic transplantation

Bone mesenchymal stem cell (BMSC)-derived exosomes (B-exos) are attractive for applications in enabling alloantigen tolerance. An in-depth mechanistic understanding of the interaction between B-exos and dendritic cells (DCs) could lead to novel cell-based therapies for allogeneic transplantation. To examine whether B-exos exert immunomodulatory effects on DC function and maturation. After mixed culture of BMSCs and DCs for 48 h, DCs from the upper layer were collected to analyze the expression levels of surface markers and mRNAs of inflammation-related cytokines. Then, before being collected to detect the mRNA and protein expression levels of indoleamine 2,3-dioxygenase (IDO), the DCs were co-cultured with B-exos. Then, the treated DCs from different groups were co-cultured with naïve CD4+ T cells from the mouse spleen. The proliferation of CD4+ T cells and the proportion of CD4+CD25+Foxp3+ T cells were analyzed. Finally, the skins of BALB/c mice were transplanted to the back of C57 mice in order to establish a mouse allogeneic skin transplantation model. The co-culture of DCs with BMSCs downregulated the expression of the major histocompatibility complex class II (MHC-II) and CD80/86 costimulatory molecules on DCs. Moreover, B-exos increased the expression of IDO in DCs treated with lipopolysaccharide (LPS). The proliferation of CD4+CD25+Foxp3+ T cells increased when cultured with B-exos-exposed DCs. Finally, mice recipients injected with B-exos-treated DCs had significantly prolonged survival after receiving the skin allograft. Taken together, these data suggest that the B-exos suppress the maturation of DCs and increase the expression of IDO, which might shed light on the role of B-exos in inducing alloantigen tolerance.

Effects of periodontal pathogen-induced intestinal dysbiosis on transplant immunity in an allogenic skin graft model

Periodontal disease can induce dysbiosis, a compositional and functional alteration in the microbiota. Dysbiosis induced by periodontal disease is known to cause systemic inflammation and may affect transplant immunity. Here, we examined the effects of periodontal disease-related intestinal dysbiosis on transplant immunity using a mouse model of allogenic skin graft in which the mice were orally administered the periodontal pathogen Porphyromonas gingivalis (Pg). For 6 weeks, the Pg group orally received Pg while the control group orally received phosphate-buffered saline solution. After that, both groups received allogenic skin grafts. 16 s rRNA analysis of feces revealed that oral administration of Pg significantly increased three short chain fatty acids (SCFAs) producing genera. SCFA (acetate and propionate) levels were significantly higher in the Pg group (p = 0.040 and p = 0.005). The ratio of regulatory T cells, which are positively correlated with SCFAs, to total CD4+ T cells in the peripheral blood and spleen was significantly greater (p = 0.002 and p < 0.001) in the Pg group by flowcytometry. Finally, oral administration of Pg significantly prolonged skin graft survival (p < 0.001) and reduced pathological inflammation in transplanted skin grafts. In conclusion, periodontal pathogen-induced intestinal dysbiosis may affect transplant immunity through increased levels of SCFAs and regulatory T cells. (198 words).

Factors involved in burn wound healing – short review

Burns represent one of the worldwide leading causes of injury. Burn wounds are associated with increased mortality and morbidity, especially the impaired quality of life due to hypertrophic scarring, scar pain and itching, paresthesia, and contractures. To properly influence the burn wound healing, it is important to establish a correct classification of the acute injury and to understand the main phase of burn wound healing process. There are various local and systemic factors that can be influenced to obtain proper healing. The most important factors include local necrosis and infection, increased local pressure and edema, anemia, hypoxia, hypotension, the presence of important chronic disease and medication, immunosuppression, nutritional status, age, and body constitution. After adequate fluid resuscitation and patient stabilization, the main step is represented by the detachment of the devitalized tissues and rapid coverage of the lesion. There are various methods to be used, like autologous or allogenic skin grafting, the use of skin substitutes, or tissue bioengineering. Knowing all these aspects, allows clinicians to properly define a therapeutic management for patients presenting severe burns.

A Survey of 1000 Respondents on the Polish Population's Knowledge and Attitudes about Tissue/Organ Donation and Transplantation in Times of Allogeneic Tissue Shortage.

Tissue-engineered human allogeneic skin grafts retrieved from a deceased donor play an important role in the therapy of extensive and deeply burned patients. However, there is a vital deficit of allogeneic skin donors, and the reserves of human allogeneic skin grafts are not sufficient. The goal of this work was to analyze the level of knowledge and attitudes of Polish society in the field of transplantation, with particular emphasis on allogeneic skin transplantation. The study used a self-made questionnaire comprised of 23 questions. 1000 respondents took part in this research. The respondents were a diverse group in terms of age, sex, education, and place of residence. The obtained results show a general positive attitude of the respondents towards the idea of transplantology. However, people with lower education presented a more negative attitude towards the donation of tissues and organs. Additionally younger people were not able to clearly declare readiness for organ procurement. What is more data analysis revealed certain gaps in more detailed knowledge and surprising attitudes. In that respect, the lack of awareness about the criteria for determining brain death could be mentioned. There was also a lack of acceptance for skin procurement in specific population groups. It can therefore be concluded that a key role in the success of the idea of transplantation in Poland is the broad and systematic education of the society.

Research Highlights

Research Highlights

Epidermolysis Bullosa: A Review of the Tissue-Engineered Skin Substitutes Used to Treat Wounds.

Skin wound healing is a crucial process for regenerating healthy skin and avoiding the undesired consequences associated with open skin wounds. For epidermolysis bullosa(EB), a debilitating group of fragile skin disorders currently without a cure, skin blistering can often be severe and heal poorly, increasing susceptibility to life-threatening complications. To prevent these, investigational therapies have been exploring the use of tissue-engineered skin substitutes(TESSs) aimed at replacing damaged skin and promoting long-term wound closure. These products have either been developed in house or commercially sourced and are composed ofallogeneic or autologous human skin cells, often with some form of bioscaffolding. They can be broadly classified based on their cellular composition: keratinocytes (epidermal substitutes), fibroblasts (dermal substitutes) or a combination of both (composite substitutes). Encouraging long-term wound healing has been achieved with epidermal substitutes. However, these substitutes have not demonstrated the same efficacy for all patients, which may be due to the molecular heterogeneity observed between EBsubtypes. Autologous compositeTESSs, which more closely resemble native human skin, are therefore being investigated and may hold promise for treating an extended range of patients. Additionally, future TESSsfor EBare focused on using gene-corrected patient skin cells, which have already demonstrated remarkable long-term woundhealing capabilities. In this review, we provide an overview of the different TESSsthat have been investigated in clinical studies to treat patients with EB, as well as their long-term woundhealing results. Where available, we describe the methods used to develop these products to inform future efforts in this field.

Current use of T-cell depleting polyclonal antibodies in liver transplant recipients: Results of a nationwide French survey

Shikonin (SHK) has multifaceted physiological functions, including antitumor, anti-inflammatory, and antimicrobial effects. Recently, SHK has been shown to affect immune responses; however, its detailed immune modulatory function in vivo remains unclear. In this study, we demonstrated that SHK not only inhibited T cell proliferation in vitro, but also intensively inhibited thymopoiesis and eliminated CD4/CD8 double-positive thymic progenitor cells in vivo. Treatment of mice with SHK resulted in immune profile alterations, which promoted myelosis in the bone marrow and increased inhibitory immune cells in central immune organs. A decrease in T cells and B cells was observed in the spleen. Using a murine allogenic skin transplantation model, we revealed that short-term treatment of recipients with SHK significantly inhibited skin graft rejection, in which the levels of myeloid-derived suppressor cells (MDSC) were markedly increased. Taken together, our study suggests that SHK can efficiently eliminate proliferating T cells and inhibit thymopoiesis while promoting the generation of MDSC, indicating its potential role in alleviating immune responses in allogeneic organ/cell transplantation.

344.9: Clinical Grade CD8+T Regulatory Cells Moving Forward in Human Transplantation

Introduction: We have demonstrated the therapeutic potential of human polyclonal and antigen-specific CAR-modified CD8+Tregs cell therapy to prevent allogeneic human skin transplantation rejection and xenogeneic GvHD in humanized NSG mice (Bézie et al., Front Immunol.2017, Blood Adv.2019). However, their potential has never been evaluated in a clinical trial. We are thus preparing the launch of a phase I/IIa human clinical trial using polyclonal CD8+Tregs cell therapy in living donors kidney transplant patients. Method: CD8+ cells were isolated from blood of healthy volunteers and patients with kidney failure by Clinimacs System then CD8+CD4-CD45RClow/-CD56- T cells were sorted by MACSQuant Tyto cell sorter. Cells were stimulated with anti-CD3 and CD28 mAbs every week and cultured in presence of rapamycin, IL-2 and IL-15. Cytotoxicity against allogeneic PBMCs was assessed by Annexin V/DAPI staining and suppression capacity was assessed in vitro on syngeneic CD4+T cells proliferation in response to allogeneic APCs and in vivo on 1.5Gy-irradiated NSG mice co-injected with human PBMCs. Results: We developed a new GMP-compatible cell manufacturing process. First, we determined a new method of isolation of CD8+Tregs from peripheral blood using a safe closed system. Next, we identified the optimal clinically compatible culture medium, refined cytokines doses and stimulation methods to preserve a high proliferation rate, phenotypic profile and suppressive function of CD8+Tregs. Using this process, we were able to efficiently expand CD8+Tregs from peripheral blood of patients with renal failure, with high purity, while preserving their regulatory function. We confirmed the absence of cytotoxicity. In addition, we showed that CD8+Tregs were phenotypically and functionally stable for 4h after conditioning, which is important for the logistic delay between cell harvest and patient infusion. Finally, we showed that CD8+Tregs persist for more than 90 days in NSG mice without inducing any signs of xenogeneic GVHD, and could withstand combined immunosuppressive drug therapy administered to the patient. Conclusion: We designed a clinically compatible manufacturing process to isolate and culture CD8+Tregs from renal failure patients that preserves their function and patient safety.

Shikonin impairs T lymphocyte proliferation and thymopoiesis while it may increase myeloid-derived suppressor cells to alleviate immune responses

Shikonin (SHK) has multifaceted physiological functions, including antitumor, anti-inflammatory, and antimicrobial effects. Recently, SHK has been shown to affect immune responses; however, its detailed immune modulatory function in vivo remains unclear. In this study, we demonstrated that SHK not only inhibited T cell proliferation in vitro, but also intensively inhibited thymopoiesis and eliminated CD4/CD8 double-positive thymic progenitor cells in vivo. Treatment of mice with SHK resulted in immune profile alterations, which promoted myelosis in the bone marrow and increased inhibitory immune cells in central immune organs. A decrease in T cells and B cells was observed in the spleen. Using a murine allogenic skin transplantation model, we revealed that short-term treatment of recipients with SHK significantly inhibited skin graft rejection, in which the levels of myeloid-derived suppressor cells (MDSC) were markedly increased. Taken together, our study suggests that SHK can efficiently eliminate proliferating T cells and inhibit thymopoiesis while promoting the generation of MDSC, indicating its potential role in alleviating immune responses in allogeneic organ/cell transplantation.

A Novel Combination of Keratinocyte and Autologous Microskin Grafting to Repair Full-Thickness Skin Loss

Introduction: The high mortality of patients with extensive deep burns is mainly attributed to the extensive burn wound and the scarce autologous skin left for wound repair. The purpose of this study was to explore how to effectively use the limited remaining autologous skin to repair the extensive deep wound. Methods: Human keratinocytes harvested from the foreskin were cultured and transfected with epidermal growth factors (EGFs) by an adenovirus vector (Ad-EGF). The expression and the biological activity of EGF in both the normal human keratinocytes and the EGF gene-modified human keratinocytes were quantified by ELISA assay and CCK-8 assay, respectively. The differentiated phenotype of epidermal cells was detected by immunofluorescence staining via CK10, CK14, and CK19 expressions. Rats were subjected to a full-thickness skin loss (3.3 cm × 3.0 cm) on the dorsum, which was repaired with the EGF gene-modified human keratinocyte suspension and autologous microskin and covered with the allogeneic skin. The wound healing was quantified, and the expression of EGF mRNA was measured by RT-PCR. Results: The EGF gene-modified human keratinocytes highly expressed EGF. CK10, CK14, and CK19 as keratinocyte differentiation markers were increased in the EGF gene-modified human keratinocytes. Wound healing was accelerated remarkably by the combination of autologous microskin grafting and EGF gene-modified human keratinocytes in vivo, and a very high EGF mRNA expression was observed in EGF gene-modified human keratinocytes groups on days 7 and 14 compared with other groups. Discussion/Conclusion: The EGF gene-modified human keratinocyte suspension may serve as promising seed cells which can effectively secrete EGF to accelerate wound repair in combination with autologous microskin grafting and reduce the autologous skin requirement for wound repair.

The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model.

BackgroundOrgan transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host’s immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models.Materials and methodsEach mouse was intraperitoneally injected with 100 μg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis.ResultsThe mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine–cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4+IL-4+ T cells and CD4+Foxp3+ T cells and decreased CD4+IFN-γ+ T cells in skin transplantation.Conclusion Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

Inhibition of miR-let-7i Induces DC Immature Cells and Improves Skin Graft Tolerance.

Dendritic cells (DC) initiate the immune response in the body. They can stimulate T cell activation, proliferation, and differentiation and ultimately participate in the immune response and the immune tolerance response. The purpose of this study was to coculture DCs and T cells and subcutaneously inject DCs transfected with miR-let-7i into rhesus monkey transplantations to verify the role of miR-let-7i in allograft immune tolerance. In vitro studies found that the expression of miR-let-7i was upregulated after inducing the maturation of DCs. The low expression of miR-let-7i inhibited the maturation of DCs, promoted the differentiation of T cells into T helper T cells 2 (Th2), and inhibited T helper T cell 1- (Th1-) driven rejection. In vivo studies also obtained similar results, and subcutaneous injection of DCs transfected with miR-let-7i inhibitor prolonged the survival time of allogeneic skin transplantation. Therefore, we conclude that inhibition of miR-let-7i inhibits DC maturation and improves the tolerance of grafted skin.

Graft-Matched Pregnancy Imparts Profound Epigenetic Changes onto Alloreactive Memory T Cells to Enforce a Phenotypic and Transcriptional State of Tolerance

Abstract Pregnancy is an immunological paradox, where the maternal immune system develops tolerance to a semi-allogeneic fetus, yet pregnancy is a major sensitizing event in clinical transplantation. We previously reported that pregnancy robustly tolerizes fetus-specific T cells, allowing for the spontaneous acceptance of fetus-matched allografts when B cells and alloantibodies are absent at the time of transplantation(1). In this study, we report that mice sensitized to 2WOVA.B/c allogeneic skin grafts (Memory) underwent successful pregnancies comparable to Naïve mice when mated with 2WOVA.B/c males. We utilized a high-dimensional multi-omics approach to characterize post-partum (PP) fetus-specific T cells from Naïve or Memory mice after semi-allogeneic pregnancies. Strikingly, pregnancy induced fetus-specific naïve and memory T cells to achieve a nearly identical phenotype, characterized by upregulation of co-inhibitory molecules FR4, CD73, PD1, and CTLA4 in CD4+FoxP3- Tconvs and upregulated PD-1, FR4 and Lag3 in CD8+ T cells. We confirmed that naïve and memory PP CD8+ T cells acquired similar transcriptional profiles via RNA-Seq; unexpectedly, pregnancy induced profound epigenetic modifications in memory, but not naïve, CD8+ T cells. In vitro studies indicated reduced TNF aproduction by memory PP CD4+ and CD8+ T cells, and experiments examining their response to fetus-matched allografts are ongoing. Our studies suggest that significant epigenetic imprinting is necessary to constrain memory T cells to preserve fetal viability during semi-allogeneic pregnancy. These findings may provide insights into controlling antigen-specific memory T cells, a major barrier to clinical transplant success

Romidepsin (FK228) improves the survival of allogeneic skin grafts through downregulating the production of donor-specific antibody via suppressing the IRE1α-XBP1 pathway.

Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.

Intermittent Exposure of Hypercapnia Suppresses Allograft Rejection via Induction of Treg Differentiation and Inhibition of Neutrophil Accumulation.

Background: In the management of major burn wounds, allogeneic skin transplantation is a critical procedure to improve wound repair. Our previous works found that intermittent exposure to carbon dioxide leads to permissive hypercapnia (HCA) and prolongs skin allograft survival. However, the modulatory effects of HCA exposure on the immune system are not well understood. Objectives: Our purpose was to investigate how intermittent exposure to HCA can effectively reduce the immune reaction to allogeneic skin graft rejection. Methods: A fully major histocompatibility complex-incompatible skin transplant from BALB/c to C57BL/6 mice model was utilized. Immune cells from splenic and draining lymph nodes were analyzed by flow cytometry. Serum proinflammatory cytokines were analyzed by ELISA. Results: Serum levels of IFN-γ, IL-2, IL-6, and TNF-α were significantly decreased in the HCA group. Additionally, the percentage of CD8+ cells in draining lymph nodes was significantly lower in HCA than in the control group. Moreover, the generation rate of FoxP3+ regulatory T cells (Tregs) from spleen naïve CD4+ T cells was increased by intermittent exposure to carbon dioxide. The infiltrated neutrophils were also eliminated by HCA. Taken together, we concluded that intermittent hypercapnia exposure could effectively suppress skin rejection by stimulating Treg cell generation and suppressing immune reactions.

The urgent need to achieve an optimal strategic stock of human allogeneic skin graft materials in case of a mass disaster in Poland.

A burn is a sudden injury which immediate or long-term consequences may be life-threatening for the patient. A mass disaster event may involve large numbers of severely burned patients. Patients of this type typically have a limited area of healthy, unburned skin from which an autologous split thickness skin graft could be collected. In a clinical situation of this type, it is necessary to use a particular skin substitute. Non-viable allogeneic human skin graft materials might be considered as the most suitable skin substitutes in the treatment of such patients. At present, Poland does not have a sufficient supply of human allogeneic skin graft materials to meet the needs arising from a sudden and unforeseen mass disaster. This study involved an analysis of selected mass disasters. From this an estimate was made from a verified casualty profile of the necessary minimum stock of human allogeneic skin graft materials. An insufficient amount of skin results from an inadequate number of skin donors, which in turn results from the current tissue donation system. Therefore, a proposal has been made for the organizational, legal and systemic changes required to improve the situation in Polish transplantology, with particular emphasis on skin donation. In order to achieve a strategic stock of human skin grafts, a tissue collecting transplantation team should be organized. The rights and obligations of the non-physician transplant team member should be extended. Proposals have been made for awareness campaigns (adverts, posters etc.) and educational schemes (educational video, lectures during transplant coordinator training, etc.). Finally, a proposal has been made for possible methods to deal with the logistic management of the allogeneic skin stock. The required, essential stock of human allogeneic skin in the event of a mass disaster has been estimated at 600,000 cm2.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10561-022-10001-z.

809 An Allogenic Fat-First Approach to Burn Reconstruction Mitigates Adhesion and Soft Tissue Deficit

IntroductionAdipose and adipose-derived stem cell therapies have met success as adjunctive treatment during burn reconstruction with well described benefit in the delayed-treatment of soft-tissue deficits. While the use of allogeneic skin is well-described, adipose tissues have typically remained autologous. Allogenic fat is not commonly used in burn care, however, in large, complex burns where autologous tissue is limited adipose may not be readily available for harvest or use. Understanding the efficacy of allogeneic tissues in this setting is critical to expand our reconstructive options. Here we describe a protocol utilizing allogeneic fat as well as examine the efficacy of this approach on burn-wound contractures, adhesions, and soft-tissue deficits.MethodsFemale, Yorkshire swine received 16, 4x4 cm full-thickness burns. After 48 hours, eschar was removed to fascia. Wounds were stratified to receive either A) No Reconstruction, B) Skin-Only, C) Fat-Only, D) Immediate-Skin, Delayed-Fat, or E) Immediate-Fat, Delayed-Skin. All fat utilized was allogeneic sourced from vendor-matched swine. At 8-weeks post-engraftment animals were sacrificed and all wounds were collected for photography, ultrasound, histology and serum studies.ResultsUse of allogeneic fat significantly improved terminal soft-tissue thickness under both immediate and delayed administration (p< 0.05). Immediate use of allogeneic fat significantly improved tissue mobility vs. untreated and skin graft controls (p< 0.05). Contracture was most significantly affected by timing of skin graft placement, however, could be further mitigated under standard delayed-fat approached with allogeneic tissue.ConclusionsHere we demonstrate use of allogeneic fat in both traditional-delayed and a fat-first approach with significant mitigation of adhesion when applied as an initial basal layer. Both immediate and delayed allogeneic fat were sufficient to improve on soft tissue deficits.

Autologous skin micro-grafts and allogenic skin allograft for mitten hand deformity treatment in epidermolysis bullosa

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; In the genetic skin blistering disorder dystrophic epidermolysis bullosa recurrent blisters and erosions on mechanically stressed areas, as the hands, result in subsequent scarring and development of pseudosyndactylies and contractures. In its most severe form, all fingers are constrained into a fist of scarred (“mitten-hand”-deformity). No causative therapy exists and only a surgical release of the fingers is possible. Hereby, the development of large skin defects is inevitable; the lack of healthy autologous skin for grafting remains a difficult challenge.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; We propose a new technique: It adopts a treatment strategy from the therapy of large extent burns and consists of a combination of autogenic and allogenic skin grafting.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; We present a beneficial outcome in 18 mitten hand deformities treated with this technique. In all patients, the initial functional outcome was improved. Nearly all patients got a considerably widened grip span and durable skin conditions post-operatively. Mean follow-up was 3 years. Typically, recurrence of the scarring occurred from 6 months to 2 years after operation.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; This is the first report on the use of the combination of autogenous pinch skin grafts with allogenic (parental) skin overlay for skin defects in EB. It provides undisturbed wound healing under a “biological dressing”, furthermore, a temporary take of the parental skin was visible. Defects were permanently covered with skin of increased mechanical stability, hand function improved remarkably. However, long term follow-up showed beginning recurrence in most of the patients comparable to recurrence rates described in the literature.&lt;/p&gt;