Full-thickness Skin Allografts Research Articles

Skin Grafting on 3D Bioprinted Cartilage Constructs In Vivo.

Background:Three-dimensional (3D) bioprinting of cartilage is a promising new technique. To produce, for example, an auricle with good shape, the printed cartilage needs to be covered with skin that can grow on the surface of the construct. Our primary question was to analyze if an integrated 3D bioprinted cartilage structure is a tissue that can serve as a bed for a full-thickness skin graft.Methods:3D bioprinted constructs (10 × 10 × 1.2 mm) were printed using nanofibrillated cellulose/alginate bioink mixed with mesenchymal stem cells and adult chondrocytes and implanted subcutaneously in 21 nude mice.Results:After 45 days, a full-thickness skin allograft was transplanted onto the constructs and the grafted construct again enclosed subcutaneously. Group 1 was sacrificed on day 60, whereas group 2, instead, had their skin-bearing construct uncovered on day 60 and were sacrificed on day 75 and the explants were analyzed morphologically. The skin transplants integrated well with the 3D bioprinted constructs. A tight connection between the fibrous, vascularized capsule surrounding the 3D bioprinted constructs and the skin graft were observed. The skin grafts survived the uncovering and exposure to the environment.Conclusions:A 3D bioprinted cartilage that has been allowed to integrate in vivo is a sufficient base for a full-thickness skin graft. This finding accentuates the clinical potential of 3D bioprinting for reconstructive purposes.

Transfusion of ethylene carbodiimide–fixed donor splenocytes prolongs survival of vascularized skin allografts

BackgroundAllograft rejection is a major obstacle to the widespread clinical application of vascularized composite allotransplantation. Recent studies revealed a noncytoreductive strategy to protect allografts by the transfusion of ethylene carbodiimide–fixed donor splenocytes (ECDI-SPs). To determine whether this approach offers advantages in protecting skin allografts, we examined the immunological protection of infusing ECDI-SPs with a 30-d administration of rapamycin on the skin allografts of mice. Materials and methodsC57BL/6 recipient mice received BALB/c donor full-thickness skin or vascularized skin transplants at day 0, along with the infusion of donor ECDI-SPs 7 d before and 1 d after allotransplantation and a 30-d course of rapamycin. Recipients received ECDI-untreated splenocytes or C3H allografts as controls. In vitro allostimulatory activity of ECDI-SPs and donor-specific ex vivo hyporesponsiveness were tested. Production of related cytokines (TGF-β, IL-10, IL-1β, and TNF-α) and expression of CD4+Foxp3+ regulatory T cells (Tregs) were also examined. ResultsTransfusion of ECDI-SPs combined with rapamycin significantly prolonged survival of full-thickness skin (median survival time [MST]: 28 d) and full-thickness skin allografts (MST: 71 d) compared with untreated splenocytes (MSTs: 11 d and 30 d) or C3H allografts (MSTs: 11 d and 38 d). This effect was accompanied by increased production of IL-10 and TGF-β, decreased production of IL-1β and TNF-α, and expansion of Tregs in vitro and in vivo. ConclusionsECDI-SP infusion combined with short-term rapamycin administration provides a promising approach to prolong the skin allograft survival.

Banking and use of glycerol preserved full-thickness skin allograft harvested from body contouring procedures

IntroductionThe use of glycerol preserved skin allograft (GPA) became a main stay in burn treatment. However, harvesting of cadaveric skin is not yet legalized in many countries including Egypt. ObjectivesTo estimate the feasibility of using skin harvested from body contouring procedures as a source of GPA and its clinical efficacy. Patients and methodsSkin harvested from body contouring procedures done in Al-Azhar university hospitals was preserved by glycerolization and used in management of burn and complicated wounds. ResultsIn the period between February 2012 and February 2013 skin was harvested from 24 abdomenoplasty cases, 6 bilateral breast reduction cases, and 1 case of thigh lift done in Al-Azhar university hospitals. This yielded about 22,000cm2 of skin preserved by glycerolization. This GPA was used in 15 excised burn wounds, in 9 cases of chronic burn wounds, and in 6 complicated wounds. Partial graft loss occurred in 3 cases and total graft loss occurred in 1 case. ConclusionThe glycerolized full-thickness skin harvested from body contouring procedures is clinically effective in burn and wound management. In the presence of regional coordination, it can serve as an abundant source for skin banking in where cadaveric skin use is not legalized.

Transplantation tolerance induced by regulatory T cells: In vivo mechanisms and sites of action

The mechanisms by which CD4 +CD25 +Foxp3 + T (Treg) cells regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer model, we analyzed the in vivo expansion, trafficking, and effector function of alloreactive T cells and donor-specific Treg cells, in response to a full-thickness skin allograft. Fluorescent-labeled CD4 +CD25 − and antigen-specific Treg cells were transferred alone or co-injected into syngeneic BALB/c-Nude recipients transplanted with skins from (C57BL/6×BALB/c) F1 donors. Treg cells divided in vivo, migrated and accumulated in the allograft draining lymph nodes as well as within the graft. The co-transfer of Treg cells did not modify the early activation and homing of CD4 +CD25 − T cells in secondary lymphoid organs. However, in the presence of Treg cells, alloreactive CD4 +CD25 − T cells produced significantly less IFN-γ and were present in reduced numbers in the secondary lymphoid organs. Furthermore, time-course studies showed that Treg cells were recruited into the allograft at a very early stage after transplantation and effectively prevented the infiltration of effector T cells. In conclusion, suppression of rejection requires the early recruitment to the site of antigenic challenge of donor-specific Treg cells, which then mainly regulate the effector arm of T cell alloresponses.

Preliminary evidence that the allogeneic response might trigger antitumour immunity in patients with advanced prostate cancer

To explore the possibility that allogeneic responses might, by chance, encompass cross-reactive T cell clones specific for neo-antigenic tumour determinants, and thereby activate antitumour immunity; such cross-reactions are well documented for antiviral immunity, and genetic instability in developing cancers generates many neo-antigenic determinants as potential targets for immune responses, but the biology inevitably favours tumour progression. Fourteen patients with hormone-refractory prostate cancer received full-thickness skin allografts from different, unrelated donors (fellow patients) until each had received six grafts. Serum prostate-specific antigen (PSA) level was used as a surrogate for tumour mass. One patient had a remarkable decline in PSA level, with levels at 1 year lower than before grafting. A second patient had stable PSA levels for almost 2 years. A third patient had stable PSA levels for 10-12 months before they resumed an exponential rise. Of four patients with PSA levels of > 10 ng/mL, three required surgery or radiotherapy for obstructive symptoms during or shortly after grafting. Transplant rejection involves mechanistically atypical T cell recognition of allogeneic major histocompatibility complex antigens, with massive polyclonal T cell activation. This unique aspect of T cell biology might represent a novel approach for initiating cross-reactive antitumour responses.

Treatment with the humanized CD154-specific monoclonal antibody, hu5C8, prevents acute rejection of primary skin allografts in nonhuman primates.

Allogeneic skin transplantation remains a rigorous test of any immune intervention designed to prevent allograft rejection. To date, no single, clinically available immunosuppressant has been reported to induce long-term primary skin allograft survival in primates. We have previously shown that treatment with the humanized CD154-specific monoclonal antibody, humanized 5C8 (hu5C8), induces long-term renal allograft survival in nonhuman primates. In this study, we evaluated the efficacy of hu5C8 in preventing primary skin allograft rejection in rhesus monkeys. Ten rhesus monkeys were transplanted with full-thickness skin allografts mismatched at both class I and class II major histocompatibility loci. Of these, two were given no treatment, five were treated with hu5C8 alone, and three received hu5C8 combined with whole blood donor-specific transfusion (DST). All recipients also received skin autografts for comparison. Animals were followed by inspection, serial biopsy, mixed lymphocyte culture, and alloantibody determination. Treatment with hu5C8 alone or hu5C8 plus DST greatly prolonged allograft survival. Rejection occurred in the untreated group within 7 days. Mean allograft survival in the monotherapy hu5C8 group was >236 days and in the DST group was >202 days; these differences were not significant. Rejection eventually occurred in most animals. Allograft survival was not correlated with the development of T cell hyporesponsiveness in mixed lymphocyte culture. Rejection was not predicted by the development of donor-specific alloantibody. These results show that treatment with the CD154-specific monoclonal antibody, hu5C8, greatly delays the onset of acute skin allograft rejection.

USE OF CTLA4-Ig IN COMBINATION WITH CONVENTIONAL IMMUNOSUPPRESSIVE AGENTS TO PROLONG ALLOGRAFT SURVIVAL

The objective of our study was to determine the effectiveness of CTLA4-Ig, a novel immunosuppressive agent, in augmenting allograft survival when combined with either cyclosporine, sirolimus, donor-specific bone marrow alone (BM), or bone marrow in conjunction with antilymphocyte serum (ALS). Full-thickness skin allografts were used in C3H to B6AF1 (class I mismatch) and AKR to C57BL/6 (complete mismatch) models. Groups of mice (n=6-14) were treated with various combinations of the following treatment protocols: murine CTLA4-Ig, L-6 control Ig, sirolimus, cyclosporine, ALS, or ALS/BM. In the class I mismatch model, L-6 control Ig had no effect whereas use of CTLA4-Ig alone resulted in a doubling of the median graft survival compared with controls. The addition of either sirolimus or cyclosporine to CTLA4-Ig increased graft survival over that achieved with CTLA4-Ig alone. CTLA4-Ig demonstrated no efficacy when used in combination with BM, ALS, or ALS/BM. CTLA4-Ig was clearly less effective in the complete mismatch model. These data suggest that CTLA4-Ig may be effective clinically in combination with cyclosporine or sirolimus but offers no additional effectiveness in combination with antilymphocyte serum with or without donor-specific bone marrow.

The role of immunosuppression in the long-term survival of tracheal allografts.

To assess the functional aspects of vascularized tracheal allograft transplant models and the long-term fate of these allografts. To examine the effects of cyclosporin A (CsA), 10 mg/kg per day, on the long-term survival of vascularized tracheal allografts and the presence and significance of host immune tolerance after cessation of immunosuppression. In a rabbit model, tracheal allografts were orthotopically transplanted after an initial period of heterotopic fascia revascularization. A full-thickness skin allograft from the same donor was used as an external monitor of the rejection process. Treatment with CsA was discontinued (group 2) or given intermittently (group 3) after an initial course of continuous CsA administration varying from 2 to 10 weeks. The first group received a continuous regimen of CsA for 10 weeks. Tracheal and skin allograft deterioration, microcirculation of tracheal transplant, mucociliary clearance, and histopathologic examination. Tracheal allografts under continuous immunosuppression with CsA, 10 mg/kg per day, showed no rejection and remained functional with preservation of mucociliary activity. After an initial course of CsA to achieve solid tracheal union, subsequent cessation and intermittent pulsing of the immunosuppressant were insufficient in maintaining indefinite graft survival. Chronic allograft rejection with simultaneous endothelial graft repopulation could be induced by intermittent pulsing of CsA, which led to prolonged allograft survival in some group 3 animals. Continuous immunosuppression is necessary to preserve an optimal morphological and functional condition of tracheal allografts.

CULTURED MOUSE KERATINOCYTE ALLOGRAFTS PRIME FOR ACCELERATED SECOND SET REJECTION AND ENHANCED CYTOTOXIC LYMPHOCYTE RESPONSE1,2

It has been reported that cultured keratinocyte (CK) allografts are not rejected in mice, unlike in other species. Several reports have suggested that mouse CK allografts are incapable of stimulating a primary alloresponse, including sensitization of recipients to alloantigens. In this study, we investigated the immunogenicity of mouse CK allografts in vivo by determining whether CK allografts primed for a second set rejection response. First, we grafted mice with either CK allografts, CK autografts, full-thickness (FT) allografts, or no graft at all. We then regrafted mice 4 weeks later with a tail skin allograft. Mice grafted with CK allografts rejected second allografts as rapidly and as vigorously as mice grafted with FT flank allografts. Next, we tested whether CK allograft primed recipients for enhanced CTL responses. We found that mice grafted with CK allografts generated a significantly enhanced CTL alloreactive response after in vitro stimulation. The response was similar to that of mice grafted with FT skin allografts. With evidence that CK allografts primed, we biopsied wounds immediately after CK allografting and, using Western immunoblotting, found that CK allografts had substantial expression of MHC class II antigens in vivo. We conclude from the results of our studies that mouse CK allografts unequivocally prime recipients to alloantigens in vivo and suggest that a possible mechanism for alloantigen priming may be CK allograft expression of MHC class II antigens.

Cultured keratinocyte grafts are recognized, but not rejected by CD8+ T cells in vivo

It has been shown in the mouse that cultured keratinocyte allografts [fully major histocompatibility complex (MHC) mismatched] survive for at least 100 days without evidence of rejection. In an attempt to analyze the immune mechanisms underlying this phenomenon we have investigated the induction of tolerance to such grafts. Primary cultures of BALB/c keratinocytes were prepared using irradiated 3T3 feeder cells, and the cultured cell sheets were grafted, using a silicone transplantation chamber, onto CBA recipients. After the cultured grafts had been in place for 4-6 weeks full-thickness tail skin from BALB/c donors was grafted onto the dorsal flank opposite the cultured graft. The median graft survival time of these full-thickness allografts was 15.5 days compared to 13 days in the control group. These data show that in the absence of Langerhans cells and MHC class II expression, keratinocytes expressing class I and minor histocompatibility antigens induce a prolonged survival of full-thickness skin allografts. The results from experiments in which T cell subsets were depleted in vivo suggest that CD8+ cells (a) recognize the class I alloantigens of the cultured graft, (b) do not reject the cultured graft and (c) do not progress further in their maturation pathway. We propose that these CD8+ T cells might have been partially primed by receiving only the first signal of activation and that they may be equivalent to "poised" cytotoxic T cells. These CD8+ cells can reject non cultured full-thickness skin grafts, and do so more effectively after removing CD4+ cells.

Immunocompetent cells in the bed of a full-thickness skin allograft transplanted at different times of day

Immunocompetent cells in the bed of a full-thickness skin allograft transplanted at different times of day

The immunosuppressive effect of concanavalin A in monkeys.

These studies were performed to evaluate the immunosuppressive effect of concanavalin A (ConA) in monkeys. Bonnet monkeys (Macaca radiata) weighing between 2.5-3.5 kg were used. Following the exchange of full thickness skin allografts in randomly selected animals, gross and histopathological studies of the rejection reaction were performed. Gross observations showed the allograft reaction in control animals to be a well defined phenomenon occurring at 9.0 +/- 0.7 days. In contrast the rejection process in animals that received i.v. ConA was a poorly defined prolonged process occurring at 14.8 +/- 3.9 days (single 75-mg dose of ConA), and 19.0 +/- 2.2 days (75 mg 1 day before and 25 mg 3 days after grafting). In monkeys that received the double dose schedule of ConA, histopathological studies showed distinct retardation of the rejection reaction with reduced lymphocytic infiltration of the graft-host junction. Control grafts showed almost complete rejection in 7-9 days while allografts on ConA-treated animals showed corresponding degrees of destruction only by the 15th day. These studies indicate that ConA does have an immunosuppressive effect in vivo in monkeys. The local and systemic toxic effects of ConA encountered in these animals have been reported and its haematological effects in vivo briefly described.

SYNERGISM IN IMMUNOSUPPRESSION

SUMMARY Intact and splenectomized mice were given full thickness skin allografts and tetanus toxoid and then treated with several immunosuppressive agents singly and in combination. Both allograft rejection and humoral antibody production were suppressed in a similar proportion of animals by similar treatment protocols. Splenectomy potentiated depression of the cellular and humoral response in mice treated with one agent. A radiosensitive cell essential to humoral and cellular immunocompetence may have been the common target affected in both types of immunosuppression.

Primary Rejection of Skin Allografts in the Anterior Chamber of the Rabbit Eye

Abstract The survival of primary skin allografts placed within the anterior chamber of rabbits was examined in temporal sequence following transplantation to normal and x-irradiated recipients. Initial “take” was successful in all instances as judged by vascularization of graft stroma and hyperplasia of skin epithelium. Starting on day 6, and thereafter to an increasing degree, mononuclear cells infiltrated the graft and graft bed, eventually obscuring this border completely. Rejection of the irisimplanted tissue was complete by day 14, though graft epithelium which had extended to cover the undersurface of the cornea persisted for a few days longer before its ultimate rejection. The present study offers direct evidence that primary full-thickness skin allografts placed within the anterior chamber succumb to a rejection process temporally and histologically similar to rejection of orthotopic skin grafts. The immunologic nature of this reaction is supported by the finding that whole body irradiation abrogates this response, and further that similarly transplanted autologous skin or lymph node fragments survive intact for at least 120 days. It is concluded that the anterior chamber is not a generally privileged site for allograft survival.

Effects of cortisol on the microvasculature of mouse skin isografts and allografts

The mouse transparent skin chamber has been improved to permit high magnification in vivo observations of the cellular and microcirculatory events within a full-thickness skin graft from the time of transplantation to ultimate rejection of the allograft. The effects of systemic cortisol have been observed to: 1. Suppress vascularization and suppress maturation of the invading graft vessels; 2. Cause tortuosity of the new vessels and cause formation of microaneurysms; 3. Produce vessel fragility with resultant petechia formation; 4. Suppress WBC sticking to allograft endothelium and minimize cellular infiltrate within allografts; and 5. Prolong the microcirculatory flow through full-thickness skin allografts for an additional 7–10 days. The primary mechanism for prolonging the blood flow through a full-thickness skin allograft appears to be a function of cortisol suppression of the inflammatory response within the microvasculature of the graft.