Mimicking Clinical Rejection Patterns in a Rat Osteomyocutaneous Flap Model of Vascularized Composite Allotransplantation.

INTRODUCTION: Graft loss in vascularized composite allotransplantation (VCA) is more often associated with vasculopathy and chronic rejection (CR) than acute cellular rejection (ACR). We present a rat osteomyocutaneous flap model using titrated tacrolimus administration that mimics the graft rejection patterns in our clinical hand transplant program. Comparison of outcomes in these models support a role for ischemia reperfusion injury (IRI) and microvascular changes in CR of skin and large-vessel vasculopathy. The potential of the surgical models for investigating mechanisms of rejection and vasculopathy in VCA and treatment interventions is presented. MATERIALS AND METHODS: Four rodent groups were evaluated: syngeneic controls (Group 1), allogeneic transient immunosuppression (Group 2), allogeneic suboptimal immunosuppression (Group 3), and allogeneic standard immunosuppression (Group 4). Animals were monitored for ACR, vasculopathy, and CR of the skin. RESULTS: Transient immunosuppression resulted in severe ACR within 2 wk of tacrolimus discontinuation. Standard immunosuppression resulted in minimal rejection but subclinical microvascular changes, including capillary thrombosis and luminal narrowing in arterioles in the donor skin. Further reduction in tacrolimus dose led to femoral vasculopathy and CR of the skin. Surprisingly, femoral vasculopathy was also observed in the syngeneic control group. CONCLUSIONS: Titration of tacrolimus in the allogeneic VCA model resulted in presentations of rejection and vasculopathy similar to those in patients and suggests vasculopathy starts at the microvascular level. This adjustable experimental model will allow the study of variables and interventions, such as external trauma or complement blockade, that may initiate or mitigate vasculopathy and CR in VCA.

Acute Rejection Rates in Vascularized Composite Allografts: A Systematic Review of Case Reports.

INTRODUCTION: Vascularized Composite Allografts (VCA) are usually performed in a full major histocompatibility complex mismatch setting, with a risk of acute rejection depending on factors such as the type of immunosuppression therapy and the quality of graft preservation. In this systematic review, we present the different immunosuppression protocols used in VCA and point out relationships between acute rejection rates and possible factors that might influence it. METHODS: This systematic review was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We systematically searched Medline (PubMed), Embase, and The Cochrane Library between November 2022 and February 2023, using following Mesh Terms: Transplant, Transplantation, Hand, Face, Uterus, Penis, Abdominal Wall, Larynx, and Composite Tissue Allografts. All VCA case reports and reviews describing multiple case reports were included. RESULTS: We discovered 211 VCA cases reported. The preferred treatment was a combination of antithymocyte globulins, mycophenolate mofetil (MMF), tacrolimus, and steroids; and a combination of MMF, tacrolimus, and steroids for induction and maintenance treatment, respectively. Burn patients showed a higher acute rejection rate (P = 0.073) and were administered higher MMF doses (P = 0.020). CONCLUSIONS: In contrast to previous statements, the field of VCA is not rapidly evolving, as it has encountered challenges in addressing immune-related concerns. This is highlighted by the absence of a standardized immunosuppression regimen. Consequently, more substantial data are required to draw more conclusive results regarding the immunogenicity of VCAs and the potential superiority of one immunosuppressive treatment over another. Future efforts should be made to report the VCA surgeries comprehensively, and muti-institutional long-term prospective follow-up studies should be performed to compare the number of acute rejections with influencing factors.

Cell-Based Therapies Induce Tolerance of Vascularized Composite Allotransplants: A Systematic Review.

INTRODUCTION: Vascularized composite allotransplantation (VCA) is the transplantation of multiple tissue types as a solution for devastating injuries. Despite the highly encouraging functional outcomes of VCA, the consequences of long-term immunosuppression remain the main obstacle in its application. In this review, we provide researchers and surgeons with a summary of the latest advances in the field of cell-based therapies for VCA tolerance. METHODS: Four electronic databases were searched: PubMed, Scopus, Cumulative Index to Nursing and Allied Health Literature , and Web of Science. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis as the basis of our organization. RESULTS: Hematopoietic stem cells prolonged VCA survival. A combination of immature dendritic cells and tacrolimus was superior to tacrolimus alone. T cell Ig domain and mucin domain modified mature dendritic cells increased VCA tolerance. Bone marrow-derived mesenchymal stem cells prolonged survival of VCAs. A combination of adipose-derived mesenchymal stem cells, cytotoxic T-lymphocyte antigen 4 immunoglobulin, and antilymphocyte serum significantly improved VCA tolerance. Ex-vivo allotransplant perfusion with recipient's bone marrow-derived mesenchymal stem cells increased VCA survival. Recipient's adipose-derived mesenchymal stem cells and systemic immunosuppression prolonged VCA survival more than any of those agents alone. Additionally, a combination of peripheral blood mononuclear cells shortly incubated in mitomycin and cyclosporine significantly improved VCA survival. Finally, a combination of donor recipient chimeric cells, anti-αß-T cell receptor (TCR), and cyclosporine significantly prolonged VCA tolerance. CONCLUSIONS: Evidence from animal studies shows that cell-based therapies can prolong survival of VCAs. However, there remain many obstacles for these therapies, and they require rigorous clinical research given the rarity of the subjects and the complexity of the therapies. The major limitations of cell-based therapies include the need for conditioning with immunosuppressive drugs and radiation, causing significant toxicity. Safety concerns also persist as most research is on animal models. While completely replacing traditional immunosuppression with cell-based methods is unlikely soon, these therapies could reduce the need for high doses of immunosuppressants and improve VCA tolerance.

Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

Sub-zero non-freezing of vascularized composite allografts in a rodent partial hindlimb model.

Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4-6 h of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore, this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by a storage solution with cryoprotective agents (CPA) developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation.

First-in-Human Whole-Eye Transplantation: Ensuring an Ethical Approach to Surgical Innovation.

As innovations in the field of vascular composite allotransplantation (VCA) progress, whole-eye transplantation (WET) is poised to transition from non-human mammalian models to living human recipients. Present treatment options for vision loss are generally considered suboptimal, and attendant concerns ranging from aesthetics and prosthesis maintenance to social stigma may be mitigated by WET. Potential benefits to WET recipients may also include partial vision restoration, psychosocial benefits related to identity and social integration, improvements in physical comfort and function, and reduced surgical risk associated with a biologic eye compared to a prosthesis. Perioperative and postoperative risks of WET are expected to be comparable to those of facial transplantation (FT), and may be similarly mitigated by immunosuppressive protocols, adequate psychosocial support, and a thorough selection process for both the recipient and donor. To minimize the risks associated with immunosuppressive medications, the first attempts in human recipients will likely be performed in conjunction with a FT. If first-in-human attempts at combined FT-WET prove successful and the biologic eye survives, this opens the door for further advancement in the field of vision restoration by means of a viable surgical option. This analysis integrates recent innovations in WET research with the existing discourse on the ethics of surgical innovation and offers preliminary guidance to VCA programs considering undertaking WET in human recipients.

A World Update of Progress in Lower Extremity Transplantation: What's Hot and What's Not.

ABSTRACT: The field of vascularized composite allotransplantation (VCA) is the new frontier of solid organ transplantation (SOT). VCA spans life-enhancing/life-changing procedures such as upper extremity, craniofacial (including eye), laryngeal, tracheal, abdominal wall, penis, and lower extremity transplants. VCAs such as uterus transplants are life giving unlike any other SOT. Of all VCAs that have shown successful intermediate- to long-term graft survival with functional and immunologic outcomes, lower extremity VCAs have remained largely underexplored. Lower extremity transplantation (LET) can offer patients with improved function compared to the use of conventional prostheses, reducing concerns of phantom limb pain and stump complications, and offer an option for eligible amputees that either fail prosthetic rehabilitation or do not adapt to prosthetics. Nevertheless, these benefits must be carefully weighed against the risks of VCA, which are not trivial, including the adverse effects of lifelong immunosuppression, extremely challenging perioperative care, and delayed nerve regeneration. There have been 5 lower extremity transplants to date, ranging from unilateral or bilateral to quadrimembral, progressively increasing in risk that resulted in fatalities in 3 of the 5 cases, emphasizing the inherent risks. The advantages of LET over prosthetics must be carefully weighed, demanding rigorous candidate selection for optimal outcomes.

Novel Strategies in Transplantation: Genetic Engineering and Vascularized Composite Allotransplantation.

INTRODUCTION: Despite the clinical success in vascularized composite allotransplantation (VCA), systemic immunosuppression remains necessary to prevent allograft rejection. Even with potent immunosuppressive regimens (tacrolimus, mycophenolate mofetil, and steroids), most patients experience several rejection episodes, often within the same year. The risk of systemic side effects must constantly be weighed against the risk of under-immunosuppression and, thus, acute and chronic rejection. In this context, genomic editing has emerged as a potential tool to minimize the need for toxic immunosuppressive regimens and has gained attention in the fields of solid organ transplantation and xenotransplantation. This strategy may also be relevant for the future of VCA. METHODS: We discuss the topic of genetic engineering and review recent developments in this field that justify investigating tools such as clustered regularly interspaced short palindromic repeats/Cas9 in the context of VCA. RESULTS: We propose specific strategies for VCA based on the most recent gene expression data. This includes the well-known strategy of tolerance induction. Specifically, targeting the interaction between antigen-presenting cells and recipient-derived T cells by CD40 knockout may be effective. The novelty for VCA is a discovery that donor-derived T lymphocytes may play a special role in allograft rejection of facial transplants. We suggest targeting these cells prior to transplantation (e.g., by ex vivo perfusion of the transplant) by knocking out genes necessary for the long-term persistence of donor-derived immune cells in the allograft. CONCLUSION: Despite the demonstrated feasibility of VCA in recent years, continued improvements to immunomodulatory strategies using tools like clustered regularly interspaced short palindromic repeats/Cas9 could lead to the development of approaches that mitigate the limitations associated with rejection of this life-giving procedure.

Tolerance Induction in Vascularized Composite Allotransplantation-A Brief Review of Preclinical Models.

Pre-clinical studies are an obligatory tool to develop and translate novel therapeutic strategies into clinical practice. Acute and chronic rejection mediated by the recipient's immune system remains an important limiting factor for the (long-term) survival of vascularized composite allografts (VCA). Furthermore, high intensity immunosuppressive (IS) protocols are needed to mitigate the immediate and long-term effects of rejection. These IS regiments can have significant side-effects such as predisposing transplant recipients to infections, organ dysfunction and malignancies. To overcome these problems, tolerance induction has been proposed as one strategy to reduce the intensity of IS protocols and to thereby mitigate long-term effects of allograft rejection. In this review article, we provide an overview about animal models and strategies that have been used to induce tolerance. The induction of donor-specific tolerance was achieved in preclinical animal models and clinical translation may help improve short and long-term outcomes in VCAs in the future.

What is needed to ensure long-term sustainability for the field of vascularized composite allotransplantation?

The field of vascularized composite allotransplantation (VCA) has demonstrated remarkable advances since its inception with some excellent long-term results in a variety of graft types. However, unlike solid organ transplantation, it has yet to become mainstream. We therefore discuss strategies on ensuring long-term sustainability by addressing continued clinical developments of VCA to improve the risk-to-benefit balance, importance of public support, improved policy and financial support, and need for a bridge to the future of transplant surgery. There has been headway on all fronts and collaboration among the VCA centers for centralization of data and incorporation of patient voices will be essential for continued progress.

Novel cell-based strategies for immunomodulation in vascularized composite allotransplantation.

PURPOSE OF REVIEW: Vascularized composite allotransplantation (VCA) has become a clinical reality in the past two decades. However, its routine clinical applications are limited by the risk of acute rejection, and the side effects of the lifelong immunosuppression. Therefore, there is a need for new protocols to induce tolerance and extend VCA survival. Cell- based therapies have emerged as an attractive strategy for tolerance induction in VCA. This manuscript reviews the current strategies and applications of cell-based therapies for tolerance induction in VCA. RECENT FINDINGS: Cellular therapies, including the application of bone marrow cells (BMC), mesenchymal stem cells (MSC), adipose stem cells, regulatory T cells (Treg) cells, dendritic cells and donor recipient chimeric cells (DRCC) show promising potential as a strategy to induce tolerance in VCA. Ongoing basic science research aims to provide insights into the mechanisms of action, homing, functional specialization and standardization of these cellular therapies. Additionally, translational preclinical and clinical studies are underway, showing encouraging outcomes. SUMMARY: Cellular therapies hold great potential and are supported by preclinical studies and clinical trials demonstrating safety and efficacy. However, further research is needed to develop novel cell-based immunosuppressive protocol for VCA.

Defining chronic rejection in vascularized composite allografts - do we have reliable surrogates to look for?

PURPOSE OF REVIEW: Chronic rejection (CR) is a major threat in the field of vascularized composite tissue allografts (VCAs) as it causes graft dysfunction and usually graft loss. Unfortunately, knowledge of CR in VCA is incomplete because of the limited number of VCA recipients, the heterogeneous nature of VCAs and the short follow-up. RECENT FINDINGS: The diagnosis of CR in VCA has relied on clinical and pathological findings. Clinical changes include graft fibrosis, dyschromia and ischemic/necrotic ulcerations. Pathological changes primarily affect allograft vessels and manifest with graft vasculopathy (i.e. myo-intimal proliferation and luminal narrowing of allograft vessels, leading to graft ischemia). Attempts are made to diagnose CR with non- or minimally-invasive techniques, such as imaging studies (ultrasound biomicroscopy, functional magnetic resonance imaging) and serum biomarkers. These techniques provide interesting results and further insight into the mechanisms of CR in VCA. SUMMARY: The diagnosis of CR in VCA still relies mainly on clinicopathological graft alterations; unfortunately, these become overt rather late during the rejection process, when reversal of CR is problematic. More recent, minimally- or non-invasive techniques have provided encouraging results, but their usefulness in the diagnosis of CR requires further studies. These data highlight the paramount importance of CR prevention.

Treg-inducing microparticles promote donor-specific tolerance in experimental vascularized composite allotransplantation.

For individuals who sustain devastating composite tissue loss, vascularized composite allotransplantation (VCA; e.g., hand and face transplantation) has the potential to restore appearance and function of the damaged tissues. As with solid organ transplantation, however, rejection must be controlled by multidrug systemic immunosuppression with substantial side effects. As an alternative therapeutic approach inspired by natural mechanisms the body uses to control inflammation, we developed a system to enrich regulatory T cells (Tregs) in an allograft. Microparticles were engineered to sustainably release TGF-ß1, IL-2, and rapamycin, to induce Treg differentiation from naïve T cells. In a rat hindlimb VCA model, local administration of this Treg-inducing system, referred to as TRI-MP, prolonged allograft survival indefinitely without long-term systemic immunosuppression. TRI-MP treatment reduced expression of inflammatory mediators and enhanced expression of Treg-associated cytokines in allograft tissue. TRI-MP also enriched Treg and reduced inflammatory Th1 populations in allograft draining lymph nodes. This local immunotherapy imparted systemic donor-specific tolerance in otherwise immunocompetent rats, as evidenced by acceptance of secondary skin grafts from the hindlimb donor strain and rejection of skin grafts from a third-party donor strain. Ultimately, this therapeutic approach may reduce, or even eliminate, the need for systemic immunosuppression in VCA or solid organ transplantation.

Special Considerations in Face Transplantation: A Systematic Review.

ABSTRACT: Vascularized composite allotransplantation of the face is an exceedingly complex procedure, requiring extensive planning and surgical precision in order to successfully manage patients with facial disfigurements. This review aims to present an overview of the salient anatomic considerations in facial transplantation, as well as give attention to unique patient populations and special considerations.A literature review was performed in search of articles pertaining to considerations in facial transplantation using the databases PubMed, Web of Science, and Cochrane. Articles selected for further review included full-text articles with an emphasis on specific anatomic defects and how they were addressed in the transplant process, as well as management of special patient populations undergoing facial transplantation. In total, 19 articles were deemed appropriate for inclusion.The use of computer-assisted technologies for the planning portion of the procedure, as well as intraoperative efficiency, has yielded favorable results and can be considered as part of the operative plan. The ultimate outcome is dependent upon the synchronization of subunits of the allograft and the desired functional outcomes, including osseous, ocular, oral, and otologic considerations. Management of specific pathology and subgroups of patients are critical aspects. Although pediatric face transplantation has not yet been performed, it is a likely a future step in the evolution of this procedure.When performing a face transplantation, many components must be considered pre-, intra-, and post-operatively. This systematic review presents specific anatomic considerations, as well as information about special patient populations within this crosssection of multidisciplinary microsurgery, psychiatry, and transplant immunology.

Abdominal Wall Vascularized Composite Allotransplantation: A Scoping Review.

BACKGROUND: Abdominal wall vascularized composite allotransplantation (AW-VCA) is a novel reconstructive technique used for large abdominal wall defects in combination with intestinal transplantation (ITx) or multivisceral abdominal transplantation (MVTx). Since the introduction of this procedure, several studies have been published reporting their experience. This study aims to present a scoping review looking at all available evidence-based medicine information to understand the most current surgical techniques and clinical outcomes. METHODS: This scoping review followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) extension for scoping reviews checklist. A comprehensive research strategy of several databases was conducted. RESULTS: A total of 31 studies were included in this review, which comprised animal, cadaveric, and human studies. In human studies, four surgical techniques with high flap survival rates and low complication rates were found. In cadaveric studies, it was shown that the use of iliofemoral cuff-based flaps provided adequate tissue perfusion to the abdominal wall graft. Also, the use of thoracolumbar nerves have been described to provide functionality to the AW-VCA and prevent long-term muscle atrophy. CONCLUSION: AW-VCA is a safe and efficient alternative for patients with large and complex abdominal wall defects. The future holds a promising evolution of a functional AW-VCA, though surgeons must face and overcome the challenge of distorted anatomy frequently present in this population. Forthcoming studies with a better level of evidence are required to evaluate functionality and differences between surgical techniques.

Vascularized composite allotransplantation: emerging psychosocial issues in hand, face, and uterine transplant.

PURPOSE OF REVIEW: Currently, several research approaches warrant further attention, given the influence of psychosocial and bioethical issues on the success of upper extremity (UETx), face (FTx), and uterine transplantation (UTx). This review will highlight recent results of psychosocial and bioethical research in the field of vascularized composite allotransplantation (VCA), discuss most recent findings, provide information to guide future research approaches, and address the importance of a multicenter research approach to develop international standards. RECENT FINDINGS: Previously published reports have tried to identify psychosocial factors that are essential to predict psychosocial outcomes and guide posttransplant treatment after VCA procedures. These issues in VCA are receiving more attention but we are still at the beginning of a systematic investigation of these domains. This review article summarizes the emerging psychosocial issues in UeTx, FTx, and UTx by including recent literature and current clinical practice. SUMMARY: Even though different VCA procedures address different domains leading to specific psychosocial issues, common aspects impacting all forms of VCA would benefit of further coordination. These domains include clinical resources, public attitude and perception, bioethical considerations, adherence and rehabilitation, motives for VCA, information needs and multidisciplinary communication, body image, domains of quality of life, coping strategies, and follow-up care.

Exosomes from donor-derived adipose mesenchymal stem cells prolong the survival of vascularized composite allografts.

Donor-derived adipose-derived mesenchymal stem cells (ADMSCs) dampen the alloimmune response and exosomes are reported to have biological activity similar to their parent cells. Here, we investigated the roles of exosomes from donor-derived ADMSCs (ADMSC-exo) in vascularized composite allotransplantation (VCA). Brown Norway-to-Lewis rat hindlimb transplantations were intravenously treated with either exosome from donor-derived ADMSCs or phosphate-buffered saline, combined with a short course of immunosuppression. We established that the treatment with ADMSC-exo prolongs the survival time of VCA grafts. Skin and muscle samples from ADMSC-exo-treated animals showed no histological signs of rejection, but samples from controls showed rejection of degree III. Comparing to the control group, a significant increase of donor cell chimerism, Tr1 and Treg, while a decrease of CD4+ T and Th1 cells were observed in the ADMSC-exo-treated group. Our findings imply that ADMSC-exo may be a valuable and safe treatment for extending VCA graft survival.

Vascularized Composite Allotransplantation: A Functional Hind Limb Model in Mice.

BACKGROUND: Vascularized composite allograft has emerged as a reconstructive option for patients who have suffered severe tissue loss. Animal models are critical for understanding the unique mechanisms of rejection in vascularized composite allograft. We present a functional mouse model of orthotopic hind limb transplantation using end-to-side anastomoses of the donor aorta and inferior vena cava to the respective recipient vessels. To the best of our knowledge, this approach has not been reported in the scientific literature. MATERIALS AND METHODS: A single surgeon performed all transplants (J.W.). A total of 13 syngeneic and 10 fully mismatched allogeneic transplants were performed without immunosuppression. Skin samples from the grafts were collected at the time of euthanasia. RESULTS: Five syngeneic mice survived for more than 90 d after transplant. All allografts displayed clinical and histologic signs of acute rejection such as a rash at the time of graft excision. The overall technical success rate of all transplants in this study was 74% (17 of 23). CONCLUSIONS: We demonstrate the feasibility of end-to-side anastomoses of the donor aorta and inferior vena cava with functional recovery of the transplant in a mouse model of orthotopic hind limb transplantation.

Is Vascularized Composite Allograft Transplantation Experimental or an Accepted Surgical Procedure: Results from a National Survey.

BACKGROUND: More than 85 patients have received over 100 hand/arm transplants and more than 35 patients have received full or partial face transplants at institutions around the world. Given over two decades of experience in the field and in the light of successful outcomes with up to 17 years follow up time, should we still consider vascularized composite allograft (VCA) as a research/clinical investigation? We present the results of a nationwide electronic survey whose intent was to gather institutional bias with regard to this question. METHODS: An 11 question survey that was developed by VCA advisory committee of American Society of Transplantation was sent to all identified Internal Review Board chairs or directors in the United States. RESULTS: We received a total of 54 responses (25.3%) to the survey. The majority (78%) of responses came from either the chairperson, director, or someone who is administratively responsible for an IRB. CONCLUSION: Though certainly not an exhaustive investigation into each institution's preference, we present a representative sampling. The results of which favor VCA as an accepted clinical procedure given the appropriate setting. Further research is needed to fully ascertain practices at each individual institution.

Effect of Static Cold Storage on Skeletal Muscle after Vascularized Composite Tissue Allotransplantation.

BACKGROUND: Prolonged cold ischemia associated with static cold storage (SCS) results in higher incidence of acute and chronic allograft rejection in solid organ transplantations. Deleterious effects of SCS on vascularized composite tissue allograft were studied with limited data on muscle structure and function. The aim of this study is to evaluate the long-term impact of SCS on muscle metabolism, structure, and force generation using a syngeneic rat hindlimb transplantation model. METHODS: Sixty-five male Lewis rats (250 ± 25 g) were distributed into five groups, including naive control, sciatic nerve denervation/repair, immediate transplantation, transplantation following static warm storage for 6 hours at room temperature, and transplantation following SCS for 6 hours at 4°C. Sciatic nerves were repaired in all transplantations. Muscle samples were taken for histology and metabolomics analysis following electromyography and muscle force measurements at 12 weeks after transplantation. RESULTS: All cold-preserved limbs remained viable at 12 weeks, whereas animals receiving limbs preserved in room temperature had no survivors. The SCS transplantation group showed a 73% injury score, significantly higher than groups receiving immediate transplants without cold preservation (50%, p < 0.05). A significant decline in muscle contractile force was also demonstrated in comparison to the immediate transplantation group (p < 0.05). In the SCS group, muscle energy reserves remained relatively well preserved in surviving fibers. CONCLUSION: SCS extends allograft survival but fails to preserve muscle structure and force.