Limb Transplantation Research Articles

Foxp3+ Treg Cells Resident within Donor Bone Marrow are Essential for Costimulation Blockade-Induced Long-Term Survival of Murine Limb Transplants

Foxp3+ Treg Cells Resident within Donor Bone Marrow are Essential for Costimulation Blockade-Induced Long-Term Survival of Murine Limb Transplants

Ex vivo Expanded Regulatory T cells Combined with Short-term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts

Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model. Methods Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data. Results Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01[GB1]). [GB2] Mixed chimerism was detected in recipients receiving this combined treatment protocol with 5.013 ± 1.23 % of CD11b+ cells being donor-derived on POD 55. Vβ – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of νβ5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of νβ5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism[GB3] with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells. Conclusion The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation by inducing sustained mixed chimerism.

Abstract 11.35 MICROSURGICAL MODELS FOR VASCULARIZED COMPOSITE ALLOTRANSPLANTS IN RATS

CONTENT: The aim of this work was to familiarize with some experimental microsurgical animal models used in the research field of Vascularized Composite Allotransplants (VCAs). All these animal models are vascularized allotransplants from Brown Norway to Lewis rats. A total 125 procedures were done: orthotopic (n = 60) and heterotopic (n = 5) hind limb transplantation, femur transplantation (n = 5), limb and contralateral femur transplantation (n = 5), knee transplantation (n = 5), heterotopic calf transplantation (n = 5), tail transplantation (n = 5), sternum transplantation (n = 5), hemiface transplantation (n=5), scalp transplantation (n = 5), ear transplantation (n = 5), penis transplantation (n = 5), heterotopic mandible transplantation (n = 5), toe to thumb transplantation (n = 5). All animals received drug therapy (FK506, MMF and Prednisone) for 8 weeks, then treatment was ceased entirely. The average success rate of transplantation was 95,2%. This study demonstrated that these experimental models could be a reliable to study the vascularized composite allotransplants.

Subcutaneous administration of a neutralizing IL-1β antibody prolongs limb allograft survival.

Cytokine‐expression profiles revealed IL‐1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL‐1β antibody in limb transplantation. Following allogenic hind‐limb transplantation, Lewis rats were either left untreated1 or treated with anti‐lymphocyte serum + tacrolimus (baseline)2; baseline immunosuppression + anti‐IL‐1β (1 mg/kg once/week, 6‐8 subcutaneous injections) into the transplanted3 or contralateral4 limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti‐IL‐1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL‐1β, IL‐4, IL‐13, IP‐10, MCP‐1, and MCP‐3 in long‐term‐survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti‐IL‐1β. Inhibition of IL‐1β with short‐term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.

T158. Sympathetic reinnervation in patient with transplanted upper limbs

Introduction Autonomic peripheral nerve reinnervation for skin vasculature and eccrine sweat glands has not been reported or analyzed before. As upper limb transplantation (ULT) remains an uncommon procedure sympathetic physiologic reinnervation process still remains a challenge and an important question to be answered in patients who undergo anastomosis of a peripheral nerve after a complete section. Methods A 52 year old male, underwent forearm transplantation 16 months after a high current electrical burn. Autonomic evaluation yearly during 4 years after transplant included: (1) evaluation of skin sympathetic response (SSR); (2) measurement of temperature with an infrared thermometer of high sensitivity (Nubee© range 50–350 °C, with one decimal point of discrimination); (3) evaluation of skin blood flow (SBF) with an infrared photopletysmograph; (4) capillary filling at the nails; (5) observation of the presence or absence of atrophic changes of the skin; (6) observation for the presence or absence of edema; (7) presence or absence of allodynia; (8) systolic blood pressure changes at the heart level and lowering the arm 60° (Portapres©); (9) presence or absence of the veno-arteriolar reflex Results SSR on both palmar surfaces was evident and reliable on the third year postransplantation. The patient clinically also referred sweating in both hands. No powder sweating was done Temperature of both hands had normal values, compared to the arms above transplant. No trophic changes were observed. The humidity of the skin appeared normal. There was no edema. No allodynia was present. The SBF demonstrated that the arteriolar vessels responded normally to stimuli such as breathing, baroreceptor stimulation, gravity pressor activation, and had normal blood pressure changes to breathing and lowering the arm. Capillary filling of the nail was normal. The veno-arteriolar reflex was present. Conclusion The eccrine sweat glands of the glabrous skin appear to be reinnervated as observed by the normal humidity, trophism of the hand, and presence of sympathetic skin response. The skin was not dry or scamous but soft and humid. The exocrine sweat glands of the non-glabrous skin of the forearm also had normal humidity and trophism which strongly suggests normal function of these glands. Piloerection could not be evaluated. The normal temperature, the absence of edema, the absence of trophic changes of the skin and more importantly the normal response of the SBF and SBP to deep breathing, postural changes and baroreceptor stimulation suggests that the arterial and venous system are reinnervated. The diffuse distributed sympathetic system seem to reinnervate skin and probably muscular vasculature after several years of a limb transplant.

Desensitization and Prevention of Antibody-Mediated Rejection in Vascularized Composite Allotransplantation by Syngeneic Hematopoietic Stem Cell Transplantation.

Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA. Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry. Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6). In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

Trans-femoral lower limb transplantation in a Sri Lankan patient: a case report and surgical technique

Trans-femoral lower limb transplantation in a Sri Lankan patient: a case report and surgical technique

Improved Cuff Technique and Intraoperative Detection of Vascular Complications for Hind Limb Transplantation in Mice.

BackgroundVascularized composite tissue allotransplantation (VCA) from a cadaveric donor has now become a clinical reality and the treatment modality of choice for patients with devastating injuries, deformities, and complex tissue defects. However, many VCA patients experience severe toxicities due to the strong immunosuppression required secondary to high antigenicity of the grafts. To improve immunosuppressive protocols for VCA, feasible and reliable preclinical models are necessary. The purpose of this study was to introduce new techniques to an established preclinical VCA model to accelerate future investigations.MethodsC57BL/6 (H-2b) and BALB/c (H-2d) mice were used to perform VCA as recipients and donors, respectively. Surgery time, success rate, associated complications, and mortality were analyzed. Blood flow in grafts was interrogated with laser speckle image (LSI).ResultsA nonsuture cuff technique was used with the abdominal aorta for end-to-end anastomosis. The cuff technique demonstrated efficiency for donor surgery (52 ± 10 minutes for donor vs. 45 ± 8 minutes for recipient surgery). Successful revascularization was achieved in 27 (90%) of 30 transplants. The majority of surgical complications occurred within 48 hours including artery occlusion, venous occlusion, cerebral stroke, and minor bleeding without mortality. LSI was useful in detecting intraoperative vascular complications with display patterns predictive of complication type.ConclusionsThe described techniques may facilitate a more efficient heterotopic hind limb transplantation mouse model of VCA.

Clinical Application of Mathematical Long Bone Ratios to Calculate Appropriate Donor Limb Lengths in Bilateral Upper Limb Transplantation.

Background: Limited methods exist to aid in deciding the appropriate donor limb lengths in bilateral upper limb amputees qualifying for vascularized composite allotransplantation. We hypothesized mathematical equations could be created using long bone length ratios, and applied to radiographs, to approximate the patient's limb length prior to amputation. Methods: A data set of 30 skeletons' unilateral upper limb long bones measured using osteometric board and calipers was used. Anatomic segment ratios were calculated based on humerus length after multivariate linear regression analysis. For clinical application testing, 5 cadavers' upper limbs were radiographed. Radiographic bone lengths were then measured along the long axis of each long bone. These measured radiographic lengths were then compared with the predicted bone lengths, generated from the skeleton data set ratios, for each cadaver. Results: The chi-square goodness-of-fit test showed excellent fit (P < .01) between the predicted and radiographically measured lengths for the 5 cadavers, and interobserver measurements showed no statistical difference. Depending on the cadaver, percent error in total limb length predicted to measure ranged from 0.9% to 2.7%. The variables to multiply an individual humerus length to calculate a given anatomic segment thus proved to be effective. Conclusions: If a bilateral upper limb amputee has 1 intact humerus, ratios to the humerus length can be reliably applied to calculate the preamputation limb length based on the patient's radiographic humerus length. These formulas are indicated for finding the appropriate limb lengths, and smaller anatomic segments, for donor-recipient matching in upper limb transplantation.

Upper limb reconstructive transplantation in military recipients

AbstractLessons from conflict have contributed to military medical advances which have significantly improved the survivability of critically injured servicemen and women. However, survival following such severe combat polytrauma is often associated with devastating tissue loss and functional deficits that challenge conventional reconstruction. Despite recent advances, upper extremity prosthetic alternatives lack the fidelity to restore the complex intrinsic and sensory function of the human hand. Upper Limb Reconstructive Transplantation, or Vascularised Composite Allotransplantation (VCA), is currently an experimental procedure offering superior anatomical and functional outcomes compared to prostheses.Military candidates for VCA usually enjoy high pre-morbid fitness, mental health resilience and support, and relatively rich provision of rehabilitation. However, co-morbidities of polytrauma, such as traumatic brain injury and post-traumatic stress disorder, may have an impact on outcomes.Since 1998, over 120 upper limb transplants have been performed worldwide. Of these, six have been performed on servicemen and women across three continents. The morbidity and mortality associated with the requirement for lifelong immunosuppression, however, continues to skew the risk-benefit considerations of these promising procedures when compared to prostheses. Thus, although the technical surgical feasibility of upper limb and other VCA such as face, abdominal wall, uterus and penis transplants has been established over the past decade, these procedures remain restricted to recipients fulfilling stringent inclusion criteria.We review the current state of VCA in military recipients, and summarise ongoing collaborative VCA research conducted by British and American military medical teams.

MicroRNAs are potential objective and early biomarkers for acute rejection of transplanted limbs in a rat model.

Limb transplantation is considered to be a treatment option for amputees. Visual skin inspection and histological assessment are used to assess rejection, but these techniques are largely subjective. Using a rat model, we examined the potential of several microRNAs (miRNAs) to be used as objective and minimally invasive biomarkers of acute rejection of transplanted limbs. Three Brown Norway rats and 15 Lewis rats were assigned to 2 groups. In the allograft group, 3 Brown Norway rats were used as the donors and 6 Lewis rats as the recipients. In the syngeneic (control) group, 9 Lewis rats were used as the donors and recipients. The hind limbs of the donors were orthotopically transplanted to the recipients. Plasma samples were obtained from all recipients before surgery and on posttransplantation days 3, 7, 10, and 14. All recipients were euthanized on day 14, and skin tissues were harvested for histological assessment. On posttransplantation days 10 and 14, the plasma expression of miRNA-146a, miRNA-155, and miRNA-182 was significantly upregulated in the allograft group compared with the syngeneic control group. Of these 3 miRNAs, miRNA-182 had the highest sensitivity and specificity; however the cutoff point for miRNA-182 was close to the baseline and miRNA-155 was considered to be most suitable for identifying rejection. Most skin samples in the allograft group were classified as exhibiting grade III rejection on day 14. Some miRNAs are upregulated during acute rejection of transplanted limbs. These miRNAs are potential biomarkers for the objective, early, minimally invasive diagnosis of rejection.

Abstract: Extended Normothermic Ex-Situ Limb Perfusion Preserves Limbs Viable and Functional Up to 44 Hours

INTRODUCTION: Ischemia and reperfusion injury remains one of the major limiting factors for the success of both replantation and vascularized composite allotransplantation. Compared to cold storage of preserved/procured organs, normothermic ex-situ perfusion is a novel approach that prolongs viability of the limb by maintaining physiologic cellular metabolism avoiding the deleterious effects of both hypoxia and cooling. This study aims to develop an ex-situ normothermic limb perfusion protocol to preserve the viability and function of amputated limbs for over 24 hours. METHODS: A total of 23 swine limbs were perfused using an oxygenated colloid solution at 38°C containing washed RBCs. The first 13 limbs were used to optimize the perfusion protocol. The subsequent 5 limbs (Group A) were perfused for 12 hours and the following 5 (Group B) as long as muscle contractility/peripheral perfusion were present. Electrolytes were balanced by partial perfusate exchanges. Limb viability was compared in the 2 groups by muscle contractility, compartment pressure, tissue oxygen saturation, indocyanine green (ICG)-angiography and thermography. RESULTS: Perfused limbs were able to retain physiological parameters and function for 12 hours in group A and up to 44 (24–44) hours in group B. Limbs in group A had lower final weight increase (0.54%±0.07 VS 14.11%±16.27) (p=0.008) and compartment pressure compared to group B (16.23 ± 7.10 VS 24.75 ± 7.79) (p=0.175). Final myoglobin and CK mean values were lower in group A compared with group B (875 ± 291.4 ng/mL VS 1010.6 ± 323.6 ng/mL and 53344 ± 14850.34 U/L VS 71881 ± 20475 ng/mL). In group B thermography and tissue oxygen saturation were significantly higher than in group A (respectively 35.37 ± 0.69°C VS 33 ± 1.44°C (p0.01) and 69.31 ± 9.3% VS 58.69 ± 8.4% (p=0.048)). Contractility and ICG-angiography were comparable in the two groups. CONCLUSION: The normothermic perfusion protocol has the potential to significantly impact outcomes in limb replantation and transplantation. Our results suggest that limbs in the 12-hours perfusion group retain better physiologic parameters at the end-point. However, the continuous optimization of the protocol allowed to maintain limb’s function and preserved peripheral perfusion for up to 44 hours.

The combination of mitomycin-induced blood cells with a temporary treatment of ciclosporin A prolongs allograft survival in vascularized composite allotransplantation.

Vascularized composite allotransplantation (VCA) is a rapidly expanding field of transplantation and provides a potential treatment for complex tissue defects. Peripheral blood mononuclear cells (PBMCs) shortly incubated with the antibiotic and chemotherapeutic agent mitomycin C (MMC) can suppress allogeneic T cell response and control allograft rejection in various organ transplantation models. MMC-incubated PBMCs (MICs) are currently being tested in a phase I clinical trial in kidney transplant patients. Previous studies with MICs in a complex VCA model showed the immunomodulatory potential of these cells. The aim of this study is to optimize and evaluate the use of MICs in combination with a standard immunosuppressive drug in VCA. Fully mismatched rats were used as hind limb donors [Lewis (RT11)] and recipients [Brown-Norway (RT1n)]. Sixty allogeneic hind limb transplantations were performed in six groups. Group A received donor-derived MICs combined with a temporary ciclosporin A (CsA) treatment. Group B received MICs in combination with a temporarily administered reduced dose of CsA. Group C served as a control and received a standard CsA dose temporarily without an additional administration of MICs, whereas Group D was solely medicated with a reduced CsA dose. Group E received no immunosuppressive therapy, neither CsA nor MICs. Group F was given a continuous standard immunosuppressive regimen consisting of CsA and prednisolone. The endpoint of the study was the onset of allograft rejection which was assessed clinically and histologically. In group A and B, the rejection-free interval of the allograft was significantly prolonged to an average of 23.1±1.7 and 24.7±1.8days compared to the corresponding control groups (p<0.01). Rejection in groups C, D, and E was noted after 14.3±1.1, 7.8±0.7, and 6.9±0.6days. No rejection occurred in control group F during the follow-up period of 100days. No adverse events have been noted. The findings of this study show that the combination of MICs with a temporary CsA treatment significantly prolongs the rejection-free interval in a complex VCA model. The combination of MICs with CsA showed no adverse events such as graft-versus-host disease. MICs, which are generated by a simple and reliable in vitro technique, represent a potential therapeutic tool for prolonging allograft survival through immunomodulation.

Physical medicine after limb transplantation. Therapeutic effects of physical procedures in the treatment of limb transplantation after cut-off by a threshing machine – Case report

Introduction. The number of accidents in agriculture involving children as the victims continues to increase year-by-year. Parents often allow children to perform certain tasks, or even entrust them to perform such tasks as a duty. The paper presents results of treatment using a 3D magnetic field in a 31-year- old patient after transplantation of the upper extremity of the arm. Amputation of the extremity had been caused by an accident (hand cut-off by a threshing machine). The therapeutic cycle for the patient constituted of 3 series of 15 daily procedures, performed for 15 minutes. There was a break of 4 weeks between sessions. Additionally, the patient performed kinesitherapy exercises in the outpatient clinic. The results obtained regarding improvement of the functional condition of the limb showed that in the complex planning of treatment, the taking into account of the physical medicine procedures should be indispensable.

The Effect of Bilateral Hand Transplantation on Perceived Quality of Life in Two Combat Veterans

Abstract Date Presented 3/31/2017 This presentation explores the perceived quality of life in two upper-extremity limb transplant recipients and compares them with a population of bilateral upper-extremity prosthetic users. Primary Author and Speaker: Kaitlyn Jones Additional Authors and Speakers: Carol A. Coté

Hand Transplantation: Current concepts and management algorithm

Introducción. El trasplante de mano ha sido uno de los retos del siglo XXI, cuyo primer caso reportado ocurrió en 1964. En este campo se han hecho estudios y avances que permitieron explorar nuevas alternativas para el manejo del paciente con trasplante de mano, por lo que diversos centros de referencia han realizado análisis basados en sus experiencias, las cuales permitieron lograr la viabilidad de este tipo de trasplante.Objetivo. Esta revisión busca dar una visión general sobre el trasplante de mano y proponer un algoritmo de manejo.Materiales y métodos. La selección de candidatos requiere una serie de criterios, tales como evaluación clínica, paraclínica y psicosocial, desarrollados por un equipo multidisciplinario. La inmunosupresión busca evitar el rechazo y los inmunosupresores deben tener los niveles séricos apropiados para reducir sus efectos adversos. Se han reportado casos de rechazo agudo clásico y atípico, donde la piel es el principal tejido blanco, y rechazo crónico, en el cual se afectan los vasos sanguíneos. El seguimiento se realiza con varias pruebas, de modo que la de oro es la biopsia de piel.Resultados. Las complicaciones del tratamiento farmacológico derivan de la toxicidad de los medicamentos y se manifiestan como alteraciones metabólicas, infecciones oportunistas y neoplasias. La rehabilitación y los aspectos sociales, como el grado de satisfacción del paciente, deben ser evaluados durante la recuperación para asegurar adherencia al tratamiento. En 2011 el registro internacional de alotransplante compuesto de mano reportó 39 casos de trasplante de extremidades superiores con resultados variables; todo esto evidencia que para lograr un resultado óptimo y viable del trasplante debe realizarse seguimiento por un equipo multidisciplinario, red de apoyo del paciente y motivación del mismo, junto con el cumplimiento del manejo farmacológico.Conclusión. Se espera que nuevas investigaciones puedan crear estrategias para desarrollar tolerancia y, de esta forma, reducir el manejo mediante inmunosupresión.

Computer-aided training sensorimotor cortex functions in humans before the upper limb transplantation using virtual reality and sensory feedback

One of the biggest problems of upper limb transplantation is lack of certainty as to whether a patient will be able to control voluntary movements of transplanted hands. Based on findings of the recent research on brain cortex plasticity, a premise can be drawn that mental training supported with visual and sensory feedback can cause structural and functional reorganization of the sensorimotor cortex, which leads to recovery of function associated with the control of movements performed by the upper limbs. In this study, authors – based on the above observations – propose the computer-aided training (CAT) system, which generating visual and sensory stimuli, should enhance the effectiveness of mental training applied to humans before upper limb transplantation. The basis for the concept of computer-aided training system is a virtual hand whose reaching and grasping movements the trained patient can observe on the VR headset screen (visual feedback) and whose contact with virtual objects the patient can feel as a touch (sensory feedback). The computer training system is composed of three main components: (1) the system generating 3D virtual world in which the patient sees the virtual limb from the perspective as if it were his/her own hand; (2) sensory feedback transforming information about the interaction of the virtual hand with the grasped object into mechanical vibration; (3) the therapist's panel for controlling the training course. Results of the case study demonstrate that mental training supported with visual and sensory stimuli generated by the computer system leads to a beneficial change of the brain activity related to motor control of the reaching in the patient with bilateral upper limb congenital transverse deficiency.

Intra-graft injection of tacrolimus promotes survival of vascularized composite allotransplantation

BackgroundImmunosuppressive therapies derived from solid organ transplantation are effective in promoting survival of vascularized composite allotransplantation (VCA), but they cause serious side effects that are difficult to justify for this non–life-saving procedure. Unlike solid organ transplantation, hand and face transplants offer the possibility of site-specific immunosuppression for reducing systemic exposure while increasing intra-graft concentrations of the drug. Therefore, in this study, we tested whether a single intra-graft injection tacrolimus could promote VCA survival. MethodsBrown Norway-to-Lewis hind limb transplantations were performed, and animals were left untreated (group I), treated with a daily injection of 1-mg/kg tacrolimus for 21 days (group 2) or injected with 7-mg tacrolimus directly into the transplanted limb on day 1 (group III). Graft rejection was monitored, and animals were sacrificed at grade 3 rejection or 200 days after transplantation. ResultsIntra-graft injection of tacrolimus significantly prolonged allograft survival as compared to untreated animals or animals treated with systemic tacrolimus. Half of the intra-graft–treated rats rejected their graft on average at day 70.5. Interestingly, the other half remained rejection-free for more than 200 days without signs of kidney or liver toxicity. In these animals, tacrolimus was detected in the VCA skin but not in the blood until day 200. Long-term survival was not linked to induction of donor-specific tolerance but to a higher level of lymphocyte chimerism. ConclusionsIntra-graft delivery of tacrolimus may promote VCA survival by increasing tissue drug availability and promoting the establishment of transient chimerism and thus long-term graft acceptance.

Rat Hindlimb Cryopreservation and Transplantation: A Step Toward "Organ Banking".

In 2016, over 5 million reconstructive procedures were performed in the United States. The recent successes of clinical vascularized composite allotransplantations, hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, due to limited availability and lifelong immunosuppression, application is limited. Long-term banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant-velocity linear temperature gradient, enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible, and stable ways to preserve composite tissue. We believe that with further research and development, cryopreservation may lead to composite tissue "banks." This may lead to a paradigm shift from few and far apart emergent surgeries to wide-scale, well-planned, and better-controlled elective surgeries.

Bone marrow-derived mesenchymal stem cells (MSCs) to enhance nerve regeneration and functional recovery in vascular composite allotransplantation (VCA)

Abstract Introduction Optimizing nerve regeneration and mitigating denervation atrophy of target muscles are the keys to achieving successful VCA such as limb and face transplants. Methods Lewis rat was anesthetized, sciatic or individual sciatic nerve branches (tibial, peroneal and sural) of the right hind-limb were transected and surgically repaired. MSC (5 million; passage ≤6) or vehicle were administered locally and intravenously. Results Rat MSCs expanded ex vivo were CD29+, CD90+, CD34−, CD31−, CD45low, MHC Class I+, Class II−, and were pluripotent. Total sensory function recovery at 2 weeks post sciatic or individual nerve repair (SNR or INR) with or without MSC was ~ 1.2 on a scale of Grade 0–3 (0=No function; 3=Normal); by 12 weeks it was 2.6–2.8 (n≥6/group). Peroneal sensory function recovery was as early as 1 week but not tibial or sural. MSC treatment accelerated sensory function onset. At 8 weeks post-INR, the sciatic nerve function index (SFI), a measure of motor function (0=Normal; −100=Nonfunctional) was −34 and −77 in MSC and vehicle groups, respectively (n≥9); with SNR it was −72 and −92 in MSC and vehicle groups, respectively. Long-term motor function (24 weeks) was apparent in MSC treated INR (SFI −63) but not in SNR (SFI −100) model. Gastrocnemius muscle atrophy was lower (P ≤0.05) in INR compared to SNR model. Significant reduction in axonal area (P ≤0.002), g ratio/axonal myelination (P ≤0.05), reduced nerve fiber density, and increased degenerating axons in the distal segment of the transected/repaired tibial nerve was observed compared to the contralateral naive tibial nerve. Histology and molecular studies are ongoing. Conclusion MSC therapy appears to promote peripheral nerve regeneration and functional recovery.