Vascularized Composite Allograft Transplantation Research Articles

Could Sentinel Skin Transplants Have Some Utility in Solid Organ Transplantation?

Accurate diagnosis of allograft rejection can be hazardous and challenging. A strategy that has emerged from experience with vascularized composite allografts (VCAs) is the use of sentinel skin transplants (SSTs)-portions of donor skin transplanted synchronously to an allograft. Work in nonhuman animal models and experience with VCAs suggest concordance between rejection occurring in the primary allograft and the SST, and that appearance of rejection in the SST may precede rejection in the primary allograft, permitting early therapeutic intervention that may improve outcomes with lower rates of chronic rejection. The encouraging findings reported in VCA transplantation raise the possibility that SST may also be useful in solid organ transplantation. Some evidence is provided by experience with abdominal wall transplantation in some intestinal and multivisceral transplant recipients. Results from those reports raise the possibility that rejection may manifest in the skin component before emergence in the intestinal allograft, providing a "lead time" during which treatment of rejection of the abdominal wall could prevent the emergence of intestinal rejection. It is plausible that these findings may be extrapolated to other solid organ allografts, especially those for which obtaining an accurate diagnosis of acute rejection can be hazardous and challenging, such as the lung or pancreas. However, more data are required to support widespread adoption of this technique.

Extending Vascularized Composite Tissue Survival Using Near-Normothermic Ex Situ Perfusion

Objective/Hypothesis: Currently, vascularized composite allografts (VCAs) are cold preserved (4°C) until transplantation. This process is time limited, as the tissue has to be revascularized within 4 to 6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation. This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury. Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours. In this study, we aimed to test this system on human forearm allografts. Material and Methods: Five human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature-controlled (30°C-33°C) ex situ perfusion system for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4 to 6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Results: Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 93 ± 2 mm Hg, perfusion flow 310 ± 20 mL/min (~6%-8% of the donor’s estimated cardiac output), and vascular resistance 153 ± 16 mm Hg/mL/min. Perfusate had an average pH of 7.43 ± 0.04, pCO2 32 ± 1 mm Hg, pO2 317 ± 18 mm Hg, and Hb 4.8 ± 0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment. Hematoxylin-eosin staining showed mild fatty infiltration on some myocytes at 24 hours. There was minimal change in fiber size, likely due to variation in age and gender between donors. Muscle architecture was preserved at the end of 24 hours perfusion. Conclusions: All limbs remained viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation. While no assumptions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area. It also has the potential to circumvent complications associated with cold preservation.

Near Normothermic Ex Situ Perfusion of Human Limbs for 24 Hours: N/A - Not a clinical study

There is no financial information to disclose. Currently vascularized composite allografts (VCA) are cold preserved (4°C) until transplantation.1Hartzell T.L. Benhaim P. Imbriglia J.E. Shores J.T. Goitz R.J. Balk M. et al.Surgical and technical aspects of hand transplantation: is it just another replant?.Hand Clin. 2011; 27: 521-530Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar This process is time limited, as the tissue has to be revascularized within 4-6 hours to minimize ischemia reperfusion (IR) injury. Normothermic perfusion was proposed as an alternative method of preservation in solid organ transplantation.2Rojas-Pena A. Reoma J.L. Krause E. Boothman E.L. Padiyar N.P. Cook K.E. et al.Extracorporeal support: improves donor renal graft function after cardiac death.Am J Transpl. 2010; 10: 1365e-1374eCrossref PubMed Scopus (33) Google Scholar This method helps to avoid complications associated with cold preservation and maintains tissue viability without inducing IR injury.3Murata S. Miniati D.N. Kown M.H. Koransky M.L. Lijkwan M.A. Balsam L.B. et al.Superoxide dismutase mimetic m40401 reduces ischemia-reperfusion injury and graft coronary artery disease in rodent cardiac allografts.Transplantation. 2004; 78: 1166-1171Crossref PubMed Scopus (27) Google Scholar Using this method, previous investigators demonstrated its potential to prolong swine forelimb allograft survival up to 24 hours.4Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. Ex-situ limb perfusion system to extend vascularized composite tissue allograft survival.Transplantation. 2015; 99: 2095-2101Crossref PubMed Scopus (37) Google Scholar, 5Ozer K. Rojas-Pena A. Mendias C.L. Bryner B. Toomasian C. Bartlett R.H. The effect of ex situ perfusion in a swine limb vascularized composite tissue allograft on survival up to 24 hours.J Hand Surg Am. 2016; 41: 3-12Abstract Full Text Full Text PDF Scopus (45) Google Scholar In this study, we aimed to test this system on human limb allografts. Four human forearms were procured from brain-dead adult donors under tourniquet control. Following elbow disarticulation, the brachial artery was cannulated. The limb was flushed with heparinized saline and connected to a temperature controlled (30-33°C) ex situ perfusion system (Figure) for 24 hours. The perfusate consisted of plasma and red blood cells with a target hemoglobin (Hb) concentration of 4-6 g/dL. Muscle biopsies (flexor carpi radialis) were obtained at 0, 12, and 24 hours. Average warm ischemia time was 76 minutes. Average arterial systolic pressure was 98±2 mmHg, perfusion flow 319±18 mL/min (∼6-8% of the donor’s estimated cardiac output), and vascular resistance 158±18 mmHg/mL/min. Perfusate had an average pH of 7.43±0.04, pCO2 32±1 mmHg, pO2 317±18 mmHg, and Hb 4.8±0.4 g/dL. Electrolytes (sodium, potassium, chloride) remained within a physiologic range. Lactate started to increase steadily throughout the experiment; however, neuromuscular electrical stimulation revealed ongoing contraction throughout the experiment and H& E staining showed mild to moderate fatty infiltration on some myocytes at 24 hours. •All limbs appeared viable after 24 hours of near-normothermic ex situ perfusion as evidenced by ongoing neuromuscular stimulation.•While no conclusions can be drawn about the long-term function of the extremity, this approach could help extend VCA transplantation to a wider geographic area.•It also has the potential to circumvent complications associated with cold preservation.

Vascularized composite allograft-specific characteristics of immune responses

Vascularized composite allograft (VCA) transplantation, or reconstructive transplantation, has revolutionized the treatment of complex tissue and functional defects. Despite arriving during an age in which the immunology of solid organ transplant rejection has been investigated in much detail, these transplants have offered new perspectives from which to explore the immunobiology of transplantation. VCAs have a number of unique molecular, cellular, and architectural features which alter the character and intensity of the rejection response. While much is yet to be clarified, an understanding of these distinct mechanisms affords new possibilities for the control of immune responses in an effort to improve outcomes after VCA transplantation.

The reversed paradigm of chimerism induction: Donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation.

To test a new approach of donor conditioning with recipient bone marrow cells (BMC) to induce tolerance in vascularized composite allograft (VCA) transplantation. Lewis rats' (recipients) BMC were stained with PKH-26. The ACI rats (donors) were conditioned with 80×106 Lewis BMC, 24 or 72 hours before VCA (groin flap) transplantation. Forty-eight VCA were performed between ACI donors and Lewis recipients. In groups I and II, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received 7-day anti-αβ-TCR/cyclosporine-A post-transplantation. In groups III and IV, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received no systemic immunosuppression. In group V, recipients received 7-day anti-αβ-TCR/cyclosporine-A post-transplantation. In group VI, recipients received no systemic immunosuppression. Assessment included evaluation of transplant viability and induction of donor-specific chimerism via flow cytometry, immunofluorescence, and PCR. Groups III, IV, and VI rejected allografts, at an average of 14 ± 5.2, 10 ± 2.7, and 8 ± 0.7 days. In groups I, II, and V, the mean survival was 80 ± 18.2 (p = 0.0002), 64 ± 27.4 (p = 0.001), and 30 ± 4.7 (p = 0.02) days. In groups I and II, donor-specific chimerism in the blood decreased from 8.8 ± 3.4% and 8.6 ± 3.4% on day 7 to 3.7 ± 1.32% (p = 0.02) and 4.7 ± 2.7% when the flaps manifested grade 3 rejection. The presence of PKH-26+ Lewis BMC was confirmed in the donor's blood, bone marrow, lymphoid organs, and liver (preconditioned at 24 and 72 hours). Donor preconditioning is a novel approach modifying recipient's responsiveness to donor allograft and prolonging the allograft survival under short-term immunosuppression. © 2015 Wiley Periodicals, Inc. Microsurgery 36:676-683, 2016.

Regulatory oversight in the United States of vascularized composite allografts.

Vascularized composite allograft (VCA) transplantation is a medically acceptable treatment for the reconstruction of major tissue loss. The advent of VCA transplantation has spurred regulatory and policy development in the United States to address the multiple clinical, ethical and legal issues that must be considered for the practice of VCA donation and transplantation to develop within the existing framework of public trust and transparency vital to the success of donation and transplantation.

Studies Introducing Costimulation Blockade for Vascularized Composite Allografts in Nonhuman Primates

Vascularized composite allografts (VCAs) are technically feasible. Similar to other organ transplants, VCAs are hampered by the toxicity and incomplete efficacy associated with conventional immunosuppression. Complications attributable to calcineurin inhibitors remain prevalent in the clinical cases reported to date, and these loom particularly large given the nonlifesaving nature of VCAs. Additionally, acute rejection remains almost ubiquitous, albeit controllable with current agents. Costimulation blockade offers the potential to provide prophylaxis from rejection without the adverse consequences of calcineurin-based regimens. In this study, we used a nonhuman-primate model of VCA in conjunction with immunosuppressive regimens containing combinations of B7-specific costimulation blockade with and without adhesion blockade with LFA3-Ig to determine what adjunctive role these agents could play in VCA transplantation when combined with more conventional agents. Compared to tacrolimus, the addition of belatacept improved rejection free allograft survival. The combination with LFA3-Ig reduced CD2(hi) memory T cells, however did not provide additional protection against allograft rejection and hindered protective immunity. Histology paralleled clinical histopathology and Banff grading. These data provide the basis for the study of costimulation blockade in VCA in a relevant preclinical model.

Procurement strategies for combined multiorgan and composite tissues for transplantation.

The purpose of this article is to identify the unique aspects of combined multiorgan and vascularized composite allograft (VCA) procurement from deceased donors and outline the steps essential for success. Transplantation of nonsolid organ composite tissues is becoming a viable option for reconstruction of massive tissue defects. With the United Network for Organ Sharing designation of VCAs as organs, placing them under the domain of the Organ Procurement and Transplantation Network, a systematic method for combined solid organ and VCA procurement is required. Several centers have reported experience with successful procurement strategies including sequential and simultaneous retrievals. The published literature describing donor screening, sequence of procurement with relation to solid organs and allocation is reviewed. With the 2013 classification of VCAs as organs, the Organ Procurement and Transplantation Network and United Network for Organ Sharing are better suited to aligning procurement and allocation policies. As VCA transplantation becomes more commonplace, protocol guidelines will ensure smooth integration with existing procurement infrastructure.

The potential role for regulatory T-cell therapy in vascularized composite allograft transplantation.

Vascularized composite allograft (VCA) transplantation restores defects to a degree not possible by conventional techniques. However, it is limited by the need for long-term immunosuppression and high rates of acute rejection directed against skin. There is therefore a need for a therapy that may shift the risk-benefit ratio in favour of VCA transplantation. Regulatory T cells (Tregs) are a subset of T cells with potent immunoregulatory properties and the potential to promote immunosuppression-free allograft survival. In this review, we consider the evidence for Treg therapy in VCA transplantation. CD4 Tregs are the best-studied immunoregulatory cell type, and a large amount of experimental and clinical data is emerging to endorse their use in VCA transplantation. Data from animal and humanized models are particularly encouraging and demonstrate the potent efficacy of Treg at preventing skin allograft rejection. Moreover, central tolerance induction techniques in VCA transplantation models are demonstrating a dependence on Tregs for graft survival. An improvement in outcomes after VCA transplantation has the potential to revolutionize the field. Several effective therapeutic strategies have demonstrated great promise experimentally, and there is now a need to assess their safety and efficacy in a clinical setting.

Simultaneous transplantation of hematopoietic stem cells and a vascularized composite allograft leads to tolerance.

We have previously demonstrated that tolerance to a vascularized composite allograft (VCA) can be achieved after the establishment of mixed chimerism. We test the hypothesis that tolerance to a VCA in our dog leukocyte antigen-matched canine model is not dependent on the previous establishment of mixed chimerism and can be induced coincident with hematopoietic cell transplantation (HCT). Eight dog leukocyte antigen-matched, minor antigen mismatched dogs received 200 cGy of radiation and a VCA transplant. Four dogs received donor bone marrow at the time of VCA transplantation (group 1), whereas a second group of four dogs did not (group 2). All recipients received a limited course of postgrafting immunosuppression. All dogs that received HCT and VCA were given donor, third-party, and autologous skin grafts. All group 1 recipients were tolerant to their VCA (>62 weeks). Three of the four dogs in group 2 rejected their VCA transplants after the cessation of immunosuppression. Biopsies obtained from the muscle and skin of VCA from group 1 showed few infiltrating cells compared with extensive infiltrates in biopsies of VCA from group 2. Compared with autologous skin and muscle, elevated levels of CD3+ FoxP3+ T-regulatory cells were found in the skin and muscle obtained from the VCA of HCT recipients. All group 1 animals were tolerant to their donor skin graft and promptly rejected the third-party skin grafts. These data demonstrated that donor-specific tolerance to all components of the VCA can be established through simultaneous nonmyeloablative allogeneic HCT and VCA transplantation protocol.

Vascularized Composite Allograft Tolerance Across MHC Barriers in a Large Animal Model

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

New Perspectives in Tolerance Induction for Vascularized Composite Allotransplantation

Although transplantation of Vascularized Composite Allografts (VCA) has become a clinical reality, complications resulting from long-term immunosuppression remain major barriers to broader indication. Ideally, induction of tolerance has been considered the Holy Grail in both solid organ and VCA transplantation. Recent advances in immunomodulatory medication and cell-based therapy exploiting donor bone marrow (BM) cells, regulatory T cells (Tregs) and mesenchymal stem cells have opened exciting opportunities for tolerance induction in VCA transplantation. This mini-review focuses on current understanding of these drugs and cell-based immunomodulatory strategies and their role(s) in tolerance induction for VCA transplantation.

Donor and procurement related issues in vascularized composite allograft transplantation

To understand the unique requirements of vascularized composite allograft (VCA) donation and procurement practices and the integral role of the established nationwide organ procurement organizations in organ procurement. The recent issuance of a Final Rule (July 2013) by the United States Secretary of Health that redefines VCAs as organs rather than tissues, opens up the potential to formalize policies and procedures, under the auspices of the Organ Procurement and Transplantation Network, that can improve VCA donation, procurement practices, develop allocation algorithms and provide transparent oversight. Improved VCA donation rates, procurement procedures and broader sharing nationwide of VCA donors will have important implications in advancing the emerging field of VCA transplantation.

A Clinically Applicable Tolerance Protocol To Induce Tolerance to Vascularized Composite Allografts without the Need for Persistent Donor Cell Chimerism

INTRODUCTION: The application of Vascularized Composite Allograft (VCA) transplantation is limited by the need for immunosuppression. Tolerance induction protocols using stem cell transplantation can eliminate this requirement but the engraftment of these cells can lead to Graft-Versus-Host-Disease (GVHD). In this current study, we have modified our protocol to allow for transient donor engraftment after VCA transplantation and with selective rejection of donor stem cells. METHODS: 5 Haploidentical canine recipients received a non-myeloablative conditioning regimen of 350 cGy TBI, mobilized donor stem cells (PBMC) and VCA transplantation followed by a limited course of immunosuppression (MMF for 56 days and Cyclosporine for 70 days). Peripheral blood chimerism was evaluated by PCR techniques weekly. Peripheral blood cytokine expression was evaluated by flow cytometry. VCA rejection was followed clinically and confirmed histologically after routine biopsies. RESULTS: All 5 animals tolerated the conditioning regimen. One rejected the PBMC at 4 weeks post transplantation and went on to reject the VCA transplant following the cessation of immunosuppression. One dog fully engrafted and remains tolerant to the VCA allograft (POD 168). 3 dogs demonstrated initial engraftment of the PBMC (6 – 14 weeks) rejected the donor cells after cessation of immunosuppression without acute rejection of their donor VCAs. One of these dogs was euthanized for persistent fevers at pod 147 with no sign of rejection. No dog developed GVHD. Early cytokine analysis in tolerant versus non-tolerant animals reveals a significantly elevated IL-4 and IL-10 compared to baseline, suggesting a Th2 profile during induction of tolerance. CONCLUSION: In this study we demonstrate that our non-myeloablative protocol allows for selective rejection of donor stem cells and elimination of GVHD risks without acute rejection of the VCA transplant. Transient donor cell chimerism or engraftment is required to induce tolerance, but after induction, maintenance of tolerance may not be dependent on chimerism. Early after conditioning the establishment of aTh2 profile appears to contributing to the anti-inflammatory environment.

Human Di-Chimeric Cells a New Approach for Tolerance Inducing Protocols in Transplantation: A Preliminary Study

Successful vascularized composite allograft (VCA) transplantation requires life-long treatment combining more than two immunosuppressive agents and pose significant risks and side effects. Cell based therapies are a new promising approach for tolerance induction that could prevent or reduce negative impact of life-long immunosuppression. Bone marrow transplantation has already been tested for solid organs, face and upper extremity transplants for modulation of immune responses. We propose a new cellular therapy based on the ex vivo created donor-recipient chimeric cells as an alternative approach to bone marrow based therapies in support of VCA. The aim of this preclinical study was to create and characterize in vitro the phenotype, genotype and viability of fused human di-chimeric cells (dCC). Fourteen ex vivo fusions of human umbilical cord blood (UCB) cells were performed. Mononuclear cells (MNCs) were isolated from UCB originating from 2 different donors. Next MNCs were stained separately by PKH26 and PKH67. Fusion procedure was performed using polyethylene glycol (PEG) technique. Double PKH26 and PKH67 stained cells were sorted out and subjected to further assessments. Flow cytometry (FC), (CD3, CD4, CD8, CD19, CD34 and CD90, viability test), confocal microscopy (CM), fluorescent lymphocytotoxicity assay (LCT), PCR-reverse sequence-specific oligonucleotide probe (PCR-rSSOP) and short tandem repeat- PCR (STR-PCR) and colony- forming unit (CFU) assay were assessed to characterize the phenotype and genotype of fused human chimeric cells. FC and CM analysis confirmed UCB fusion and creation of human dCC. Using LCT assay we determined that human dCC are sharing HLA class I and class II antigens specific for both types of UCB donors used for fusion. Results of the LCT test were confirmed by rSSOP and STR assay which revealed that fused dCC were in fact originating (39-51%) from each of the UCB donors. After fusion 96-99% of cells were viable. Preliminary phenotype characterization showed expression of all assessed markers on the surface of dCC. CFU assay confirmed the presence and functionality of dCC mature progenitor comparable to untreated cord blood progenitor cells. We successfully confirmed feasibility of ex vivo fusion of UBC cells leading to creation of human fused dCC. We characterized cell phenotype and viability. This unique concept of di-chimeric cell therapy introduces new applications in transplant surgery. The ultimate goal is to induce tolerance in VCA transplants.

Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning

We demonstrate herein that combination treatment with regulatory T cells (Tregs) and vascularized bone marrow transplantation (VBMT) can achieve stable mixed chimerism and long-term transplantation tolerance to vascularized composite allografts (VCA) without requiring cytoreductive recipient conditioning in rats. An appreciable number of Tregs of recipient origin was shown at the interface between recipient and transplanted VCA tissues, implicating a significant role for Tregs in protecting VCA from rejection. This cytoreduction-free protocol using co-treatment with Tregs and VBMT warrants further investigation toward potential clinical application for VCA transplantation.

Commentary on “Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning”

Transplantation of vascularized composite allografts (VCAs) could significantly affect the liver of the approximately 7 million Americans every year. These Americans would benefit from transplantation because they had undergone or need to undergo reconstructive surgery as the result of oncologic procedures, congenital anomalies, and trauma [ [1] Gander B. Brown C.S. Vasilic D. et al. Composite tissue allotransplantation of the hand and face: a new frontier in transplant and reconstructive surgery. Transplant Int. 2006; 19: 868 Crossref PubMed Scopus (77) Google Scholar ]. Just as important, it may also help bring us a step closer to the “Holy Grail” of transplantation, which is the induction of donor-specific immunologic tolerance. The reason is because, to achieve a state of tolerance, the most promising approach appears to be the creation of mixed hematopoietic chimerism [ [2] Bradley J.A. Induction of transplant tolerance through mixed hematopoietic chimerism. Am J Transplant. 2012; 12: 1073 Crossref PubMed Scopus (3) Google Scholar ]. The significance of VCA transplantation lies in the fact that by definition, VCA already contain hematopoietic tissue, which can lead to chimerism with reduced conditioning. Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioningJournal of Surgical ResearchVol. 178Issue 2PreviewCotreatment with regulatory T cells (Treg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether Treg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. Full-Text PDF