Vascularized Bone Marrow Transplantation Research Articles

Preclinical Studies in Vascularized Composite Allotransplantation

Introduction: Since 1998, when the first hand transplant was performed, Vascularized Composite Allotransplantation (VCA) field represents a new dimension in reconstructive surgery, offering a viable treatment option for injuries and defects that involve multiple layers of functional tissue, impossible to repair using conventional surgical techniques. Worldwide experience includes allotransplantation of upper and lower extremities, face, trachea, larynx, abdominal wall, uterus and penis, with promising clinical results. The major challenge remains at the immunologic level, translational ongoing studies being focused to develop less toxic immunosuppressant drugs and possibly achieve donor-specific tolerance. Materials and Methods: We conducted a series of preclinical studies of vascularized composite allotransplantation in preparation for further translation to clinical applications of this field. Our experience includes experimental vascularized allotransplantation models in rats exploring technical particularities and also analyzing the role of microchimerism in immunomodulation and promoting of the immune tolerance. Also, we performed a cadaveric study in order to explore the surgical techniques required in abdominal wall allotransplantation and the use of iliac crest as a potential microsurgical model of vascularized bone marrow transplantation(VBMT). Results: Working on small animal models allows the familiarization with microsurgical techniques in experimental composite tissue allotransplantation and evaluation of important immunological aspects: rejection dynamics, immunosuppression protocol testing and induction of immunologic tolerance. The surgical procedures performed in cadaveric models shown the feasibility of the abdominal wall allotransplantation procedure which may be translated to future clinical practice in association with organ transplantation. It may be possible to develop an alternative approach for iliac crest microsurgical transplant as tolerogenic strategy in solid organs or vascularized composite allotransplantation. Conclusion: Preclinical experience represents a mandatory step in developing a vascularized composite allotransplantation program, translating the knowledge obtained from experimental animal research and cadaveric studies to further clinical practice, ensuring optimal results for patients with complex tissue defects.

Vascularized Bone Marrow Cellular Depletion or Discontinuity Abrogates Protection of Vascularized Composite Allografts in Nonhuman Primates.

Background.Vascularized composite allografts (VCA) have demonstrated good clinical outcomes dependent on chronic immunosuppression. Previous work by our group and others supports that cotransplanted vascularized bone marrow (VBM) as a component of VCA offers immunologic protection to prolong graft survival. We aimed to characterize the requirements and potential mechanisms of VBM-mediated protection of VCA by modifying grafts through various strategies.Methods.Experimental groups of mismatched cynomolgus macaque recipients received VCA transplants modified by the following approaches: heterotopic separation of the VCA and VBM components; T-cell depletion of either donor or recipient; irradiation of donor VCA; and infusion of donor bone marrow. All groups received standard immunosuppression with tacrolimus and mycophenolate mofetil.Results.Experimental modifications to donor, recipient, or graft all demonstrated short-graft survivals (31 d). Chimerism levels without bone marrow infusion were transient and minimal when detected and were not associated with prolonged survival. Donor bone marrow infusion increased levels of chimerism but resulted in alloantibody production and did not improve graft survival.Conclusions.VCA graft survival is significantly reduced compared with previously reported VCA with VBM transplants (348 d; P = 0.01) when the hematopoietic niche is removed, altered, or destroyed via irradiation, depletion, or topographical rearrangement. These experimental manipulations resulted in similar outcomes to VCA grafts without cotransplanted VBM (25 d). These data support the presence of a radiosensitive, T-cell population within the VBM compartment not reconstituted by reinfusion of bone marrow cells.

The intragraft vascularized bone marrow component plays a critical role in tolerance induction after reconstructive transplantation

The role of the vascularized bone marrow component as a continuous source of donor-derived hematopoietic stem cells that facilitate tolerance induction of vascularized composite allografts is not completely understood. In this study, vascularized composite tissue allograft transplantation outcomes between recipients receiving either conventional bone marrow transplantation (CBMT) or vascularized bone marrow (VBM) transplantation from Balb/c (H2d) to C57BL/6 (H2b) mice were compared. Either high- or low-dose CBMT (1.5 × 108 or 3 × 107 bone marrow cells, respectively) was applied. In addition, recipients were treated with costimulation blockade (1 mg anti-CD154 and 0.5 mg CTLA4Ig on postoperative days 0 and 2, respectively) and short-term rapamycin (3 mg/kg/day for the first posttransplant week and then every other day for another 3 weeks). Similar to high-dose conventional bone marrow transplantation, 5/6 animals in the vascularized bone marrow group demonstrated long-term allograft survival (>120 days). In contrast, significantly shorter median survival was noted in the low-dose CBMT group (~64 days). Consistently high chimerism levels were observed in the VBM transplantation group. Notably, low levels of circulating CD4+ and CD8+ T cells and a higher ratio of Treg to Teff cells were maintained in VBM transplantation and high-dose CBMT recipients (>30 days) but not in low-dose VBM transplant recipients. Donor-specific hyporesponsiveness was shown in tolerant recipients in vitro. Removal of the vascularized bone marrow component after secondary donor-specific skin transplantation did not affect either primary allograft or secondary skin graft survival.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

AB108. Vascularized bone marrow transplantation with renal transplantation

Since the first case of kidney transplant succeeded in 1954, the technology is relatively mature in renal transplant. After operation one year and five years of survival rate can reach more than 90% and 80%, respectively. But it still has to face rejection. The traditional way is to apply immune inhibitors to prevent rejection. But the side effects brought by the immunosuppressant seriously affects patients’ quality of life. So the induction of immune tolerance has become a research hotspot.Vascularized bone marrow transplantation (VBMT) is developed on the basis of bone marrow transplantation (BMT). Its characteristic is with marrow stromal environment, which not only can provide cell nutrition, promoting cell growth, also can adjust and control function of lymphocytes. Possible explanation is that the complete bone marrow stromal cells produce different cytokines. These factors may have effects on hematopoietic stem cells, and promote their ability of proliferation differentiation. The other aspect, the bone itself is the part of the bone marrow stromal microenvironment, and osteoblast has a proliferous and differentiated effect on hematopoietic stem cells. At the same time, the bone marrow stromal cells can produce extracellular matrix, which can pass through specific surface receptors, promoting the information transfer between hematopoietic cells, stabilizing growth factor between the matrix and the hematopoietic cells. And many scholars have proved it in the unique effect of establishing the chimera and inducing immune tolerance.Therefore, we can try to build VBMT in rats model of kidney transplantation, to explore the effect of VBMT in the induction of immune tolerance in renal transplantation, and research the mechanism.

A novel rat full-thickness hemi-abdominal wall/hindlimb osteomyocutaneous combined flap: influence of allograft mass and vascularized bone marrow content on vascularized composite allograft survival.

Vascularized bone marrow transplantation (VBMT) appears to promote tolerance for vascularized composite allotransplantation (VCA). However, it is unclear whether VBMT is critical for tolerance induction and, if so, whether there is a finite amount of VCA that VBMT can support. We investigated this with a novel VCA combined flap model incorporating full-thickness hemiabdominal wall and hindlimb osteomyocutaneous (HAW/HLOMC) flaps. Effects of allograft mass (AM) and VBMT on VCA outcome were studied by comparing HAW/HLOMC VCAs with fully MHC-mismatched BN donors and Lewis recipients. Control groups did not receive treatments following transplantation. Treatment groups received a short course of cyclosporine A (CsA), antilymphocyte serum, and three doses of adipocyte-derived stem cells (POD 1, 8, and 15). The results showed that all flaps in control allogeneic groups rejected soon after VCAs. Treatment significantly prolonged allograft survival. Three of eight recipients in HLOMC treatment group had allografts survive long-term and developed donor-specific tolerance. Significantly higher peripheral chimerism was observed in HLOMC than other groups. It is concluded that the relative amount of AM to VBMT is a critical factor influencing long-term allograft survival. Accordingly, VBMT content compared with VCA mass may be an important consideration for VCA in humans.

Long-Term Tolerance of Vascularized Composite Allografts Is Induced By Vascularized Bone Marrow Transplantation Under Costimulation Blockade-Based Therapy.

Long-Term Tolerance of Vascularized Composite Allografts Is Induced By Vascularized Bone Marrow Transplantation Under Costimulation Blockade-Based Therapy.

New Findings in Anatomy of Blood Supply to Rat Femur Isolated Vascularized Bone Marrow Transplantation Model

New Findings in Anatomy of Blood Supply to Rat Femur Isolated Vascularized Bone Marrow Transplantation Model

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression. This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring. Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression. This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring. Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A vascularized bone marrow transplantation model in rat

Objective To establish a simple and reliable vascularized bone marrow transplantation (VBMT) model as a new method for bone marrow transplantation.Methods The femoral bones were transplanted into the recipient inguinal regions and their nutrient vessels were anastomosed with recipients vessels.Twenty Lewis rats were divided into 3 groups.In syngeneic group,femoral bone transplantation was performed between Lewis rats.In both rejection and immunosuppression groups,BN rats were used as donors and Lewis rats as recipients.Rats in rejection group received no treatment,and those in immunosuppression group were given cyclosporine A after allotransplantation.The viability of the all grafts was evaluated grossly and morphologically,and the chimerism was determined by using flow cytometry.Results The bone grafts in syngeneic group remained viable and the bone marrow maintained normal morphologically at 30th day post-transplant,but the grafts in rejection group were clearly rejected at 7th day after transplantation,and the number of bone marrow cells was decreased sharply with almost no chimerism.In accord with syngeneic grafts,the grafts in immunosuppression group showed normal appearance of the viable tissue.Donor-specific chimerism was confirmed in peripheral blood at 1st,2nd,3rd,and 4th week posttransplant,and the chimerism level was detected at (4.7 ± 2.0) ％,(2.2 ± 1.2) ％,(1.8 ± 0.9) ％ and (1.5 ± 0.3) ％ respectively.Conclusion Vascularized femoral bone grafting is a simple and reliable method for the transplantation of bone marrow,and may provide a useful tool for the study of immune tolerance. Key words: Vascularized bone marrow transplantation; Chimerism; Immune tolerance; Femoral bone transplantion

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.

Vascularized Bone Marrow Transplant Vs Conventional Bone Marrow Cells Transplantation in Conditionated Rats

Vascularized Bone Marrow Transplant Vs Conventional Bone Marrow Cells Transplantation in Conditionated Rats

Role of Donor-Specific Regulatory T Cells in Long-Term Acceptance of Rat Hind Limb Allograft

BackgroundVascularized bone marrow transplantation (VBMT) is widely accepted as an efficient means of establishing chimerism and inducing tolerance. However, the mechanism underlying is poorly understood. Recently, regulatory T cells (Tregs) have been shown to play an important role in regulating immune responses to allogeneic antigens. In this study, we explored the role of Tregs in the induction of tolerance in an allogeneic hind limb transplantation model.Methodology/Principal FindingsForty-eight Lewis rats were divided into 6 groups. They received isografts and allografts from Brown-Norway hind limbs. Recipients in groups 1 and 2 received isografts and those in the other groups received allografts. The bone components of donor limbs were kept intact in groups 1, 3, and 5 but removed before transplantation into groups 2, 4, and 6. Tapered cyclosporin A (CsA) was administered to recipients in groups 5 and 6 after transplantation. During the 100-day observation period, all isografts survived, but the allografts in groups 3 and 4 were rejected within 8 to 12 days. CsA-treated intact allografts survived rejection-free for more than 100 days, and CsA-treated allografts lacking bone elements were rejected within 2 months. Stable peripheral chimerism and myeloid chimerism were observed in group 5. Declining peripheral chimerism and a lack of myeloid chimerism were observed in group 6. Donor-specific Tregs were exclusively detected in both peripheral blood and in the spleens of long-term recipient rats in group 5, with an increased FoxP3 mRNA expression in the allografts. This was further demonstrated to be responsible for donor-specific hyporeactivity by in vitro one-way mixed lymphocyte reaction (MLR).Conclusion/SignificanceBone components in the allogeneic hind limbs can induce myeloid chimerism and donor-specific Tregs may be essential to tolerance induction. The bone-removal hind limb model may be a suitable counterpart to the induction of tolerance in the study of limb 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

Composite osseomusculocutaneous sternum, ribs, thymus, pectoralis muscles, and skin allotransplantation model of bone marrow transplantation

Cellular and vascularized bone marrow cells have been used to induce donor-specific chimerism in various models of composite tissue allotransplantation. Although thymus transplantation has been reported in the literature, the effect of thymus transplantation on chimerism levels in vascularized bone containing composite tissue allotransplantation has not been reported. In this study, a new method for composite vascularized sternal bone marrow transplant model is descried that can be applied to augment chimerism after transplantation. A total of seven composite osseomusculocutaneous sternum, ribs, thymus, pectoralis muscles, and skin transplantations were performed in two groups. The first group (n = 5) was designed as an allotransplantation group and the second group (n = 2) was designed as an isotransplantation group. Composite osseomusculocutaneous sternum, ribs, thymus, and pectoralis muscles allografts were harvested on the common carotid artery and external jugular vein and a heterotopic transplantation was performed to the inguinal region of the recipient rat. Cyclosporine A monotherapy was administered in order to prevent acute and chronic allograft rejection. Animals sacrificed when any sign of rejection occurred. The longest survival was 156 day post-transplant. Assessment of bone marrow cells within sternum bone component and flow cytometry analysis of donor-specific chimerism in the peripheral blood of recipients were evaluated. Our results showed that this composite allograft carried 7.5 × 10(6) of viable hematopoietic cells within the sternum component. At day 7 post-transplant chimerism was developed in T-cell population and mean level was assessed at 2.65% for RT1(n) /CD4 and at 1.0% for RT1(n) /CD8. In this study, a new osseomusculocutaneous sternum, ribs, thymus, pectoralis muscle, and skin allotransplantation model is reported which can be used to augment hematopoietic activity for chimerism induction after transplantation.

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

BackgroundCotreatment 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. MethodsRecipient Lewis rats treated (day 0) with or without naturally sorted Treg (3 × 106) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days −1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically. ResultsThe combination of Treg and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12–18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without Treg treatment. Graft-versus-host disease did not occur in the VBMT recipients. ConclusionsCotreatment with Treg and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive Treg-VBMT protocol has potential for clinical application in VCAs.

Combined procedure of vascularized bone marrow transplantation and mesenchymal stem cells graft – An effective solution for rapid hematopoietic reconstitution and prevention of graft-versus-host disease

Hematopoietic stem cell transplantation (HSCT) is a a standard therapeutic option for several diseases. The success of the procedure depends on quality and quantity of transplanted cells and on stromal capacity to create an optimal microenvironment, that supports survival and development of the hematopoietic elements. Conditions associated with stromal dysfunction lead to slower/insufficient engraftment and/or immune reconstitution. A possible solution to this problem is to realize a combined graft of hematopoietic stem cells along with the medular stroma in the form of vascularized bone marrow transplant (VBMT). Another major drawback of HSCT is the risk of graft versus host disease (GVHD). Recently, mesenchymal stromal cells (MSC) have demonstrated the capacity to down-regulate alloreactive T-cell and to enhance the engraftment. Cotransplantation of MSC could be a therapeutic option for a better engraftment and GVHD prevention.

Improving the Safety of Tolerance Induction: Chimerism and Cellular Co-Treatment Strategies Applied to Vascularized Composite Allografts

Although vascularized composite allografts (VCAs) have been performed clinically for a variety of indications, potential complications from long-term immunosuppression and graft-versus-host disease remain important barriers to widespread applications. Recently it has been demonstrated that VCAs incorporating a vascularized long bone in a rat model provide concurrent vascularized bone marrow transplantation that, itself, functions to establish hematopoietic chimerism and donor-specific tolerance following non-myeloablative conditioning of recipients. Advances such as this, which aim to improve the safety profile of tolerance induction, will help usher in an era of wider clinical VCA application for nonlife-saving reconstructions.