Role Of Vascularized Bone Marrow Within Vascularized Composite Allografts In Tolerance Induction

[Backgroud]Severe face and limb injuries frequently present with multiple tissues defect with loss of the bone and deep soft tissue. Traditional reconstructive procedures provide a limited coverage for such wounds and result in an unsatisfactory outcome. Vascularized composite allograft(VCA), with adequate tissues recovered from a deceased donor, can prevent such limitations. It provides a “like to like” tissue for defect repair without causing donor-site morbidity. Encouraging functional and aesthetic outcomes can be readily achieved via VCA. Since the success of first human hand transplantation in 1998 and first face transplantation in 2005, VCA has been translated from research concepts into clinical practice. Over 100 hand and more than 30 face allotransplantation have been performed to date. Most of them gained satisfactory form and function, which impelled the development of vascularized composite allotransplantation in field of plastic surgery. However, unlike solid organ transplantation, VCA is just a life-changing procedure but not a life-giving procedure. The difference has limited its popularization and application for the ethical concerns about life-long exposure of otherwise healthy individuals to high-dose and multidrug immunosuppression. How to reduce or eliminate the use of immunosuppressive drugs has become a key rate-limiting factor for VCA development.Bone marrow cell transplantation(BMT), as an effective way for tolerance induction, may be the only solution to this problem. It has been successfully applied in clinical kidney transplantation without maintenance immunosuppression in three different research center, showing great potential in tolerance induction. Various publications have been reported on the study of tolerogenic mechanism. Certain VCAs, such as hand and face, themselves include a vascularized bone morrow component, which has been reported to promote tolerance for limb and face transplantation in rats. It may be seen as an effective way of bone marrow transplantation to induce tolerance. VBM contains hematopoietic stem cells(HSCs) as well as syngeneic microenvironment that are able to sustain HSCs survival. Full engraftment is achieved immediately after transplantation with no requirement of cell isolation and homing process. So it is widely thought that VBMT may provide a continuous supply of donor BMCs and promote tolerance induction. However, few studies focus on this unique form of bone marrow transplantation. Our studies aim at investigating the role of vascularized bone marrow within VCA so as to find a new way to induce immune tolerance for VCA.To study this unique form of bone marrow transplantation, we should first establish an appropriate VBMT model. Traditional VBMT model is the rat hind-limb transplant, which simulated clinical hand allografts and enabled us to evaluate both immunologic feature and functional rehabilitation after transplantation. However, the hind-limb transplant is technically complicated with the requirement of osteotomy and intramedullary fixation. Various complications such as bleeding, infection are frequently encountered, which increases the difficulty of experiments. The main purpose of our studies was to investigate the immunological aspects of vascularized bone marrow but not the functional results, so we attempt to replace the hind-limb with a similar model that contains all components of a limb allografts but is technically simpler and reliable. Its establishment may provide a useful tool for the study of vascularized bone marrow.The VBM not only contains the content of HSCs but also has a variety of stromal cells, such as endothelial cell, fibroblasts, adipose cell, osteoblast cells et al, which are vital for the function of transplanted bone marrow cells. Numerous studies have demonstrated that transplantation of microenvironment together with marrow stem cells permitted stable marrow stem cell engraftment, and bone fragments under the kidney capsule or flushed femur in subcutaneous tissues facilitated successful treatment of intractable autoimmune disease. Comparable results can also be obtained by simultaneous transplantation of stromal cells and hematopoietic cells. However, little is known about whether VBMT can function as hematopoiesis or not? Whether VBMT is a more effective technique than i.v.BMT in either reconstitution of recipient hematopoiesis or induction of tolerance is also unknown? These questions are what we aim to answer in this study.The ultimate goal of this study is to induce tolerance via VBMT with a nonmyeloblative conditioning regimen so as to explore a feasible way to induce tolerance for clinical VCA patients. Generally, for a successful BMC transplantation, myeloablative conditioning is required to “make room” for donor bone marrow in addition to subsequent T cell depletion agents to prevent rejection of the allogeneic cells. However, myeloablative conditioning would inevitably lead to serious complications such as myelosuppression, aplasia, pancytopenia et al, which are appearantly unacceptable for healthy individuals. VBM has its own “space” along with its own microenvironment, such that myeloablative conditioning of the recipient may not be requisite. This phenomenon may reduce the toxicity of the conditioning regimen. Therefore, we plan to design a nonmyeloblative conditioning regimen including two infusions of the antilymphocyte serum and one set of thymic irradiation with no requirement of total body irradiation. This conditioning regimen may offer a platform to study the immunologic benefit of VBM in VCA. [Objectives]1) To establish a simple and reliable VBMT model; 2) To investigate the potential role of VBM in hematopoietic reconstitution for myeloablative recipients and make comparison with traditional intravenous transplantation of bone marrow cells suspension; 3) To study the effect of vascularized bone marrow on survival of vascularized composite allografts and explore its possible mechanisms. [Methods]1) Establishment of VBMT models: The femur was taken as the carrier of bone marrow. Femur osteomyocutaneous flaps from Lewis rats were transplanted into syngeneic recipients’ inguinal region, and their nutrient vessels were anastomosed with recipient vessels. This model was consisted of a groin flap and a partial femur, which was therefore termed femur osteomyocutaneous flap. Before transplantation, anatomic study was first performed to determine the courses of pedicle vessels. Then the basic operating procedure to havest a free vascularized femur was formulated based on the anatomic results. The femoral artery and vein were taken as the vessel pedicle. To confirm the feasibility and safty of the model, methylene blue injection was used to evaluate the blood perfusion of grafted tissues. Gross and histological examination were also evaluated after transplantation for the viability of grafts to further verify the feasibility of this model. Finally, some modifications were made on established model so as to create some new VBMT models for the subsequent studies.2) Evaluation of hematopoietic potential of VBM-1: Based on previously established VBMT model, a vascularized femur without inclusion of skin and subcutaneous tissue was used as the carrier of BMCs. Lewis rats were irradiated with 5Gy X-ray and repopulated with GFP positive BMCs(SD background) introduced by either intravenous infusion(i.v.BMT) or vascularized bone marrow(VBM) approach. Cyclosporine A was used to inhibit rejection and graft versus host disease. The number of peripheral blood cells, the colony-forming unites of spleen and the reconstitution of bone marrow were compared between the i.v.BMT and VBMT groups. The origin of regenerated cells was determined by flow cytometry anlysis and bone marrow smears.3) Evaluation of hematopoietic potential of VBM-2: To rule out the possible effects of rejection, graft versus host disease and gene differences on hematopoiesis for the use of outbred rats, we repeated the BMT experiments between syngeneic Lewis rats. The recipients still received 5 Gy irradiation but no immunosuppressant. Peripheral blood cells, the colony-forming unites of spleen and the reconstitution of bone marrow were evaluated again among the three groups.4) Evaluation of tolerogenic potential of VBM within VCA: Fully MHC mismatched Lewis and BN rats were used as recipients and donors respectively. All Lewis rats received a vascularized groin flap from donor as the rejection monitor. It combined with transplantation of a vascularized femur or isolated bone marrow cells suspension or saline. Meanwhile, all rats were preconditioned with antilymphocyte serum, cyclosporine A and thymic irradiation before transplantation. Comparison of flap survival and chimerism was made in rats with and without the inclusion of a vascularized femur. Histologic sections of grafted flap were evaluated for rejection and bone specimens for viability of transplanted femur components. Possible mechanism on tolerance induction through VBM was also explored by flow cytometry analysis of the cell phenotype within VBM. [Results]1)(1)The mean operative time of femur osteomyocutaneous flap transplantation was(159.0±8.3) min with the harvesting time of(68.0±4.8) min and the ischemia time of(55.8±6.8) min.(2)The methylene blue injection showed rich blood supply of isolated femur osteomyocutaneous flap, confirming the feasibility of this model to some extent.(3)All flaps survived the transplant procedure uneventfully with pink skin and hair regrowth.(4)Hemotoxylin and eosin stain of transplanted flaps and bone revealed the normal appearance of viable skin and bone marrow, which verifed the feasibility of this model.(5)Based on this model, two modified femur transplant model were further developed. They were combined VBM model consisting of whole femur and groin flap, or whole femur transplantation model without inclusion of flap.2)(1)The results of hematopoietic reconstitution showed that both VBMT and i.v.BMT group brought about faster recovery of leukocytes and platelets in comparison with the control group(P<0.05). The majority of regenerated cells were found to be GFP positive, indicating that the regenerated cells were primarily donor-derived BMC but not the remaining recipient BMCs.(2)More spleen colonies and higher spleen index were observed in recipients of VBMT than that of sham control(P<0.05). Histological examination of the spleen in VBMT group showed numerous lymphocytes in the white pulp and red pulp area in contrast to the sham control where few nucleated cells were seen in spleen sections.(3)Histological appearances of bone marrow also showed scattered BMCs in recipient of VBMT, but the bone marrow in control group had almost been occupied by vacuolar tissues. Results of BMC counts corresponded with histological examination that the number of BMCs in control group was a magnitude lower than those in VBMT and i.v.BMT groups. Regenerated BMCs in recipient bone marrow were confirmed again to be donor-derived, since over 90% of BMCs were GFP positive in flow cytometry analysis of recipient bone marrow and bone marrow smears showed marked GFP expression of BMCs under fluorescence microscopy.(4) After excision of transplanted femur, both WBC count and platelet count decreased sharply, indicating the important role of VBM in assisting the long-term maintenance of hematopoiesis in VBMT recipients.(5)However, VBMT demonstrated no significant advantages over i.v.BMT in hematopoiesis, though it was effective in reconstitution of host hemopoietic system. Earlier recovery of platelets at day 5 was observed in VBMT group, but it didn’t accelerate the recovery of WBCs compared to i.v.BMT. For bone marrow reconstitution, it even demonstrated delayed reconstitution of host bone marrow in early stage after VBMT(<10 days).(6)Similar experiments on synergetic Lewis rat BMT model further confirmed the equal hematopoietic capability of VBMT and i.v.BMT.3)(1)The conditioning regimen used in our experiment was effective in T cell ablation, which provide a good platform for tolerance induction.(2)Based on this regimen, significantly prolonged survival of the flap was seen in the animals receiving vascularized femurs, with a mean survival time of 78.8±13.0 days, which was significantly different to the BM infusion group(60.4±1.7 days) and control group(58.6±1.3)(P<0.05).(3)Continuous and stable chimerism of 5.81%±1.98% was detected for the first 60 days in recipient of femur with the loss of chimerism on 75 days after transplant. In contrast, animals transplanted with BM cells or the flap alone demonstrated almost no chimerism in either peripheral blood or immune organs.(4)VBM enhanced engraftment of donor cells into recipient spleen, bone marrow and lymph nodes, but at no stage were donor cells found in recipient thymus.(5)Bone marrow within transplanted femur was unable to provide an ongoing source of donor cells and was gradually replaced by recipient origin cells.(6)Bone marrow in VCA may have started rejecting prior to the skin allograft or at least at the same time as the skin allograft. [Conclusions]Our studies presented some important findings: 1) Three simple and reliable VBMT models were established based on vascularized femur, and their establishment will provide useful tools for the study of vascularized bone component within VCA. 2) Our study confirmed that vascularized bone marrow transplantation was an effective way to transplant bone marrow that can reconstituted hematopoietic system of myeloablative recipients. 3) Our studies supported an immunomodulatory role of VBM in VCA that generates donor cells, induces macrochimerism and improves the survival outcome. 4) VBM in VCA cannot provide a continuous source of donor stem cells, and donor bone marrow within VBM was gradually replaced by recipient origin cells with the duration of time.In conclusion, VBM has an immunological benefit on VCA survival, which may provide a new way to induce tolerance for VCA.