Recipient‐derived angiogenesis with short term immunosuppression increases bone remodeling in bone vascularized composite allotransplantation: A pilot study in a swine tibial defect model

Current vascularized composite allotransplantation (VCA) transplantation protocols rely upon life‐long immune modulation to maintain tissue perfusion. Alternatively, bone‐only VCA viability may be maintained in small animal models using surgical angiogenesis from implanted autogenous vessels to develop a neoangiogenic bone circulation that will not be rejected. This study tests the method's efficacy in a large animal model as a bridge to clinical practice, quantifying the remodeling and mechanical properties of porcine tibial VCAs. A segmental tibial defect was reconstructed in Yucatan miniature swine by transplantation of a matched tibia segment from an immunologically mismatched donor. Microsurgical repair of nutrient vessels was performed in all pigs, with simultaneous intramedullary placement of an autogenous arteriovenous (AV) bundle in Group 2. Group 1 served as a no‐angiogenesis control. All received 2 weeks of immunosuppression. After 16 weeks, micro‐CT and histomorphometric analyses were used to evaluate healing and remodeling. Axial compression and nanoindentation studies evaluated bone mechanical properties. Micro‐CT analysis demonstrated significantly more new bone formation and bone remodeling at the distal allotransplant/recipient junction and on the endosteal surfaces of Group 2 tibias (p = 0.03). Elastic modulus and hardness were not adversely affected by angiogenesis. The combination of 2 weeks of immunosuppression and autogenous AV‐bundle implantation within a microsurgically transplanted tibial allotransplant permitted long‐term allotransplant survival over the study period of 16 weeks in this large animal model. Angiogenesis increased bone formation and remodeling without adverse mechanical effects. The method may allow future composite‐tissue allotransplantation of bone without the risks associated with long‐term immunosuppression. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1242–1249, 2017.

     

Autogenous Arteriovenous Bundle Implantation Maintains Viability Without Increased Immune Response in Large Porcine Bone Allotransplants

BackgroundTransplantation of living allogeneic bone segments may permit reconstruction of large defects, particularly if viability is maintained without immunosuppression. Development of a new autogenous osseous blood supply accomplishes this goal in rodent experimental models. This study evaluates potential systemic and local inflammatory responses to this angiogenesis in a large-animal model.MethodsVascularized allogeneic tibia segments were transplanted orthotopically into matched tibial defects in Yucatan minipigs. Microvascular anastomoses of bone nutrient artery and vein were supplemented by intramedullary placement of an autogenous arteriovenous (AV) bundle in group 1. Group 2 served as a no-angiogenesis control. A 3-drug immunosuppression regimen was withdrawn after 2 weeks. During the 20-week survival period, periodic leukocyte counts and inflammatory cytokine levels were measured. Thereafter, osteocyte survival was quantified and transplant rejection graded by histologic examination and quantitative real-time polymerase chain reaction of immunologic markers.ResultsBoth groups developed an initial systemic response, which resolved after 4 to 6 weeks. No differences were seen in blood cytokine levels. Interleukin 2 expression was diminished in group 1 tibiae. As expected, nutrient pedicles had thrombosed without sustained immunosuppression, occluded by intimal hyperplasia. In group 1, angiogenesis from the autogenous AV bundle resulted in significantly less osteonecrosis (P = .04) and fibrosis (P = .02) than group 2 allotransplants.ConclusionsSystemic immune responses to large-bone allotransplants were not increased by generation of an autogenous osseous blood supply within porcine tibial bone allotransplants. Implanted AV bundles diminished inflammation and fibrosis and improved bone viability when compared to no-angiogenesis controls.     

Repopulation of vascularized bone allotransplants with recipient‐derived cells: Detection by laser capture microdissection and real‐time PCR

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (±2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real‐time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex‐determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77–97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex‐mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1514–1520, 2009     

Host-derived angiogenesis maintains bone blood flow after withdrawal of immunosuppression

A novel method of living bone allotransplantation combining microvascular repair of the nutrient circulation, implantation of host-derived arteriovenous (AV) bundles, and short-term immunosuppression is described. We hypothesized that neoangiogenesis from the implanted vessels would maintain graft viability and circulation after withdrawal of FK506 (Tacrolimus) immunosuppression. Vascularized femoral transplantation was performed between DA and PVG rats. In addition to microsurgical pedicle anastomoses, a saphenous AV bundle from the recipient animal was implanted in the medullary space. Ninety-seven rats were randomly allocated to groups differing in immunosuppression and AV bundle patency. Implanted vessels significantly improved capillary density and bone blood flow in nonimmunosuppressed and immmunosuppressed groups, respectively. A lower incidence of spontaneous AV bundle thrombosis was found with Tacrolimus treatment. More viable osteocytes were seen at 4 weeks when the AV bundle was patent. Further investigations may confirm host-derived neoangiogenesis as an alternative to tolerance induction or immunosuppression in bone allotransplantation.     

Host-derived neoangiogenesis with short-term immunosuppression allows incorporation and remodeling of vascularized diaphyseal allogeneic rabbit femur transplants

The purpose of this study was to demonstrate that living bone allotransplants can incorporate, remodel, and maintain mechanical properties without long-term immunosuppression in a fashion comparable to living autotransplants. For this, viability is maintained by repair of nutrient vessels and neovascularization from implanted host-derived vasculature. Microsurgically revascularized femoral diaphysis allotransplants were transferred from young male New-Zealand-White (NZW) into 4 groups of male Dutch-Belted (DB) rabbits. Short-term immunosuppression by tacrolimus (IS, groups 4 and 5) and host-derived neovascularization (NV) from implanted fascial flaps was used to maintain viability (groups 3 and 5) as independent variables. Group 2 received neither IS nor NV. Vascularized pedicled autotransplants were orthotopically transplanted in group 1. After 16 weeks, transplants were evaluated using radiologic, histologic, biomechanical, and histomorphometric parameters. Vascularized bone allotransplants treated with both short-term IS and host-derived NV (group 5) healed in a fashion similar to pedicled autotransplants (group 1). Their radiographic scores were higher than other groups. Groups with patent fascial flaps (3 and 5) showed significantly greater neoangiogenesis than ligated controls (2 and 4). Tacrolimus administration did not affect neoangiogenesis. Elastic modulus and ultimate stress were significantly greater in autogenous bone than in allotransplanted femora. Biomechanical properties were not significantly different among allotransplants. Bone turnover was decreased with IS, but increased with NV by the implanted fascial flaps. Living allogeneic femoral allotransplants treated with short-term IS and host-derived neoangiogenesis can lead to stable transplant incorporation in this rabbit model. The combination of both factors optimizes bone healing. Transplant mineralization is improved with neoangiogenesis but diminished with IS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 763–770, 2009     

Knee joint transplantation combined with surgical angiogenesis in rabbits--a new experimental model

Purpose: We have previously described a means to maintain bone allotransplant viability, without long‐term immune modulation, replacing allogenic bone vasculature with autogenous vessels. A rabbit model for whole knee joint transplantation was developed and tested using the same methodology, initially as an autotransplant. Materials/Methods: Knee joints of eight New Zealand White rabbits were elevated on a popliteal vessel pedicle to evaluate limb viability in a nonsurvival study. Ten additional joints were elevated and replaced orthotopically in a fashion identical to allotransplantation, obviating only microsurgical repairs and immunosuppression. A superficial inferior epigastric facial (SIEF) flap and a saphenous arteriovenous (AV) bundle were introduced into the femur and tibia respectively, generating a neoangiogenic bone circulation. In allogenic transplantation, this step maintains viability after cessation of immunosuppression. Sixteen weeks later, X‐rays, microangiography, histology, histomorphometry, and biomechanical analysis were performed. Results: Limb viability was preserved in the initial eight animals. Both soft tissue and bone healing occurred in 10 orthotopic transplants. Surgical angiogenesis from the SIEF flap and AV bundle was always present. Bone and joint viability was maintained, with demonstrable new bone formation. Bone strength was less than the opposite side. Arthrosis and joint contractures were frequent. Conclusion: We have developed a rabbit knee joint model and evaluation methods suitable for subsequent studies of whole joint allotransplantation. © 2011 Wiley Periodicals, Inc. Microsurgery, 2012.     

A modified vascularized whole knee joint allotransplantation model in the rat

Previous papers have shown surgical neoangiogenesis to allow long‐term bone allotransplant survival without immunosuppression. Whole joint composite tissue allotransplants (CTA) might be treated similarly. A novel rat knee CTA model is described for further study of the roles of neoangiogensis in joint allotransplant survival and adjustment of immunosuppression. Microvascular knee CTA was performed in nine rats across a major histocompatibility barrier with both pedicle repair and implantation of host‐derived arteriovenous (“a/v”) bundles. In the control group (N = 3), the pedicle was ligated. Immunosuppression was given daily. Joint mobility, weight‐bearing, pedicle patency, bone blood flow, and sprouting from a/v bundles were assessed at 3 weeks. All but the nonrevascularized control knees had full passive motion and full weight bearing. One nutrient pedicle thrombosed prematurely. Blood flow was measurable in transplants with patent nutrient pedicles. Implanted a/v bundles produced new vascular networks on angiography. This new rat microsurgical model permits further study of joint allotransplantation. Patency of both pedicles and implanted a/v bundles was maintained, laying a foundation for future studies. © 2010 Wiley‐Liss, Inc. Microsurgery, 2010.     

Fibroblast growth factor‐2 and vascular endothelial growth factor mediated augmentation of angiogenesis and bone formation in vascularized bone allotransplants

We previously demonstrated recipient‐derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long‐term immunosuppression. The effect of cytokine delivery to enhance this process is studied. Vascularized femur transplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(d,l ‐lactide‐co‐glycolide) microspheres loaded with buffer (N = 11), basic fibroblast growth factor (FGF2) (N = 10), vascular endothelial growth factor (VEGF) (N = 11), or both (N = 11) were inserted intramedullarly alongside a recipient‐derived arteriovenous bundle. FK‐506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric, and alkaline phosphatase measurements were performed. Bone blood flow was greater in the combined group than control and VEGF groups (P = 0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (P < 0.05). Bone viability, growth, and alkaline phosphatase activity did not vary significantly between groups. Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants. © 2014 Wiley Periodicals, Inc. Microsurgery 34:301–307, 2014.     

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.     

Effects of Surgical Angiogenesis on Segmental Bone Reconstruction With Cryopreserved Massive‐Structural Allografts in a Porcine Tibia Model

Cryopreserved bone allografts (CBA) used to reconstruct segmental bone defects provide immediate structural stability, but are vulnerable to infection, non‐union and late stress fracture as the majority of the allograft remains largely avascular. We sought to improve the bone vascularity and bone formation of CBAs by surgical angiogenesis with an implanted arteriovenous (AV) bundle, using a porcine tibial defect model. Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA) mismatched Yucatan minipigs to reconstruct a 3.5 cm segmental tibial defect. A cranial tibial AV‐bundle was placed within its intramedullary canal to induce angiogenesis. The AV bundle was patent in eight pigs and ligated in a control group of eight pigs. At 20 weeks neo‐angiogenesis was evaluated by micro‐angiography. Bone formation was measured by quantitative histomorphometry and micro‐computed tomography. Seven of eight AV‐bundles in the revascularized group were patent. One had thrombosed due to allograft displacement. Total vascular volume was higher in the revascularized allografts compared to the ligated group (p = 0.015). Revascularized allografts had increased levels of bone formation on the allograft endosteal surface compared to the ligated control group (p = 0.05). Surgical angiogenesis of porcine tibial CBAs by intramedullary implantation of an AV‐bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation on allograft endosteal surfaces. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1698–1708, 2019     

Allogenic transplants of bone revascularized by microvascular anastomoses: a preliminary study

An area of experimental bone grafting that needs further study is the use of free vascularized allografts of bone. In 35 outbred mongrel dogs, the viability of vascularized bone allografts with and without azathioprine immunosuppression was compared to vascularized autogenous bone grafts. Viability was assessed by histologic techniques, fluorochrome bone labeling, and electron microscopy. Autogenous vascularized bone grafts remained viable, and it was concluded that microvascular technique was not the limiting factor in attaining survival of the grafts. The behavior of autogenous vascularized bone grafts with and without the influence of azathioprine was similar. Allogenic vascularized bone transplants uniformly failed at a period between 2 and 3 weeks. Immunosuppression with azathioprine did not appreciably affect survival of the osteocytes. However, the host response to the foreign tissue was slightly modified. The clinical ramifications of bone transplantations in humans are not analogous to the clinical situation of transplantation of other organs. If vascularized bone transplants are performed in humans, a relatively safe form of immunosuppression is necessary. This study suggests that azathioprine alone does not offer sufficient immunosuppression to insure viability of the vascularized transplant.     

Allogenic transplants of bone revascularized by microvascular anastomoses: A preliminary study

An area of experimental bone grafting that needs further study is the use of free vascularized allografts of bone. In 35 outbred mongrel dogs, the viability of vascularized bone allografts with and without azathioprine immunosuppression was compared to vascularized autogenous bone grafts. Viability was assessed by histologic techniques, fluorochrome bone labeling, and electron microscopy. Autogenous vascularized bone grafts remained viable, and it was concluded that microvascular technique was not the limiting factor in attaining survival of the grafts. The behavior of autogenous vascularized bone grafts with and without the influence of azathioprine was similar. Allogenic vascularized bone transplants uniformly failed at a period between 2 and 3 weeks. Immunosuppression with azathioprine did not appreciably affect survival of the osteocytes. However, the host response to the foreign tissue was slightly modified. The clinical ramifications of bone transplantations in humans are not analogous to the clinical situation of transplantation of other organs. If vascularized bone transplants are performed in humans, a relatively safe form of immunosuppression is necessary. This study suggests that azathioprine alone does not offer sufficient immunosuppression to insure viability of the vascularized transplant.     

Surgical angiogenesis: a new approach to maintain osseous viability in xenotransplantation

Chung Y‐G, Bishop AT, Giessler GA, Suzuki O, Platt JL, Pelzer M, Friedrich PF, Kremer T. Surgical angiogenesis: a new approach to maintain osseous viability in xenotransplantation. Xenotransplantation 2010; 17: 38–47. © 2010 John Wiley & Sons A/S. Abstract: Background: Large segmental osseous defects are challenging clinical problems. Current reconstructive methods, using non‐viable allografts, vascularized autografts or prostheses have significant rates of serious complications and failure. These include infection, stress fracture and non‐union (frozen structural allogenic bone); loosening and implant failure (prosthetic replacement); limited availability, poor match of size and shape and donor site morbidity (vascularized autograft bone). In the future, microvascular transplantation of living allogenic or xenogenic bone could solve some of these issues, combining the advantages of living bone autografts (capability of primary osseous healing, remodeling, and fracture resistance) with the ability to match size and shape, provide immediate stability and avoid donor site morbidity. Xenotransplants would be particularly attractive, as they could be readily available, if long‐term bone survival could be achieved without unacceptable morbidity. Here, we present a preliminary study to evaluate a new and unique method to maintain xenogenic bone circulation without need for long‐term immune modulation that depends upon generation of a neo‐angiogenic circulation within the transplanted bone from recipient‐derived vessels. Thus, only short‐term immunosuppression would be required to achieve bone survival. Methods: One hundred and forty‐one hamster femora were microsurgically transplanted to rats, restoring nutrient vessel circulation with standard microvascular anastomoses. At the same time, a host‐derived arteriovenous bundle (AVB) was placed within the medullary canal. Two independent variables were evaluated: use of tacrolimus/cyclophosmamid immunosuppression (IS) and patency of the implanted AVB. Rats were therefore randomized to four groups; group 1—no IS + patent AVB; group 2—no IS + ligated AVB; group 3—IS + patent AVB; group 4—IS + ligated AVB. Rats were sacrificed after 1 or 2 weeks. We evaluated bone blood flow (microsphere entrapment), neoangiogenesis (microangiography and quantification of capillary density), bone necrosis rate (osteocyte counts) and nutrient pedicle rejection (microsurgical anastomotic patency). Statistical Analysis was performed with two‐way ANOVA with Bonferroni adjustment. Differences were considered significant when P < 0.05. Results: Capillary density was significantly increased with a patent intramedullary AVB (groups 1/3) compared to groups with ligated AVBs (groups 3/4). Capillary sprouting was predominantly restricted to the endosteal layer. Most nutrient pedicles (78.7%) stayed patent in groups with IS (groups 3 and 4). Consequently, bone blood flow was significanty higher in groups 3 and 4 compared to groups 1 and 2. Similary, a patent AV bundle improved flow in group 1 when compared to group 2. The bone necrosis rate was not influenced by the presence of patent AVBs but was significantly reduced in groups 3 and 4. Conclusions: Surgical angiogenesis occurs when patent arteriovenous bundles are placed in the medullary canal of xenogenic bone and leads to increased bone blood flow. Bone viability was not significantly influenced by neoangiogenesis. Although capillary sprouting was restricted to the endosteal layer in this short term study, more complete cortical revascularization might be observed in a long‐term study. Such a study should further evaluate whether these new vessels supply sufficient blood flow to maintain long‐term bone viability and allow remodeling.     

Effect of rhBMP‐2 and VEGF in a vascularized bone allotransplant experimental model based on surgical neoangiogenesis

We have demonstrated survival of living allogeneic bone without long‐term immunosuppression using short‐term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study, bone morphogenic protein‐2 (rhBMP‐2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra‐medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP‐2 or rhBMP‐2 + VEGF. FK‐506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP‐2 + VEGF) group than rhBMP‐2 and control groups (p < 0.05). Osteoblast counts were also higher in the rhBMP‐2 + VEGF group (p < 0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p = 0.059). Local administration of VEGF and rhBMP‐2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 561–566, 2013     

Tunneled superficial inferior epigastric artery (SIEA) myocutaneous/vascularized femur chimeric flaps: a model to study the role of vascularized bone marrow in composite allografts

The role of vascularized bone marrow in promoting composite allograft survival can be assessed by intrinsically chimeric flaps. In this study, we introduce a significant modification to a previously described rat model of combined superficial inferior epigastric artery (SIEA) myocutaneous/vascularized femur transplantation. We previously noted autocannibalization in orthotopic myocutaneous SIEA allotransplants, which complicated clinical and histologic evaluation of rejection. We therefore designed syngeneic experiments in eight Lewis (RTl(1) ) rat pairs to explore the feasibility of tunneling the SIEA component of chimeric SIEA myocutaneous/vascularized femur flaps to the recipient dorsum. Vascularized SIEA myocutaneous/femur transplants survived in their entirety to POD 63 study endpoint with patent anastomoses in seven of eight (87.5%) transplants as confirmed clinically, histologically, and via near-infrared fluorescent angiography. Tunneling of the SIEA component of SIEA myocutaneous/vascularized femur flaps to the recipient dorsum can be achieved with high success rate and acceptable operative times, and is a technically easy method to study the role of vascularized bone marrow in composite allografts. This modification facilitates SIEA component monitoring, removes it from constant contact with cage bedding, and places it in a location where autocannibalization is unlikely.     

Short-term immunosuppression and surgical neoangiogenesis with host vessels maintains long-term viability of vascularized bone allografts.

Currently available methods to reconstruct large skeletal defects have limitations. These include nonunion and stress fractures in structural allografts, and inability to match the size, shape, and/or strength of most recipient sites using vascularized fibular autografts. Prosthetic diaphyseal replacements may loosen or produce periprosthetic fractures. Transplantation of living allogenic bone would enable matching donor bone to the recipient site, combined with the desirable healing and remodeling properties of living bone. We propose a novel method by which the transplantation of such tissue might be done without the risks of life-long immunosuppression, using surgical neoangiogenesis to develop a new host-derived osseous blood supply. We performed vascularized femoral allografts from 86 female Dark Agouti donor rats to male Piebald Virol Glaxo recipients across a major histocompatibility (MHC) barrier. In addition to microvascular reconstruction of the nutrient vessel, we surgically implanted a host arteriovenous (AV) bundle into the medullary canal to promote host vessel neoangiogenesis. Independent variables included patency of the implanted AV bundle, and use of 2 weeks' FK-506 immunosuppression. After 18 weeks, bone blood flow was measured, and neoangiogenic capillary density quantified. Bone blood flow and capillary density were significantly greater in transiently immunosuppressed recipients with a patent AV pedicle. We conclude that neoangiogenesis from implanted host-derived AV-bundles, combined with short-term immunosuppression maintains blood flow in vascularized bone allografts, and offers potential for clinical application.     

The effects of different schedules of total-body irradiation in heterotopic vascularized bone transplantation. An experimental study in the Lewis rat

To evaluate the effects of irradiation on heterotopically placed vascularized knee isografts, a single dose of 10 Gy of total-body irradiation was given to Lewis donor rats. Irradiation was delivered either 2 or 6 days prior to harvesting or subsequent transplantation, and evaluated at 1, 2, and 4 weeks after grafting. Irradiation caused endothelial depopulation of the graft artery, although vascular pedicle patency was maintained throughout the study. Bone graft viability and mineralization were normal. Dramatic changes in the bone marrow were seen that included an increase of its fat content (P less than 0.001), and a concomitant decrease in bone marrow-derived immunocompetent cells. These changes were more prominent in recipients of grafts from day -6 irradiated donor rats. Total-body irradiation did not prejudice the use of vascularized bone grafts, and exhibited an associated immunosuppresant effect over the vascular endothelium and bone marrow. This may be a further rational conditioning procedure to avoid recipient manipulation in vascularized bone allotransplantation.     

Transplantation of a vascularized rabbit femoral diaphyseal segment: mechanical and histologic properties of a new living bone transplantation model

A new vascularized bone transplantation model is described, including the anatomy and surgical technique of isolating a rabbit femoral diaphyseal segment on its nutrient vascular pedicle. The histologic and biomechanical parameters of pedicled vascularized femoral autotransplants were studied following orthotopic reimplantation in the resulting mid-diaphyseal defect. Vascularized femur segments were isolated in 10 rabbits on their nutrient pedicle, and then replaced orthotopically with appropriate internal fixation. Postoperative weightbearing and mobility were unrestricted, and the contralateral femora served as no-treatment controls. After 16 weeks, the bone flaps were evaluated by x-ray (bone healing), mechanical testing (material properties), microangiography (quantification of intraosseous vasculature), histology (bone viability), and histomorphometry (bone remodeling). Bone healing occurred by 2 weeks, with further callus remodeling throughout the survival period. Eight transplants healed completely, while two had a distal pseudarthrosis. Microangiography demonstrated patent pedicles in all transplants. Intraosseous vessel densities were comparable to nonoperated (control) femora. We found ultimate strength and elastic modulus to be significantly reduced when compared to normal controls. Viable bone, increased mineral apposition rate, and bone turnover were demonstrated in all transplants. The method described, and the data provided will be of value for the further study of isolated segments of living bone, and in particular, for investigations of reconstruction of segmental bone loss in weight-bearing animal models. This study also provides important normative data on living autologous bone flap material properties, vascularity, and bone remodeling. We intend to use this method and data for comparison in subsequent studies of large bone vascularized allotransplantation.     

Vascularized Bone Marrow‐Based Immunosuppression Inhibits Rejection of Vascularized Composite Allografts in Nonhuman Primates

Vascularized composite allograft (VCA) transplantation (also referred to as composite tissue allotransplantation) has demonstrated clinical success in cases of hand, arm and face transplantation despite prior belief that skin provides an insurmountable barrier to allograft rejection. These overall good outcomes are facilitated by substantial immunosuppressive requirements in otherwise healthy patients, yet still demonstrate frequent rejection episodes. We developed a nonhuman primate model of facial segment allotransplantation to elucidate the unique pathophysiology and immunosuppressive requirements of VCA with addition of concomitant vascularized bone marrow (VBM). Heterotopically transplanted facial segment VCA with VBM treated only with tacrolimus and mycophenolate mofetil (MMF) demonstrated prolonged rejection‐free survival, compared to VCA without VBM that demonstrated early rejection episodes and graft loss. While VCA with VBM demonstrated sporadic macrochimerism, acute and chronic rejection and graft loss occurred after discontinuation of immunosuppression. These data support an immunomodulatory role of VBM in VCA that reduces immunosuppressive requirements while providing improved outcomes.     

Experimental study on vascularized bone allografts for reconstruction of massive bone defects

To study the healing mechanism of vascularized bone allografts under short-term as well as long-term immunosuppression with cyclosporin A, experimental vascularized intercalary bone allograft transplantation was carried out between inbred rats using the tibiofibula graft model. Bone scintigram and radiographs were used as an indicator for early detection of rejection after transplantation and bone union. In vascularized bone allografts under long-term immunosuppression with cyclosporin A, early bone union and continuous incorporation were similar to that observed in vascularized bone autograft transplantation. When administration of cyclosporin A was discontinued before completion of bone union, the graft was rejected and bone union was delayed. Apparent swelling on the operated limb associated with a decrease in bone scintigram uptake suggested the occurrence of rejection of the allograft. Vascularized bone allograft transplantation is useful for reconstruction of massive bone defects only if immunosuppressants are used and maintained at least until bone union is obtained. © 1994 Wiley-Liss, Inc.