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. ß

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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.

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 (AE2 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. ß

ABSTRACT:We have previously shown experimental transplantation of living allogeneic bone to be feasible without long-term immunosuppression by development of a recipient-derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient-derived arteriovenous (a/v) bundle. FK-506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGFþVEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGFþVEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGFþVEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression. ß