Dolph Houben

128 CHAPTER 6 Vascularized composite allotransplantation (VCA) bone represents a potential alternative source of structural bone segments with both the desirable size and shape of banked cryopreserved bone and the many advantages of autogenous bone [24] . Currently, VCAs rely on the use of life-long immunotherapy to maintain vascular pedicle patency and tissue viability. The cost of drugs and monitoring are significant, as are the risks of immunosuppressive drug therapy for a lifetime. Risks include graft-versus-host disease, opportunistic infections, malignancy, metabolic diseases, and end-organ toxicity [25-28] . We have demonstrated bone-only vascularized composite allotransplants to survive long-term, heal and remodel without sustained immunosuppression by surgical implantation of autogenous arteriovenous bundles or fascial flaps within bone. The result is the generation of an autogenous neoangiogenic circulation and subsequent active healing and remodeling of the allotransplant by autogenous circulation-derived cells [11, 29-31] . In this report, a segmental tibial defect model in Yucatan minipigs was developed as a pre-clinical model, to demonstrate similar outcomes and confirm findings obtained in laboratory rats and rabbits. Movement of cells into the bone (transplant chimerism) and from bone into other tissues (mixed chimerism) has been demonstrated following bone VCA in rat femora, made possible by sex- mismatched organ transplantation [14, 32, 33] . Detection of transplant chimerism from whole segments of VCA bone is of less interest than sampling small areas of bone where active bone remodeling is occurring post-transplant. Laser capture microdissection allows such samples to be obtained and have shown new bone to be primarily autogenous in the rat femoral model [32] . In this porcine study, large structural tibial VCAs used to reconstruct segmental defects demonstrate similar findings. Linage studies in calcified tissue are challenging. Decalcification of bone followed by formalin fixation and paraffin embedding (FFPE) resulted in loss of fluorochromes used to label areas of active bone formation and degraded DNA. While frozen bone sections were not similarly affected, they did not adhere as effectively as FFPE specimens to PEN membranes needed for LCM. Reduced adhesion makes capturing very small areas, for example containing a single or a few osteocytes difficult (Fig. 1A). Nevertheless, sampling areas primarily between the double fluorochrome labels demonstrated no remaining allogenic ( SRY ) DNA in seven of eleven samples. This finding serves to confirm that much of the active remodeling and healing occurring in these large bone VCAs is the result of autogenous, likely circulation-derived bone formation. Due to the expense, technical difficulty, and surgical complexity of these large animal experiments, group size and survival period were necessarily limited. Nevertheless, our findings are of importance as the only large animal bone allotransplantation study in the literature. It is fundamentally different from the porcine hindlimb study of Kuo et al. [34] , due to the marked differences in antigenicity between bone and the soft tissue components included in their study. Previously, analysis of areas of new bone formation in rat femur VCAs by laser capture microdissection has shown the majority of osteocytes to be autogenous in origin rather than of allogeneic lineage at 18 weeks post-transplant [32] . These data were obtained by qPCR analysis of the SRY gene after female-to-male sex-mismatched bone allotransplants. Although some allogeneic osteocytes may survive due to their relatively sequestered location in lacunae of