Dolph Houben

116 CHAPTER 6 Abstract Background: Bone allotransplant viability can be maintained long-term by implanting arteriovenous (AV) bundles, creating an autogenous neoangiogenic circulation. Only short-term immunosuppression is required. This study investigates the origin of viable osteocytes observed in areas of active bone remodeling in orthotopically transplanted tibiae in a Yucatan mini-pig model. Methods: Segmental tibial defects created in female Yucatan minipigs (N=14) were reconstructed with a matched vascularized composite allotransplant from a male donor. The circulation was microsurgically restored, with simultaneous autogenous AV-bundle implantation in group 1 (N=7). A ligated AV-bundle was implanted as a no-angiogenesis control in group 2 (N=7). After 20-weeks, repopulation of the allotransplant was assessed by real-time qPCR measurement of relative copy numbers of a Y chromosome-specific gene ( SRY ) and an autosomal housekeeping gene, ribosomal protein L4 ( RPL4 ). A lower SRY/RPL4 ratio demonstrates replacement of male allogeneic cells with female, autogenous cells in the sample. Genomic DNA was extracted from cross-sections of the allotransplant, liver and spleen. Additionally, areas of new bone formation within the allotransplant were sampled by laser capture microdissection. A comparison was made between groups as well as male control samples. RNA was extracted from bone as well, as a measure of metabolically active cells. Results: Laser-captured areas of new bone formation in animals with both normal and ligated AV-bundles were found to have significantly lower relative copy numbers of SRY (p=0.03) than control specimens from male bone, indicating replacement by female (autogenous) bone-forming cells. Analysis of an entire segment of the allotransplant from Group 1 was similarly reduced (p=0.04), unlike that from Group 2. RNA expression of SRY was observed in both groups. No chimerism could be found in non-bone tissues (liver and spleen). Conclusion: We observed a significant level of transplant chimerism in areas of new bone formation sampled by laser capture microdissection. The migration of autogenous cells including osteocytes was seen in both groups. Survival of some allogeneic (male) cells was also demonstrable. No microchimerism was found in liver and spleen.