Dolph Houben

176 CHAPTER 9 The extent of surgical neo-angiogenesis and the effect of it on transplant viability and gene expression was evaluated in Chapter 5 . In this chapter, we found significantly greater amounts of medullary vessels in the patent AV bundle group 20-weeks after transplantation. Endosteal osteocyte counts were highest in this group. A significant increase in the expression of endothelial growth factor like-6 (EGFL-6) was observed, with a positive correlation with measured vessel volumes. The sex-mismatched nature of our large animal model allowed for evaluating the extent of transplant chimerism by investigation of the SRY gene with RT-qPCR. Additionally, further development of Laser Capture Microdissection techniques combined with fluoroscopy allowed precise sampling of osteocytes. In Chapter 6 , we studied cell lineage within sex-mismatched bone VCAs by LCM (Laser Capture Microdissection) and RT-qPCR. Analyses of areas of new bone formation showed significant levels of microchimerism, demonstrating new bone formation to result from the migration of autogenous cells. Some allogenic male donor cells survived, evidenced by RNA analyses. No systemic chimerism was found in liver and spleen, indicating that induction of donor specific tolerance was not important. Only two weeks of immunosuppressive triple therapy was used in our large animal model. After cessation of the immunosuppression, recipient rejection of the VCA would be expected. In Chapter 7 , we measured the systemic immune response and effect of AV bundle implantation on local immune status of the allotransplant 20 weeks after transplantation. Thrombosis of the allogenic vascular pedicle occurred 4-6 weeks after transplantation, as found in Chapter 4. Histologic evaluation of the allogenic pedicle confirmed loss of pedicle patency due to intimal hypertrophy, as expected without immune modulation. Necrosis of the bone does not occur due to the development of a neo-angiogenic autogenous blood supply during the initial period of drug therapy. In this study, there was no adverse systemic effect of this process, studied by periodic peripheral blood cytokine determinations during a 20-week survival period. The allotransplants themselves demonstrated less inflammation, less necrosis and improved viability with the addition of an autogenous AV bundle. All implanted patent AV bundles in were patent 20 weeks after transplantation and showed significant spouting into the allotransplant as demonstrated in Chapter 5. Histologic evaluation of the AV bundle showed a normal patent arterial wall. In conclusion, this novel approach to bone allotransplantation seems to have significant advantages over conventional VCA methods proven in a pre-clinical experimental animal model. The data found throughout chapters 4-7 are consistent with those found in previous studies using the same novel method to maintain transplant viability in small animal bone-only VCA [13- 19] . The porcine model has distinct advantages for allogenic tissue transplantation research in order to retrieve pre-clinical translatable results. Their size, anatomy, physiology and immunology are well known and comparable to human. Our porcine tibia defect model has proven to be a great asset for VCA research and produces consistent and reproducible results [20-22] . Living bone allotransplantation with short-term immunosuppression and AV bundle implantation holds therefore clinical potential using our novel method.