Dolph Houben

108 CHAPTER 5 Previous published studies [2] [1] have provided results indicating that long-term graft viability can be maintained by surgical induced neo-angiogenesis and short-term immunosuppression. They transplanted vascularized allogenic femoral segments in a subcutaneous pocket in a rat model. Our current results on the effects of implanting autogenous AV-bundles corroborate these findings. The significant new finding of our current studies is that we are able to reconstruct a tibia defect in a weight-bearing pre-translational large animal model. Maintaining transplant viability in a large animal model has been previously described in a feasibility study [4] . The prior pre-clinical model with a 16-week survival period, quantified the same remodeling parameters as we did in our current model. In this study, we did observe three non-unions at the distal interface. The authors believe this could due to the early mobilization and ambulation of the animals directly post-operative. In clinical setting these distal non-unions would be prevented by longer post- operative immobilization until healing has occurred. If a distal non-union would still occur, we could manage this complication with an additional cancellous bone graft. The implantation of the AV-bundle within the bone in group 1 was hypothesized to develop a neoangiogenic autologous circulation in the allotransplant during the immunosuppressive period, as we have seen in rat and rabbit bone VCA allotransplants [1-3, 21, 22] . This is expected to maintain bone viability despite the expected thrombosis of the allogenic nutrient pedicle following stoppage of drug immunosuppression. We would further expect group 2 VCAs to have little or no endosteal cortical bone remodeling and less viable bone and marrow, again due to pedicle thrombosis but without the benefit of autogenous vascular angiogenesis. Evaluation of total and cortical bone vessel volume must include the substantial effect of well-vascularized tissue surrounding the VCA segment on the periosteal surface. The effect of the AV bundle in group 1, placed within the medullary canal, will be seen primarily in the medullary space and its primary effect expected to be on endosteal bone surfaces, at least in the first few weeks or months following transplantation. This is in fact what we found; the increased medullary vessel volume in group 1 had a significant positive effect on bone vitality on the endosteal surface of the allotransplant. The effect becomes less obvious when summed with the total bone and cortical bone volume. Angiogenesis is required for bone development, growth, and repair. It is influenced by the local bone environment that involves cross-talk between endothelial cells and adjacent bone cells [18] . The role of EGFL6 has been described in a calvarial osteoblastic cell culture (ex vivo) [18] . EGFL-6 plays an important role in this cross-talk and promotes endothelial cell migration and angiogenesis. Although gene expression literature on angiogenesis markers after allotransplantation is scare, we have shown a significantly higher expression of EGFL-6 in our patent AV-bundle group. The higher EGFL6 expression may have contributed to a higher medullary vessel volume. CD34 is a transmembrane protein in vascular associated tissue, therefore it is a parameter of the amount of vascular tissue when quantified by RT-qPCR. Quantification of total vessel volume showed a slightly higher volume of vessels (0.40%) in the patent AV-bundle group (Group1). Logically, one would expect the higher vessel volume would result in higher CD34 expression. Our results show a 1.36 higher CD34 expression in the patent AV-bundle group, but we could not appreciate a statistical correlation between CD34 and the higher vessel volume. VEGFA is considered an important neo-angiogenesis growth factor. We could not appreciate a positive effect of surgical angiogenesis on the expression of VEGFA . HIF1A should have a positive effect on VEGFA expression [23] , although we could not appreciate this effect.