Dolph Houben

109 Neoangiogenensis, transplant viability and molecular analysis of bone VCA 5 The discovery of the RANKL/OPG system and its role in the regulation of bone resorption has been relatively well described in literature since the 1990’s [16, 17, 24] . In our results, the implantation of an AV-bundle does not seem to have a positive biological effect on bone resorption when we look at the RANKL/OPG interplay and the excretion of CTSK by osteoclasts when we compare them between the two intervention groups. We monitored putative bone anabolic activity using BGLAP gene expression as a biomarker. Overall, our molecular biomarkers for bone homeostasis were not statistically different upon implantation of AV-bundles. It is possible that the inability to detect differences in molecular bone parameters is due to technical issues, including biological considerations in the acquisition of RNA samples and technical complexities of RT-qPCR. For example, RNA was obtained by pulverizing a complete section of the transplant without separating medullary bone from cortical bone which may differ in bone turn over. Further research should analyze the bone homeostasis, bone remodeling and neo-angiogenesis by gene expression using endosteal and periosteal surfaces separately. Our previous results in small animals have shown neo-angiogenesis from the AV-bundle reached the outer cortex in bone clearing studies [1, 2, 25] . Bone clearing protocols to visualize the contrast agentmacroscopically havegenerally failed in larger animal modelsdue to the thickness of thebone. Microangiographic quantification of the vasculature with micro-CT provides a good alternative, although it is a demanding and slow process. Microfil is useful, as it fills the microvasculature. Unfortunately, it has the same density as mineralized bone expressed in Hounsfield units. Micro- CT after decalcification provides reasonably good imaging of microvasculature in bone but is technically unable to follow the contrast agent from the AV-bundle through the cortical bone to its periosteal surface (Fig. 2). Ideally, we would have been able to follow the AV bundle through the transplant to the periosteal surface. This is a limitation of our study; future research should be performed with a higher magnification micro-CT. Alternatively, when we look at the evaluation of the microangiography and histology of the transplant, microfil reached into both endosteal and periosteal surface of the bone in group 1. It is probable that some vessels are from vascular ingrowth from the surrounding soft tissue.