Dolph Houben

20 CHAPTER 1 Aim 9: To measure the extent of new bone formation and correlate bone formation to measures of angiogenesis (Chapter 4). Areas of new bone formation will be identified by fluorochrome labeling. We will quantify the amount of new bone formation and resorption by histomorphometric analyses. Aim 10: To study gene expression associated with the formation of new bone and a neo- angiogeneic circulation (chapter 5) . We will quantify gene expression in the allotransplant by RNA extraction and subsequent reverse transcript qPCR analyses for multiple genes associated with bone formation, resorption, remodeling and neo-angiogenesis. Hypothesis 3: New bone formation in transplanted allogeneic bone is the result of transplant chimerism Aim 11: To measure the lineage of osteocytes in areas of new bone formation after transplantation and viability (chapter 6). Transplanted (male donor bone) specimens removed from female recipient animals will be studied. Areas of new bone formation will be identified by fluorochrome labeling. We will micro dissect these areas of new bone formation by use of Laser Capture Microdissection (LCM) technologies and extract g-DNA. With the use of real-time qPCR we can amplify the genomic DNA and quantify the SRY gene (Y-chromosome specific) in areas of new bone formation. If no SRY can be amplified, the new bone formation is recipient-derived. Aim 12: To measure repopulation of the allotransplant by recipient derived cells due to autologous AV bundle implantation (Chapter 6). Complete sections of the allotransplant will be pulverized, and g-DNA and RNA extracted to quantify the SRY gene and RPL-4 to test the repopulation rate and viability of the remaining male donor cells. Aim 13: To record levels of chimerism in peripheral tissues and determine if any systemic chimerism may have resulted in a state of donor specific tolerance (Chapter 6). Liver and spleen specimens will be obtained 20 weeks after bone-only VCA transplantation and extraction of g-DNA. Real-time qPCR will be used to quantify the relative copy number of SRY gene (Y-chromosome specific) compared to RPL-4. This determines if there was a level of systemic chimerism after bone only VCA.