Dolph Houben

19 Introduction and Outline of the Thesis 1 Hypothesis 2: Bone VCA viability can be maintained in a large animal model using surgical angiogenesis and short-term immunosuppression without tolerance induction or other permanent immune modulation. Aim 3: To successfully reconstruct segmental tibial bone loss by microsurgical transplantation of tibial VCA’s + autologous surgical angiogenesis in Yucatan mini pigs (Chapter 4). We will orthotopically transplant bone-only VCAs across a major histocompatibility barrier using sex-mismatched Yucatan miniature swine, implanting an AV-bundle within the medullary canal to induce recipient-derived angiogenesis (surgical angiogenesis). Two weeks of immune suppressive triple therapy will be used. We will implant a patent AV-bundle in group 1 and a ligated AV bundle in group 2 as a no angiogenesis control. In both groups a microsurgical repair of the nutrient vessel is achieved in the recipient combined with ridged internal fixation of the allotransplant. Aim 4: To study allotransplant viability after cessation of the immunosuppression (Chapter 5). We will histologically evaluate allotransplant sections and quantify osteocyte viability 20 weeks after transplantation. With the use of Micro-CT analyses, we will quantify a micro angiography of the allotransplant as a measure for neo-angiogenesis. Aim 5: To study allogeneic pedicle patency as a function of time (Chapter 4). We will use Doppler ultrasound during the survival period of the animals to check the allogeneic pedicle patency. We will measure the systolic and diastolic peak velocities in m/s. Aim 6: To measure bone healing, ambulation and bone material properties after transplantation and cessation of immune modulation (Chapter 4). We will observe weight bearing on the operated leg after transplantation on daily basis, measure bone healing scores by taking radiographs at evaluation time points (0,2,4,6,10,20 weeks), and will use biomechanical evaluation methods as axial compression testing and cyclic reference point indentation to measure bone material properties. Additionally, with the use of Micro-CT analyses, we will measure the bone micro architecture, bone mineral density, and quantify union 20 weeks after transplantation. Aim 7: To study histological changes in the allogenic pedicle, 20 weeks after transplantation (Chapter 7). We will harvest, embed, cut and stain (Elastica-Van Giesson) the allogeneic pedicle after sacrifice of the animal. Thereafter we will histologically evaluate the microscopic changes of the allogenic pedicle. Aim 8: To study the underlying systemic and local immune responses to bone VCA with and without autologous AV bundle implantation (Chapter 7). We will obtain peripheral blood at evaluation time points (0,1,2,4,6,10,20 weeks) to monitor the systemical immune response by blood cell counts and multiplex cytokine analyses in both intervention groups.