Dolph Houben

118 CHAPTER 6 Methods Transplantation Surgery Vascularized bone allotransplantation was performed in a porcine tibial defect model. The Yucatan miniature swine with identical blood type and known swine leukocyte antigen haplotypes (SLA) were provided by Sinclair Bioresources, LLC. Male donors and female recipients were paired to result in an SLA mismatch and thus subsequent VCA rejection. A 3.5cm vascularized tibia segment was transplanted from the male donor into a matched hindlimb defect of a female recipient. Rigid internal fixation was made with dual locked plates. The nutrient blood supply was repaired with microsurgical anastomoses of both artery and vein. An autogenous cranial tibial arteriovenous (AV) bundle was simultaneously implanted within the medullary canal to create an autogenous neoangiogenic blood supply in group 1 (n=7) (Fig. 1). In group 2 (N=7), the arteriovenous bundle was ligated as a no-angiogenesis control. All animals received a 2-weeks’ immunosuppression triple therapy regimen as previously described to maintain initial circulation through the microsurgically-repaired allogeneic nutrient blood supply during AV bundle angiogenesis [8] . Subsequent VCA survival relied upon both the neoangiogenic endosteal circulation and ingrowth of vessels from periosteal surface contact, as the allogenic vascular pedicle had thrombosed [9] . The complete surgical procedure has been previously described [10-12] . The experiment was terminated after a 20-week survival period. At fourteen and four days prior to sacrifice, Calcein (Sigma, St Louis, MO 20mg/kg, IM) and Oxytetracycline Hydrochloride (Vetrimycin, Boise, ID, 20mg/kg, IM) were respectively administered in order to detect new bone formation during the 10-day interval by fluorescence microcopy. Two pigs with surgical complications were excluded from the study. This study was approved by the Institutional Animal Care and Use Committee and performed according to established and National Institutes of Health (NIH) guidelines. Sacrifice procedure All animals were sacrificed after the survival period, as recommended by the Panel on Euthanasia of the Veterinary Medical Association and performed according to the NIH guidelines under the direction of the Institutional Animal Care and Use Committee. The allotransplant was harvested in a sterile fashion from the tibia and two 2mm cross-sections removed with a sterile cooled oscillating bone saw. Soft tissues were removed to obtain bone sections. Liver and spleen samples were acquired as non-bone tissues. All samples were snap-frozen in liquid nitrogen and stored at -80⁰C. Tissue sections were subsequently used for laser capture microdissection or nucleic acid extraction.