Dolph Houben

136 CHAPTER 7 Methods Experimental design This study was approved by the Institutional Animal Care and Use Committee and was performed according to established National Institutes of Health guidelines. Orthotopic vascularized bone allotransplantation was performed in fourteen Yucatan miniature swine divided into 2 equal groups (Sinclair Bioresources, LLC). An additional 7 animals served as donors for two tibiae each, matched by age (mean 5.8 months), size (15-35kg) and blood type (type A). Donor and recipient animals were mismatched by pre-operative swine leukocyte antigen (SLA) haplotyping. Pre-operative DNA sequence haplotyping ensured a mismatch of five to ten class I and II SLA markers. Microsurgical tibial transplantation was performed in all animals as previously described [20] , transplanting a 3.5 cm tibial segment orthotopically into a matched defect. Rigid internal fixation was made with dual locked plates. The nutrient blood supply was restored with microsurgical anastomoses of both artery and vein. Simultaneously, an autogenous cranial tibial arteriovenous (AV) bundle was raised and placed within the VCA medullary canal in Group 1 (Fig. 1). It was placed but ligated as a no-angiogenesis control in Group 2. A central venous line was maintained for intravenous drug administration and blood draws during the first two weeks. Immunosuppressive therapy was given for a period of 14 days, including Tacrolimus 0.6-1.5mg/ kg (Sandoz Inc. Princeton, NJ), Mycophenolate Mofetil 30-60mg/kg (Mylan Institutional Inc., Rockford, IL) and Methylprednisolone sodium succinate (Pfizer Inc., New York, NY). Tacrolimus and Mycophenolate were administrated orally and Methylprednisolone intravenously. Doses were adjusted as needed to maintain therapeutic levels (Tacrolimus: 5.0-15.0 ng/ml, Mycophenolate 1.0-3.5 mcg/ml), monitored by blood draws taken every other day. The methylprednisolone initial dose was 500 mg, tapered over 2 weeks (100-12.5mg/day). Antibiotic prophylaxis was given with Enrofloxacin 7.5mg/kg IM (Baytril, Bayer Healthcare LLC, Shawnee Mission, KS) and Ceftiofur 5mg/kg IM (Excede, Pfizer Inc, New York, NY). Full weight-bearing was allowed immediately. Pain was monitored daily and treated with appropriate analgesics (Carprofen an Buprenorphine). The animals were individually housed and received standard pig feed and water ad libitum. At 2, 4, 6, 10 and 20 weeks, the pigs were anesthetized with Tiletamine HCL + Zolazepam HCL 5 mg/ kg IM (Telazol, Zoetis Inc, Kalamazoo, MI.) and Xylazine 2 mg/kg IM (Xylamed, Bimeda-MTC, Cambridge ON, Canada) for wound inspection and blood draws. Drug immunotherapy was stopped, and the central venous catheter removed on postoperative day 14. The survival period was 20 weeks.