Dolph Houben

99 Neoangiogenensis, transplant viability and molecular analysis of bone VCA 5 VCA Transplantation The proximal tibia was exposed through an identical anterolateral hindleg incision. A segment of tibia was removed with the same cutting jig, from the same location as the donor VCA. A second incision in the medial thigh was used to expose the superficial femoral artery and vein, tunneling between the two incisions subcutaneously for pedicle passage. The VCA segment was placed into the defect and stabilized with dual locked compression plates (Fig.1C), followed by end-to-side arterial and end-to-end venous anastomoses to the superficial femoral vessels. Surgical induced neo-angiogenesis was achieved by implanting an autogenous cranial tibia arteriovenous bundle (AV-bundle) into the medullary canal. Group 1 had a patent AV-bundle and in group 2 a ligated AV-bundle as a control. All animals were randomly divided into groups and received two weeks of immunosuppression postoperatively. This consisted of Tacrolimus 0.6-1.5 mg/kg (Sandoz Inc., Princeton, NJ, orally), Mycophenolate Mofetil 30-60 mg/kg (Mylan Institutional Inc., Rockford, IL, orally) and Methylprednisolone sodium succinate 500mg start-up dose (Pfizer Inc., NY, NY) intravenous for two weeks. Methylprednisolone was tapered over the immunosuppressive period. Immunosuppression levels were monitored by blood draws taken every other day from the central venous catheter. Dose adjustments were made to maintain a therapeutic level of Tacrolimus (between 5.0-15.0 ng/ml) and Mycophenolate (between 1.0- 3.5mcg/ml). Only short-term immunosuppression was used, to test the ability of autogenous angiogenesis to maintain allotransplant viability long-term. All animals received prophylactic antibiotic therapy, appropriate analgesics and were monitored by staff veterinarians. The animals were individually housed, for a planned 20-week survival period. Unrestricted weight-bearing was allowed directly after the procedure. Sacrifice procedure A 20-week survival period was used in this study, chosen based upon our previous experience as sufficient to demonstrate substantial healing of the VCA segment as well as angiogenesis and resulting bone remodeling from a patent autogenous neoangiogenic blood supply [4] . Practical considerations do not permit the many months or even years likely required for complete remodeling in a large animal model, nor multiple time points with large numbers of animals at each survival period. After the 20-week survival period all animals were anesthetized with Telazol + Xylazine IM, and euthanized with intravenous administration of Pentobarbital Sodium 0.22 ml/kg (Vortech, Dearborn, MI) as recommended by the Panel on Euthanasia of the American Veterinary Medical Association and performed according to NIH guidelines under the direction of the Institutional Animal Care and Use Committee. Both femoral arteries of the animal were dissected proximately, cannulated and flushed with heparin and saline. Later microangiographic analysis of neo-angiogenesis was enabled by injection with a contrast agent (Microfil, MV-122, Flow Tech, Carver, MA). After 45 min of curing, both the experimental and contralateral normal tibiae were harvested with sterile technique. A 5mm proximal segment was used for histology, and a more distal 2mm segment for PCR. Decalcification and micro CT angiography was performed on a 20mm mid-VCA section. The contralateral tibia was harvested, scanned and analyzed in the same manner for control purposes; we will refer to this as normal bone.