Dolph Houben

158 CHAPTER 8 Materials and Methods A series of preliminary anatomical studies in porcine cadavers were performed to define the vascular anatomy of the knee blood supply in the pig, and develop techniques to harvest the allotransplant, microsurgically restore blood flow, stably internally fix the femoral and tibial skeleton, implant autogenous vessels into femur and tibia, and provide adequate soft tissue coverage of the reconstruction. Subsequently, three sex-mismatched vascularized whole knee joint allotransplantations with autogenous AV-bundle implantation and muscle coverage were performed with a 10-week survival period. Anatomic study Six fresh porcine cadaveric hind limbs were obtained from three outbred pigs. The animals were a mean 25 kg of weight. The hind limbs were dissected to define the vascular anatomy of the knee and surrounding soft tissues. The femoral artery was cannulated, and the limb flushed with heparinized saline 10000 USP/L (0.9% Sodium Chloride, Baxter Healthcare Corporation, Deerfield, IL) (Heparin Sodium Injection, Fresenius Kabi LLC, Lake Zurich, IL). It was next injected with Ward’s red latex (Wards’s, Rochester, NY). After curing overnight, the lower limbs were dissected and vessel topography relevant for vascularized whole joint allotransplantation defined, including the vascular supply to the gracilis muscle for wound coverage use. Rigid internal fixation of whole knee joints knee has been described in humans with intramedullary nail fixation [1, 5, 7] . We tested and developed the means to use veterinary locked intramedullary nails for both femoral and tibial fixation in these hind limbs. We determined that prolonging the vascular pedicle to include the entire femoral artery and vein, mobilized from the inguinal ligament to the knee greatly facilitated knee VCA transplantation, permitting end-to-side arterial and end-to-end venous anastomoses to the femoral vessels in the recipient hind limb. Feasibility study Three recipient outbred female farm pigs were used in the feasibility study. Three outbred male farm pigs were used as donors to enable lineage studies. Donor and recipient were otherwise matched in size, age, and blood type. All animal procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) approval. Knee VCA harvest On the day of surgery, the male donor was anesthetized with Tiletamine HCL + Zolazepam HCL (Telazol, Zoetis Inc, Kalamazoo, MI, 5 mg/kg IM), Xylazine (Xylamed, Bimeda-MTC, Cambridge ON, Canada, 2 mg/kg IM) and euthanized with Pentobarbital Sodium (Vortech Dearborn MI, 0.22 ml/kg IV). The right limb of the donor was prepped and draped in a sterile fashion. The complete hind limb was then covered with Ioban 2 Antimicrobial Incise drapes (3M Health Care products, St. Paul, MN). One large anteromedial longitudinal skin incision over the knee was made, curved medially proximal toward the femoral vessels distal to the inguinal ligament. The superficial femoral artery and vein were identified in the interval between the gracilis and vastus medialis muscles. They were mobilized to serve as the transplant vascular pedicle, ligating all small muscular branches