Dolph Houben

148 CHAPTER 7 Discussion Large segmental bone defects are often reconstructed today with structural cryopreserved allografts, and vascularized bone autografts, used alone or in combination. Allografts provide immediate strength and stability but remain largely non-viable. Significant rates of non-union, infection and late stress-fracture are the result [24] . Vascularized bone autografts may hypertrophy in response to mechanical load, heal more readily and resist infection [25] . They are generally poorly matched to defect size and shape and result in some donor site morbidity [26] . Bone-only VCAs may in the future offer the best of both methods: with immediate stability and strength if matched to the defect, yet with similar healing and remodeling potential as vascularized bone autografts. Few have been used clinically, as VCAs require life-long immunosuppression, with the previously-described risks, periodic evaluation and expense. The novel method we have described permits bone-only VCA survival without prolonged drug therapy. Porcine animal models have distinct advantages for allogenic tissue transplantation research in order to retrieve pre-clinical translatable results. Their size, anatomy, physiology and immunology are well known and comparable to man. Furthermore, as blood type and swine leukocyte antigen (SLA) haplotypes have been well studied [27, 28] . Experimental transplantation with manipulation of these variables in these large animals provides better insight into potential clinical utility than studies of smaller rodents [20, 29] . Long-term monitoring of systemic immune responces after transplantation can be accomplished by hematology studies and cytokine detection. Immunosuppresive drug levels are performed as routine clinical tests. The immune response within the bone allotransplant at 20 weeks was also analyzed by RT=qPCR. Segments of each transplanted bone were pulverized, and specific messenger RNA sequences were used to investigate the specific nature of the immunologic activity. Previous investigators have found the need for decalcification to render other methods such as in-situ hybridization or immunohistochemical staining unreliable. A mild patchy lymphocytic infiltration was found in both groups, most likely the result of an inflammatory response to the allogeneic material. Quantitative RT-qPCR analyses of messenger RNA further identified the presence of immunologic markers within the allotransplant, although not significantly elevated compared to normal bone 20 weeks after transplantation. These results are important to better understand the immune status of the allotransplant over time. Vascularized bone and joint allotransplantation has seldom been performed clinically. The largest case series is that of Hofmann, who has performed three vascularized femoral diaphysis and five vascularized whole knee joint transplantations [30, 31] . All patients were matched for bloodtype, no attempt was made to match HLA markers. Maintainance immunosuppression varied in type and length, and included cyclosporin A, azathioprine, anti-thymocyte globulin, and mythylprednisolone. Rejection of vascular tissue has been well understood from solid-organ transplantation literature, when this rejection occurs it leads to intima hypertrophy. This type of vasculopathy can lead to transplant ischemia and eventual loss of the allotransplant [23] . Eventually, all of the complete knee joint transplantation cases have failed due to infection, rejection or chronic allotransplant vasculopathy [4, 23, 30-32] . The two of the vascularized femoral diaphysis failed,