Visser, Noortje J.

Segmental bone loss reconstruction using frozen bone allografts has a great advantage over amputation. Unfortunately, frozen allografts are avascular, and their use often results in infection, non-union, or fracture. We have attempted to overcome these complications by revascularizing the bone using gene therapy. Segmental tibial bone transplants were performed in 16 Yucatan minipigs. An arteriovenous (AV) bundle was placed intramedullary inside the allografts, which were transfected with growth factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in 8 out of the 16 pigs. Survival time was set at 20 weeks. Bone remodeling and biomechanical properties were tested using histology, compression testing, bone mineralization, and gait analysis. In the growth factor group, five out of eight pigs developed cutaneous vascular tumors, causing excessive bleeding, which led to the exclusion of one pig. Bone remodeling was significantly more present in the VEGF group compared to the control group. This correlated with the amount of pressure the pig allowed on the operated leg for 10 weeks, which was also significantly more (p = 0.029) than the control. Both bone mineralization (p = 0.156) of the area inside the cortex and the amount of stress the allograft could endure before breaking (p = 0.078) seemed to be no different for the VEGF group compared to its healthy contralateral side, which was the control group. We have shown that growth factor-induced frozen allograft bone revascularization using an intramedullary AV bundle results in significantly more bone remodeling and better biomechanical bone properties. However, we encountered benign vascular cutaneous tumors in five out of eight pigs, making the clinical application of this technique impossible at this time.

AbstractSegmental bone defects are often performed with cryopreserved allografts. They provide immediate stability, but risk nonunion, infection and late stress fracture. Improving the rate and extent of bone revitalization may improve results. Angiogenesis from surgically placed arteriovenous (AV) bundles improves bone blood flow and vitality in cryopreserved rat femora, augmented by vasculogenic growth factors. This study tests the same principal in Yucatan mini‐pigs with a tibial diaphyseal defect, combining surgical angiogenesis with angiogenic gene therapy within cryopreserved orthotopically‐placed allografts. Tibial diaphyseal defects were reconstructed with cryopreserved allografts and rigid internal fixation in 16 mini pigs. Half of the cranial tibial AV bundles placed within the allograft medullary canal were transfected with an adeno‐associated virus containing vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF) genes (AAV9.VEGF.PDGF). Bone remodeling, angiogenesis, and allograft healing were assessed. During the postoperative survival period 5 of 8 transfected animals developed cutaneous benign vascular lesions at sites remote from the operated hindlimb, causing excessive bleeding. Within the allograft, both medullary (p = 0.013) and cortical (p = 0.009) vascular volumes were higher and vessels more mature than nontransfected allografts. Bone turnover (p = 0.013), bone mineralization (p = 0.018), bone healing (p = 0.008) and graft incorporation (p = 0.006) were all significantly higher in the gene therapy group. In a large animal tibial defect model, gene therapy of implanted AV bundles improved revascularization, remodeling and healing of cryopreserved allografts used for limb reconstruction. However, benign vascular lesions causing excessive bleeding developed in 5 out of 8 pigs transfected with AAV containing genes for VEGF and PDGF. This unforeseen complication makes vasculogenic gene therapy unacceptable for clinical use.

AbstractCryopreserved bone allografts(CBA) are susceptible to infection, nonunion, and late stress fracture. Although surgical revascularization by intramedullary implantation of an arteriovenous bundle (AV bundle) generates a neoangiogenic blood supply, there is potential for vascular ingrowth‐mediated bone resorption to weaken the graft. For this reason, we have evaluated changes in CBA mechanical properties of structural tibial allografts with and without surgically induced angiogenesis. Cryopreserved tibia bone allografts were transplanted to reconstruct a 3.5 cm segmental tibial defect in 16 Yucatan mini pigs. Surgical revascularization was performed in half by implantation of a cranial tibial AV bundle, (revascularization group). A control group of identical size had a ligated AV bundle implanted, (ligated group). At 20 weeks micro‐computed tomography (CT) measured bone mineral density (BMD) as well as bone union. Reference point indentation (RPI) compared cortex material properties, and axial compression determined the allotransplant compressive modulus. Seven of eight tibiae in the angiogenesis group were healed at both junction points at 20 weeks. Only four of eight tibiae healed in the ligated control group. There was no significant difference between the revascularization and ligated control groups in BMD and axial compression test. Similarly, RPI parameters were statistically equal. In paired comparisons with contralateral tibias, however, some RPI values were significantly worse in the ligated control group tibiae. This study demonstrates no adverse effect of surgical angiogenesis on cryopreserved structural bone allograft biomechanical properties in a large animal orthotopic segmental tibial defect model. These data suggest the potential value of surgical angiogenesis in clinical limb‐sparing reconstructive surgery.

ABSTRACTCryopreserved bone allografts (CBA) used to reconstruct segmental bone defects provide immediate structural stability, but are vulnerable to infection, non‐union and late stress fracture as the majority of the allograft remains largely avascular. We sought to improve the bone vascularity and bone formation of CBAs by surgical angiogenesis with an implanted arteriovenous (AV) bundle, using a porcine tibial defect model. Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA) mismatched Yucatan minipigs to reconstruct a 3.5 cm segmental tibial defect. A cranial tibial AV‐bundle was placed within its intramedullary canal to induce angiogenesis. The AV bundle was patent in eight pigs and ligated in a control group of eight pigs. At 20 weeks neo‐angiogenesis was evaluated by micro‐angiography. Bone formation was measured by quantitative histomorphometry and micro‐computed tomography. Seven of eight AV‐bundles in the revascularized group were patent. One had thrombosed due to allograft displacement. Total vascular volume was higher in the revascularized allografts compared to the ligated group (p = 0.015). Revascularized allografts had increased levels of bone formation on the allograft endosteal surface compared to the ligated control group (p = 0.05). Surgical angiogenesis of porcine tibial CBAs by intramedullary implantation of an AV‐bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation on allograft endosteal surfaces. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1698–1708, 2019

Objective: The treatment of segmental bone defects remains challenging as complications frequently occur with the currently available methods, including vascularized autograft, prosthetic replacement, bone transport, and cryopreserved bone allograft (CBA). Although the use of CBA is comparatively simple, it lacks donor site morbidity and provides immediate stability; however, the grafts remain largely necrotic, resulting in high failure rates due to nonunion, infection, and stress fractures. Revitalizing the CBA has the potential to solve these problems. Previous studies in small animal models have shown the use of surgical revascularization to induce neoangiogenesis and improve bone viability in the CBA. The purpose of this study is to investigate if surgical revascularization enhances bone circulation and bone remodeling in a tibial defect orthotopic reconstruction model, placing a cryopreserved allograft in a Yucatan mini pig. Methods: Cryopreserved tibial bone allografts were transplanted in swine leukocyte antigen (SLA)-type-mismatched Yucatan mini pigs after creating a 3.5-cm segmental bone defect in the tibia. The anterior tibial arteriovenous bundle (AV-bundle) was inserted into the intramedullary canal. Eight pigs received a patent AV-bundle (revascularized group), 8 pigs received a proximally ligated AV-bundle (control group), and the contralateral side was used as an untreated control. The graft was fixated with a locking compression plate to provide a weightbearing construction. After 20 weeks, the pigs were killed and the tibia was removed and analyzed. Neoangiogenesis was evaluated by quantifying vascular volumes using the microcomputed tomography. Bone remodeling was measured by quantitative histomorphometry and micro-computed tomography. Results: Seven AV-bundles in the revascularized group were patent and 1 bundle was thrombosed due to partial dislocation of the graft. Total vascular volume was higher in the revascularized allografts (127 mm 3 ) compared with both the control group (40 mm 3 , P = .003) and the contralateral side (29 mm 3 P = .000). All patent bundles showed neovascularization extending into the cortical bone. Revascularized allografts had increased bone remodeling in the inner cortical area of the graft compared with the non-revascularized grafts (bone formation rate: 381 ± 60 µm 3 /µm 3 /year vs 299 ± 143 µm 3 /µm 3 /year, P = .05). In both the revascularized group and the control group, the bone formation rate exceed the contralateral side: In the revascularized grafts, the bone formation was 305% of contralateral side, and in the non-revascularized grafts, the bone formation was 179% of the contralateral side. There was no statistical significant difference in outer cortical bone formation between the revascularized and the non-revascularized allografts. Conclusion: Surgical revascularization of porcine tibial CBAs by implantation of an AV-bundle creates an enhanced autogenous neoangiogenic circulation and accelerates active bone formation.