Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL-and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.In contrast to soft tissue organ transplantation (i.e., heart, liver, kidney), which must be live from a histocompatible donor, structural musculoskeletal grafts (i.e., bone, ligament) are often derived from allogenic cadavers. Although this convenience makes structural allografts readily available, the utility of these transplants is limited by their lack of viability. This is most evident from experimental and clinical studies showing that fresh vascularized autogenous grafts are vastly superior to allograft in terms of healing and remodeling 1,2 . Structural bone grafts used to heal critical defects and bone fusions undergo a repair and remodeling process that closely resembles fracture healing 3 . In live autograft healing, cells from both the graft and the host contribute to mediate bony union 4,5 . In contrast, healing of a diaphyseal defect that has been allografted can only be accomplished by invasion of the graft by host tissue to obtain a cortexCorrespondence should be addressed to E.M.S. (edward_schwarz@urmc.rochester.edu).. COMPETING INTERESTS STATEMENT The authors declare competing financial interests (see the Nature Medicine website for details). to-cortex union 6 . Following union, autografts continue to remodel as a result of osteoclastic resorption of necrotic or disused cortical bone that is followed by osteoblastic formation of new woven bone, which is later remodeled into stronger lamellar bone. In this way, autografts are sustained through normal bone homeostasis. In contrast, once creeping callus from the host calcifies on the cortex of an allograft, healing ceases, leaving a large segment of necrotic bone that is unable to respond to normal mechanical loading. Thus, structural allografts have a limited life span because microfractures that occur in them over time cannot be remodeled and repaired, and negative outcomes include a 25-35% failure rate from infection, nonunion and fracture 7,8 . NIH Public AccessTwo central issues that must be addressed to improve structural allografting are elucidatio...

Structural bone allografts often fracture due to their lack of osteogenic and remodeling potential. To overcome these limitations, we utilized allografts coated with recombinant adeno-associated virus (rAAV) that mediate in vivo gene transfer. Using beta-galactosidase as a reporter gene, we show that 4-mm murine femoral allografts coated with rAAV-LacZ are capable of transducing adjacent inflammatory cells and osteoblasts in the fracture callus following transplantation. While this LacZ vector had no effect on allograft healing, bone morphogenetic protein signals delivered via rAAV-caAlk2 coating induced endochondral bone formation directly on the cortical surface of the allograft by day 14. By day 28 there was evidence of remodeling of the new woven bone and massive osteoclastic resorption of the cortical surface of the rAAV-caAlk2-coated allografts only. Micro-CT analysis of rAAV-LacZ- vs rAAV-caAlk2-coated allografts after 42 days of healing demonstrated a significant increase in new bone formation (0.67 +/- 0.21 vs 2.49 +/- 0.40 mm(3); P < 0.005). Furthermore, the 3D micro-CT images of femurs grafted with rAAV-Alk2-coated allografts provided the first evidence that complete bridging of bone around a cortical allograft is possible. These results indicate that cell-free, rAAV-coated allografts have the potential to revitalize in vivo following transplantation.