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...

To further understand the cellular and molecular mechanisms underlying cortical bone graft healing, we have developed a novel mouse femur model that permits quantitative and molecular analysis of structural bone graft healing. A 4 mm mid-diaphyseal femoral segment was removed and replaced by either immediate implantation of a fresh autograft, a frozen, genetically identical isograft or a frozen allograft from a different strain of mouse, which was secured with a 22-gauge metal intramedullary pin. Healing was evaluated by radiology, histomorphometry, and in situ hybridization. Autograft repair occurred by endochondral bone formation at the host-graft junction and by intramembranous bone formation along the length of the graft bed at 2 weeks, with maturation and remodeling apparent by 4 weeks. Bone repair in allografts and isografts completely relied on endochondral bone formation at the host-graft cortical junction, with absence of periosteal bone formation along the length of the graft, suggesting that live periosteal cells from the donor tissue are necessary for this response. This small animal model of structural bone grafting can be used to evaluate tissue-engineered allografts and novel bone graft substitutes using quantitative and molecularly defined outcome measures.

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification.

Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cellbased therapy. Furthermore, recombinant adeno-associated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light-activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200 J/m 2 . Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 h after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 h after irradiation and required only a 10-min exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFb1 protein from rAAV-TGFb1-IRES-eGFP infected to HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.

Nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX) activity, reduced pain and are commonly used in patients with skeletal injury. In this article we will also present data to show that selective COX-2 inhibitor delays allograft healing and incorporation. In contrast, local delivery of prostaglandin E2 (PGE2) enhanced bone formation at cortical bone graft junction. A 4-mm mid-diaphyseal segmental femoral defect was created and then repaired by frozen bone allograft of the same size. A 22-gauge metal pin was placed in the intramedullary cavity to stabilize the bone graft. Healing was evaluated weekly by X ray and by a semiquantitative histomorphometric analysis at 5 weeks postsurgery. Celecoxib (25 mg/kg/day) and Ketorolac (4 mg/kg/day) were administered daily for 2 weeks or 5 weeks. PGE2 was infused locally at a dose of 800 nmol/kg per day via osmotic minipump for 4 weeks. Inhibition of cyclooxygenase by daily administration of the Celecoxib or Ketorolac for 5 weeks reduced new bone ingrowth by about 60% (P < 0.05). The percentage of bony bridging in both drug-treated groups was significantly decreased at 5 weeks. Temporal administration of Celecoxib for 2 weeks also significantly reduced bone formation by 45% and withdrawal of the Celecoxib only led to slight recovery of bone formation at the graft side. In contrast to the inhibitory effects of NSAIDS, PGE2 infusion at the cortical bone junction increased bone formation by about twofold. These results demonstrated that COX-2 is essential for bone allograft incorporation. Furthermore, our data support the notion that COX-2-dependent PGE2 produced at the early stage of bone healing is prerequisite for efficient skeletal repair.