A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP-2-producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene-enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.Introduction: The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization. Materials and Methods: Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express ␤-galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell-based, adenovirus-mediated gene therapy approach to engraft BMP-2-producing bone marrow stromal cells onto devitalized allografts. Results: X-gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative CT analyses showed a 3-fold increase in new bone callus formation and a 6.8-fold increase in neovascularization for BMP-2/stromal cell-treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP-2/stromal cell-treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft. Conclusions:The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.

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...

Objective The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. In this study, we have investigated the role of β-catenin signaling in IVD tissue function. Methods β-catenin protein levels were measured in disc samples derived from patients with disc degeneration and normal subjects by immunohistochemistry (IHC). To generate β-catenin conditional activation (cAct) mice, Col2a1-CreERT2 transgenic mice were bred with β-cateninfx(Ex3)/fx(Ex3) mice. Changes in disc tissue morphology and function were analyzed by micro-CT, histology and real-time PCR assays. Results We found that β-catenin protein was up-regulated in disc tissues from patients with disc degeneration. To assess the effects of increased β-catenin on disc tissue we generated β-catenin cAct mice. Overexpression of β-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old β-catenin cAct mice which were associated with significant changes in the cells and extracellular matrix of disc tissues and growth plate. Gene expression analysis demonstrated that activation of β-catenin could enhance Runx2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of the Mmp13 or Adamts5 under β-catenin cAct background, or treatment of β-catenin cAct mice with a specific MMP13 inhibitor, ameliorated the mutant phenotype. Conclusions β-catenin signaling pathway plays a critical role in disc tissue function.

The presence of live periosteal progenitor cells on the surface of bone autografts confers better healing than devitalized allograft. We have previously demonstrated in a murine 4 mm segmental femoral bone-grafting model that live periosteum produces robust endochondral and intramembraneous bone formation that is essential for effective healing and neovascularization of structural bone grafts. To the end of engineering a live pseudo-periosteum that could induce a similar response onto devitalized bone allograft, we seeded a mesenchymal stem cell line stably transfected with human bone morphogenic protein-2/beta-galactosidase (C9) onto devitalized bone allografts or onto a membranous small intestinal submucosa scaffold that was wrapped around the allograft. Histology showed that C9-coated allografts displayed early cartilaginous tissue formation at day 7. By 6 and 9 weeks, a new cortical shell was found bridging the segmental defect that united the host bones. Biomechanical testing showed that C9-coated allografts displayed torsional strength and stiffness equivalent to intact femurs at 6 weeks and superior to live isografts at 9 weeks. Volumetric and histomorphometric micro-computed tomography analyses demonstrated a 2-fold increase in new bone formation around C9-coated allografts, which resulted in a substantial increase in polar moment of inertia (pMOI) due to the formation of new cortical shell around the allografts. Positive correlations between biomechanics and new bone volume and pMOI were found, suggesting that the biomechanical function of the grafted femur relates to both morphological parameters. C9-coated allograft also exhibited slower resorption of the graft cortex at 9 weeks than live isograft. Both new bone formation and the persistent allograft likely contributed to the improved biomechanics of C9-coated allograft. Taken together, we propose a novel strategy to combine structural bone allograft with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering.

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.