Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1 ؊/؊ mice. Vertebral bone volume and strength of TNF-Tg/Smurf1 ؊/؊ mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1 ؊/؊ mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.Osteoporosis and fragility fractures are common and preventable complications of rheumatoid arthritis (RA).2 For example, one study has reported that 53.3% of RA patients had osteoporosis and 19.3% had vertebral fractures, rates that are much higher than those of the general population (1). These complications are thought to occur through inflammatory stimulation of osteoclast bone resorption and inhibition of osteoblast function (2) mediated by cytokines, such as TNF (3-5). TNF and other cytokines are overproduced by various cells in the inflamed joints of RA patients, which lead to severe local erosion of cartilage and bone, as well as periarticular osteopenia and systemic osteoporosis.TNF is a strong inhibitor of osteoblast functions in vitro, but the molecular mechanisms that mediate the inhibitory effects of TNF on osteoblasts have not been fully investigated. TNF inhibits the recruitment of osteoblast progenitors, reduces expression of genes produced by mature osteoblasts, and promotes osteoblast apoptosis through the NF-B pathway (6 -10). It decreases the expression and DNA binding activity of runt-related transcription factor 2 (Runx2), which is partially through suppression of Runx2 gene transcription and destabilization o...

Freeze-dried recombinant adeno-associated virus (rAAV) coated structural allografts have emerged as an approach to engender necrotic cortical bone with host factors that will persist for weeks following surgery to facilitate revascularization, osteointegration, and remodeling. However, one major limitation is the nonporous cortical surface that prohibits uniform distribution of the rAAV coating prior to freeze-drying. To overcome this we have developed a demineralization method to increase surface absorbance while retaining the structural integrity of the allograft. Demineralized bone wafers (DBW) made from human femoral allograft rings demonstrated a significant 21.1% (73.6+/-3.9% versus 52.5+/-2.6%; p<0.001) increase in percent surface area coating versus mineralized controls. Co-incubation of rAAV-luciferase (rAAV-Luc) coated DBW with a monolayer of C3H10T1/2 cells in culture led to peak luciferase levels that were not significantly different from soluble rAAV-Luc controls (p>0.05), although the peaks occurred at 60h and 12h, respectively. To assess the transduction efficiency of rAAV-Luc coated DBW in vivo, we first performed a dose response with allografts containing 10(7), 10(9) or 10(10) particles that were surgically implanted into the quadriceps of mice, and assayed by in vivo bioluminescence imaging (BLI) on days 1, 3, 5, 7, 10, 14, and 21. The results demonstrated a dose response in which the DBW coated with 10(10) rAAV-Luc particles achieved peak gene expression levels on day 3, which persisted until day 21, and was significantly greater than the 10(7) dose throughout this time period (p<0.01). A direct comparison of mineralized versus DBW coated with 10(10) rAAV-Luc particles failed to demonstrate any significant differences in transduction kinetics or efficiency in vivo. Thus, surface demineralization of human cortical bone allograft increases its absorbance for uniform rAAV coating, without affecting vector transduction efficiency.

The use of processed structural allografts for treatment of massive segmental defects in long bones can be complicated by poor incorporation and remodeling of the devitalized graft, foreign-body reaction and micro-damage accumulation which often leads to catastrophic graft failure [1]. It is therefore useful to develop a bioengineered, biodegradable scaffold that is able to stimulate healing of the defect region. The use of bioengineered scaffolds has been limited due to their poor mechanical strength that does not permit withstanding large in vivo loads and due to their poor osteoinductive properties. We therefore investigated the use of rigid polylactic acid/beta-tricalcium phosphate (PLA/βTCP) composites used in conjunction with osteoinductive factors such as growth hormones (parathyroid hormone (PTH)) and growth factors (bone morphogenic protein-2 (BMP-2) & vascular endothelial growth factor (VEGF)) to stimulate bone formation and vessel ingrowth in the segmental defect region. We examined the physical characteristics of the scaffolds, and evaluated their osteoinductive potential in a clinically-relevant mouse model of a femoral segmental defect with or without PTH treatment. Finally, we used an ectopic bone formation model to assess the efficacy of the scaffold in site-specific delivery of bone anabolic factors.

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