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Abstract 3249 Variations in the amino acid sequence of the beta globin gene (HBB) chain can result in the production of variant hemoglobins. Sickle cell anemia is caused by homozygosity for a single nucleotide mutation in codon 6 of HBB, GAG>GTG, resulting in the substitution of valine for glutamic acid (glu6val). Bone pathologies are among the myriad complications of sickle cell disease and are a common cause of pain and morbidity. These pathologies include osteonecrosis, osteoarthritis and vaso-occlusive infarct. Chronic vaso-occlusion in sickle cell disease patients leads to ischemia and necrotic lesions while bone marrow hyperplasia contributes to the softening of trabecular bone, and overall bone loss. The mechanism underlying these structural changes in sickle bone are not fully understood. Bone constantly undergoes a self-renewal process and adapts to damage through remodeling. The remodeling process is highly regulated and carried out by bone forming cells (osteoblast) and bone resorption cells (osteoclast). Disease states can alter the remodeling capacity of bone and compromise its structure and quality. To determine the effect of sickle cell disease on bone, we used microcomputed tomographic (microCT) analysis, real time PCR and immunohistochemistry to characterize the bone phenotype in a transgenic mouse model (Townes) of sickle cell disease. Osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), while proliferating osteoblasts show alkaline phosphatase (AP) activity. Relative levels of TRAP and AP mRNA were measured in RNA derived from the bone of 10 week and 21 week old mice. TRAP and AP were elevated (P MicroCT analysis of the femoral trabecular morphology of the epiphysis and metaphysis also showed an age-dependent effect on bone. In addition, there were significant differences observed on the basis of sex. At 10 weeks, there were no significant differences observed between trait and sickle mice, however, there was a trend towards lower values in sickle mice. Although these differences were not statistically significant, an increase in the sample size might lead to more significant findings. Comparison of the 10 week old mice to the 21 week old mice showed a general decrease in trabecular number, and an increase in trabecular spacing, which may be indicative of bone loss. This difference was significant in the female trait mice. Although the trend in the female sickle mice was noted, a full statistical analysis was not possible due to the small sample size of the 21week old female sickle mice. Sickle cell disease and its impact on bone are highly variable. Our preliminary result suggest that age and sex may play a role and form the basis for further study. Figure 1, A and B. Expression levels of AP and TRAP mRNA in RNA derived from bone in trait and sickle mice. Figure 1, A and B. Expression levels of AP and TRAP mRNA in RNA derived from bone in trait and sickle mice. (A) AP and TRAP show relative and elevated expression in 10 week and 21 weeks old respectively trait mice compared to sickle mice. Results are shown as mean +/− SD (n=3 for each). (B). Alkaline phosphatase significantly reduces in sickle mice at 21weeks. (*) denotes statistical significance. Figure 2. Figure 2. Representative 3D thickness maps for femoral metaphyseal trabecular volumes of interest. Top panel shows trait mice at 10 and 21 weeks respectively. Bottom panel shows sickle mice at 10 and 21 weeks respectively. Measurements are depicted in pseudocolor scale - thicker = red, thinner = blue/green. Disclosures: No relevant conflicts of interest to declare.

This study investigated the potential use of stem cell-synthesized extracellular matrix (ECM) as a bone graft replacement therapy. ECM was synthesized by both mesenchymal stem cells (MSCs) and amniotic fluid stem cells (AFS cells) and decellularized via freeze-thaw cycling and DNase treatment. A series of in vitro studies were performed to evaluate the effect of the ECM on cell attachment, proliferation, and differentiation. Reseeded MSCs were able to attach to and proliferate on both ECM types in both 2D and 3D culture. In 2D, cells cultured on both ECM types showed increased levels of calcium deposition. In 3D, both ECM types increased the mineralized matrix production of reseeded MSCs, with the ECM produced by the AFS cells inducing a greater amount of mineralized matrix formation than the MSC synthesized ECM. Both ECM types were used to treat a rat femoral segmental defect in vivo. Each ECM type increased the rate of bridging of the defect when compared to collagen coated scaffolds. However, the presence of the ECM did not have a significant effect on the volume of mineralized matrix within the defect site in this study.

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.

SummaryIntermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

anti-catabolic in human articular chondrocytes and especially inhibits NF-kB-mediated catabolic events, whereas Wnt/b-catenin signaling in mouse articular chondrocytes stimulates catabolic events, and that (b) AnxA6 via stimulating NF-kB activity and inhibiting Wnt/b-catenin signaling plays a major role in human cartilage degradation during OA pathology. Our findings suggest that targeting AnxA6 function, especially its modulatory function of the NF-kB andWnt/b-catenin signaling pathways may be a more effective way to control these signaling pathways and their interactions during OA pathology than targeting these individual pathways directly.