NLRP3 is involved in long bone edification and the maturation of osteogenic cells.

Overexpression of the nucleotide-binding leucine-rich repeat protein 3 (NLRP3) inflammasome in chronic auto-immune diseases leads to skeletal anomalies, with severe osteopenia due to the activation of osteoclasts. Reproducing this phenotype in Nlrp3 knock-in mice has provided insights into the role of NLRP3 in bone metabolism. We studied the role of NLRP3 in physiological bone development using a complete Nlrp3 knock-out mouse model. We found impaired skeletal development in Nlrp3-/- mice, resulting in a shorter stature than that of Nlrp3+/+  mice. These growth defects were associated with altered femur bone growth, characterized by a deficient growth plate and an osteopenic profile of the trabeculae. No differences in osteoclast recruitment or activity were observed. Instead, Nlrp3-/- femurs showed a less mineralized matrix in the trabeculae than those of Nlrp3+/+  mice, as well as less bone sialoprotein (BSP) expressing hypertrophic chondrocytes. In vitro, primary osteoblasts lacking NLRP3 expression showed defective mineralization, together with the downregulation of BSP expression. Finally, follow-up by micro-CT highlighted the role of NLPR3 in bone growth, occurring early in living mice, as the osteopenic phenotype diminishes over time. Overall, our data suggest that NLRP3 is involved in bone edification via the regulation of hypertrophic chondrocyte maturation and osteoblast activity. Furthermore, the defect appeared to be transitory, as the skeleton recovered with aging.

Sclerostin Deficiency Promotes Reparative Dentinogenesis.

In humans, the SOST gene encodes sclerostin, an inhibitor of bone growth and remodeling, which also negatively regulates the bone repair process. Sclerostin has also been implicated in tooth formation, but its potential role in pulp healing remains unknown. The aim of this study was to explore the role of sclerostin in reparative dentinogenesis using Sost knockout mice ( Sost-/-). The pulps of the first maxillary molars were mechanically exposed in 3-mo-old Sost-/- and wild-type (WT) mice ( n = 14 mice per group), capped with mineral trioxide aggregate cement, and the cavities were filled with a bonded composite resin. Reparative dentinogenesis was dynamically followed up by micro-computed tomography and characterized by histological analyses. Presurgical analysis revealed a significantly lower pulp volume in Sost-/- mice compared with WT. At 30 and 49 d postsurgery, a large-forming reparative mineralized bridge, associated with osteopontin-positive mineralization foci, was observed in the Sost-/- pulps, whereas a much smaller bridge was detected in WT. At the longer time points, the bridge, which was associated with dentin sialoprotein-positive cells, had expanded in both groups but remained significantly larger in Sost-/- pulps. Sclerostin expression in the healing WT pulps was detected in the cells neighboring the forming dentin bridge. In vitro, mineralization induced by Sost-/- dental pulp cells (DPCs) was also dramatically enhanced when compared with WT DPCs. These observations were associated with an increased Sost expression in WT cells. Taken together, our data show that sclerostin deficiency hastened reparative dentinogenesis after pulp injury, suggesting that the inhibition of sclerostin may constitute a promising therapeutic strategy for improving the healing of damaged pulps.

Centrosome fine ultrastructure of the osteocyte mechanosensitive primary cilium.

The centrosome is the principal microtubule organization center in cells, giving rise to microtubule-based organelles (e.g., cilia, flagella). The aim was to study the osteocyte centrosome morphology at an ultrastructural level in relation to its mechanosensitive function. Osteocyte centrosomes and cilia in tibial cortical bone were explored by acetylated alpha-tubulin (AαTub) immunostaining under confocal microscopy. For the first time, fine ultrastructure and spatial orientation of the osteocyte centrosome were explored by transmission electron microscopy on serial ultrathin sections. AαTub-positive staining was observed in 94% of the osteocytes examined (222/236). The mother centriole formed a short primary cilium and was longer than the daughter centriole due to an intermediate zone between centriole and cilium. The proximal end of the mother centriole was connected with the surface of daughter centriole by striated rootlets. The mother centriole exhibited distal appendages that interacted with the cell membrane and formed a particular structure called "cilium membrane prolongation." The primary cilium was mainly oriented perpendicular to the long axis of bone. Mother and daughter centrioles change their original mutual orientation during the osteocyte differentiation process. The short primary cilium is hypothesized as a novel type of fluid-sensing organelle in osteocytes.

Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia.

INTRODUCTION: Alcohol is known to decrease bone mineral density (BMD) and to induce trabecular microarchitecture deterioration. However, little is known about the effects of chronic alcohol consumption on osteocytes in situ. The aim of this study was to assess the effects of a high alcohol dose on osteocytes in an alcohol-induced osteopenia model. MATERIALS AND METHODS: 24 male Wistar rats, 2-months old were separated in 2 groups: Control (C) or Alcohol (A35). The rats in the A35 group drank a beverage composed of 35% ethanol v/v mixed to water for 17 weeks. BMD was assessed by DXA, while the microarchitecture was analyzed using µCT. Bone remodeling was studied measuring serum concentration of osteocalcin, NTx and TRAP. Bone marrow adiposity, osteoblastic lineage differentiation, osteocyte morphology and apoptosis were assessed using bright field, epifluorescence, transmission electron and confocal microscopy. RESULTS: BMD, trabecular thickness, TRAP and NTx concentration were significantly decreased in A35, while cortical thickness was thinner. There were 10 fold more cells stained with cleaved caspase-3, and 35% more empty lacunae in A35, these data indicating a large increase in osteocyte apoptosis in the A35 group. The number of lipid droplets in the marrow was increased in A35 (7 fold). Both the osteocyte apoptosis and the fat bone marrow content strongly correlated with femur BMD (p=0.0017, r = -0.72 and p=0.002, r = -0.70) and whole body BMD. CONCLUSION: These data suggest that low BMD is associated with osteocyte apoptosis and bone marrow fat content in alcohol-induced osteopenia.

Exercise and zoledronic acid on lipid profile and bone remodeling in ovariectomized rats: a paradoxical negative association?

Exercise (EXE) and amino-bisphosphonates (BP) are both considered as useful strategies in the prevention of post-menopausal bone loss. Exercise reduces lipid levels, and BP may induce increase in high-density lipoprotein cholesterol (HDL-C). We hypothesized that combined effects of BP and exercise would produce a better improvement of lipid profile. We studied the specific and combined effects of zoledronic acid (Z) and EXE on lipid profile and bone remodeling in mature ovariectomized (OVX) rats. Six-month old female rats were randomly assigned to either a sham-ovx group (n = 12) or one of four OVX groups (n = 12): vehicle-treated sedentary (OVX); OVX + EXE (OVX-E, running on a treadmill for 12 weeks); OVX + Z (20 microg/kg, i.v.), (OVX-Z); OVX + Z+EXE (OVX-ZE). Total cholesterol (TC), HDL-C and bone remodeling markers were measured at baseline and at the end of the study. We demonstrated that both Z and EXE prevented the increase in bone resorption resulting from OVX, and individually improved the atherosclerotic risk index. Therapy with Z resulted in significant increase (39.00 +/- 0.03 vs. 53.6 +/- 0.01 mg/dl; +37.4%, P < 0.05) in serum concentration of HDL-C and a non significant decrease in TC (135.30 +/- 0.03 vs. 144.80 +/- 0.05 mg/dl; -5.8%) in the OVX-Z group compared to the OVX group. Post-menopausal women have elevated risk of CVD and bone resorption, hence, these data ultimately demonstrate (except for the elevated ratio in the combined group) that exercise and zoledronic acid are useful in minimizing the impact of these two processes in women and the combination of the two may be clinically relevant.

Osteocyte: the unrecognized side of bone tissue.

INTRODUCTION: Osteocytes represent 95% of all bone cells. These cells are old osteoblasts that occupy the lacunar space and are surrounded by the bone matrix. They possess cytoplasmic dendrites that form a canalicular network for communication between osteocytes and the bone surface. They express some biomarkers (osteopontin, beta3 integrin, CD44, dentin matrix protein 1, sclerostin, phosphate-regulating gene with homologies to endopeptidases on the X chromosome, matrix extracellular phosphoglycoprotein, or E11/gp38) and have a mechano-sensing role that is dependent upon the frequency, intensity, and duration of strain. DISCUSSION: The mechanical information transmitted into the cytoplasm also triggers a biological cascade, starting with NO and PGE(2) and followed by Wnt/beta catenin signaling. This information is transmitted to the bone surface through the canalicular network, particularly to the lining cells, and is able to trigger bone remodeling by directing the osteoblast activity and the osteoclastic resorption. Furthermore, the osteocyte death seems to play also an important role. The outcome of micro-cracks in the vicinity of osteocytes may interrupt the canalicular network and trigger cell apoptosis in the immediate surrounding environment. This apoptosis appears to transmit a message to the bone surface and activate remodeling. The osteocyte network also plays a recognized endocrine role, particularly concerning phosphate regulation and vitamin D metabolism. Both the suppression of estrogen following menopause and chronic use of systemic glucocorticoids induce osteocyte apoptosis. On the other hand, physical activity has a positive impact in the reduction of apoptosis. In addition, some osteocyte molecular elements like sclerostin, connexin 43, E11/gp38, and DKK1 are emerging as promising targets for the treatment of various osteo-articular pathologies.