Sclerostin (Scl) negatively regulates bone formation and favors bone resorption. Osteocytes, the cells responsible for mechanosensing, are known as the primary source of Scl and are a key regulator of bone remodeling through the induction of receptor activator of NF-κB ligand (RANKL). However, the spatiotemporal patterns of Scl in response to mechanical stimuli and their regulatory mechanisms remain unknown. We investigated the regulatory dynamics of the SOST/Scl expression generated by orthodontic tooth movement (OTM) in vivo and in vitro. In 8-wk-old male mice, coil springs were used to move the first molar mesially for 0, 1, 5, or 10 d. A regional histogram and the distribution patterns of the Scl expression showed that the Scl expression in the alveolar bone was increased on the compression side and peaked on day 5, with a gradual increase in the degree of significance. On day 10, the expression around the periodontal ligament (PDL)-alveolar bone boundary returned to the control level. Conversely, the expression of Scl on the tension side was only significantly decreased on day 1. Compressive force biphasically modulated the SOST/Scl expression in the isolated human PDL and thereby upregulated osteocytic SOST via paracrine activation in an osteocyte-PDL co-culture system designed to mimic OTM. This system did not affect the RANKL or OPG expression in osteocytes, suggesting that the bone resorption pathways are acted upon in a PDL-dependent and osteocyte-independent manner through RANKL/OPG signaling. Moreover, sclerostin neutralizing antibody significantly attenuated the upregulation of SOST that was induced by compressive force. In conclusion, our results provide evidence to support that factors secreted by the PDL, including SOST/Scl, control alveolar bone remodeling through osteocytic SOST/Scl in OTM.
The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation.Introduction: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied. Materials and Methods: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions. Results: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes. Conclusions: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.
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