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.

Human periodontal ligament (hPDL) fibroblasts are thought to receive mechanical stress (MS) produced by orthodontic tooth movement, thereby regulating alveolar bone remodeling. However, the role of intracellular calcium ([Ca ²⁺ ] _i )‐based mechanotransduction is not fully understood. We explored the MS‐induced [Ca ²⁺ ] _i responses both in isolated hPDL fibroblasts and in intact hPDL tissue and investigated its possible role in alveolar bone remodeling. hPDL fibroblasts were obtained from healthy donors' premolars that had been extracted for orthodontic reasons. The oscillatory [Ca ²⁺ ] _i activity induced by static compressive force was measured by a live‐cell Ca ²⁺ imaging system and evaluated by several feature extraction method. The spatial pattern of cell‐cell communication was investigated by Moran's I , an index of spatial autocorrelation and the gap junction (GJ) inhibitor. The Ca ²⁺ ‐transporting ionophore A23187 was used to further investigate the role of [Ca ²⁺ ] _i up‐regulation in hPDL cell behavior. hPDL fibroblasts displayed autonomous [Ca ²⁺ ] _i responses. Compressive MS activated this autonomous responsive behavior with an increased percentage of responsive cells both in vitro and ex vivo . The integration, variance, maximum amplitude, waveform length, and index J in the [Ca ²⁺ ] _i responses were also significantly increased, whereas the mean power frequency was attenuated in response to MS. The increased Moran's I after MS indicated that MS might affect the pattern of cell‐cell communication via GJs. Similar to the findings of MS‐mediated regulation, the A23187‐mediated [Ca ²⁺ ] _i uptake resulted in the up‐regulation of receptor activator of NF‐κB ligand (Rankl) and Sost along with increased sclerostin immunoreactivity, suggesting that [Ca ²⁺ ] _i signaling networks may be involved in bone remodeling. In addition, A23187‐treated hPDL fibroblasts also showed the suppression of osteogenic differentiation and mineralization. Our findings suggest that augmented MS‐mediated [Ca ²⁺ ] _i oscillations in hPDL fibroblasts enhance the production and release of bone regulatory signals via Rankl/Osteoprotegerin and the canonical Wnt/β‐catenin pathway as an early process in tooth movement–initiated alveolar bone remodeling.—Ei Hsu Hlaing, E., Ishihara, Y., Wang, Z., Odagaki, N., Kamioka, H. Role of intracellular Ca ²⁺ ‐based mechanotransduction of human periodontal ligament fibroblasts. FASEB J. 33, 10409–10424 (2019). www.fasebj.org

Background: In this study, we investigated the effect of the mechanical loading history on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in MLO-Y4 osteocyte-like cells. Methods: Three hours after MLO-Y4 osteocytes were seeded, a continuous compressive force (CCF) of 31 dynes/cm² with or without additional CCF (32 dynes/cm²) was loaded onto the osteocytes. After 36 h, the additional CCF (loading history) was removed for a recovery period of 10 h. The expression of RANKL, OPG, RANKL/OPG ratio, cell numbers, viability and morphology were time-dependently examined at 0, 3, 6 and 10 h. Then, the same additional CCF was applied again for 1 h to all osteocytes with or without the gap junction inhibitor to examine the expression of RANKL, OPG, the RANKL/OPG ratio and other genes that essential to characterize the phenotype of MLO-Y4 cells. Fluorescence recovery after photobleaching technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells. Results: The expression of RANKL and OPG by MLO-Y4 osteocytes without a loading history was dramatically decreased and increased, respectively, in response to the 1-h loading of additional weight. However, the expression of RANKL, OPG and the RANKL/OPG ratio were maintained at the same level as in the control group in the MLO-Y4 osteocytes with a loading history but without gap junction inhibitor treatment. Treatment of loading history significantly changed the capacity of GJIC and protein expression of connexin 43 (Cx43) but not the mRNA expression of Cx43. No significant difference was observed in the cell number or viability between the MLO-Y4 osteocyte-like cells with and without a loading history or among different time checkpoints during the recovery period. The cell morphology showed significant changes and was correlated with the expression of OPG, Gja1 and Dmp1 during the recovery period. Conclusion: Our findings indicated that the compressive force-induced changes in the RANKL/OPG expression could be habituated within at least 11 h by 36-h CCF exposure. GJIC and cell morphology may play roles in response to loading history in MLO-Y4 osteocyte-like cells.