Mechanical stretch is known to promote osteoblast differentiation in vitro and accelerate bone regeneration in vivo, whereas the relevant mechanism remains unclear. Recent studies have shown the importance of reciprocal interactions between mammalian target of rapamycin (mTOR) and nuclear factor kappa B (NF-κB; two downstream molecules of Akt) in the regulation of tumor cells. Thus, we hypothesize that mTOR and NF-κB as well as their interconnection play a critical role in mediating stretch-induced osteogenic differentiation in osteoblasts. We herein found that mechanical stretch (10% elongation at six cycles/min) significantly promoted the expression of osteoblast differentiation-related markers (including ALP, BMP2, Col1α, OCN, and Runx2) in osteoblast-like MG-63 cells, accompanied by increased mTOR phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. Blockade of mTOR by antagonist or small interfering RNA suppressed osteogenesis-related gene expression in response to mechanical stretch, whereas inhibition of NF-κB further increased stretch-induced osteoblast differentiation. Moreover, inhibition of mTOR decreased the phosphorylation of NF-κB, and blockade of NF-κB reduced the mTOR activation in MG63 cells under mechanical stretch. Coinhibition of mTOR and NF-κB abolishes the alteration of osteogenic differentiation induced by single mTOR or NF-κB inhibition under mechanical stretch, which is equivalent to the noninhibition level for osteoblasts under mechanical stretch. The expression levels of osteogenic differentiation in osteoblasts after inhibition of Akt were similar to those after co-inhibition of mTOR and NF-κB under mechanical stretch. This study for the first time reveals the reciprocal interconnection between mTOR and NF-κB in osteoblasts under mechanical stretch and indicates that mTOR and NF-κB as well as their interactions play a key role in the regulation of cellular homeostasis of osteoblasts in response to mechanical stretch. These findings are helpful for enriching our basic knowledge of the molecular mechanisms of osteoblast mechanotransduction, and also providing insight

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMF) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMF resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 weeks remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number or osteoclast-specific gene expression (TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a and Wnt10b), but not non-canonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3β and β-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/β-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration.

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Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well‐recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO‐Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO‐Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG‐induced apoptosis in MLO‐Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG‐incubated MLO‐Y4 cells were observed after mechanical vibration via the F‐actin fluorescence staining. The real‐time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β‐catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor‐κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme‐linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor‐α and RANKL in the supernatant of HG‐treated MLO‐Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

Chronic high‐altitude hypoxia induces irreversible abnormalities in various organisms. Emerging evidence indicates that hypobaric hypoxia markedly suppresses bone mass and bone strength. However, few effective means have been identified to prevent such bone deficits. Here, we assessed the potential of pulsed electromagnetic fields (PEMFs) to noninvasively resist bone deterioration induced by hypobaric hypoxia. We observed that exogenous PEMF treatment at 15 Hz and 20 Gauss (Gs) improved the cancellous and cortical bone mass, bone microstructure, and skeletal mechano‐properties in rats subjected to chronic exposure of hypobaric hypoxia simulating an altitude of 4500 m for 6 weeks by primarily modulating osteoblasts and osteoblast‐mediated bone‐forming activity. Moreover, our results showed that whereas PEMF stimulated the functional activity of primary osteoblasts in hypoxic culture in vitro, it had negligible effects on osteoclasts and osteocytes exposed to hypoxia. Mechanistically, the primary cilium was found to function as the major electromagnetic sensor in osteoblasts exposed to hypoxia. The polycystins PC‐1/PC‐2 complex was identified as the primary calcium channel in the primary cilium of hypoxia‐exposed osteoblastic cells responsible for the detection of external PEMF signals, and thereby translated these biophysical signals into intracellular biochemical events involving significant increase in the intracellular soluble adenylyl cyclase (sAC) expression and subsequent elevation of cyclic adenosine monophosphate (cAMP) concentration. The second messenger cAMP inhibited the transcription of oxygen homeostasis‐related hypoxia‐inducible factor 1‐alpha (HIF‐1α), and thus enhanced osteoblast differentiation and improved bone phenotype. Overall, the present study not only advances our understanding of bone physiology at high altitudes, but more importantly, proposes effective means to ameliorate high altitude‐induced bone loss in a noninvasive and cost‐effective manner. © 2023 American Society for Bone and Mineral Research (ASBMR).