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The underlying mechanisms of action of osteogenic differentiation of rat osteoblasts induced by exposure to a 50 Hz pulsed magnetic field should be investigated.
In a previous study, the authors showed that 50 Hz pulsed magnetic field exposure could stimulate proliferation and osteogenic differentiation of rat osteoblasts and that the existence of a primary cilium is crucial for this effect (Yan et al. 2015). However, the mechanisms of action of this effect are not clear. Rat calvarial osteoblasts were isolated from 10 neonatal rats and divided into different groups. For short term effects: exposure to the pulsed magnetic field for 1) 5 min, 2) 15 min, 3) 30 min, 4) 60 min and 5) 120 minutes. Cells without exposure (0 min) were used as the control group. For long-term effects, cells were cultured in osteogenic culture medium and divided into the following groups: exposure to the pulsed magnetic field for 6) 3 days, 7) 6 days and 8) 12 days. For each of groups 6-8, a separate control group was used. Bone morphogenetic proteins (BMPs) have diverse functions in embryonic development and play crucial roles in osteogenesis. The BMP-Smad1/5/8 signaling pathway is important for osteogenic cell differentiation. To study the mechanisms of action, cilia formation was prevented in cells via treatment with siRNA (small interfering RNA) to induce a gene knockdown (effect similar to knockout). Knockdown of bone morphogenetic proteins receptor II (BMPRII) was also conducted with the help of siRNA. Moreover, noggin, an antagonist of BMPs, and LDN-193189, an inhibitor of BMPRI kinases, were used.
細胞機能: mechanisms of action of magnetic field-induced osteogenic differentiation
分子生合成: protein expression of phosphorylated Smad1/5/8 (proteins of the BMP-Smad1/5/8 signal pathway), BMPRII, BMPRIA and BMPRIB (BMP receptors) (immunohistochemical FITC stain, confocal microscopy), protein expression of BMP2, phosphorylated Smad1/5/8, Smad1/5/8, BMPRIA, BMPRIB, BMPRII and other osteogenesis markers COL-1, Runx-2 and Osx (Western blot), gene expression of IFT88 and BMPRII (validation of siRNA gene knockdown, real-time RT-PCR)
細胞機能: enzyme activity of alkaline phosphatase (marker for early osteogenic differentiation; only in groups 6 and 7, commercial kit), osteoblast maturation (formation of mineralized nodules; only in group 8, Alizarin Red S stain, microscopy)
In cells with normal primary cilia formation, the protein expression of BMP2 and phosphorylated Smad1/5/8 was significantly increased in cells after 5 minutes of exposure (group 1) and peaked after 30 minutes of exposure (group 3) compared to the control group, indicating an activation of the BMP-Smad1/5/8 pathway. Furthermore, protein expression of BMP receptors BMPRII and BMPRIB was significantly increased after 15 minutes (group 2) and 120 minutes (group 5) compared to the control group, and receptors were localized at the bases of primary cilia. Osteogenic differentiation and maturation of cells were significantly increased after 3, 6 and 12 days (groups 6-8) compared to the control groups. These promoting effects were negated by co-exposure with inhibitors LDN-193189 and noggin, confirming the BMP-Smad1/5/8 signal pathway in osteoblast differentiation and maturation. Elimination of primary cilia via gene knockdown abolished the magnetic field-induced upregulation of BMPRII and its ciliary localization. Knockdown of BMPRII expression had no effects on primary cilia but significantly decreased the promoting effect of the magnetic field on osteoblastic differentiation and maturation. The authors conclude that exposure to a 50 Hz pulsed magnetic field might stimulate osteogenic differentiation of rat osteoblasts by primary cilium-mediated upregulation of BMPRII expression and subsequently activation of the BMP-Smad1/5/8 signaling pathway.