Department of Ultrasound, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Department of Ultrasound, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, China.
Biotechnol J. 2024 Aug;19(8):e2400310. doi: 10.1002/biot.202400310.
The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.
低强度脉冲超声（LIPUS）可诱导骨髓间充质干细胞（BMSCs）向成骨分化。然而，LIPUS 刺激的分子机制尚不清楚。本研究旨在探讨 LIPUS 促进 BMSCs 成骨分化的可能分子机制。通过碱性磷酸酶（ALP）活性定量、茜素红 S 染色、ALP 染色和建立颅骨缺损模型来评估成骨效果。通过免疫荧光观察微丝和瞬时受体电位 melastatin 7（TRPM7）的表达。通过 Western blot 检测 LIPUS 单独或 LIPUS 联合细胞骨架干扰药物（细胞松弛素 D [CytoD]或 Jasplakinolide [JA]）作用下 F-肌动蛋白/G-肌动蛋白和成骨相关蛋白的水平。采用定量实时逆转录聚合酶链反应（qRT-PCR）检测 Trpm7mRNA 的表达。此外，还通过 Western blot 验证了腺病毒 Trpm7 敲低。结果表明，LIPUS 促进了体内骨形成。在体外成骨诱导下，LIPUS 诱导的 BMSCs 成骨伴随着微丝的解聚和重排以及 TRPM7 水平的升高。通过干扰细胞内肌动蛋白动力学，CytoD 增强了 LIPUS 的促成骨作用并增加了 TRPM7 水平，而 JA 抑制了 LIPUS 的促成骨作用并降低了 TRPM7 水平。此外，Trpm7 的敲低抑制了 LIPUS 诱导的 BMSCs 成骨促进作用。LIPUS 介导的细胞骨架微丝的瞬态解聚和重排可以影响 TRPM7 的表达，进而促进 BMSCs 的成骨。本研究为进一步探索 LIPUS 作为机械应力促进 BMSCs 成骨分化的分子机制提供了方向。
