Adequate bone volume is required for osseointegrated implants to restore lost teeth and oral function. Several studies have demonstrated potential advantage of stem cells in regenerative medicine using osteoblasts. The periosteum is composed of osteoblasts, fibroblasts, and osteoprogenitor cells. It may be an alternative source for bone tissue engineering because of easy isolation and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has proved successful in recoveries from nonunions, delayed unions, and fracture of the bone in both animal experiments and clinical treatments. The study was to investigate the influence of LIPUS on the osteogenic differentiation in murine periosteum-derived cells (PDCs) and the underlying mechanism of LIPUS. PDCs were treated daily with LIPUS for 20 min up to 21 days with 3 MHz frequency, 30 mW/cm² intensity, and pulse repetition frequency of 1 kHz. The effects of LIPUS on cell proliferation and viability were investigated. Osteogenic differentiation was analyzed by alkaline phosphatase (ALP)-positive cell staining, ALP activity assay, mineralized nodule formation, real-time reverse transcription-polymerase chain reaction, as well as western blotting. The results indicated that ultrasound stimulation did not significantly affect the proliferation of PDCs. But LIPUS significantly increased ALP activity on day 7 and markedly promoted formation of mineralized nodules on day 21. mRNA expression of ALP and osteocalcin was significantly upregulated by stimulation with LIPUS. LIPUS enhanced gene expression of both bone morphogenetic protein-2 (BMP-2) and osterix only in the presence of osteogenic medium. LIPUS stimulation did not affect Smad 1 and Smad 5 protein expression, but significantly upregulated protein levels of BMP-2 and phosphor-Smad 1/5/9 in PDCs. Thus, LIPUS stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of BMP-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition. Therefore, LIPUS might have potential to promote osteogenesis in PDCs.

中文翻译：

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低强度脉冲超声刺激体外骨膜细胞的成骨分化。

骨整合种植体需要足够的骨量来恢复丢失的牙齿和口腔功能。几项研究已经证明了干细胞在使用成骨细胞的再生医学中的潜在优势。骨膜由成骨细胞、成纤维细胞和骨祖细胞组成。由于在体内和体外易于分离和快速增殖，它可能是骨组织工程的替代来源. 在动物实验和临床治疗中，低强度脉冲超声 (LIPUS) 已证明可成功治愈骨不连、延迟愈合和骨折。本研究旨在探讨 LIPUS 对小鼠骨膜衍生细胞 (PDCs) 成骨分化的影响及 LIPUS 的潜在机制。PDC 每天用 LIPUS 处理 20 分钟，最多 21 天，频率为 3 MHz，30 mW/cm ²强度，脉冲重复频率为 1 kHz。研究了 LIPUS 对细胞增殖和活力的影响。通过碱性磷酸酶 (ALP) 阳性细胞染色、ALP 活性测定、矿化结节形成、实时逆转录聚合酶链反应以及蛋白质印迹分析成骨分化。结果表明，超声刺激对PDCs的增殖没有显着影响。但 LIPUS 在第 7 天显着增加 ALP 活性，并在第 21 天显着促进矿化结节的形成。 LIPUS 刺激显着上调 ALP 和骨钙素的 mRNA 表达。LIPUS 仅在成骨培养基存在的情况下增强骨形态发生蛋白 2 (BMP-2) 和 osterix 的基因表达。LIPUS 刺激不影响 Smad 1 和 Smad 5 蛋白表达，但显着上调 PDC 中 BMP-2 和磷-Smad 1/5/9 的蛋白水平。因此，LIPUS 刺激增加了正常培养基中的早期成骨分化，并通过成骨培养基中的经典 Smad 信号通路进一步增强了 BMP-2 的表达和随后的 osterix 表达，导致矿物质沉积。因此，LIPUS 可能具有促进 PDC 成骨的潜力。导致矿物同位。因此，LIPUS 可能具有促进 PDC 成骨的潜力。导致矿物同位。因此，LIPUS 可能具有促进 PDC 成骨的潜力。