Wang Xiaxia, Dong Shaojie, Dong Qianqian, Sun Xuefei
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, China; Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, Department of Endodontics, College of Stomatology, Xi'an Jiaotong University, Xi'an, China.
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, China; Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, Department of Endodontics, College of Stomatology, Xi'an Jiaotong University, Xi'an, China.
Int Dent J. 2025 Jun;75(3):1885-1896. doi: 10.1016/j.identj.2025.01.018. Epub 2025 Feb 17.
Mechanical stimuli signals regulate the odontogenic differentiation of dental pulp stem cells (DPSCs), but they are difficult to apply in clinical treatment. Piezo1, a specific mechanically activated ion channel that mediates mechanical transduction, may serve as a potential target for regulating mechanical signals. In the present study, we aimed to investigate the function and potential molecular mechanisms of Piezo1 in the odontogenic differentiation of DPSCs.
Piezo1 expression in human dental pulp and DPSCs was detected by immunofluorescence or immunohistochemistry (IHC). The mechanotransduction of Piezo1 ion channels in DPSCs was determined by fluid shear stress (FSS) detection of calcium fluorescence intensity and whole-cell patch clamp detection. The role of Piezo1 in the odontogenic differentiation of DPSCs was detected by alizarin red staining and Western blotting under hydrostatic pressure (HP). The expression and distribution of the downstream molecules Piezo1, CaN, and YAP were detected through coimmunoprecipitation (co-IP), immunocytochemistry (ICC), and Western blot analysis.
The Piezo1 protein was positively expressed in human dental pulp samples, especially in the odontoblast layer. Increased Piezo1 expression was also detected after odontogenic differentiation of DPSCs in vitro. The fluorescence intensity of intracellular calcium ions (Cai) increased rapidly with treatment with FSS or Yoda1 (a Piezo1-specific agonist) but did not significantly change after treatment with GsMTx4 (a Piezo1 antagonist) or BAPTA (an extracellular calcium ion chelating agent). A whole-cell patch clamp was used to record the inward current induced by mechanical stimulation of the DPSCs. After Yoda1 treatment, the peak current increased, but the currents nearly completely disappeared after pretreatment with GsMTx4. In addition, we found that blocking CaN or YAP reversed the ability of HP to promote DPSC odontogenic differentiation. Co-IP and ICC revealed that the CaN and YAP proteins colocalized and bound to each other in DPSCs.
These findings indicated that the Piezo1 ion channel mediates the mechanical transduction of DPSCs. In addition, Piezo1 promotes odontogenic differentiation of DPSCs through the Ca/CaN/YAP signalling axis under HP, which provides effective intervention targets for mechanical stimulation-mediated regulation of reparative dentin and vital pulp preservation.
机械刺激信号可调节牙髓干细胞（DPSCs）的成牙本质分化，但难以应用于临床治疗。Piezo1是一种介导机械转导的特异性机械激活离子通道，可能成为调节机械信号的潜在靶点。在本研究中，我们旨在探讨Piezo1在DPSCs成牙本质分化中的功能及潜在分子机制。
采用免疫荧光或免疫组织化学（IHC）检测Piezo1在人牙髓和DPSCs中的表达。通过流体剪切力（FSS）检测钙荧光强度和全细胞膜片钳检测来确定DPSCs中Piezo1离子通道的机械转导。在静水压力（HP）下，通过茜素红染色和蛋白质免疫印迹法检测Piezo1在DPSCs成牙本质分化中的作用。通过免疫共沉淀（co-IP）、免疫细胞化学（ICC）和蛋白质免疫印迹分析检测下游分子Piezo1、钙调神经磷酸酶（CaN）和Yes相关蛋白（YAP）的表达及分布。
Piezo1蛋白在人牙髓样本中呈阳性表达，尤其在成牙本质细胞层。体外DPSCs成牙本质分化后，Piezo1表达也升高。用FSS或Yoda1（一种Piezo1特异性激动剂）处理后，细胞内钙离子（Cai）荧光强度迅速增加，但用GsMTx4（一种Piezo1拮抗剂）或BAPTA（一种细胞外钙离子螯合剂）处理后无明显变化。采用全细胞膜片钳记录DPSCs机械刺激诱导的内向电流。Yoda1处理后，峰值电流增加，但用GsMTx4预处理后电流几乎完全消失。此外，我们发现阻断CaN或YAP可逆转HP促进DPSC成牙本质分化的能力。Co-IP和ICC显示，CaN和YAP蛋白在DPSCs中共定位并相互结合。
这些发现表明，Piezo1离子通道介导DPSCs的机械转导。此外，Piezo1在HP作用下通过Ca/CaN/YAP信号轴促进DPSCs的成牙本质分化，这为机械刺激介导的修复性牙本质调节和牙髓活力保存提供了有效的干预靶点。
