Lwin Hnin Yu, Tiskratok Watcharaphol, Kyawsoewin Maythwe, Manokawinchoke Jeeranan, Termkwanchareon Chutimon, Limjeerajarus Nuttapol, Limjeerajarus Chalida Nakalekha, Egusa Hiroshi, Osathanon Thanaphum, Limraksasin Phoonsuk
Center of Excellence for Dental Stem Cell Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand.
Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand.
Int J Mol Sci. 2025 Jun 13;26(12):5667. doi: 10.3390/ijms26125667.
This study aimed to investigate the effects of shear stress on osteogenic differentiation of human dental pulp stem cells (hDPSCs). The hDPSCs were subjected to shear stress for 24 h before osteogenic induction for 21 days. The mRNA expression of osteogenic markers such as RUNX2, OSX, ALP, COL1A1, OCN, and OPN was evaluated by real-time RT-PCR. Alkaline Phosphatase (ALP) activity and Alizarin Red S (ARS) staining were investigated to confirm osteogenic differentiation and mineralization of hDPSCs, respectively. The protein expression of osterix was shown by immunofluorescence staining and Western blotting. RNA sequencing was performed to investigate how shear stress affects the osteogenic differentiation of hDPSCs, which was validated through p38 inhibitor (SB203580) treatment. Real-time RT-PCR revealed that shear stress enhanced osteogenic marker-gene expression. The increased osterix protein expression was detected on Day 14 in the shear-stress loading group compared to the static group. Shear stress enhanced ALP activity and mineralization, observed on Days 14 and 21. A volcano plot exhibited up- and downregulated genes, while the p38 inhibitor markedly inhibited osteogenic differentiation of hDPSCs triggered by shear stress. In conclusion, shear stress promotes the osteogenic differentiation of hDPSCs through the p38 mitogen-activated protein kinase signaling pathway.
本研究旨在探讨剪切应力对人牙髓干细胞（hDPSCs）成骨分化的影响。在进行21天的成骨诱导之前，将hDPSCs施加剪切应力24小时。通过实时逆转录聚合酶链反应（RT-PCR）评估成骨标志物如RUNX2、OSX、碱性磷酸酶（ALP）、I型胶原蛋白（COL1A1）、骨钙素（OCN）和骨桥蛋白（OPN）的mRNA表达。分别研究碱性磷酸酶（ALP）活性和茜素红S（ARS）染色，以确认hDPSCs的成骨分化和矿化。通过免疫荧光染色和蛋白质印迹法显示osterix的蛋白质表达。进行RNA测序以研究剪切应力如何影响hDPSCs的成骨分化，并通过p38抑制剂（SB203580）处理进行验证。实时RT-PCR显示剪切应力增强了成骨标志物基因的表达。与静态组相比，在剪切应力加载组的第14天检测到osterix蛋白表达增加。在第14天和第21天观察到剪切应力增强了ALP活性和矿化。火山图展示了上调和下调的基因，而p38抑制剂显著抑制了剪切应力触发的hDPSCs的成骨分化。总之，剪切应力通过p38丝裂原活化蛋白激酶信号通路促进hDPSCs的成骨分化。
