Shear stress regulates osteogenic differentiation of human dental pulp stem cells via p38 pathway

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 hours 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. Overall design: RNA-sequence profiling of control (hDPScs) and shear stress applied hPDSCs for 24 hours at day 0 of osteogenic differentiation

本研究旨在探讨剪切应力对人牙髓干细胞（human dental pulp stem cells，hDPSCs）成骨分化的影响。实验中将hDPSCs施加剪切应力处理24小时，随后进行21天成骨诱导培养。采用实时逆转录聚合酶链反应（real-time RT-PCR）检测RUNX2、OSX、ALP、COL1A1、OCN、OPN等成骨标志物的mRNA表达水平；分别通过碱性磷酸酶（Alkaline Phosphatase，ALP）活性检测与茜素红S（Alizarin Red S，ARS）染色，验证hDPSCs的成骨分化与矿化能力；通过免疫荧光染色及蛋白质印迹法（western blotting）检测Osterix的蛋白表达水平。本研究开展RNA测序以探究剪切应力对hDPSCs成骨分化的调控机制，并通过p38抑制剂（SB203580）处理验证该调控效应。实时RT-PCR结果显示，剪切应力可上调成骨标志物基因的表达；与静态培养组相比，剪切应力加载组在第14天即可检测到Osterix蛋白表达上调；剪切应力可增强ALP活性与矿化能力，该现象在第14天及第21天均可观察到。火山图展示了差异表达的上调基因与下调基因，而p38抑制剂可显著抑制剪切应力诱导的hDPSCs成骨分化。综上，剪切应力可通过p38丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路促进hDPSCs的成骨分化。整体实验设计：在成骨分化第0天，对对照组（hDPSCs）及施加24小时剪切应力的hDPSCs进行RNA测序分析。