Retinoic Acid Promotes Osteogenic Differentiation of Mesenchymal Stem Cells in the Mouse Facial Suture Under Mechanical Stretching via <i>Piezo2</i>

Trans-sutural Distraction Osteogenesis (TSDO) is a common surgical method for treating facial hypoplasia in secondary cleft lip and palate. Its mechanism involves utilizing mechanical traction to induce the differentiation of mesenchymal stem cells（MSCs） within the facial cleft into osteoblasts. Retinoic acid (RA) is involved in various life processes and promotes osteogenic differentiation under low-dose conditions. However, the impact of RA treatment on stem cells differentiation under mechanical force is unclear. To investigate this mechanism, we performed cell stretching experiment on MSCs that isolated from the mouse facial suture for 2 hours and 3 days alone or supplemented with RA. RNA-seq data showed that mechanical stretching induced the expression of osteogenic-related transcription factors. More importantly, the induction of genes involved in TGFβ/ BMP and WNT signaling pathways was significantly increased upon RA and mechanical stretching co-treatment. Simultaneously, Gene Ontology (GO) enrichment analysis revealed that differentially expressed genes (DEGs) in these co-treatment samples were enriched in the biological process “osteogenic differentiation”. Transcriptomic profiling revealed Piezo2 (mechanosensitive ion channel) as a differentially expressed gene (DEG, p &lt; 0.0001; Log2 (Fold Change) = 4.91). This finding was corroborated by in vitro mechanostimulation assays and in vivo RA-treated murine models, which exhibited 8.38-fold upregulation of Piezo2 mRNA (p &lt; 0.001) compared to untreated controls. After knockout of the Piezo2 gene in MSCs, the cells were subjected to in vitro stretching under osteogenic induction conditions. The formation of calcium nodules in the retinoic acid (RA) treatment group was significantly enhanced compared with the control group. These results indicate that RA putatively promotes osteogenic differentiation under mechanical stretching via Piezo2.