Matrix Stiffness Orchestrates Mesenchymal Stem Cell Lineage Commitment Toward Osteogenesis and Adipogenesis Through the PIEZO1/SP1/STC2 Axis

Mesenchymal stem cells (MSCs) are critical for bone regeneration, and their osteogenic and adipogenic lineage balance is intricately regulated by cellular mechanotransduction. Through single‐cell RNA sequencing reanalysis of the public dataset GSE166824, primary mouse MSC functional assays, and in vivo genetic and bone defect models, this study identifies a novel PIEZO1/specificity protein 1 (SP1)/stanniocalcin 2 (STC2) signaling axis that drives matrix stiffness‐dependent MSC lineage commitment. Stiff matrices robustly direct MSCs toward osteogenic differentiation while inhibiting adipogenesis by activating the mechanosensitive ion channel PIEZO1. PIEZO1 activation triggers Ca 2 + influx, leading to calcium/calmodulin‐dependent protein kinase II (CaMKII)‐dependent SP1 activation. SP1 shows enrichment at the promoter region of Stc2 and upregulates its expression. The secreted protein STC2 functions downstream of PIEZO1 on stiff matrices while retaining pro‐osteogenic and anti‐adipogenic activity when PIEZO1 remains inactive. In a mouse femoral bone defect model under hindlimb unloading, both stiff gelatin methacryloyl (GelMA) hydrogels and exogenous STC2 administration enhance early‐stage bone formation on day 7. Conversely, Prrx1 ‐lineage‐specific inducible conditional knockout (iCKO) of Piezo1 abrogates the regulatory effects of matrix stiffness. These findings establish the PIEZO1/SP1/STC2 axis as a pivotal mechanosensitive signaling pathway for MSC fate determination, offering novel molecular targets for mechanically optimized bone regenerative biomaterials.