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 Ca2 + 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.