The Piezo1/Ca²⁺/NOTCH2 axis mediates matrix stiffness-induced osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells

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Human bone marrow-derived mesenchymal stem cells (hMSCs) are key seed cells with multi‑lineage differentiation potential, and their osteogenic differentiation capacity forms the basis of bone regeneration. Extracellular matrix stiffness acts as a potent biophysical cue directing hMSC osteogenic lineage commitment, yet the underlying mechanotransduction pathways remain poorly defined. hMSCs were cultured on hydrogels with defined stiffness (1 kPa and 40 kPa). Osteogenic differentiation was assessed by ALP and ARS staining, RT-qPCR, and western blot. Intracellular Ca²⁺ levels were measured using Fluo-4 AM. Piezo1 function was interrogated by siRNA knockdown and pharmacological activation (Yoda1). Notch signaling involvement was tested using the γ-secretase inhibitor DAPT, and Ca²⁺ dependence was assessed with BAPTA-AM. Stiff (40 kPa) hydrogels promoted hMSC osteogenic differentiation compared to soft (1 kPa) hydrogels. Piezo1 expression and channel activity were significantly upregulated under stiff conditions, and Piezo1 knockdown abolished stiffness-induced osteogenesis. Mechanistically, matrix stiffness triggered Piezo1-mediated Ca²⁺ influx, which activated NOTCH2 (but not NOTCH1) signaling, leading to NICD2 release and upregulation of osteogenic genes. Ca²⁺ chelation with BAPTA-AM blocked Yoda1-induced NOTCH2 activation, confirming Ca²⁺-dependence. This study uncovers a Piezo1/Ca²⁺/NOTCH2 mechanotransduction axis that translates matrix stiffness into hMSC osteogenic differentiation. Modulating this pathway could represent a promising approach to improve stem cell‑based bone regeneration.