Substrate stiffness modulates osteogenic differentiation of BMMSCs via the hedgehog signaling pathway

Substrate stiffness is a critical biophysical cue regulating mesenchymal stem cell (MSC) fate, yet the underlying mechanisms remain incompletely understood. Here, we investigated how substrate stiffness modulates the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and the involvement of the Hedgehog (Hh) signaling pathway in this process. Polydimethylsiloxane (PDMS) substrates with tunable stiffness (soft: 32.73 ± 3.74 kPa; medium: 57.59 ± 5.65 kPa; stiff: 147.4 ± 11.04 kPa) were fabricated and functionalized with arginine-glycine-aspartic acid (RGD) peptides to mimic the mechanical microenvironment of bone tissue. BMMSCs cultured on stiff substrates exhibited enhanced cell spreading and proliferation compared to those on soft substrates. Osteogenic induction experiments revealed that stiff substrates significantly upregulated alkaline phosphatase (ALP) expression and calcium nodule formation after 7 and 21 days, respectively. Mechanistically, the Hh pathway was activated on stiff substrates at day 3. Inhibition of Hh signaling using GANT61 impeded stiffness-induced effects, reducing cell spreading, proliferation, and osteogenic differentiation. These findings demonstrate that substrate stiffness promotes BMMSCs osteogenesis in a Hh signaling-dependent manner, providing new insights into the mechanobiology of bone regeneration and informing the design of stiffness-optimized biomaterials for tissue engineering applications.