Substrate Stiffness Regulates the Osteogenesis of PDLSCs Via ERK-Mediated YAP Nuclear Translocation

Objectives: Periodontitis is an inflammatory disease and may lead to the progressive destruction of alveolar bone. Periodontal ligament stem cells (PDLSCs) exhibit significant regenerative potential, and their osteogenic differentiation is influenced by the biophysical microenvironment, particularly substrate stiffness. However, the mechanistic pathways through which stiffness modulates PDLSCs osteogenesis remain incompletely understood. This study aimed to elucidate the role of ERK1/2 and YAP signalling pathways in regulating PDLSCs osteogenic differentiation in response to substrate stiffness. Methods: Gelatin methacryloyl (GelMA) hydrogels with varying stiffness were fabricated to culture PDLSCs. Cell viability, spreading, and osteogenic differentiation were evaluated through live/dead staining, immunofluorescence, qRT-PCR, Western blot analysis, and alizarin red staining. The involvement of ERK1/2 and YAP signalling pathways was investigated using specific inhibitors. An in vivo rat periodontitis model was developed to validate in vitro findings, with periodontal ligament stiffness and alveolar bone resorption assessed via nanoindentation and micro-computed tomography (micro-CT). Results: PDLSCs cultured on stiffer substrates exhibited enhanced osteogenic differentiation, as evidenced by upregulated expression of osteogenic markers (RUNX2 and OCN) and increased mineralization. Stiff substrate promoted ERK1/2 phosphorylation and YAP nuclear translocation. Inhibition of ERK1/2 reduced YAP nuclear localization and suppressed osteogenic marker expression. In vivo, periodontitis-associated reductions in periodontal ligament stiffness correlated with decreased YAP nuclear translocation and significant alveolar bone resorption. YAP inhibition mirrored these effects, underscoring its critical role in osteogenesis. Conclusions: Substrate stiffness regulates PDLSCs osteogenesis via ERK-mediated YAP nuclear translocation. These findings advance our understanding of the mechanotransduction mechanisms underlying PDLSCs differentiation and highlight potential therapeutic targets for periodontal regeneration.