Resveratrol modulates Hedgehog signaling pathway and increases the osteogenic commitment of periodontal ligament mesenchymal cells.

Human periodontal ligament mesenchymal cells (PDLCs) are promising for regenerative therapies but show heterogeneous osteogenic commitment that limits bone regeneration. To attempt to overcome this limitation, we focus on the Hedgehog (Hh) signaling pathway, which is crucial for osteoblastic differentiation as the mechanistic focus of the study and as a strategy to overcome this heterogeneous osteogenic commitment. Resveratrol, a naturally occurring polyphenol, exhibits osteoinductive properties, but its effects on osteogenesis in PDLCs with low osteogenic potential (l-PDLCs) remain unclear. We aimed to investigate whether resveratrol could activate the transcriptional program of Hh signaling in l-PDLCs, as the mechanistic focus of the study and as a strategy to overcome the heterogeneous osteogenic commitment, thereby enhancing mineralization in vitro. Cell viability and osteogenic potential were analyzed under different concentrations and treatment protocols. RNA sequencing, gene expression analysis, transcriptional correlation, and molecular docking were performed in l-PDLCs cultured for 10 days under osteogenic medium (OM) or pretreated for 3 days with resveratrol followed by 10 days of induction (OM/Resv). Pretreatment with 0.1 μM resveratrol exhibited a two-fold increase in mitochondrial metabolism and mineral deposition in vitro. Although multiple osteogenic pathways were modulated, Hh signaling was notably activated, with SHH, DHH, IHH, GLI1 and PTCH1 significantly enriched in OM/Resv compared to OM. Importantly, IHH and SP7 were upregulated and strongly correlated after resveratrol treatment. Resveratrol pretreatment during osteogenic induction upregulated GLI1, PTCH1, and SMO, while downregulating RAB23 (p ≤ 0.01), corroborating RNA-seq results. Molecular docking predicted 23 interactions between resveratrol and RAB23, a negative regulator of Hh signaling, suggesting competition with GDP at the RAB23 active site. Resveratrol enhanced proliferation and osteogenic differentiation in l-PDLCs. Activation of osteogenic markers via the Hh pathway and RAB23 downregulation suggests its role in bone regeneration. These findings provide insights into resveratrol-induced osteogenesis and support its potential as a bioactive modulator of osteogenic pathways in PDLCs for bone regenerative therapies.