Modulation of Osteogenic Differentiation in Human Dental Pulp Stem Cells by EphrinB2 Signaling

Current treatments for dental pulp diseases, such as root canal therapy, fail to regenerate functional pulp tissue, highlighting the need for innovative regenerative strategies. Human dental pulp stem cells (hDPSCs) hold significant potential for pulp regeneration due to their multipotency, but the molecular pathways governing their differentiation remain poorly understood. This study investigates the role of EphrinB2 signaling in regulating osteogenic differentiation of hDPSCs and identifies EphB4 as its key mediator. Using lentiviral overexpression of EphrinB2 combined with a periodontal ligament stem cell-derived decellularized extracellular matrix (PDLSC-DECM) scaffold, we demonstrated that EphrinB2 significantly enhances early osteogenic differentiation, as evidenced by upregulated expression of RUNX2, BMP2, and IGF, alongside increased alkaline phosphatase (ALP) activity. Strikingly, inhibition of EphB4 (via TNYL-RAW peptide) suppressed osteogenesis, whereas EphB2 inhibition had no effect, indicating EphB4-specific regulation. Notably, late-stage mineralization markers (OCN, DSPP, DMP1) remained unaffected, suggesting EphrinB2-EphB4 signaling primarily drives early osteogenic commitment. These findings contrast with reports in other cell types where EphB2 dominates, underscoring cell type-specific receptor usage. Our work establishes EphrinB2-EphB4 as a critical axis for hDPSC osteogenesis and provides a scaffold-integrated strategy to advance dental pulp regeneration. This study lays the foundation for EphrinB2-targeted therapies to overcome the limitations of conventional dental treatments and promote functional tissue repair.