Transgenic high expression of EphB4 in canine periodontal ligament stem cells modulates osteogenic differentiation and migration in cPDLSCs

Abstract Background Signaling molecules play a critical role in regulating stem cell-mediated bone regeneration. In this study, we investigated the effect of EphB4 gene transfection on the osteogenic differentiation and migratory capacity of canine periodontal ligament stem cells (cPDLSCs). Using a lentiviral vector, we established stable EphB4-overexpressing cPDLSCs and evaluated their functional properties in vitro and regenerative potential in vivo. Results Our results demonstrated that EphB4 transfection significantly enhanced the osteogenic capability of cPDLSCs, as evidenced by elevated expression of osteogenic markers—osteocalcin, Runx2, and collagen type I—along with increased mineral deposition. Furthermore, EphB4 overexpression strongly promoted cell migration, indicating roles in enhancing cell homing and recruitment. Mechanistic analyses indicated that these effects were associated with greater EphB4 phosphorylation, suggesting activation of forward signaling pathways. In a canine alveolar bone defect model, transplantation of EphB4-modified cPDLSCs led to significantly augmented bone regeneration compared with control groups. Micro-computed tomography analysis revealed greater bone volume/total volume ratio, increased trabecular number and thickness, and reduced trabecular separation. Histological and immunohistochemical analyses confirmed greater expression of osteogenic proteins in the EphB4 group; no significant differences were observed between the two control groups (untreated cPDLSCs and empty vector-transduced cPDLSCs). Conclusions These findings collectively indicate that EphB4 overexpression potentiates the osteogenic and migratory properties of cPDLSCs, thereby promoting alveolar bone repair in vivo. Our results highlight the therapeutic potential of EphB4 for periodontal and bone regeneration applications.