EphrinB2 signaling regulates osteogenic/odontogenic differentiation of dental pulp stem cells

Abstract

Objectives: Because previous studies demonstrated conclusively that EphrinB2 signaling play crucial roles in the osteogenic differentiation of osteoblasts, mesenchymal stem cells and periodontal ligament fibroblasts; this study therefore investigated the role of this signaling pathway in the osteogenesis/odontogenesis of DPSCs. Methods: The endogenous expression levels of EphrinB2 and its cognate receptors EphB2 and EphB4 in DPSCs were analyzed by qRT-PCR and Western blotting after 7, 14 and 21 days of osteo/odontogenic induction culture. Subsequently, we investigated whether supplementation of recombinant EphrinB2-Fc within the induction milieu can enhance the osteo/odontogenic differentiation of DPSCs. The underlying molecular mechanisms were also investigated. Results: Endogenous gene and protein expression levels of EphrinB2, EphB2 and EphB4 were upregulated in induced versus uninduced DPSCs, over 21 days of osteo/odontogenic induction. Preliminary investigation of a concentration range (0, 0.5, 1 and 2 µg/ml) of recombinant EphrinB2-Fc showed that 0.5 µg/ml was optimal for enhancing the osteo/odontogenic differentiation of DPSCs over an induction duration of 14 days. Subsequently, more comprehensive qRT-PCR analysis with 0.5 µg/ml EphrinB2-Fc revealed significant upregulation of several key osteogenic marker genes in treated versus untreated DPSCs after 21 days of osteo/odontogenic induction. By 7 days of induction, DPSCs treated with 0.5 µg/ml EphrinB2-Fc exhibited significantly more calcium mineralization (Alizarin red S staining) and alkaline phosphatase activity than the untreated control. Conclusions: EphrinB2 signaling plays a key role in the osteo/odontogenic differentiation of DPSCs.