Transgenic expression of EphrinB2 in PDLSCs modulates osteogenic differentiation via signaling crosstalk between EphrinB2 and EphB4 in PDLSCs and between PDLSCs and pre-osteoblasts within co-culture

Abstract

Objectives: Transduction of PDLSCs to overexpress ephrinB2 will be carried out to investigate: (i) how signaling crosstalk between ephrinB2 and EphB4 within PDLSCs might increase the osteogenic capacity of this cell type, and (ii) how ephrinB2/EphB4 signaling might facilitate interaction between PDLSCs and pre-osteoblasts within co-culture, which may in turn enhance osteogenesis. Methods: PDLSCs were transfected with transgenic (hEfnB2-GFP-Bsd) vector or empty vector (GFP-Bsd). Vector-PDLSCs, EfnB2-PDLSCs, MC3T3-E1, and co-cultures of vector-PDLSCs and MC3T3-E1 and EfnB2-PDLSCs and MC3T3-E1 were subjected to osteogenic induction. The osteogenic differentiation of EfnB2-PDLSCs, vector-PDLSCs, and co-cultures were assessed by RT-PCR, ALP assay, and Alizarin-red S staining. Protein expression levels of ephrinB2, EphB4, and phosphorylated ephrinB2, EphB4 were analyzed by western blot, immunoprecipitation, and co- immunoprecipitation assays. Results: ALP assay and Alizarin-red S staining demonstrated higher ALP activity and more mineralization with EfnB2-PDLSCs versus vector-PDLSCs and with co-culture of EfnB2-PDLSCs and MC3T3-E1 versus vector-PDLSCs and MC3T3-E1. RT-PCR revealed that the expression of human odonto/osteogenic markers (ALP, RunX2, COL1, and DSPP) were enhanced significantly in EfnB2-PDLSCs compared to vector-PDLSCs, and that expression of mouse odonto/osteogenic markers (Runx2, COL1, OCN) were significantly higher in co-culture of EfnB2-PDLSCs and MC3T3-E1 versus vector-PDLSCs and MC3T3-E1. The EphB4 receptor was activated through phosphorylation during osteogenic differentiation. Conclusions: Our data indicate that transgenic expression of ephrinB2 in PDLSCs could promote osteogenic differentiation via stimulating phosphorylation of ephrinB2 and EphB4, thus regulating cell communication within PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.