The enhanced osteogenic differentiation of human periodontal ligament stem cells and M2 polarization of macrophages may be mediated by EphB4/ephrinB2 signaling pathway: a study of their direct co-culture

Background Periodontal tissue regeneration can be achieved by periodontal ligament stem cells (PDLSCs) through its regulating the immune system. However, the specific signal or molecular mechanism remains unreported. The interaction between MSCs and macrophages (Mφ) has been the focus of the research in recent years. The objective of this study is to examine the effect of direct co-culture of human periodontal ligament stem cells (hPDLSCs) and macrophages on the osteogenic differentiation of hPDLSCs and the polarization of macrophages, and also to explore the role of the EphB4/ephrinB2 signaling pathway in the interaction of co-cultured hPDLSCs and macrophages. Methods hPDLSCs isolated from human periodontal ligament were co-cultured with non-activated M0 macrophages (M0-Mφ) induced from THP-1. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) staining and assay, as well as Alizarin red staining (ARS) were carried out to evaluate hPDLSCs osteogenic differentiation. qRT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) were employed to detect the expression of macrophage polarization-related factors. Western Blot was utilized to detect the expression of EphB4, ephrinB2, ERK1/2 and STAT3. Results When M0-Mφ was directly co-cultured with hPDLSCs at a ratio of 5:1, the co-culture system significantly promoted the osteogenic differentiation of hPDLSCs, as demonstrated by enhanced ALP staining/activity, ARS mineralization and upregulated mRNA expression of osteogenic markers (Runx2, ALP, OCN, and OPN). Meanwhile, the co-culture system markedly increased anti-inflammatory factor expression (TGF-β1 and IL-10) and decreased the pro-inflammatory factors (TNF-α and IL-1β), indicating enhanced polarization of alternatively activated macrophages (M2-Mφ). The mRNA and protein expression of EphB4 and ephrinB2 increased significantly with the time extension of the two cells’ co-culture. However, pharmacological interruption of EphB4/ephrinB2 signaling pathway resulted in the inhibition of hPDLSC osteogenic differentiation, M2 macrophage polarization, and p-STAT3 expression in the co-culture system. Conclusions The EphB4/ephrinB2 pathway may mediate the osteogenic differentiation of hPDLSCs and the polarization of M2-Mφ in the co-culture system. Its regulatory effect on the osteogenic differentiation of hPDLSCs may be achieved through the STAT3 signaling pathway.