Bidirectional ephrinB2‑EphB4 signaling regulates the osteogenic differentiation of canine periodontal ligament stem cells.

The aim of the present study was to evaluate the effect of ephrinB2 gene‑transfected canine periodontal ligament stem cells (cPDLSCs) on the regulation of osteogenic differentiation. cPDLSCs were transfected with a transgenic null‑control green fluorescent protein (GFP) vector (termed Vector‑cPDLSCs) or with NFNB2 GFP‑Blasticidin (termed EfnB2‑cPDLSCs). Subsequently, the osteogenic differentiation of Vector‑cPDLSCs and EfnB2‑cPDLSCs was assessed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), alkaline phosphatase (ALP) assay and Alizarin Red S staining. The migratory abilities of cPDLSCs, Vector‑cPDLSCs and EfnB2‑cPDLSCs were also assessed. Following osteogenic induction of Vector‑cPDLSCs and EfnB2‑cPDLSCs, the protein expression levels of collagen I, Runt‑related transcription factor 2, osteocalcin, ephrin type‑B receptor 4 (EphB4), phospho‑EphB4, ephrinB2 and phospho‑ephrinB2 were analyzed by western blot assays. Following gene transfection, the RT‑qPCR and western blotting results revealed that the mRNA and protein expression levels of ephrinB2, respectively, were significantly increased in EfnB2‑cPDLSCs compared with that in Vector‑cPDLSCs (P<0.05). ALP and Alizarin Red S staining assays revealed increased ALP activity and mineralization nodules, respectively, in EfnB2‑cPDLSCs. Cell proliferation and migration assays revealed that EfnB2‑cPDLSCs exhibited enhanced proliferation and migration compared with Vector‑cPDLSCs (P<0.05). In conclusion, the findings of the current study indicated that ephrinB2 gene‑modified cPDLSCs exhibited enhanced osteogenic differentiation, with the ephrinB2 reverse signaling and EphB4 forward signaling pathways serving a key role in this process. Furthermore, ephrinB2 gene modification was observed to promote the migration and proliferation of cPDLSCs.