The Role of EphrinB2–EphB4 Signalling Pathway in Regeneration of Inflammatory Bone Defect

Abstract

The important role of the EphrinB2–EphB4 signalling pathway in bone remodelling has been demonstrated, while its effect on inflammatory bone defect regeneration remains poorly understood. This study was to assess the effect of EphB4–EphrinB2 signalling on inflammation-mediated bone defect repair in murine models. The modelling method of inflammation-mediated bone defect in mice was established by intraperitoneally injecting different concentrations of TNF-α. Then, three randomly assigned groups were administered vehicle (PBS, control), EphrinB2 siRNA, and EphB4 siRNA into a 1.5-mm diameter mandibular bone defect with 5 μg/kg TNF-α intraperitoneally injected every 2 days. The gene expression of osteogenic differentiation markers Runx2, Osterix, ALP, OCN and BSP in healing tissue of the bone defect was examined by quantitative real-time polymerase chain reaction (PCR). Runx2 and BSP protein expressions were examined by western blot, and the decalcified tissues were subjected to histological examination. Compared with the control group, the EphB4 siRNA group mice exhibited lower levels of osteogenic differentiation markers and higher levels of the osteoclastogenic marker. H&E staining, TRACP staining and bone histomorphometry showed that the bones were thinner and the number of giant osteoclasts in the EphB4 siRNA group was higher compared with the control group, whereas there were no significant differences in osteoblastic and osteoclastic differentiation between EphrinB2 siRNA mice and control mice. In conclusion, the EphrinB2–EphB4 signalling pathway plays a critical role in the inflammation-induced bone defect repair process; selective inhibition of EphB4 using siRNA results in decreased bone formation and increased bone resorption under high inflammatory circumstances in vivo.