Osteocytes exposed to titanium particles inhibit osteoblastic cell differentiation via connexin 43/β-catenin pathway

AbstractBackground Periprosthetic osteolysis (PPO) induced by wear particles is the most severe complication of total joint replacement, however, the mechanism of PPO remains unknown. Previous studies have shown that osteocytes play an important role in wear particle-induced osteolysis. In this study, we investigated the effects of connexin 43 (Cx43) on the regulation of osteocyte to osteoblast differentiation. Methods In vivo, a murine model of calvarial osteolysis induced by titanium (Ti) nanoparticles was established. The osteolysis characteristic and osteogenesis markers in the osteocyte-selective deficiency of Cx43 (cKO) mice and wild-type (WT) mice were observed. In vitro, osteocytic cell line MLO-Y4 was treated with Ti nanoparticles. β-catenin were detected when Cx43 of MLO-Y4 cell was silenced or overexpressed. Co-culture of MLO-Y4 cells with MC3T3-E1 osteoblastic cells was used to observe the effects of Ti-treated osteocyte on osteoblast differentiation. Co-immunoprecipitation detection of Cx43 and β-catenin binding in MLO-Y4 cells and MC3T3-E1 cells. Results In vivo, the calvarial osteolysis induced by Ti particles was partially attenuated in the cKO mice. The expression of β-catenin, Runx2, osterix, alkaline phosphatase (ALP), and osteocalcin (OCN) increased significantly in the femurs of cKO mice. In vitro. Ti particles decreased β-catenin expression and increased Cx43 expression. Silencing of Cx43 increased the β-catenin expression in MLO-Y4 cells and over-expressed Cx43 decreased the β-catenin expression. In the co-culture model, as a result of Cx43 silencing in the MLO-Y4 cells, the MC3T3-E1 cells displayed decreased Cx43 expression, increased β-catenin expression, activation of Runx2, and promotion of osteoblastic differentiation. Cx43 silencing in MLO-Y4 cells attenuated the inhibitory effects of the Ti treatment of MLO-Y4 cells on the osteoblastic differentiation of MC3T3-E1 cells. Finally, Cx43 expression was found to be negatively correlated to the activity of the Wnt signaling pathway through binding of β-catenin to the nucleus. Conclusion In conclusion, the results of our study suggest that Ti nanoparticles increased Cx43 expression in osteocytes and osteocytes may participate in the regulation of osteoblast function via the Cx43/β-catenin pathway during periprosthetic osteolysis.