Osteocytes Exposed to Titanium Particles Inhibit Osteoblastic Cell Differentiation via Connexin 43

Periprosthetic osteolysis (PPO) induced by wear particles is the most severe complication of total joint replacement; however, the mechanism behind PPO remains elusive. Previous studies have shown that osteocytes play important roles in wear-particle-induced osteolysis. In this study, we investigated the effects of connexin 43 (Cx43) on the regulation of osteocyte-to-osteoblast differentiation. We established an in vivo murine model of calvarial osteolysis induced by titanium (Ti) particles. The osteolysis characteristic and osteogenesis markers in the osteocyte-selective Cx43 (CKO)-deficient and wild-type (WT) mice were observed. The calvarial osteolysis induced by Ti particles was partially attenuated in CKO mice. The expression of beta-catenin and osteogenesis markers increased significantly in CKO mice. In vitro, the osteocytic cell line MLO-Y4 was treated with Ti particles. The co-culturing of MLO-Y4 cells with MC3T3-E1 osteoblastic cells was used to observe the effects of Ti-treated osteocytes on osteoblast differentiation. When Cx43 of MLO-Y4 cells was silenced or overexpressed, beta-catenin was detected. Additionally, co-immunoprecipitation detection of Cx43 and beta-catenin binding in MLO-Y4 cells and MC3T3-E1 cells was performed. Finally, beta-catenin expression in MC3T3-E1 cells and osteoblast differentiation were evaluated after 18 alpha-glycyrrhetinic acid (18 alpha-GA) was used to block the intercellular communication of Cx43 between MLO-Y4 and MC3T3-E1 cells. Ti particles increased Cx43 expression and decreased beta-catenin expression in MLO-Y4 cells. The silencing of Cx43 increased the beta-catenin expression, and the over-expression of Cx43 decreased the beta-catenin expression. In the co-culture model, Ti treatment of MLO-Y4 cells inhibited the osteoblastic differentiation of MC3T3-E1 cells and Cx43 silencing in MLO-Y4 cells attenuated the inhibitory effects on osteoblastic differentiation. With Cx43 silencing in the MLO-Y4 cells, the MC3T3-E1 cells, co-cultured alongside MLO-Y4, displayed decreased Cx43 expression, increased beta-catenin expression, activation of Runx2, and promotion of osteoblastic differentiation in vitro co-culture. Finally, Cx43 expression was found to be negatively correlated to the activity of the Wnt signaling pathway, mostly through the Cx43 binding of beta-catenin from its translocation to the nucleus. The results of our study suggest that Ti particles increased Cx43 expression in osteocytes and that osteocytes may participate in the regulation of osteoblast function via the Cx43 during PPO.

Automated summary

This study investigated the regulation of osteocyte-to-osteoblast differentiation by connexin 43 (Cx43). The results showed that calvarial osteolysis induced by titanium particles was partially attenuated in mice lacking Cx43. Silencing or overexpressing Cx43 affected beta-catenin expression, and Cx43 binding to beta-catenin inhibited the Wnt signaling pathway. These findings suggest that osteocytes may regulate osteoblast function through Cx43 during periprosthetic osteolysis.