Our present study showed that anti-RANKL antibody can significantly inhibit the bone loss in peri-implantitis and additional miR-146a treatment will enhance this inhibition through its anti-inflammation effects via TLR2/4 signaling. This is the first report in a murine model of peri-implantitis to demonstrate that anti-RANKL antibody and miR-146a together can significantly reduce bone resorption and inflammation in peri-implantitis, suggesting a potential therapeutic strategy for peri-implantitis patients. Moreover, the data showed that anti-bone loss effects of anti-RANKL antibody are independent of TLR2 and TLR4 and anti-RANKL antibody alone did not affect peri-implant inflammatory infiltration, suggesting that RANKL modulation of bone loss is downstream of peri-implant inflammation and more directly towards bone resorption. However, anti-RANKL antibody + miR-146a treatment showed significantly stronger inhibition of bone loss than anti-RANKL antibody alone treatment, indicating that the suppression of inflammation can be used to reduce peri-implantitis bone loss by removing additional RANKL-independent etiology and pathogenesis of bone loss. These data (Fig. 1–5, Supplemental Table S1) showed that anti-inflammatory miR-146a enhance anti-RANKL-induced inhibition of peri-implant bone resorption through the regulation of TLR2/4 signaling and inhibition of TNF-α expression. Thus, the ideal osteoimmunological treatment for peri-implantitis should include both direct anti-osteoclastogenesis and anti-inflammation components.

As innate immune recognition receptors, TLR family plays a central role in innate immunity, inflammation, cell survival, and proliferation [5, 26, 37, 38]. TLR2 and TLR4 are essential signaling proteins in progression of inflammation and related bone metabolism in periodontitis [28, 29, 39, 40]. However, little is known about the functions of TLR2 and TLR4 signaling in peri-implantitis. In the present study, we investigated the changes in bone resorption, gingival TNF-α (pro-inflammatory marker) mRNA levels, and gingival soluble RANKL protein levels in ligation-induced experimental peri-implantitis in WT and TLR2/4 KO mice with or without anti-RANKL antibody alone treatment and anti-RANKL antibody + miR-146a treatment. The results showed that anti-bone loss effects of anti-RANKL antibody are TLR2/4 independent but anti-inflammation and related anti-bone loss effects of miR-146a are TLR2/4 dependent, suggesting that TLR2/4 signaling is crucial for RANKL-independent, inflammation-induced bone loss in peri-implantitis.