In our ligature-induced experimental peri-implantitis mouse model, teeth extraction, implant placement, ligation placement, and gingival injection will be performed in a 12-week process (Fig. 1). 86.67% (52 out of 60) of implants in TLR2/4 KO mice achieved osteointegration (no mobility when touched by needles, no obvious bleeding upon probing) after being placed for 4 weeks, which has no significant difference with WT mice success rate 81.67% (Table 1). The gingival tissue surrounding the implant appeared healthy (pink in color, no obvious swelling, no bleeding upon probing) 28 days after implant placement in WT and TLR2/4 KO mice, but gingival tissue turned red in color and displayed apparent swelling 3 days after the ligature was placed. Furthermore, the ligation group showed a significantly higher bone resorption compared with the non-ligation group in WT mice, for both 2D imaging analysis (p = 0.0021, Fig. 2a, b) and μCT analysis (p = 0.0012, Fig. 2d), and in TLR2/4 KO mice for both 2D imaging analysis (p = 0.0039, Fig. 2a, c) and μCT analysis (p = 0.0023, Fig. 2e). Taken together, these results indicate that ligature successfully induced inflammation and bone resorption in this mouse model of peri-implantitis.

Anti-RANKL antibody alone significantly reduced bone loss compared with the ligation only group in both WT and TLR2/4 KO mice; however, injection of miR-146a in addition to anti-RANKL antibody significantly enhanced the inhibition of bone loss in WT mice but not in TLR2/4 KO mice (Fig. 2b–e). Significantly higher number of osteoclasts were observed in the ligation group vs. non-ligation group in WT mice and TLR2/4 KO mice in TRAP staining (Fig. 3a–c), which is consistent with the results of 2D and 3D bone loss analysis (Fig. 2b–e). Moreover, the anti-RANKL+miR-146a treatment groups showed significantly lower number of osteoclasts compared with the anti-RANKL treatment group in WT mice but not in TLR2/4 KO mice (Fig. 3b, c), which is also consistent with the results of bone loss analysis (Fig. 2b–e). Taken together, miR-146a in addition to anti-RANKL antibody can further reduce bone loss in WT mice but is ineffective when TLR2 and TLR4 are deficient, suggesting miR-146a anti-bone loss effects in peri-implantitis are TLR2/4 dependent.