Wild-type (WT) C57BL/6 and TLR2 KO and TLR4 KO mice in C57/BL6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). TLR2 and TLR4 double KO mice (TLR2/4 KO) were crossbreed from TLR2 KO and TLR4 KO mice and confirmed by genotyping. All the animal-associated protocols were reviewed and approved (#17-022) by the Institutional Animal Care and Use Committee of the Forsyth Institute. All the mice used in the study were maintained in specific pathogen-free units. Mice were fed a soft diet ad libitum for the duration of the experiment. Forty-eight WT mice and forty-eight TLR2/4 KO mice were used in this study, and the mice were randomly divided into 4 groups in WT and TLR2/4 KO mice as follows: (1) implant only (n = 12), (2) implant + ligation (n = 12), (3) implant + ligation+ anti-RANKL antibody (n = 12), and (4) implant + ligation + anti-RANKL antibody + miR-146a (n = 12).

The procedures of tooth extraction and implant placement were as previously described [5]. Briefly, all the mice had their left maxillary first and second molars extracted at 4 weeks old with 6 weeks of healing time after the tooth extraction. Drinking water with antibiotics (sulfamethoxazole and trimethoprim, 850 μg/170 μg per mL) was used for 2 weeks to decrease the possibility of infection after tooth extraction. Then, the maxillary alveolar bone was drilled with the 0.3-mm-diameter carbide micro-hand drill, and a smooth-surface, screw-shaped titanium implant (1 mm in length and 0.5 mm in diameter, D. P. Machining) was screwed clockwise into the bone through the drilled site until torque could be achieved. Implants were allowed to heal for 4 weeks, and the antibiotic water was given during the first week after implantation as described above. Four weeks after implant placement, experimental peri-implantitis was initiated with a 7-0 silk ligature tied around the implants. The ligation day was recorded as day 0, and the ligature remained in place for 2 weeks. For group 3 and group 4, each mouse received palatal gingival injections of 2 μL of anti-RANKL antibody (500 μg/mL, Peprotech, Rocky Hill, NJ) or miR-146a (100 nM, GeneCopoeia, Rockville, MD) on the mesial and distal gingival papillae of implant by a 31-gauge double-beveled MicroFine needle (Becton, Dickinson) as these doses were established from our previous publications [24, 28]. The injections for animals were administered three times on days 3, 6, and 9, and all the mice were euthanized by CO₂ inhalation on day 14. All the procedures, including tooth extraction, implant placement, ligature placement, and injection, were performed using an optical microscope (S6D Stereozoom, Leica).