Results : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
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.
Serial posts:
- Abstract : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Abstract : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Background : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Background : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Methods : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Methods : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Methods : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [3]
- Results : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Results : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Discussion : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Discussion : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Conclusions : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Availability of data and materials : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Abbreviations : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- References : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- References : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- References : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [3]
- References : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [4]
- Acknowledgements : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Funding : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Author information : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [1]
- Author information : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling [2]
- Ethics declarations : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Additional information : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Supplementary information : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
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- About this article : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling
- Table 1 Success rate (SR) of osseointegrated implants 4 weeks after implant placement : RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through
- Fig. 1. Mouse model of ligature-induced experimental peri-implantitis. (a) Tooth extraction: left maxillary first and second molars extracted at 4 weeks old and the tooth extraction socket healed well with smooth gingiva surface after 6 weeks post-extraction. Implant placement: implant was put in alveolar bone without flap elevation. Ligature placement: at 4 weeks post-implant, 7-0 ligatures were applied under the fixture head. Gingival injection: injections for animals were administered three times on days 3, 6, and 9 during 14 days ligation period. Sample collection: 14 days post-ligation, the gingival tissues and the skulls were collected. (b) Images depicting processing steps of the experimental design (scale bar, 500 μm) : RANKL blockade alleviates peri-implant
- Fig. 2. Anti-RANKL and anti-RANKL+miR-146a treatments decreased ligature-induced bone resorption with different patterns in experimental peri-implantitis of WT and TLR2/4 KO mice. Buccal side images of the defleshed skulls were taken of the control (non-ligation) group, ligation (non-treatment) group, ligation with anti-RANKL antibody (ligation+AR) treatment group, and ligation with anti-RANKL antibody + miR-146a (ligation+A+MiR) treatment group in WT mice and TLR2/4 KO mice (a) (scale bar, 500 μm). The bone resorption area based on these images was measured and analyzed for WT mice (b) and TLR2/4 KO mice (c) (mean ± SD, n = 6, *p < 0.05, **p < 0.01, SEM, standard error of difference between two means). Three dimension (3D) images from μCT were collected and analyzed for WT mice (d) and TLR4 KO mice (e) (mean ± SD, n = 6, *p < 0.05, **p < 0.01) : RANKL blockade alleviates peri-implant
- Fig. 3. Anti-RANKL and anti-RANKL+miR-146a treatments decreased TRAP-positive cell quantities with different patterns in experimental peri-implantitis of WT and TLR2/4 KO mice. TRAP-positive cells (red color) with 3 or more nuclei were considered osteoclasts and were shown in the control group, ligation group, ligation with anti-RANKL antibody treatment group, and ligation with anti-RANKL antibody + miR-146a treatment group in WT mice and TLR2/4 KO mice (a) (Im, implant; Av, alveolar bone; scale bar, 100 μm). The quantities of TRAP-positive cells were analyzed in each group of WT mice (b) and TLR2/4 KO mice (c) (mean ± SD, n = 6, **p < 0.01) : RANKL blockade alleviates peri-implant
- Fig. 4. Anti-RANKL and anti-RANKL+miR-146a treatments decreased the inflammatory cell infiltration of the implant gingival tissues with different patterns in experimental peri-implantitis of WT and TLR2/4 KO mice. HE staining of the gingival tissue around implants were performed in the control group, ligation group, ligation with anti-RANKL antibody treatment group, and ligation with anti-RANKL antibody + miR-146a treatment group in WT mice and TLR2/4 KO mice (a) (scale bar, 100 μm). Inflammatory cell numbers were measured and analyzed in each group of WT mice (b) and TLR2/4 KO mice (c) (mean ± SD, n = 6, **p < 0.01) : RANKL blockade alleviates peri-implant
- Fig. 5. Anti-RANKL and anti-RANKL+miR-146a treatments decreased gingival mRNA expression of TNF-α and RANKL with different patterns in experimental peri-implantitis of WT and TLR2/4 KO mice. Gingival tissues around ligatured implants and non-ligation implants were excised and processed for RT-qPCR analysis to determine mRNA level of TNF-α of WT mice (a) and TLR2/4 KO mice (b) (mean ± SD, n = 6, *p < 0.05, **p < 0.01) and mRNA level of RANKL of WT mice (c) and TLR2/4 KO mice (d) (mean ± SD, n = 6, *p < 0.05, **p < 0.01). : RANKL blockade alleviates peri-implant