Fig. 6.

Combination treatment of CD40L, IL-21 and anti-Tim1 reduced bone loss and increased gingival IL-10 expression and mRNA level and decreased RANKL mRNA level in mouse experimental periodontitis. Silk ligatures were tied around maxillary secondary molars of both sides in C57/BL6 mice on day 0 and CD40L (1 μg/ml)+ IL-21 (1 μg/ml)+ anti-Tim1 (5 μg/ml) combination or PBS was injected on days 3, 6 and 9. Maxilla were collected on day 14 and the lingual and buccal sides alveolar bone resorption areas were pictured (A) and analyzed as bone resorption area/mm² at the magnification of 30× (C) (means ± SD, n = 5 mice per group, *p < 0.05, **p < 0.01). TRAP staining was performed on tissue sections and images of periodontal tissues were analyzed at the magnification of 200× (B). The number of multinucleated TRAP+ cells along the alveolar bone surface were counted and compared between control group and CD40L +IL-21 + anti-Tim1 combination treatment group (D) (means ± SD, n = 5 mice per group, **p < 0.01). Gingival IL-10 protein expression were measured by ELISA in control and CD40L + IL-21+ anti-Tim1 combination treatment group (E) (mean ± SD, n =5 mice per group, *p < 0.05). Gingival IL-10 (F) and RANKL (G) mRNA levels were determined by real-time PCR in control and CD40L + IL-21+ anti-Tim1 combination treatment group respectively (mean ± SD, n =5 mice per group, *p < 0.05).