Effect of avocado/soybean unsaponifiables on periodontal repair in rats with arthritis and induced periodontitis

Abstract

Objective: This study aimed to evaluate the effect of avocado/soybean unsaponifiables (ASU) on periodontal repair in rats with induced periodontitis and arthritis.

Methodology: Forty-five rats were submitted to periodontitis induction by insertion of ligatures into the upper second molars, maintained for 15 days. These animals were randomly allocated to 3 groups according to the presence of induced arthritis (ART) and the application of the ASU: Control (CTR) group-healthy animals, where saline solution was administered; ART-animals with induced arthritis, where saline solution was administered; ART/ASU-animals with induced arthritis, where ASU (0.6 mg/ kg) was administered. The drugs were administered daily by gavage and the animals were euthanized after 7, 15 and 30 days of the ligature removal. Bone resorption, inflammatory infiltrate composition and marker proteins expression of the differentiation and formation of osteoclasts (RANKL and TRAP) were assessed.

Results: The ART/ASU group presented higher bone volume than the ART group at 7 and 30 days after the ligature removal. Furthermore, the ART group presented higher quantity of inflammatory cells and expression of TRAP and RANKL than the other groups.

Conclusion: ASU administration improves the repair of periodontal tissues in an experimental periodontitis model in rats with induced arthritis.

Figure 1. Flowchart of the experiment. Two systemic challenges were achieved with mBSA within 7 days. Fourteen days after the last systemic challenge, the application of the antibody against mBSA within the knee joints was started every 7 days. On the day of the second intra-articular challenge, the induction of periodontal disease was performed by ligatures that were held in place for 15 days. After removing the ligatures, the animals were submitted to euthanasia at 7, 15 and 30 days

Figure 2. Descriptive analysis of the right posterior paw joints of animals from different groups: A) CTR, B) ART and C) ART/ASU. The arthritis induction promoted alterations in the cartilage articulation, since it was possible to note hyperplasia in the region associated with bone resorption in the subchondral area (black arrow), as observed in Figure B. In Figure C, it can be noticed that the ASU application partially prevented the articular alterations; however, it was still possible to notice changes in the morphology of the articular meniscus (Black Arrow) (Original augmentation 100x-HE). M, meniscus; IC, intra articular cartilage; SB, subchondral bone

Figure 3. 3D-images from micro-CT scans representing all groups at the 7-day period, and the representative graphs of the analyses. A) CTR, B) ART, C) ART/ASU: It is possible to notice the greater degree of bone resorption in the ART group in relation to the other groups. The use of ASU reduced bone resorption in animals with induced arthritis; however, this effect was not enough to reach the same condition of the CTR group. D) BV/TV (%) of the inter-radicular region; E) BV/TV (%) of the interproximal regions; and F) the linear distance from the cementum-enamel junction (CEJ) to the top of the crestal bone (CB) in mm. The CTR group presented higher bone volumes and shorter distance from the CEJ-CB than the ART group. In addition, a greater bone volume was observed in the interproximal regions in the ART/ ASU group in relation to the ART group. All of these differences occurred at the 7-day period. *p<0.05; ***p<0.001 – Significant differences between the groups – One-way ANOVA complemented by Tukey‘s test; Different letters represent different levels of significant differences within each group – One-way ANOVA complemented by Tukey‘s test

Figure 4. Representative histological images and graphs from the stereometric analysis. A) Interproximal region image representative of the ART group at the period of 7 days, and B) Furcation region image representative of the ART group at the period of 7 days. In both images, the presence of inflammatory infiltrate (black arrows) can be noticed as associated with destruction of bone and reduction of extracellular matrix component and fibroblasts. C) Representative image of the interproximal region of the CTR and ART/ASU groups at the 7-day period; and D) the representative image of the furcation region of the CTR and ART/ASU groups at the 7-day period. The presence of the inflammatory infiltrate may also be noted in these groups, but to a lesser extent than in the ART group. E) Fibroblasts; F) Inflammatory cells; G) Extracellular connective tissue matrix; and H) Blood vessels. In general, the CTR and ART/ASU groups presented more fibroblasts and less inflammatory cells than the ART group at the 7-day period. *p<0.05; **p<0.01; ***p<0.001 – Significant differences between the groups–One-way ANOVA complemented by Tukey‘s test; Different letters represent different levels of significant differences within each group – One-way ANOVA complemented by Tukey‘s test (Original augmentation 100x-HE). B, bone; CT, connective tissue; iCT, inflammatory infiltrate; T, tooth

Figure 5. Representative images and graphs of the analysis of TRAP and RANKL expression. A) Negative control; B) TRAP expression in the ART group at 7 days; C) TRAP expression in the CTR group at 7 days. It was shown that the TRAP expression was verified in osteoclast cells that were in the vicinity of the bone tissue (black arrows); D) RANKL expression in the ART group at 7 days; E) RANKL expression at the ART/ASU group at 7 days. The RANKL expression was noted in cells close to the bone tissue or associated with cells in the vicinity of the blood vessels (Black arrows). F) TRAP: Compared with the CTR group at 7 days, a higher expression of TRAP positive cells was shown in the ART group. *p<0.05 – Significant differences between the groups – One-way ANOVA complemented by Tukey‘s test; Different letters represent different levels of significant differences within each group – One-way ANOVA complemented by Tukey‘s test. G) RANKL: Compared with the ART/ASU group, a higher expression of RANKL was observed in the ART group at the period of 7 days. *p<0.05 – Significant differences between the groups – Kruskall-Wallis complemented by Dunn's test; Different letters represent different levels of significant differences within each group – Kruskall-Wallis complemented by Dunn‘s test. (Original augmentation 200x)