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.


Introduction
The supporting periodontium is the tissue responsible for the insertion of the tooth in the maxillary bones, and this tissue is composed of cementum, periodontal ligament and the alveolar bone. 1 The destruction of the supporting periodontium occurs during the progression of periodontal disease  [5][6][7][8][9] This action on the proliferation of connective tissue associated with the reduction of biological pro-inflammatory mediators expression has raised interest in the use of ASU in periodontal treatment. 10,11 Pre-clinical studies showed that ASU improved the periodontal repair and treatment outcomes of induced periodontitis, 10,11 as well as improving the osseointegration patterns of implants; 12 these effects were related with the reduction of proinflammatory biological mediators expression, such as Interleukin 1 beta (IL-1β), Receptor activator of nuclear factor kappa-Β ligand (RANKL), and Tartrateresistant acid phosphatase (TRAP), 10 and enhanced in the expression of growth factors, such as Bone morphogenetic protein 2 (BMP2) and Transforming growth factor beta 1 (TGFβ1). 12 It has been proposed that the PD treatment with host response modulators has a more expressive effect in patients who present some systemic condition that changes the progression of tissue destruction or the response to periodontal therapy. 2,13 For example, compared to healthy individuals, patients with ART have more severe PD, with higher levels of periodontal inflammation, more dental loss, higher frequency of sites with advanced insertion loss and deeper periodontal pockets; 14,15 thus, patients with ART and PD can benefit from anti-arthritic therapy with ASU, because this drug has a mechanism for blocking inflammatory processes associated with the stimulation of connective tissue proliferation. 5

Groups
The animals were randomly allocated in 3 groups

Immunohistochemistry analysis
The histological sections were mounted on silanized slides, followed by a routine laboratory procedure for deparaffinization and rehydration. Then, the endogenous peroxidase was inactivation performed by applying 3% hydrogen peroxide for 30 minutes, and then blocking the non-specific epitopes was performed by applying the bovine albumin protein   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 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.  14,15,17 It is important to note that, compared to the ART group, the greater amount of bone volume found in the ART/ASU group corroborates the previous findings showing that ASU promotes an improvement in periodontal repair, although not at the same level as that found in healthy animals in an earlier study. 10 It is important to note that the mainly statistical differences between the groups occurred at the early periods of evaluation, which may be related with the progressive periodontal repair observed after the ligature removal in this model. 10 Associated with the results of the bone volume analysis, it was observed that the ART group presented more inflammatory cells and greater expression of TRAP and RANKL than the CTR and ART/ASU groups.
The ASU administration has been shown to reduce the expression of pro-inflammatory biological mediators (e.g., IL-1β, TNFα, PGE2, iNOS, and MMPs) in in vitro studies with chondroblasts 18,19 and fibroblast cells. 20 These findings have also been demonstrated in preclinical studies in rats, where it was found that ASU administration promoted improved periodontal repair in healthy animals and was associated with reduced expression of important biological mediators of bone resorption, i.e., TRAP, RANKL, and IL-1β. 10,11 In addition to the anti-inflammatory effect, ASU has an important effect on connective tissue proliferation

Conclusion
Considering the results obtained in this study, it can be concluded that, compared to the ART group animals, the use of ASU promoted a significant improvement in periodontal bone repair in animals with experimental arthritis, which was expressed by increased bone volume, reduction in the number of inflammatory cells and reduction of the RANKL and TRAP expression.