Evaluation of induced apical periodontitis in osteoporotic mice submitted or not to treatment with alendronate

Abstract

There is little data available in the literature about the osteoporosis impact and bisphosphonates administration in apical periodontitis development and progression (Xiong et al. 2007; 2010). Considering that osteoporosis is a disease that affects worldwide the life's quality of many patients and the vast majority of these patients taking alendronate, a strong bisphosphonate, the aim of this study will be to evaluate induced apical periodontitis in osteoporotic mice submitted or not to treatment with alendronate. After appreciation of this research project by the Ethics Committee on Animal Use, School of Dentistry of Ribeirão Preto - USP, will be used 40 female mice C57BL / 6, 6-8 weeks of age. The animals will be randomly divided into 5 groups: Group I - Sham (sham surgery) (n = 5 animals), Group II - Sham and induced apical periodontitis (n = 5), Group III - animals subjected to OVX (ovariectomy) and induced apical periodontitis (n = 10), Group IV - OVX, induced apical periodontitis and treatment with sodium alendronate (ALD ) (n = 10) and Group V - OVX, induced apical periodontitis and treatment with distilled water (n = 10). The animals in groups IV and V will receive ALD or distilled water by gavage, 21 days after OVX and until the euthanasia moment. Each week the animals will be weighed to control so that a new solution will be made with the appropriate concentration of ALD according to average weight obtained. After 21 days of surgery (sham or OVX) the first lower molars of groups II, III, IV and V will be submitted to coronal access, the pulp tissues will be exposed to oral cavity with aid of spherical dental drill at low speed and the canals will remain exposed to oral microbiota for inducing apical periodontitis. After the experimental period of 21 days, it will also be performed microbiological samples of the root canals contents to the identification and the quantification of 40 species of micro-organisms in the root canals through DNA probes (checkerboard DNA-DNA hybridization) and the animals will be euthanized in a CO2 chamber. Tibias representing all groups will be removed from surgical and the proximal tibial metaphysis will be analyzed with a dualenergy X-ray densitometer to record the Bone Mineral Density (BMD) in g/cm2. The left jaws will be removed and submitted to histotechnical process. The representative sections of each group will be stained with hematoxylin and eosin (HE) and subjected to conventional optical microscopy analysis, to describe the pulp tissues and apical and periapical areas. In same time will be performed a morphometric analysis to evaluate the extension of apical periodontitis in fluorescence microscopy. Then, sequential samples will be evaluated by histoenzimology for TRAP activity to identify and counting osteoclasts. Brown and Brenn staining for the bacteria presence and location and immunohistochemistry for osteoclastogenesis markers (RANK, RANKL and OPG) and cathepsin K will be also performed. The right jaw will be removed and submitted to gene expression detection of pro-inflammatory cytokines (IL-1², IL-6, IL-10 and TNF-±) and matrix metalloproteinases (MMP-1, MMP-8 and MMP-13) by means of real-time polymerase chain reaction (RT-PCR). The results will be statistically analyzed with a significance level of 5%. (AU)