Impact of type 2 diabetes and metformin treatment on ligature-induced bone loss, alveolar bone healing and ex vivo cytokines production: study in rats

Abstract

The aim of the present project is to evaluate the influence of type 2 diabetes mellitus on ligature-induced alveolar bone loss, bone healing after exodontia and the ex vivo cytokine production in non-diabetic and diabetic rats treated or not with metformin. One hundred twelve Wistar rats (n=14/group) were assigned to one of the following groups: Non-Diabetics without periodontics (ND), Non-Diabetic with Periodontitis (NDP), Non-Diabetic without Periodontitis and Treated (NDT), Non-Diabetic with Periodontitis and Treated (NDPT), Diabetics without Periodontitis (D), Diabetics without Periodontitis and Treated (DT), Diabetic with Periodontitis (DP), Diabetics with Periodontitis and Treated (DPT). Type-2 diabetes will be induced by fructose ingestion and streptozotocin administration. Animals from non-diabetic groups will receive only water while animals from the diabetic groups will receive water with fructose at the beginning of the study. At 14th day of experimental period, animals of the diabetic groups will be injected with streptozotocin and animals from the non-diabetic groups will receive only citrate buffer (vehicle). At 54th day after the beginning of experimental period and 40th day after diabetes induction, NDP, NDPT, DP, DPT groups will receive a ligature that will be maintained during 30 days. On 69th day after the beginning of the experiment, 55th day after diabetes induction and 15th day after ligature placement, the groups NDT, NDPT, DT, DPT will be treated with metformin (40mg/Kg of body weight) by oral via until the end of the experimental period. All animals will be submitted to tooth extraction at eight days before the end of experimental period i.e. 84th day. After euthanasia, the jaws and the maxillae will be removed, fixed, decalcified and submitted to routine histological procedure, while a spleen cells suspension will be obtained of the animals from all experimental groups. These cells will be in vitro stimulated with LPS from Escherichia coli. Subsequently the supernatant will be collected for immune-inflammatory mediators analysis: TNF-±, IFN-³, IL-12, IL-4, TGF-², IL-10, IL-6 and IL-17. Serial histological sections of the mandible will be stained to evaluate of the: alveolar bone loss and the stained cells for tartrate resistant acid phosphatase (TRAP), osteoprotegerin (OPG), receptor activator of NF-º² ligand (RANKL), osteopontin and osteocalcin. The maxillae will be removed to evaluat the bone healing after exodontia by histometry and gene expression of Sox9, RunX2 and Osterix. The results will be submitted to normality test to selected parametric (ANOVA) or non-parametric tests (Kruskal Wallis). The significance level for all analyses will be established at 5% (p<0.05). (AU)