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Efficacy of instrumentation rotary with different auxiliary chemical substances in neutralizing the toxic effects of lipotheicoic acid (LTA) from Enterococcus faecalis in root canals


Gram-positive bacteria release as an important virulence factor lipoteichoic acid (LTA), which is structurally and immunologically similar to lipopolysaccharide (LPS) of Gram-negative bacteria. The aim of this study is to evaluate the efficacy of rotary instrumentation with nickel-titanium instruments associated with different auxiliary chemical substances (1% sodium hypochlorite, 2% chlorhexidine gel and glycolic extract of propolis 12%) and intracanal medications to neutralize the cytotoxic effects of LTA from Enterococcus faecalis in root canals, analyzing the production of nitric oxide, G-CSF and cytokines (IL-1², IL-6, IP-10, TNF-± and MIP- 1±) by macrophages (RAW 264.7). Will be used 144 single-rooted human teeth with standardized size of 16 mm. Root canals will be prepared until the instrument BR2 (25/0.04) and the specimens will be distributed on plates (n = 12). After sterilization (Co60 gamma radiation), will be performed three repeated inoculations every 24 hours, 10 ¼L of solution of E. faecalis LTA in root canals. After the root canals will be prepared with 5 NiTi rotary instruments (BioRaCe system), according to manufacturer's specifications, the following sequence: BR3 (25/0.06), BR4 (35/0.04), BR5 (40/0.04); BR6 (50/0.04) and BR7 (60/0.02). According to the auxiliary chemical substance used, the specimens will be divided into 4 experimental groups (n = 36): a) Group 1: 1% sodium hypochlorite b) Group 2: 2% chlorhexidine gel + saline; c) Group 3 : glycolic extract of propolis 12% d) group 4 (control): saline. Then be applied EDTA (3 min) and each group will be divided into three subgroups (n = 12) according to the dressing (MIC): A) calcium hydroxide with saline solution, B) 2% chlorhexidine gel (CLX ) C) + CLX calcium hydroxide. The MIC will remain for 14 days. In total, three collections will be made of the root canal: 1) immediately after instrumentation; 2nd) after EDTA, 3rd) after removal of the MIC. These samples will be used to verify if the treatment protocols have the ability to neutralize the cytotoxic effects of LTA. To this end, macrophages (RAW 264.7) will be activated with the samples collected from the root canals, and after 24 hours, the supernatants will be used to verify the production of nitric oxide (Griess method), G-CSF and cytokines (IL-1², IL -6, IP-10, TNF-± and MIP-1±) by enzyme immunoassay (ELISA). The results will be statistically analyzed (ANOVA and Tukey's test, 5%). (AU)

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