In vitro action of intracanal medication to detoxify the effects of Enterococcus faecalis LTA in nitric oxide, G-CSF, IP-10 e MIP-1a by macrophages

Abstract

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 intracanal medications to neutralize the cytotoxic effects of LTA from Enterococcus faecalis in root canals, analyzing the production of nitric oxide, G-CSF, IP-10, and MIP- 1a by macrophages (RAW 264.7). Will be used 48 single-rooted human teeth with a 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 of solution of E. faecalis LTA (10 µL) in root canals, every 24 hours. After the root canals will be prepared with 5 NiTi rotary instruments (BioRaCe system), according to the 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) using apirogenic physiologic solution as irrigant. After EDTA application (3 min), the root will be divided into four groups (n = 12) according to the intracanal medication: CH) calcium hydroxide with saline solution; CLX) 2% chlorhexidine gel; CLX + CH) chlorhexidine + calcium hydroxide; Controle) apirogenic physiologic solution. The intracanal medication will remain for 14 days. The root canals will be irrigated with a physiologic solution and the samples will be obtained. These samples will be used to verify if the treatment protocols have the ability to neutralize the cytotoxic effects of LTA. 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, IP-10, and MIP-1a by enzyme immunoassay (ELISA). The results will be statistically analyzed (ANOVA and Tukey's test, 5%).(AU)