The main virulence factor of gram-negative bacteria is represented by the release of endotoxin (LPS) presented in the outer layers of their cell walls. The accumulation of these bacterial components in the root canal and periradicular tissues should stimulate macrophages present in the area to produce cytokines such as IL-1 and TNF-± which are potent stimulators of bone resorption. The aim of this study is to isolate and identify gram-negative anaerobic species through biochemical methods and PCR from root canals of teeth with necrotic pulp and apical periodontitis, evaluating the effect of chemomechanical preparation (CMP) with 2% chlorhexidine gel and 17% EDTA, using or not ultrasound. The reduction of microorganisms and LPS, correlating the microbiota and endotoxin content with the expression of pro-inflammatory cytokines IL-1a, IL-1b, TNF± and PGE-2. Twenty-four root canals were selected with pulp necrosis and presence of periapical lesion. Endotoxins and microbiological samples will collect Samples were collected before and after CMP, before and after using 17% EDTA, with or without application of ultrasound, and finally after seven days of the first session. Samples for cytokines quantification will also collect by passing a paper point 1 mm beyond the apex, after the PQM and after 7 days, The microbiological samples will be diluted, plated and incubated for further isolation and identification. PCR will be performed using specific primers. A limulus amebocyte lysate (LAL) assay will be used to quantify endotoxins Cytokines collected from apex will be quantified using specific kits. The values obtained will be tabulated and statistically analyzed.
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