Análise microbiológica, de endotoxinas, de citocinas proinflamatórias e de metaloproteinases em infecções endodônticas primárias com lesões periapicais

Author(s)

Daniel Rodrigo Herrera

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2014-02-14

Examining board members

Brenda Paula Figueiredo de Almeida Gomes; Carlos Estrela; Antonio Paulino Ribeiro Sobrinho; Francisco José de Souza Filho; Alexandre Augusto Záia

• Advisor: Brenda Paula Figueiredo de Almeida Gomes

Abstract

Lipopolysaccharides (LPSs, known as endotoxins) present on the outer layers of Gram-negative bacterial envelope, and released during bacteria multiplication and death, can egress into periradicular tissues, acting as one of the most potent stimuli for immunocompetent cells in the release of inflammatory mediators and matrix metalloproteinases (MMPs). Thus, the objectives of this study were: 1) To evaluate the influence of primarily infected root canal contents (bacterial and endotoxin contents) on host-immune response by interleukin (IL)-1?, IL-1?, tumor necrosis factor ? (TNF-?), prostaglandin E2 (PGE2), matrix metalloproteinases (MMP) MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 production and to correlate their levels with clinical features (chapter 1); 2) To investigate the influence of 17% ethylenediaminetetraacetic acid (EDTA) ultrasonic activation after chemo-mechanical preparation (CMP) on eliminating/reducing oral bacterial lipopolysaccharide (LPS, known as endotoxins) and cultivable bacteria in teeth with pulp necrosis and apical periodontitis (chapter 2); 3) To investigate the endotoxin levels from primary endodontic infection before and after CMP, and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of MMPs (chapter 3). Methods: Root canal content samples were taken from 24 primarily infected root canals with apical periodontitis by using sterile/apyrogenic paper points. Samples were taken at different clinical times: S1- before CMP; S2- after CMP; S3- after EDTA: G1- with ultrasonic activation (n=12) and G2- without ultrasonic activation (n=12). PCR technique (16S rRNA) was used for the detection of the target bacteria. Culture techniques were used to determine the number of colony-forming units (CFU). Limulus Amebocyte Lysate (LAL) was used to measure endotoxin. Cytokines and MMPs levels were measured by enzyme-linked immunosorbent assay (ELISA) from samples that were taken passing 2 mm through the root apex after CMP. The levels of MMP-2 and MMP-9 gelatinolytic activity were measured using the zymography technique. The Pearson coefficient was used to correlate the amount of endotoxins with cytokine and MMP levels. Clinical data were set as dependent variables and correlated by individual correlation with each cytokine and MMP level. Friedman¿s and Wilcoxon tests was used to compare the amount of bacteria and endotoxin contents at each clinical time. Data obtained from gelatinolytic activity was analysed using Anova and Tukey¿s tests. The significance levels always were set at 5% (p<0,05). Results: IL-1?, IL-1?, TNF-? and PGE2, as well as, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 were detected in all samples. A correlation between endotoxin levels with cytokines and MMPs production was found (p<0.05). Root canal exudation was positively correlated with high levels of TNF-?, and symptomatic teeth were correlated with IL-1?, PGE2 and MMP-8 (p<0.05). CMP were effective in reducing endotoxins and bacterial load (p<0.05). Higher values of endotoxin reduction were achieved when EDTA received ultrasonic activation compared with the no-activation group (p<0.05). No differences were found in the bacterial load reduction after EDTA when comparing G1 (99.98%) and G2 (99.93%) (p>0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (p<0.05). No gelatinolytic activity of MMP-9 was observed. Conclusion: 1) Primarily infected root canal infection contents are potent stimuli factor for host-immune response by the production of IL-1?, IL-1?, TNF-?, PGE2, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13. 2) Although CMP was effective in reducing bacteria and endotoxins, it was not able to eliminate them from all root canals analyzed. The ultrasonic activation of EDTA was efficient in reducing even more the endotoxins levels in the root canals of teeth with pulp necrosis and apical periodontitis. 3) Root canal content from primary endodontic infection showed gelatinolytic activity for MMP-2 (AU)

• FAPESP's process: 10/13498-9 - Microbiological analysis and quantification of endotoxins and proinflammatory cytokines from primarily infected root canals with apical periodontitis
• Grantee: Daniel Rodrigo Herrera Morante
• Support Opportunities: Scholarships in Brazil - Doctorate