Investigation of LPS pathobiology in endodotic infections and its susceptibility to different therapeutic modalities

Author(s)

Ariane Cassia Salustiano Marinho

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2016-11-18

Examining board members

Brenda Paula Figueiredo de Almeida Gomes; Daniel Pinto de Oliveira; Fábio Renato Manzolli Leite; Frederico Canato Martinho; Rogerio de Castilho Jacinto

• Advisor: Brenda Paula Figueiredo de Almeida Gomes

Abstract

Clinical strategies must be performed in order to achieve major reduction of the microbial load, as well as the elimination and/or inactivation of lipopolysaccharides (LPS) and other toxic products and, therefore, reducing the inflammatory potential of endodontic content to periapical tissues. The objectives of this study were: 1); To assess the effectiveness of Reciproc (R25) system compared to different rotary systems in removing bacteria and endotoxin from contaminated root canals (Article 1); To investigate the influence of the interaction of antimicrobial substances used in endodontic therapy and Limulus amebocyte lysate (LAL) substrate in order to identify interfering substances for endotoxin measurement in clinical samples (Article 2); To monitor the effectiveness of the chemol-mechanical preparation (CMP) by testing 2.5% NaOCl and 2% Chlorhexidine (CHX) gel 2% (using saline [SS] as a control,) after the use of 17% EDTA and calcium hydroxide paste, elucidating the contribution of each step of the endodontic therapy in the reduction of endotoxins (article 3);To determine target bacterial species and endotoxin levels in primary endodontic infections and the inflammatory potential of endodontic content against macrophages through IL1- ß e TNF-? production. Elimination of LPS was assessed by the chemo-mechanical preparation (CMP) with 2.5% NaOCl or 2% Chlorhexidine (CHX) gel and after the use of 17% EDTA and intracanal dressing, using saline solution (SS) as a control (Article 4); To investigate the influence of auxiliary chemical substances of endodontic therapy and calcium hydroxide dressings on the structure and bioactivity of bacterial lipopolysaccharide, and thus to elucidate the occurrence of Lipid A/LPS structural alterations and their further functional response in TLR-4 (Article 5)-. Methods: Samples were collected from root canals using sterile/ apirogenic paper points. PCR (16S rDNA), counting of colony forming units and LAL method were performed. IL1- ß e TNF-? produced by macrophages were measured by ELISA. Structural analysis of the lipid A/LPS was conducted by means of MALDI-TOF mass spectrometry and silver staining, respectively. TLR-4 activation was performed to determine the immunostimulatory potential of LPS after different treatments. Results: Article 1- No statistical differences were found between the Reciproc R25 and the different rotary systems for bacterial and endotoxin removal, respectively: Reciproc (99.34% and 91.69%); GII - Mtwo (99.86% and 83.11%); GIII- ProTaper (99.93% and 78.56%); and GIV- FKG RaceTM (99.99% and 82.52%) (p> 0.01). Article 2- Hydrogen peroxide 30%, Otosporin® and triple antibiotic paste caused inhibition/ enhancement of endotoxin levels in all dilutions. Article 3- Irrespective of the irrigant, CMP was able to significantly reduce LPS levels: NaOCl 2.5% (99.65%) (GI), CHX 2% (94.27%) (GII), and SS (96.79%) (GIII) (p <.05). Calcium hydroxide paste for 30 days contributed to enhance endotoxin reduction: NaOCl 2.5% (90%) (GI), CHX 2% (88.8%) (GII) and SS (85.7%) (GIII, p <.05). Article 4- Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30) and Prevotella nigrescens (11/30) were the most prevalent bacterial species in primary endodontic infections. Endotoxins were present in 100% of root canals. CMP drastically reduced endotoxins (p<.05). 2.5% NaOCl associated to rotary instrumentation obtained the highest reduction (NaOCl 2,5%= 0,09 EU/mL; CHX 2%= 0,81 EU/mL; SS= 4,81 EU/mL). After rinse with 17% EDTA and intracanal dressing no statistical differences were found between groups (p >.05). IL-1? and TNF-? were produced by macrophages stimulated with initial endodontic content. A proportional decrease of cytokine levels was found after each root canal procedure (p <0.05). Article 5- 5.25% NaOCl and Ca(OH)2 induced both loss of peak of lipid A and LPS detection, while no structural alteration was observed after LPS treatment with other auxiliary chemical substances. In parallel, LPSs treated with 5.25% NaOCl and Ca(OH)2 were significantly less potent in activating TLR4 when compared to other substances (p <0.0001). It was concluded that: Reciproc (R25) was as effective as the rotary multifile systems for bacteria and endotoxin removal from contaminated root canals (Article 1); The performance characteristics - accuracy and reproducibility- of the turbidimetric kinetic assay for quantification of endotoxins are influenced by the interaction of LAL substrate with Hydrogen peroxide 30%, Otosporin® and triple antibiotic paste (Article 2); High endotoxin reduction occurs after chemical-mechanical preparation and after the use of calcium hydroxide paste (Article 3). Additional endodontic procedures enhance endotoxin reduction (Article 3). Consequently, it was found a progressively lower activation of macrophages for IL-1? and TNF-? production (Article 4); 5.25% NaOCl and calcium hydroxide paste were capable of inducing loss of lipid A peaks and LPS detection, rendering a lower immunostimulatory potential against TLR-4 (Article 5) (AU)

• FAPESP's process: 13/02402-9 - Microbiological and LPS investigation and their antigenic activity in teeth with pulpal necrosis and irreversible pulpitis in pro-inflammatory cytokine production
• Grantee: Ariane Cássia Salustiano Marinho
• Support Opportunities: Scholarships in Brazil - Doctorate