Investigation of microorganisms and endotoxins in primary endodontic infection and evaluation of antigenicity infectious content against macrophages by the levels of pro-inflammatory cytokines

Author(s)

Frederico Canato Martinho

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2011-06-05

Examining board members

Brenda Paula Figueiredo de Almeida Gomes; Milton de Uzeda; Helena Rosa Campos Rabang; Ezilmara Leonor Rolim de Sousa; Alexandre Augusto Záia

• Advisor: Brenda Paula Figueiredo de Almeida Gomes

Abstract

Gram-negative bacteria (G-ve) and its by-product [Lipopolysaccharide (LPS) - endotoxin] are capable to stimulate cells to release proinflammatory cytokines that lead to tissue destruction. The aims of this study were: 1) to determine which of the quantitative methods, namely, chromogenic endpoint, chromogenic kinetic and turbidimetric kinetic ones, best fit for the analysis of primary endodontic infections (chapter 1); 2) to investigate the microbial profile and the levels of endotoxin found in primary root canal infection with apical periodontitis (PEIAP), and to determine their antigenicity against macrophages through the levels of IL-1ß and TNF-alpha, evaluating their relationship with clinical and radiographic findings (chapter 2); 3) to investigate target G-ve bacteria species and endotoxin in PEIAP, determining their antigenicity against macrophages through the levels of PGE2 and evaluated their relationship with clinical findings (chapter 3); 4) investigation of Treponema spp. and endotoxin in primary endodontic infection and evaluation of the antigenicity of the infectious content against RAW 264.7 macrophages by the levels of IL-6 and IL-10 production (chapter 4); 5) to evaluate the antigenic activity of LPS purified from P. gingivalis and F. nulceatum isolated from infected root canals on macrophages cells (RAW 264.7) by the levels of IL-1? and TNF-?. (chapter 5); 6) to compare the efficacy of chemomechanical preparation with 2.5% NaOCl and 2% CHX-gel on eliminating oral bacterial LPS in teeth with PEIAP (chapter 6); 7) to investigate the ability of chemo-mechanical preparation (CMP) with 2.5% NaOCl + 17% EDTA and rotary NiTi system Mtwo® in removing endotoxin from PEIAP (chapter 7); 8) Comparison of different clinical sequences of NiTi rotary files Mtwo® in the removal of endotoxin from infected root canals (chapter 8). Methods: Samples were taken from root canals with PEIAP with paper points. PCR technique (16S rDNA) was used for the detection of the target bacteria. Limulus Amebocyte Lysate (LAL) was used to measure endotoxin. The amounts of IL-1ß, TNF-alpha and PGE2 in macrophages supernatants were measured by enzyme-linked immunosorbent assay - Duoset-kit (ELISA). Results: The KQCL and Turbidimetric -assay yielded a median value of endotoxin of 7.49 EU/mL and 9.19 EU/mL respectively, significantly different from the endpoint-QCL (34.20 EU/mL) (p<0.05). Prevotella nigrescens (13/21) was the most frequently Gram-negative bacteria species detected. Tooth with radiolucent area ? 2mm was related to Treponema denticola. Correlation was found between the number of Gram-negative bacteria and the levels of IL-1ß, TNF-alpha and PGE2 (p<0.05). Increased levels of endotoxin were followed by TNF-alpha release (p<0.05). Higher levels of IL-1ß (p<0.05) and endotoxin contents were related to the larger size of radiolucent area. Elevated levels of PGE2 were found in teeth with tenderness to percussion and pain on palpation. Endotoxin was detected in 100% of the root canals investigated. Higher percentage value of endotoxin reduction was found in 2.5% NaOCl (57.98%) when compared to 2% CHX-gel (47.12%) (p<0.05) using manual K-files. After chemo-mechanical preparation with 2.5% NaOCl and rotary NI-TI files endotoxin was significantly reduced to 98.06% (p<0.05). Conclusion: 1) Quantitative kinetic-turbidimetric and kinetic-chromogenic LAL methods are best fitted for analysis of endotoxin in root canal infection, both being more precise and allowing better reproducibility compared to the endpoint-QCL assay; 2) The antigenicity of the endodontic contents is not related to only the amount of endotoxin found in root canal, but also with the number of different species of Gram-negative bacteria involved in the infection. Moreover larger size (? 2mm) of radiolucent area was related to IL-1ß and endotoxin; 3) Additive effect between the number of G-ve bacterial species involved in endodontic infection regarding the induction of pro-inflammatory cytokine by macrophage cell. Moreover, teeth with clinical symptomatology were related to higher levels of endotoxin and PGE2 secretion; 4) a wide variety of Treponema species do play a role in primary endodontic. Moreover, the bacterial endodontic contents, particularly the levels of endotoxin present in root canals, were a potent stimuli for the production IL-6 and IL-10 in macrophages; 5) LPS of the P. gingivalis and F. nucleatum isolated from root canal infection is involved in the induction of IL-1 ? and TNF-?, which are pleiotropic inflammatory mediators, that can play a role in the initiation of the upregulation of the inflammatory response and can also stimulate the production of secondary mediators involved in tissue destruction; 6) 2.5% NaOCl and 2% CHX-gel were not effective on eliminating endotoxin from the primarily infected root canals using manual K-files; 7) CMP with 2.5% NaOCl + 17% EDTA and rotary NiTi files was effective in reducing 98.06% of endotoxin from PEIAP; 8) substantial reduction of endotoxin contents was achieved by using the Mtwo® sequences finished in APS #40.04 or #25.07 (AU)