Detection and function of lipopolysaccharide and its purified lipid A after treatment with auxiliary chemical substances and calcium hydroxide dressings used in root canal treatment

Author(s)

Marinho, A. C. S. ^[1] ; To, T. T. ^[2] ; Darveau, R. P. ^[2] ; Gomes, B. P. F. A. ^[1]

Affiliation

^[1] State Univ Campinas UNICAMP, Piracicaba Dent Sch, Endodont Div, Dept Restorat Dent, Av Limeira 901 Bairro Areiao, BR-13414903 Piracicaba, SP - Brazil

^[2] Univ Washington, Sch Dent, Dept Periodont, Seattle, WA 98195 - USA

• Document type: Journal article
• Source: International Endodontic Journal; v. 51, n. 10, p. 1118-1129, OCT 2018.
• Web of Science Citations: 4

Abstract

AimTo investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide {[}Ca(OH)(2)] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4). MethodologyFusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH)(2) paste for various periods (1h, 24h, 7days, 14days and 30days); (iii) combination of substances: (a) 2.5% NaOCl (1h), followed by 17% EDTA (3min) and Ca(OH)(2) (7days); (b) 2% CHX (1h), afterwards, 17% EDTA (3min) followed by Ca(OH)(2) (7days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level. ResultsMatrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P.001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P<.0001). As for Ca(OH)(2), lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24h. LPS treated with Ca(OH)(2) was a weak TLR4 activator (P<.0001). From 24h onwards, no significant differences were found amongst the time periods tested (P>0.05). The addition of Ca(OH)(2) for 7days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4. Conclusion5.25% NaOCl and Ca(OH)(2) dressings from 24h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4. (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
• FAPESP's process: 15/23479-5 - Microbiomes and immunobiological aspects in endodontic infections
• Grantee: Brenda Paula Figueiredo de Almeida Gomes
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 13/17928-6 - Analysis of Porphyromonas gingivalis lipopolysaccharide when presente in a bacterial comunity
• Grantee: Ariane Cássia Salustiano Marinho
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate