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. (2nd April 2018)

• Record Type: Journal Article
• Authors: Marinho, A. C. S.; To, T. T.; Darveau, R. P.; Gomes, B. P. F. A.
• Is Part Of: International endontic journal. Volume 51:Number 10(2018)
• Page Start: 1118
• Page End: 1129
• Publication Date: 2018-04-02
• Subjects: chlorhexidine -- endodontics -- ethylenediaminetetraacetic acid -- lipid A -- lipopolysaccharide -- sodium hypochlorite; Endodontics -- Periodicals; 617.6342
• DOI: 10.1111/iej.12920
• Languages: English
• ISSNs: 0143-2885

Abstract

Abstract: Aim: To 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). Methodology: Fusobacterium 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 (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). 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‐wayanova with Tukey–Kramer post‐hoc tests at the 5% significance level. Results: Matrix‐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 24 h. LPS treated with Ca(OH)2 was a weak TLR4 activator ( P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested ( P > 0.05). The addition of Ca(OH)2 for 7 days 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. Conclusion: 5.25% NaOCl and Ca(OH)2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4.