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

Aim To investigate the influence of auxiliary chemical substances ( ACS s) and calcium hydroxide [Ca( OH ) 2 ] dressings on lipopolysaccharides ( LPS )/lipid A detection and its functional ability in activating Toll‐like receptor 4 ( TLR 4). Methodology Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS : 5.25%, 2.5% and 1% sodium hypochlorite solutions (Na OC l), 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% Na OC l (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. TLR 4 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. 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 TLR 4 activation when compared to ACS ( P  ≤ .001). 5.25% Na OC l 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% Na OC l‐treated LPS did not activate TLR 4 ( 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 TLR 4 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% Na OC l or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR 4. Conclusion 5.25% Na OC l 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 TLR 4.