Chemomechanical preparation influences the microbial community and the levels of LPS, LTA and cytokines in combined endodontic-periodontal lesions: A clinical study.

BACKGROUND: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]), before and after chemomechanical preparation (CMP) of the root canals (RC) and their associated periodontal pockets (PP) in teeth with combined EPL. MATERIALS: Samples were taken from 10 RC and PP, before and after CMP. The microbiomes (next-generation sequencing, V3-V4 hypervariable region of the 16S rRNA gene), microbiome diversity (bioinformatics analyses), LPS (limulus amebocyte lysate), LTA, IL-1ß, and TNF-α (ELISA) were evaluated. A statistical analysis was performed with significance level set at 5%. RESULTS: The most abundant phyla in both sites were Firmicutes and Proteobacteria. Comparative studies of bacterial genera species revealed that some increased and others decreased after CMP at both sites. A 3% reduction in Gram-negative bacteria (RC) and a 4% increase in Gram-positive bacteria (PP) were detected. LPS levels were 4.4 times higher in PP than in the RC. LTA was detected in all samples investigated. Higher levels of IL-1ß and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1ß and TNF-α were reduced in both sites. CONCLUSION: The microbial community in the RC and PP in teeth with combined EPL indicated a similarity between both sites. CMP effectively reduced the microbial load and the LPS levels from teeth with EPL, and consequently diminished the cytokine levels. The reduction in LTA levels in the RC and PP proved challenging.

Accuracy of Turbidimetric Limulus Amebocyte Lysate Assay for the Recovery of Endotoxin Interacted with Commonly Used Antimicrobial Agents of Endodontic Therapy.

INTRODUCTION: This study was conducted to investigate whether the interaction between the turbidimetric limulus amebocyte lysate (LAL) substrate for endotoxin measurement and the substances/antimicrobial agents used in endodontic therapy can lead to the inhibition/enhancement of endotoxin recovery. METHODS: Ten microliters of a suspension of Escherichia coli endotoxin (O55:B55) was inoculated and kept in contact for 1 hour with different substances categorized as follows: group 1: auxiliary chemical substances: 5.25% and 2.5% sodium hypochlorite solutions, 2% chlorhexidine (CHX) (gel and solution), 1% Natrosol gel (Drogal Chemicals and Pharmaceuticals Ltd, Piracicaba, SP, Brazil), 17% EDTA, 10% citric acid, 3% hydrogen peroxide, 5% sodium thiosulfate, and 0.5% Tween 80 associated with 0.07% soy lecithin (Drogal Chemicals and Pharmaceuticals Ltd) and group 2: intracanal medications: neomycin/polymyxin B/hydrocortisone (Otosporin; Glaxo Wellcome, Rio de Janeiro, RJ, Brazil); calcium hydroxide (Ca[OH]2); Ca(OH)2 + 2% CHX gel; Ca(OH)2 + 2% CHX gel + zinc oxide eugenol; Ca(OH)2 + camphorated paramonochlorophenol (Calen; S.S. White, Rio de Janeiro, RJ, Brazil); triple antibiotic paste; mineral trioxide aggregate (MTA); and iodoform. Positive and negative controls consisted of root canal hemorrhagic exudate and pyrogen-free sterile water, respectively. All samples were diluted up to a 10:4 dilution. Each dilution was individually examined by the turbidimetric LAL assay. Collected data were analyzed through performance characteristics of the LAL assay such as linearity, coefficient of variation percentage, and product positive control (PPC) values. RESULTS: Correlation coefficient (≥0.980) and coefficient of variation percentage (<10%) of the standard curve in triplicate showed the tests' linearity. Spike recovery of auxiliary chemical substances achieved PPC values ranging from 50%-197%, showing no interferences with LAL substrate. Conversely, 3% hydrogen peroxide achieved product inhibition in which endotoxin values were underestimated even after the 10:4 dilutions. Regarding intracanal medicaments, neomycin/polymyxin B/hydrocortisone also inhibited endotoxin detection in all dilutions investigated (PPC values <50%). In contrast, Ca(OH)2 + 2% CHX gel + ZOE as well as triple antibiotic paste led to the enhancement of endotoxin detection in which endotoxin values could not be validated by the turbidimetric kinetic LAL assay (PPC value >200%). CONCLUSIONS: The performance characteristics of the kinetic turbidimetric assay for endotoxin measurement, such as precision and reproducibility, are modulated by the interaction of the LAL substrate with the substances/antimicrobial agents used in endodontic therapy.

Proinflammatory Activity of Primarily Infected Endodontic Content against Macrophages after Different Phases of the Root Canal Therapy.

INTRODUCTION: This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1ß and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. METHODS: Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1ß/TNF-α measurement using enzyme-linked immunosorbent assay. RESULTS: Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1ß and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1ß and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). CONCLUSIONS: Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages.

Clinical investigation of the efficacy of chemomechanical preparation with rotary nickel-titanium files for removal of endotoxin from primarily infected root canals.

INTRODUCTION: This clinical study was conducted to investigate the ability of chemomechanical preparation with 2.5% NaOCl + 17% ethylenediaminetetraacetic acid (EDTA) and rotary nickel-titanium (NiTi) system in removing endotoxin from primary root canal infection with apical periodontitis. METHODS: Twenty-one root canals with necrotic pulps were selected. Samples were collected before (s1) and after chemomechanical preparation (s2). The limulus amebocyte lysate (LAL) assay was used to quantify endotoxins. RESULTS: The LAL assay indicated that endotoxins were present in 100% of the root canals investigated (19/19) before (s1) and after chemomechanical preparation (s2). Analyses of the quantitative data revealed that the endotoxin content was significantly reduced at s2 (98.06%) compared with that at s1 (P < .05). CONCLUSIONS: Our findings indicate that chemomechanical preparation with 2.5% NaOCl + 17% EDTA and rotary NiTi files was effective in reducing endotoxin load in the root canal infection from primarily infected teeth with apical periodontitis.