Clinical evaluation of culturable bacteria, endotoxins and lipoteichoic acid in teeth with vital normal pulp tissues.

The objective of this study was to investigate the presence of culturable bacteria, endotoxins (LPS) and lipoteichoic acid (LTA) levels in teeth with normal vital pulp (NVP) with intact crowns (IC) and those with coronal restoration (CR) limited to the enamel level. A total of 20 teeth indicated for endodontic treatment due to prosthetic reasons were selected. Samples were collected from the root canals. The levels of cultivable bacteria, LPS and LTA were assessed. Statistical analyses were performed at significance level set at 5%. None of the teeth presented microbial growth. In the IC group, the LPS levels were limited to the lowest concentration of LPS. On the contrary, higher LPS and LTA levels were detected in teeth with CR. It was concluded that teeth with NVP and IC were negative for bacteria, LPS and LTA; while teeth with CR were positive for bacterial virulence factors.

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.

Efficacy of 6% Sodium Hypochlorite on Infectious Content of Teeth with Symptomatic Irreversible Pulpitis.

INTRODUCTION: The objective of this study was to monitor the effects of chemomechanical preparation (CMP) performed with 6% sodium hypochlorite and calcium hydroxide-based intracanal medication (ICM) on the levels and diversity of bacteria, endotoxins (lipopolysaccharides [LPS]), and lipoteichoic acid (LTA) in root canals of teeth with symptomatic irreversible pulpitis. METHODS: Samples were collected from 10 teeth with symptomatic irreversible pulpitis before CMP (S1), after CMP (S2), and after ICM (S3). The levels of bacteria, LPS, and LTA were assessed by using checkerboard DNA-DNA hybridization, LAL Pyrogent 5000, and enzyme-linked immunosorbent assay, respectively. Wilcoxon test, repeated-measures analysis of variance, and Tukey post hoc test were used for statistical analysis at a significance level of 5%. RESULTS: Forty species were detected at S1. Two species were eliminated after CMP and 5 after ICM. Resistant and pain-related species were detected in the root canals. Higher levels of culturable bacteria were detected at S1. However, CMP and ICM effectively reduced the microbial load in the root canals. Higher levels of LPS and LTA were detected at S1. CMP was effective in reducing both LPS and LTA (P < .05). ICM produced additional reduction in the levels of LPS (P > .05) and LTA (P < .05). CONCLUSIONS: Chemomechanical preparation using 6% sodium hypochlorite and calcium hydroxide-based intracanal medication were effective in reducing the levels of bacteria, LPS, and LTA in teeth with vital pulp and irreversibly inflamed pulp.

Influence of Bacterial Profiles in Cytokine and Clinical Features of Endodontic Disease.

INTRODUCTION: We verified the association between selected bacterial profiles and levels of cytokines, chemokines, and the expression of signs and symptoms of primary endodontic infection with apical periodontitis. METHODS: Samples were collected from 21 root canals, and macrophages were stimulated for 24 hours. Tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-10, IL-12p70, interferon gamma, and chemokine (C-C motif) ligand 2 (CCL2) were measured using cytometric bead array. We investigated the overlapping networks between cytokines and chemokines with regression analysis. Checkerboard DNA-DNA hybridization was used to assess 40 target bacteria species. Using factor analysis, bacterial species aggregated in 2 factors. The association of bacteria species-based factors on cytokine and chemokine levels and clinical features was estimated with regression analysis. RESULTS: A negative relationship between IL-10 (anti-inflammatory cytokine) and CCL2, TNF-α, and IFN-γ (proinflammatory cytokines) (all P < .05) was observed. CCL2 was positively correlated with TNF-α (P < .01). Thirty-eight bacteria species were detected in primary endodontic infection with apical periodontitis. The first bacteria species-based factor was associated with the size of the radiolucent area (coefficient = 15.42) and tenderness to percussion/pain on palpation (coefficient = 20.79). The second factor was associated with CCL2 levels (coefficient = 1.28). CONCLUSIONS: Different bacterial profiles can be differentially related to the expression of inflammatory proteins and the experience of clinical features.

Microbiological analysis of endodontically treated teeth with apical periodontitis before and after endodontic retreatment.

OBJECTIVE: To characterize the microbiota of teeth with endodontic treatment failure by 16S ribosomal RNA genetic sequencing (GS) and PCR at the different phases of the endodontic retreatment and to associate the presence of specific bacteria with clinical and radiographic features in teeth with apical periodontitis. MATERIALS AND METHODS: Twenty infected root canals of single-rooted teeth were selected. Samples were collected with sterile paper points before chemo-mechanical preparation (CMP) (S1), after CMP (S2) and after 30 days of intracanal medication (ICM) (S3). Microbial identification was performed using GS and PCR. Tukey-Kramer post hoc test and post hoc ANOVA were used for intergroup analysis. Paired t test and repeated-measures ANOVA were applied for intragroup analysis, at a significance level of 5%. RESULTS: A total of 89 strains were identified using GS. Sixty-five strains were recovered in S1 and 15 strains in S2, and 9 strains remained in S3. Enterococcus faecalis was the most predominant bacteria. Gram-positive cocci bacteria predominated. Gram-negative species were also detected. Using species-specific PCR primers to detect seven species, the most prevalent ones at all the phases of the endodontic retreatment were E. faecalis and Porphyromonas gingivalis. However, Parvimonas micra and P. gingivalis were associated with previous pain, P. gingivalis was associated with tenderness to percussion and E. faecalis, Fusobacterium nucleatum and P. gingivalis were associated with periapical lesion > 3 mm. CONCLUSIONS: In conclusion, the microbiota of persistent infection is polymicrobial with predominance of E. faecalis and P. gingivalis in all phases of the endodontic retreatment, regardless of the method used for microbial identification. Associations were found between specific bacteria and clinical/radiographic features. CLINICAL RELEVANCE: The characterization of the bacteria present at all phases of the endodontic retreatment is important for the monitoring of the effectiveness of the techniques used and to better understand the susceptibility of these species to the disinfection agent used during the procedures.

Investigation of Filifactor alocis in primary and in secondary endodontic infections: A molecular study.

OBJECTIVE: Identification of specific bacteria in root canals (RCs) in distinct clinical conditions can support the comprehension of pathological processes. Thus, the objective of this clinical study was to investigate the presence of F. alocis in RCs of teeth with primary endodontic infection (PEI) and with persistent/secondary endodontic infection (SEI) by using molecular techniques. It was also aimed to associate its presence with the clinical features. In addition, the levels of F. alocis as well as the total bacterial cells in the samples were also quantitated. DESIGN: One hundred teeth (50 PEI and 50 SEI) were included. Microbial samples were performed using sterile paper points and assessed by using nested PCR and quantitative Real Time PCR (qPCR). The prevalence of F.alocis in RCs from PEI and SEI were compared by chi-square analysis. Fisher´s exact test or Pearson Chi-square, when appropriate, was used to test associations between clinical and radiographic features and the presence of F. alocis. Significance level was set at 5%. RESULTS: F. alocis was detected in 23 and 28 (PEI) and 12 and 11 (SEI) RCs using Nested PCR and qPCR, respectively. Statistically significant associations were found between the presence of F. alocis and PEI, pain, wet canals, swelling, abscess and purulent exudate (P < 0.05). Total bacterial count was similar in both conditions (P > 0.05). CONCLUSIONS: PEI harbour a significantly higher number of F. alocis than those with SEI. Filifactor alocis was significantly associated with clinical features in primary endodontic infections. Total bacterial count was similar in both clinical conditions.

Clinical Investigation of Microbial Profile and Levels of Endotoxins and Lipoteichoic Acid at Different Phases of the Endodontic Treatment in Teeth with Vital Pulp and Associated Periodontal Disease.

INTRODUCTION: The aim of this in vivo study was to investigate the microbial profile as well as the levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) at different phases of endodontic treatment in teeth with vital pulp and associated periodontal disease. METHODS: Ten patients were selected for this clinical study. Samples were taken from periodontal pockets (PPs) and root canals (RCs) using sterile paper points before and after chemomechanical preparation and after intracanal medication. For microbiological analysis, nested polymerase chain reaction and checkerboard DNA-DNA hybridization were used. Levels of LPS and LTA were assessed using limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively. Data were statistically analyzed at a significance level of 5%. RESULTS: Bacterial DNA from 17 of the 17 species investigated was detected in samples of PPs, whereas 6 of the 17 species were not present in the initial samples of RCs using nested polymerase chain reaction. In the initial samples, 38 of 40 probes were detected in PPs, whereas 12 of 40 probes were detected in RCs using checkerboard DNA-DNA hybridization. Overall, endodontic procedures were efficient in modifying the microbiota of PPs and RCs. Levels of LPS and LTA were reduced after the endodontic procedures, although higher concentrations of both had been found in PPs compared with RCs. CONCLUSIONS: The microbiota of PPs and RCs in teeth with vital pulp and associated periodontal disease is polymicrobial, with the presence of gram-negative, gram-positive, facultative, and strict anaerobes. Chemomechanical preparation and calcium hydroxide-based intracanal medication allowed the reduction of infectious content in both sites.

Microbiota of periodontal pockets and root canals in induced experimental periodontal disease in dogs.

AIM: To investigate the relationship between the microbiota of periodontal pockets (PP) and root canals (RC) in dogs submitted to experimental periodontal disease (ExPD). METHODS: ExPD was induced by combining cotton and wire ligatures. After 125 days, microbiological samples were collected from PP and RC. Strains isolated from 19 teeth were submitted to DNA extraction, 16S rRNA gene amplification and gene sequencing. Pearson's χ2 - and Fisher's exact tests and McNemar's test were used when appropriate. RESULTS: The number of species in PP was greater than in RC, with prevalence of obligate anaerobes and Gram-negative bacteria. In the PP predominated Fusobacterium necrophorum, Porphyromonas gingivalis, Prevotella loescheii, Campylobacter gracilis and Veillonella parvula. In the RC samples, 9 had microbial growth, with predominance of the following genera: Staphylococcus, Streptococcus and Neisseria. Eight genera were common to both sites in the same tooth. PP presented a greater number of species than the RC. No significant difference was observed in the species found in PP and RC in the same tooth. CONCLUSION: Microbial composition of the RC could be modulated by the presence of periodontal disease, especially in cases of severe periodontal destruction. RC microbiota was less complex and diverse than the PP.

Anti-inflammatory potential of a carvedilol gel in the pulpal tissue of rats after dental bleaching: A histopathological evaluation.

AIM: Carvedilol is an antioxidant that decreases inflammation in periodontitis. The hydrogen peroxide (H2 O2 ) of bleaching gel causes inflammation and necrosis of the dental pulp. In the present study, we evaluated the anti-inflammatory potential of carvedilol in the pulp of rats after bleaching. METHODS: The right upper molars of rats received 35% H2 O2 (1× 30 minutes), and the left upper molars were used as the control. Half of the rats received carvedilol gel (1× 10 minutes), forming the following groups: bleached, bleached followed by carvedilol (bleached+carvedilol), and control. After 2 and 30 days (N = 7 hemi-maxillae/group), the rats were killed for histological evaluation, and statistical tests were performed (P < 0.05). RESULTS: After 2 days, the bleached group showed necrosis in the occlusal third of the coronal pulp, and in the bleached+carvedilol group, severe inflammation (P > 0.05), both different from the control (P < 0.05). In the middle third, the bleached group showed severe inflammation, and the bleached+carvedilol group showed moderate inflammation (P > 0.05), with the only difference between the bleached and control groups (P < 0.05). In the cervical third, the bleached group showed moderate inflammation, and the bleached+carvedilol group showed mild inflammation (P > 0.05). The difference again was only between the bleached and control groups (P < 0.05). At 30 days, there was no inflammation and a marked amount of tertiary dentin in bleached teeth (P > 0.05). CONCLUSION: Carvedilol gel has the potential of minimizing H2 O2 damage, especially in deep regions of the dental pulp of rats after bleaching.