Quantitative analysis of culturable bacteria, levels of endotoxins, inflammatory mediators and substance P in teeth with symptomatic irreversible pulpitis and in teeth with vital normal pulp tissues.

AIM: To comparatively analyse the levels of culturable bacteria, endotoxins (LPS), tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and substance P in teeth with symptomatic irreversible pulpitis (SIP) and vital normal pulp (VNP) tissues. METHODOLOGY: Thirty-two patients were included (20 teeth with SIP and 12 teeth with VNP tissues) in this cross-sectional study. Samples were collected from the full length of the root canals (microbial analysis) and periapical tissues (2 mm beyond the apex for immunological analysis), using sterile absorbent paper points. The levels of culturable bacteria (culture method), endotoxins (LAL Pyrogent 5000), TNF-α, IL-1ß and substance P (ELISA) were assessed. The Mann-Whitney test was used for comparisons between the levels of CFU/mL, LPS, TNF-α, IL-1ß and substance P in the SIP and VNP groups. The statistical analysis was performed with the significance level set at 5%. RESULTS: Culturable bacteria were recovered from all teeth with SIP. On the other hand, no positive cultures were observed in the VNP tissues group (p > .05). The levels of LPS were approximately four times higher in teeth with SIP than in teeth with VNP tissues (p < .05). Higher levels of TNF-α and substance P were detected in teeth with SIP (p < .05). On the other hand, no difference in the levels of IL-1ß was detected between the two groups (p > .05). CONCLUSION: Teeth with symptomatic irreversible pulpitis present higher levels of culturable bacteria, endotoxins, TNF-α and substance P than those with vital normal pulp tissues. On the other hand, the levels of IL-1ß were similar in teeth from both groups suggesting reduced implications of this inflammatory mediator in the early stages of infection.

Bacteria and virulence factors in periapical lesions associated with teeth following primary and secondary root canal treatment.

AIM: This cross-sectional study aimed to investigate the microbial profile and to quantify the levels of endotoxins (LPS) and lipoteichoic acid (LTA) present in periapical lesions associated with root filled teeth and those that had received root canal retreatment. It also aimed to investigate the association between microorganisms and their virulence factors with clinical and radiographic features. METHODOLOGY: Patients with periapical lesions in teeth with post-treatment endodontic disease following primary root canal treatment (n = 19) and unsuccessful root canal retreatment (n = 13) were treatment planned for endodontic microsurgery, where the periapical lesions were collected. Clinical and radiographic data were also collected. For microbiological analysis, nested polymerase chain reaction was used to detect 17 bacterial species. Levels of LPS and LTA were determined using limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively. The Student t-test or Wilcoxon-Mann-Whitney tests were applied to compare the data on LPS and LTA with clinical and radiographic features. The associations between the clinical and radiographic features and the bacterial species were analysed using the Fisher's exact test. A significance level of 5% was adopted. RESULTS: Bacterial DNA, LPS and LTA were detected in all samples. Parvimonas micra was the most commonly detected species in all groups, followed by Enterococcus faecalis, Fusobacterium nucleatum and Porphyromonas endodontalis. The type of endodontic treatment, whether a primary root canal treatment or retreatment, was not associated with the presence of any bacterial species in periapical lesions. The levels of LPS and LTA in periapical lesions of root filled teeth were not significantly different from those that had been retreated. Associations between the levels of LPS and LTA with clinical signs and symptoms were found. No association was found between specific bacteria and clinical features. CONCLUSION: Periapical lesions associated with teeth after primary root canal treatment and retreatment had similar polymicrobial composition. The levels of LPS and LTA in periapical lesions associated with teeth after primary root canal treatment and retreatment were similar, and both were associated with the same symptomatology.

Efficacy of reciprocating and ultrasonic activation of 6% sodium hypochlorite in the reduction of microbial content and virulence factors in teeth with primary endodontic infection.

AIM: To evaluate in a clinical trial the efficacy of reciprocating and ultrasonic activation of 6% sodium hypochlorite (NaOCl) in the microbial composition and reduction in microbial load as well as in levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in teeth with primary endodontic infections. METHODOLOGY: Samples were collected from 24 root canals with pulp necrosis and periapical lesions, before and after chemo-mechanical canal preparation. The teeth were randomly divided according to the activation protocol as follows: control group without activation (WA, n = 8), reciprocating activation group using Easy Clean tip (EC, n = 8) and ultrasonic activation group using Irrisonic insert (US, n = 8). Microbiological specimens were processed using a culture technique and microbiota composition was analysed using the checkerboard technique. The levels of LPS and LTA were quantified using limulus amebocyte lysate (LAL) and enzyme-linked immunosorbent assay (ELISA), respectively. The Fisher's exact test, Kruskal-Wallis, Dunn's and Wilcoxon's test with a significance level of P < 0.05 were used for statistical analysis. RESULTS: All initial specimens had growth of viable bacteria in fastidious anaerobe agar (FAA), with an average of 105  CFU mL-1 , whereas only one case had such growth after chemo-mechanical canal preparation. LPS and LTA were recovered in 100% of the cases. Chemo-mechanical canal preparation significantly decreased the levels of LPS and LTA (P < 0.05), but no significant differences were found between the groups (P > 0.05). Through the checkerboard technique, bacteria were found in 100% of the initial specimens with concentrations between <105 and 106 . The most frequently identified microorganisms were Prevotella nigrescens and Enterococcus hirae. After chemo-mechanical canal preparation, many species were not detected in any of the three groups tested. A significant reduction occurred in Group US, followed by Groups EC and WA. CONCLUSIONS: Activation of 6% NaOCl reduced the levels of LPS and LTA with no differences between the groups. However, ultrasonic activation was associated with a greater reduction in microbial load within root canals.

Clinical efficacy of EDTA ultrasonic activation in the reduction of endotoxins and cultivable bacteria.

AIM: This clinical study was conducted to investigate the influence of 17% ethylenediaminetetraacetic acid (EDTA) ultrasonic activation after chemomechanical preparation (CMP) on eliminating/reducing oral bacterial lipopolysaccharides (known as endotoxins) and cultivable bacteria in teeth with pulp necrosis and apical periodontitis. METHODOLOGY: Samples were taken from 24 root canals at several clinical periods: S1 - before CMP; S2 - after CMP; S3 - after EDTA: G1 - with ultrasonic activation (n = 12) and G2 - without ultrasonic activation (n = 12). Root canals were instrumented using Mtwo rotary files. Culture techniques were used to determine the number of colony-forming units (CFU). Limulus amebocyte lysate (LAL) was used to measure endotoxin levels. Friedman's and Wilcoxon signed-rank tests were used to compare the amount of bacteria and endotoxin levels in each period (P < 0.05). RESULTS: Endotoxins and cultivable bacteria were recovered in 100% of the initial samples (S1). CMP was effective in reducing endotoxins and bacterial load (all with P < 0.05). Higher values of endotoxin reduction were achieved with EDTA ultrasonic activation [G1, 0.02 EU mL-1 (range 0.01-0.75)] compared with the no activation group [G2, 1.13 EU mL-1 (range 0.01-8.34)] (P < 0.05). Regarding bacterial reduction, no statistically significant difference was found in S3, regardless of the group (G1, G2, P > 0.05). CONCLUSIONS: Chemomechanical preparation was effective in reducing bacteria and endotoxins, but could not completely eliminate them. The ultrasonic activation of EDTA was effective in further reducing endotoxin levels in the root canals of teeth with pulp necrosis and apical periodontitis.

Root canal content from primary endodontic infection and upregulation of gelatinases in fibroblast cells.

AIM: To investigate endotoxin levels from primary endodontic infections before and after chemomechanical preparation (CMP) and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of matrix metalloproteinases (MMPs). METHODOLOGY: Twenty-four root canals with primary endodontic infection and apical periodontitis were selected. Samples were collected using paper points before (S1) and after chemomechanical preparation (CMP) (S2). The limulus amebocyte lysate assay was used for endotoxin measurement. Fibroblasts were stimulated with root canal contents for 24 h. Supernatants of cell cultures stimulated with root canal contents were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity using the zymography technique. Friedman and Wilcoxon tests were used to compare the amount of endotoxin before (S1) and after CMP (S2) (P < 0.05). Data obtained from gelatinolytic activity were analysed using anova and Tukey's tests (P < 0.05). RESULTS: Endotoxin was recovered in 100% of the samples. There was a significant reduction in endotoxin levels after CMP (P < 0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (P < 0.05). Root canal contents of initial samples (S1) induced significantly greater MMP-2 expression by fibroblasts when compared to S2 and the nonstimulated group (P < 0.05). No gelatinolytic activity of MMP-9 was observed in S1, S2 and control group. CONCLUSIONS: Root canal contents from primary endodontic infections had gelatinolytic activity for MMP-2. Moreover, CMP was effective in reducing endotoxin levels and their antigenicity against fibroblasts on gelatinolytic activity.

4% articaine buccal infiltration versus 2% lidocaine inferior alveolar nerve block for emergency root canal treatment in mandibular molars with irreversible pulpits: a randomized clinical study.

AIM: To compare the anaesthetic efficacy of inferior alveolar nerve blocks (IANB) with 1.8 mL of 2% lidocaine (LI) to a buccal infiltration (BI) with 1.8 mL of 4% articaine (AR), both with 1 : 100 000 epinephrine, in patients with symptomatic irreversible pulpits in a randomized controlled trial. METHODOLOGY: Volunteers presenting at the Emergency Centre (FOP-UNICAMP) were randomly divided into two groups (30 for AR and 20 for LI). Operator and patient were not blinded. Success was recorded when complete pain-free treatment was achieved after a single injection (IANB or BI) or when one supplemental injection was needed for emergency endodontic procedures. Success rate of supplemental injection was evaluated between and within groups using Fisher's exact test and chi-square test. RESULTS: A higher success rate (P = 0.03/Fisher's exact test) was observed with AR (40%) than with LI (10%). No significant difference was found when a single injection plus one supplemental injection was compared between groups (P = 1.0; AR = 70%; LI = 80%). However, supplemental injection increased the anaesthetic success rates (AR, P = 0.04; LI, P = 0.0001) within groups. CONCLUSIONS: Single anaesthesia techniques (IANB or BI) were not able to achieve pain-free emergency endodontic treatment. Supplemental anaesthetic techniques should be considered prior to treatment procedures in order to increase success rate (consort: registration number - NCT01912755/Fapesp: #2009/10834-4).

Quantification of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation.

This clinical study was conducted to quantify cultivable bacteria and endotoxin in root canals with post-treatment apical periodontitis by correlating their levels with clinical features and to evaluate the effect of chemo-mechanical preparation (CMP) with 2 % chlorhexidine gel + 17 % EDTA on bacterial and endotoxin removal/elimination. Moreover, target strict Gram-negative anaerobic bacteria were investigated by polymerase chain reaction (PCR). Fifteen teeth with post-treatment apical periodontitis were sampled before (s1) and after (s2) CMP. Culture techniques determined the number of colony-forming units (CFU). PCR (16S rDNA) and limulus amebocyte lysate (LAL) assay were used for bacterial and endotoxin detection, respectively. Prevotella nigrescens (4/15), Prevotella intermedia (2/15), and Tannerella forsythia (2/15) were the most frequently detected species. Endotoxin was recovered in 100 % of the samples. At s1, bacteria and endotoxin were detected at a median value of 5.14 × 10(3) CFU/mL and 3.96 EU/mL, respectively. Higher levels of endotoxin were related to a larger size of radiolucent area (>5 mm) (p < 0.05). CMP was more effective in reducing bacteria (99.61 %) than endotoxin (60.6 %) (both p < 0.05). Our findings indicated that the levels of endotoxin found in infected root canals were related to a larger size of radiolucent area in the periapical region. Moreover, CMP was effective in reducing both bacterial and endotoxin contents in post-treatment apical periodontitis.

Evaluation of cytotoxicity and up-regulation of gelatinases in fibroblast cells by three root repair materials.

AIM: To investigate the effects of root repair materials on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in 3T3 fibroblasts. METHODOLOGY: Fibroblasts (3T3, 3 × 10(5) cells per well) were incubated with elutes of calcium hydroxide (Biodinâmica, Ibiporã, PR, Brazil), EndoBinder (Binderware, São Carlos, SP, Brazil) and mineral trioxide aggregate (MTA) (Angelus, Londrina, PR, Brazil) for 24 h. The cytotoxicity of all root repair materials was determined using the MTT assay. Supernatants of cell cultures incubated with materials were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and Tukey's test. RESULTS: Cells secreted MMP-2 after 24 h with calcium hydroxide inducing significantly greater MMP-2 expression in relation to the control and the other root repair materials (P < 0.05). The cytotoxicity results revealed that there was no significant difference in the cell viability of MTA, EndoBinder and the control group. However, there was a significantly reduced cell viability of 3T3 fibroblasts in association with calcium hydroxide (P < 0.05). CONCLUSIONS: Calcium hydroxide was associated with significantly less cell viability when compared with EndoBinder and MTA. All materials had gelatinolytic activity for MMP-2 with calcium hydroxide being associated with the greatest activity.

Evaluation of cytotoxicity and up-regulation of gelatinases in human fibroblast cells by four root canal sealers.

AIM: To investigate the effects of root canal sealers on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in human fibroblasts. METHODOLOGY: Human fibroblasts (MRC5, 3×10(5) cells per well) were incubated directly or indirectly with AH Plus, Endomethasone N, Pulp Canal Sealer EWT or Sealapex for 30 min, 1, 4 or 24 h (time-points). The cytotoxicity of all root canal sealers was determined by counting viable cells using the trypan blue exclusion assay. Supernatants of cell cultures incubated with root sealers directly or indirectly were collected after each time-point to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and the Tukey's tests. RESULTS: Cells secreted MMP-2 after periods of 4 and 24 h; however, there were no significant differences between the sealers. Secretion of gelatinases was elevated by root canal sealers in direct contact with the cell monolayer when compared to indirect contact (P < 0.05). At the time-points tested, no gelatinolytic activity could be detected in the control group without the sealers. The cytotoxicity results revealed that all sealers were cytotoxic in both contact forms. Sealapex had the lowest cytotoxicity and AH Plus the most cytotoxicity. CONCLUSIONS: All root canal sealers induced the expression of MMP-2 in MRC5 fibroblasts. AH Plus had the highest cytotoxicity amongst the tested sealers, but all were associated with cytotoxic effects.

Filling of artificial lateral canals and microleakage and flow of five endodontic sealers.

AIM: To evaluate the flow characteristics of AH Plus, Epiphany Root Canal Sealant, Endométhasone, Pulp Canal Sealer (EWT) and Sealapex and their ability to fill artificial lateral canals and prevent microleakage. METHODOLOGY: Flow of the sealers was analysed using the American Dental Association (ADA) 57 and the International Standards Organization (ISO) 6,876 specifications. Two lateral canals were produced in the middle and apical third of 64 roots using 0.1 mm cylindrical drills. Lateral condensation of gutta-percha or Resilon and one or other of the sealers were used to fill the root canals. Buccal-lingual digital radiographs were exposed. After the sealer had set, the roots were immersed in Indian ink and cleared in methyl salicylate. The extent of filling and dye penetration were measured on the buccal and lingual root surfaces under 30 x magnification with a stereoscope. RESULTS: AH Plus, Epiphany and Pulp Canal Sealer (EWT) complied with ADA 57 and ISO 6,876 specifications. Sealapex complied with the ADA 57 specification but not with ISO 6,876. Endométhasone did not comply with either specification. Filling of lateral canals was similar for the five sealers tested. Dye leakage demonstrated that AH Plus, Epiphany and Sealapex permitted less leakage than Pulp Canal Sealer (EWT) (P < 0.05). CONCLUSIONS: All the sealers flowed into the 0.1 mm artificial lateral canals. AH Plus, Epiphany and Sealapex allowed less linear leakage than Pulp Canal Sealer (EWT). The flow of Endométhasone did not comply with either ADA 57 or ISO 6,876 specifications and Sealapex did not comply with ISO 6,876.