B. P. F. A. Gomes

T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype

INTRODUCTION Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. METHODS Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. RESULTS Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. CONCLUSION Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.

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 Tukeys 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.

Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture

BACKGROUND/AIM The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.

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 Tukeys 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.

In vitro evaluation of the effectiveness of the chemomechanical preparation against Enterococcus faecalis after single- or multiple-visit root canal treatment

The purpose was to assess the elimination of Enterococcus faecalis in vitro in human mandibular premolars after chemomechanical preparation with or without the use of a calcium hydroxide dressing. After 60 days of contamination with E. faecalis, the root canals were prepared using the Crown-Down technique combined with 2% chlorhexidine gel irrigation. Then, the specimens were divided into two experimental groups, treated in a single visit or in multiple visits, and two control groups. The multiple-visit group received a dressing with calcium hydroxide for 14 days (Calen) and the single-visit group did not receive any medication. In the two control groups, the canals were filled with BHI after chemomechanical preparation with 2% chlorhexidine gel or distilled water. Microbial samples were taken from the root canals for colony forming unit count for each phase of the treatment using sterile paper points inside the root canal lumen. Data were ranked and analyzed by the Kruskal-Wallis statistical test. The residual microbial colonies were then assessed. The results showed that chemomechanical preparation using 2% chlorhexidine gel with no intra-canal dressing reduced by 100% the E. faecalis contamination of the root canal lumen. The calcium-hydroxide group that received the 14-day intra-canal dressing allowed a small number of bacteria to grow between visits, but without statistical differences between groups.

Microbiological profile and antimicrobial susceptibility pattern of infected root canals associated with periapical abscesses

The aim of this investigation was to identify microorganisms from root canals with periapical abscesses and assess the susceptibility of specific anaerobic bacteria to selected antimicrobials and their β-lactamase production. Sixty root canals were microbiologically investigated. The susceptibility of Anaerococcus prevotii, Fusobacterium necrophorum, F. nucleatum, Parvimonas micra, and Prevotella intermedia/nigrescens to antimicrobials was evaluated with the Etest, whereas β-lactamase production was assessed with nitrocefin. A total of 287 different bacterial strains were recovered, including 201 strict anaerobes. The most frequently strict isolated anaerobes were A. prevotii, P. micra, and F. necrophorum. The selected bacteria were susceptible to all the tested antibiotics, except A. prevotii and Fusobacterium species to azithromycin and erythromycin, as well as A. prevotii and F. necrophorum to metronidazole. None of the microorganisms produced β-lactamase. Gram-positive anaerobic bacteria predominated in the root canals with periapical abscesses. All microorganisms tested were susceptible to benzylpenicillin, amoxicillin, amoxicillin + clavulanate, cefaclor, and clindamycin, producing no β-lactamase.

Thickness of dentine in mesial roots of mandibular molars with different lengths

AIM To measure the minimum thickness of the distal (furcal) root dentine associated with the buccal and lingual canals of the mesial roots of mandibular first molars with different lengths. METHODOLOGY The mesial roots of 285 mandibular first molars were allocated into three groups according to their length: group I - long (24.14 mm +/- 0.85), group II - medium (22.10 mm +/- 0.65) and group III - short (19.97 mm +/- 0.75). The minimum thickness of the distal (furcal) root dentine associated with the buccal and lingual canals of the mesial roots 2 mm below the furcation was measured. The distance between the buccal and lingual canals, and the depth of concavity in the distal surface of the mesial roots were also measured. anova and Tukey-Kramer were used to test for significant differences among the groups. RESULTS The minimum thickness of the distal wall of the mesiobuccal canal was significantly different (P < 0.05) between group I (long) and III (short), with long teeth having the smallest mean values. No significant difference was found in the thickness of the distal wall of the mesiolingual canal among the groups studied (P > 0.05). The shortest distance between the mesiobuccal and the mesiolingual canals was observed in group III (P < 0.05). The distal (furcal) concavity was deeper in group I (P < 0.05) when compared with the other groups. CONCLUSION There was a significant difference in the minimum thickness of the distal (furcal) root wall of the mesiobuccal canal of mandibular first molars 2 mm below the furcation between group I (long) and group III (short) teeth. The thinnest walls were found in the longest teeth. The deepest concavities in the distal (furcal) walls of the mesial roots were found in the longest roots.

Ex vivo antimicrobial activity of several bleaching agents used during the walking bleach technique

AIM To investigate ex vivo the antimicrobial activity of a paste of sodium perborate associated with various vehicles comparing it with 37% carbamide peroxide and 35% hydrogen peroxide. METHODOLOGY The antimicrobial activity of these agents was evaluated against three microorganisms: Enterococcus faecalis, Streptococcus mutans and Candida albicans. One millilitre of each tested substance was placed on the bottom of wells of 24-well cell culture plates. Six wells were used for each time period and group. Two millilitres of the microbial suspension was ultrasonically mixed for 10 s with the bleaching pastes and placed in contact with them for 10, 30, 45 s; 1, 3, 5, 10, 20, 30 min; and 1 and 2 h. After each period of time, 1 mL from each well was transferred to tubes containing 2 mL of freshly prepared brain heart infusion agar + neutralizers. Agar plates were inoculated in appropriate gaseous conditions. Data were analysed statistically by the Kruskal-Wallis test with the level of significance set at P < 0.05. RESULTS In all groups containing chlorhexidine (groups 3, 5 and 7), the antimicrobial activity of the bleaching paste was significantly increased when compared with groups with other kinds of vehicle (groups 1, 2, 4, 6 and 8). For all tested groups, the most resistant microorganism was E. faecalis. CONCLUSIONS Chlorhexidine when used as a vehicle for sodium perborate enhanced its antimicrobial activity.

Radiographic prevalence of root canal ramifications in a sample of root canal treatments in a Brazilian Dental School

The aim of this study was to radiographically investigate the presence of root canal ramifications found after endodontic treatment, and to determine any relationship between their presence and the type of the auxiliary chemical substance used. The study evaluated 1,470 endodontic treatments performed by final year undergraduate students at the Dental School of Piracicaba, State University of Campinas (UNICAMP), SP, Brazil, during the period from 1998 to 2000. The X-rays taken during treatment were evaluated in order to establish the presence of ramifications of the root canal system. The initial X-ray did not show the presence of any canal ramifications. After filling, X-rays showed only 3 ramification types: 3.06% of lateral canals, 2.99% of apical deltas, and 0.1% of interradicular canals. The maxillary premolars showed the highest number of lateral canals (n = 13), followed by mandibular premolars (n = 10) and maxillary incisors (n = 10). Apical deltas were mostly found in mandibular molars (n = 14), followed by maxillary incisors (n = 9). Only mandibular molars had interradicular canals. The detection of ramifications increased with the use of EDTA. However, no statistically significant relationship was found between the type of auxiliary chemical substance used and the number of root canal ramifications detected after root canal filling. It was concluded that the frequency of root canal ramifications found radiographically was low in treatments performed by undergraduate students.

Seal capability of interim post and core crown with temporary cements.

The purpose of the present study was to evaluate the in vitro seal capability of interim post and core crown restorations. Eighty teeth were selected and divided into 8 groups. Four experimental groups received interim posts and core crowns. Half of each group was decoronated at the cementum-enamel junction, groups PCCH and PCZO. The other half was sectioned 2 mm coronal to the cementum-enamel junction, groups PCrZO and PCrCH. The interim post and core crowns were luted with Rely X Temp NE, groups PCrZO and PCZO; Hydro C was used for Groups PCrCH and PCCH. The control groups, PC and PCr, received uncoated post and core crowns; groups OTg and OT were left without interim post and core crowns and were totally open. Infiltration was accessed by dye exposure followed by demineralization of the teeth. The length of the infiltration was measured using digital images taken from the specimens. The images were inserted into the Image Tool 3.0 software. Kruskal-Wallis analysis of variance and Dunns multiple comparison method were used to test for significant differences among test groups (P < .05). Groups PCrZO and PCrCH showed the least dye penetration, followed by groups PCZO and PCCH. Teeth restored with interim post and core crowns will be subject to leakage. Ethics Committee: 095/2008.