In vitro treatment of Enterococcus faecalis with calcium hydroxide impairs phagocytosis by human macrophages.

OBJECTIVE: Monocyte-derived macrophages (MDMs) ability to phagocytize and produce nitric oxide (NO) was tested against root-canal strains of Enterococcus faecalis submitted to alkaline stress. Root-canal strains were also compared with urine Enterococci. MATERIALS AND METHODS: Enterococcus faecalis were stressed with alkaline-BHI broth and incubated in vitro at a cell/bacteria ratio of 1:5. Phagocytosis was analyzed by fluorescence microscopy using acridine orange stain, and NO concentration was measured in supernatants. RESULTS AND CONCLUSIONS: Alkaline-stress significantly impaired MDMs phagocytosis of E. faecalis strains analyzed, except in ATCC4083 isolated from a pulpless tooth, but NO production was unchanged. Comparison of different strains showed the urine isolate had higher NO levels than root canal strains. Alterations in the bacterial cell wall structures after alkaline-stress possibly made bacteria less recognizable and phagocytized by MDMs but did not affect their ability to activate NO production. Furthermore, root canal strains elicited different responses by immune cells compared with strains from urine. Clinically, impaired phagocytosis of E. faecalis could contribute to their persistence in root canal systems previously treated with calcium hydroxide.

Intratubular disinfection with tri-antibiotic and calcium hydroxide pastes.

OBJECTIVE: The aim of this study was to compare the in vitro intradentinal antimicrobial ability of the calcium hydroxide and tri-antibiotic pastes. MATERIALS AND METHODS: Standard bovine dentin tubes were sterilized and then infected with Enterococcus faecalis by a new contamination protocol of great depths of dentin. The specimens were filled with the medications, divided into two test-groups: calcium hydroxide (Group 1) and tri-antibiotic (Group 2) pastes. After 15 days, the teeth were evaluated by microbiological culture and confocal laser scanning microscopy (CLSM) with viability dye assay LIVE/DEAD inside dentinal tubules. In experiment of culture, the bacterial collection of the dentin fragments was done for counting the colony-forming units. RESULTS: The tri-antibiotic paste had a slightly greater antimicrobial effect; however, there was no statistical difference between the groups. CONCLUSIONS: It was concluded that the tri-antibiotic paste and the calcium hydroxide paste exercise the same effect on intra-tubular decontamination against E. faecalis. So, due the multiples advantages, the calcium hydroxide paste can be the choice for dentinal decontamination in regenerative procedures.

Can the Concentration of Citric Acid Affect Its Cytotoxicity and Antimicrobial Activity?

BACKGROUND: There has been no unanimity concerning the ideal concentration of citric acid for safe use in clinical practice. This study evaluated the cytotoxicity and the antibacterial activity in infected dentinal tubules of 10% and 1% citric acid (CA) solutions. METHODS: The cytotoxicity of CA solutions in DMEM (diluted 1/10, 1/100) was assessed in L-929 fibroblasts. A broth macrodilution method (MIC and MBC) was used to assess CA antibacterial concentration. The antimicrobial activity of CA solutions was also evaluated after their final rinse inside root canals in previously Enterococcus faecalis-contaminated dentinal tubules. Ten infected dentine samples were rinsed for 5 min with 5% NaOCl and subsequently with 1% citric acid for 3 min. Another 10 were rinsed with 5% NaOCl and 10% citric acid for 3 min; the remaining four specimens were utilized as positive controls. Two uncontaminated specimens were used as negative controls. After LIVE/DEAD BacLight staining, the samples were assessed using CLSM to analyze the percentage of residual live and dead cells. RESULTS: Both undiluted and diluted CA solutions showed severe toxicity; no changes from normal morphology were displayed when diluted 1/100. The MIC and MBC of CA were 6.25 mg/mL and 12.50 mg/mL, respectively. CA solutions demonstrated significantly low levels of bacterial counts than the positive control group, reporting a value of 9.3% for the 10% solution versus the 1% solution (35.2%). CONCLUSIONS: Despite its valuable antimicrobial properties, the cytotoxic effects of citric acid should be considered during endodontic treatment.

Impact of irrigation protocols with some chelators and mechanical agitation on intratubular decontamination.

This study evaluated main canal and intratubular decontamination using different irrigation solutions followed by adjunctive agitation steps for infected root canals. Sixty-eight lower incisors were contaminated with Enterococcus faecalis and allocated to groups according to canal treatment (n = 10): G1, NaOCl followed by ethylenediaminetetraacetic acid (EDTA); G2, a mixture of NaOCl with hydroxyethylidene bisphosphonate (HEBP); and G3, NaOCl followed by EDTA-T (EDTA with sodium lauryl ether sulfate). All three groups of teeth were agitated with passive ultrasonic irrigation (PUI) using saline solution, whereby G4, G5, and G6 were prepared as above, and agitation was performed using an XP-Endo Finisher instrument. Microbiological samples were collected from the root canals with paper points at three times: before and after chemomechanical preparation and after agitation. The colony-forming units (CFU)/mL count was determined, and bacterial intratubular viability was analyzed via confocal laser scanning microscopy using Live/Dead staining. Statistical analysis was performed using a Kruskal-Wallis test followed by Dunn tests. A Friedman test was applied for colony-counting data (α = 0.05). CFU/mL counting indicated equally effective decontamination in the experimental groups (p > 0.05). According to microscopy images, the use of irrigation solutions followed by agitation with the XP-Endo Finisher yielded better results. Moreover, NaOCl+EDTA-T followed by XP-Endo Finisher resulted in significantly lower viability than in the PUI-activated groups (p < 0.05). The cervical and medium thirds of the specimens presented similar results. Overall, NaOCl+EDTA-T exhibited the best intratubular antibacterial activity, mainly for canals that were subsequently agitated using XP-Endo Finisher.

Antibacterial Efficacy of Triple Antibiotic Medication With Macrogol (3Mix-MP), Traditional Triple Antibiotic Paste, Calcium Hydroxide, and Ethanol Extract of Propolis: An Intratubular Dentin Ex Vivo Confocal Laser Scanning Microscopic Study.

INTRODUCTION: This study aimed to assess the effectiveness of antibacterial activity of medications used in regenerative endodontic treatment. METHODS: Sixty-seven dentin cylinders of single-rooted teeth were contaminated with a culture of Enterococcus faecalis (ATCC 29212; American Type Culture Collection, Manassas, VA) for 5 days. Samples were divided into 1 control group and the following experimental groups according to the medication applied: traditional triple antibiotic paste (TAP), clindamycin-modified TAP (mTAP), triple antibiotic medication with macrogol (3Mix-MP), clindamycin-modified 3Mix-MP (m3Mix-MP), calcium hydroxide (CH), and ethanol extract of propolis (EEP). After 14 days, the medications were removed, and the samples were submitted to confocal laser scanning microscopic analysis to quantify the percentage of viable bacteria. The distribution of data was confirmed by the Shapiro-Wilk test. The Kruskal-Wallis and Dunn tests were used for intergroup comparisons, and the Wilcoxon test was used for comparison between superficial and deep antibacterial efficacy for the same medication. The level of significance was set at P < .05. RESULTS: 3Mix-MP and m3Mix-MP presented significantly higher antibacterial efficacy compared with the other tested medications (P < .05), except for mTAP. mTAP was more effective than TAP (P < .05). The antibacterial efficacy of EEP and CH did not differ significantly from TAP and mTAP (P > .05). All medications showed effective antibacterial action compared with the control group (P < .05). CONCLUSIONS: 3Mix-MP and m3Mix-MP, which present extremely high concentrations of antibiotics (1500 mg/mL), were not more effective than mTAP at the concentration recommended by the American Association of Endodontists (5 mg/mL). Moreover, CH and EEP were as effective as TAP and mTAP.

Can kinematics, file diameter, and PUI influence the intracanal decontamination and apical bacterial extrusion?

The present study investigated the intracanal decontamination and apical extrusion of bacteria and debris from root canals instrumented with rotary and reciprocating systems (ProDesign Logic or ProDesign R), with different file diameters and using conventional syringe irrigation (CSI) or passive ultrasonic irrigation (PUI). Eighty extracted mandibular premolars were contaminated with Enterococcus faecalis and randomly assigned to eight experimental groups according to the root canal instrumentation and irrigation technique employed (n = 10): G1: Prodesign Logic 25.06; G2: Prodesign R 25.06; G3 and G4 were instrumented with the same single-file systems, respectively, using 35.05 diameters and CSI. G5, G6, G7, and G8 were instrumented like the previous groups, but with PUI. Apically extruded debris during instrumentation was collected into pre-weighed microtubes. The weight of the empty microtube was subtracted from the final weight to establish the amount of extruded debris. Bacteria from root canals and extruded debris were collected for a microbiological evaluation of colony forming units (CFU/mL). For statistical analyses, the Mann-Whitney and Kruskal-Wallis followed by the Dunn's tests were used (α = 0.05). All instruments caused extrusion of debris. For irrigation techniques, PUI promoted greater debris and bacterial extrusion (p < 0.05). The CFU/mL count indicated that the instrumentation of the experimental groups were equally effective in the decontamination of the root canal (p > 0.05). The systems tested (regarding file diameter and kinematics) were associated with similar amounts of apically extruded debris and root canal decontamination. PUI was associated with greater debris and bacterial extrusion.

Antimicrobial Activity and Physicochemical Properties of Antibiotic Pastes Used In Regenerative Endodontics.

The purpose of this study was to evaluate the pH, solubility and antimicrobial action of Calcium Hydroxide Paste (CH), Double Antibiotic Paste (metronidazole+ciprofloxacin-DAP), calcium hydroxide added to DAP (CH/DAP) and Triple Antibiotic Paste (metronidazole + ciprofloxacin+minocycline-TAP). pH (n=10) were measured by pHmeter. Root canals of acrylic teeth (n=10) were filled with the above-mentioned intracanal-dressings, immersed in ultrapure water, and solubility was measured by the difference between the initial and final volume (7,15 and 30 days) by using micro-computed tomography. Enterococcus faecalis biofilm was induced on bovine dentin disc surfaces (n=20), and treated with the pastes for 7 days. Percentage bacterial viability was verified by confocal microscope, with LIVE/DEAD dye. CH and CH/DAP presented the highest pH values. Regarding solubility, after 7 days, antibiotic groups presented significant volume loss. CH and CH/DAP showed no statistical difference compared with the Control in antimicrobial action against E. faecalis biofilm. However, TAP and DAP presented a significant percentage reduction in bacterial population. Due to high solubility of the pastes, renewing antibiotic dressings every 7 days, or using the medications for this period in regeneration protocols is recommended. DAP is indicated for killing E. faecalis in biofilm because it has antimicrobial action similar to TAP. Adding Calcium Hydroxide to DAP significantly decreased its antimicrobial action. In spite of its the low solubility and high pH values, the CH paste showed a low level of antimicrobial action against E. faecalis in biofilm.

Effect of hydroalcoholic extract of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on the viability and activity of microcosm biofilm and on enamel demineralization.

OBJECTIVES: The aim of this study was to assess the effect of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves hydroalcoholic extracts on viability and metabolism of a microcosm biofilm and on enamel demineralization prevention. METHODOLOGY: Microcosm biofilm was produced on bovine enamel using inoculum from pooled human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. The biofilm was daily-treated with the extracts for 1 min. At the end, it was analyzed with respect to viability by fluorescence, CFU counting and extracellular polysaccharides (phenol-sulphuric acid colorimetric assay) and lactic acid (enzymatic assay) production. The demineralization was measured by TMR. The data were compared using ANOVA or Kruskal-Wallis (p<0.05). RESULTS: M. urundeuva All. at 100, 10 and 0.1 µg/mL and Q. grandiflora Mart. at 100 and 0.1 µg/mL reduced biofilm viability similarly to positive control (chlorhexidine) and significantly more than the negative-vehicle control (35% ethanol). M. urundeuva at 1000, 100 and 0.1 µg/mL were able to reduce both lactobacilli and mutans streptococci CFU counting, while Q. grandiflora (1000 and 1.0 µg/mL) significantly reduced mutans streptococci CFU counting. On the other hand, the natural extracts were unable to significantly reduce extracellular polysaccharides and lactic acid productions neither the development of enamel carious lesions. CONCLUSIONS: The extracts showed antimicrobial properties on microcosm biofilm, however, they had no effect on biofilm metabolism and caries protection.

Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel.

Titanium tetrafluoride (TiF4) is known for interacting with enamel reducing demineralization. However, no information is available about its potential antimicrobial effect. OBJECTIVES: This study evaluated the antimicrobial and anti-caries potential of TiF4 varnish compared to NaF varnish, chlorhexidine gel (positive control), placebo varnish and untreated (negative controls) using a dental microcosm biofilm model. MATERIAL AND METHODS: A microcosm biofilm was produced on bovine enamel previously treated with the varnishes, using inoculum from human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. All experiments were performed in biological triplicate (n=4/group in each experiment). Factors evaluated were: bacterial viability (% dead and live bacteria); CFU counting (log10 CFU/mL); and enamel demineralization (transverse microradiography - TMR). Data were analysed using ANOVA/Tukey's test or Kruskal-Wallis/Dunn's test (p<0.05). RESULTS: Only chlorhexidine significantly increased the number of dead bacteria (68.8±13.1% dead bacteria) compared to untreated control (48.9±16.1% dead bacteria). No treatment reduced the CFU counting (total microorganism and total streptococci) compared to the negative controls. Only TiF4 was able to reduce enamel demineralization (ΔZ 1110.7±803.2 vol% µm) compared to both negative controls (untreated: ΔZ 4455.3±1176.4 vol% µm). CONCLUSIONS: TiF4 varnish has no relevant antimicrobial effect. Nevertheless, TiF4 varnish was effective in reducing enamel demineralization under this model.

Do different strains of E. faecalis have the same behavior towards intracanal medications in in vitro research?

The aim of this study was to evaluate the antimicrobial action of different endodontic pastes against Enterococcus faecalis ATCC 29212, isolated from the urinary tract, and compare the action with E. faecalis ATCC 4083, isolated from the root canal. For this purpose, dentin blocks were infected for 21 days with both bacteria at different time-intervals to ensure there would be no cross contamination. After this period, blocks were immersed in the test medications for 7 days, according to the following groups: CH/S, CH/P, CH/CMCP, CH/CHX, CH/DAP and TAP. Images of the samples were captured with a confocal microscope and the percentage of live cells was computed by means of the Bioimage program. The ATCC 29212 strain was shown to be more resistant to CH/SS, Calen, CH/DAP, and TAP than the ATCC 4083 strain. The antimicrobial action of the medications against each strain were divergent concerning the order of susceptibility. The authors concluded that the strains behaved in a different manner: in general, those extracted from the urinary tract were more resistant to the tested medications. Therefore, when E. faecalis must be used for in vitro research in endodontics, we suggest the use of ATCC 4083 strain to obtain results that are closer to the clinical reality.

Effect of two methods of irrigant agitation on the temperature and cleanliness of sodium hypochlorite associated or not with a chelator: irrigant agitation and influence in temperature

Aim: This study investigated the influence in vitro of different sodium hypochlorite (NaOCl) agitation protocols associated or not with DualRinse (HEDP) on the temperature of the solution. Methods: Forty-eight premolars were instrumented and their apical third sealed to allow a closed irrigation system. The teeth remained immersed in a basin of warm water (37°C). The teeth were divided into the groups: G1 (NaOCl+Passive Ultrasonic Irrigation (PUI)), G2 (NaOCl/HEDP + PUI), G3 (NaOCl + EasyClean (EC)) and G4 (NaOCl/HEDP + EC). The canals were filled with the respective solutions and after 180 seconds the first temperature measurement was taken (T0). Then, the solutions were agitated, following the different protocols, for 60 seconds and a new measurement was performed (T60). The temperature was measured using a digital thermometer for type "K" sensors that was inserted into the middle third of the teeth. At the end of the measurements, the teeth were sectioned and prepared for scanning electron microscopy. The dentinal wall of middle third was graded according to the amount of debris and smear layer remaining on the walls. The results were analyzed using ANOVA test and Tukey's multiple comparisons (p<0.05). Results: G1 and G2 had an average increase in temperature of 1.1°C and 1.65°C, respectively (p>0.05). EasyClean caused a decrease in the temperature of the solutions in both groups, without a significant statistical difference with T0 (p>0.05). Regarding cleaning, it was only possible to observe clean dentinal tubules in the groups with the chelator. PUI discretely increased the temperature of the solution, regardless of the solution. The opposite effect was observed after activation with EasyClean. Conclusion: The association of NaOCl with a chelating agent promoted the cleaning of the dentinal tubules

Intradentinal antimicrobial action and filling quality promoted by ultrasonic agitation of epoxy resin-based sealer in endodontic obturation.

MATERIAL AND METHODS: The aim of this study was evaluate the influence of ultrasonic activation (UA) of AH Plus to improve canal and isthmus filing, and analyse the antimicrobial effect against Enterococcus faecalis within dentinal tubules. Thirty mesial roots of mandibular first molars were selected and divided into 2 groups (n = 15): with and without UA of the sealer. Then the root canals were filled by using the single cone technique, and the specimens were sectioned at 2, 4 and 6 mm from the apex for stereomicroscope and confocal laser scanner microscopy (CLSM) analysis. In addition, 30 bovine incisors were contaminated with Enterococcus faecalis and divided into 3 groups (n = 10). The specimens were obturated by using the single cone technique with (G1) and without (G2) UA of the sealer and G3 as the control group. All were sectioned into 6 mm-long cylinders and stained with LIVE/DEAD to assess bacterial viability by CLSM. RESULTS: The UA of the sealer significantly reduced the presence of unfilled areas in the canal and isthmus area in all sections (p<0.05), and there was a significant increase in sealer penetration in both canals and isthmuses (p<0.05). As regards gaps, a significant reduction was found at 2 and 6 mm in the isthmus area of the UA group (p<0.05). Moreover, UA of the sealer significantly reduced bacterial viability in the superficial dentine when compared with the other groups (p<0.05). CONCLUSION: Ultrasonic activation of the AH Plus sealer promoted a better quality of root canal filling and increased the intratubular penetration of sealer, especially in the isthmus area. Additionally, ultrasonic activation of the sealer increased the intradentinal antimicrobial action against Enterococcus faecalis, mainly in the superficial dentine of the root canal.

Effect of ultrasound streaming on the disinfection of flattened root canals prepared by rotary and reciprocating systems.

New technical and scientific developments have been advocated to promote the success of the endodontic treatment. In addition to rotary and reciprocating systems, irrigating solution agitation has been suggested and passive ultrasonic irrigation (PUI) is the most used. OBJECTIVE: To evaluate, in vitro, the effect of ultrasound streaming (US) in the disinfection of flattened root canal systems prepared by the ProTaper, BioRaCe and Reciproc systems, utilizing the microbiological culture. METHODOLOGY: Extracted human mandibular incisors (n=84) were used. Suspensions of Enterococcus faecalis (ATCC 29212) were standardized and inserted along with the teeth immersed in brain-heart infusion (BHI) broth. The contamination was made following a protocol during 5 days. The teeth were randomly divided into six groups: G1, ProTaper Universal; G2, ProTaper Universal with US; G3, BioRaCe; G4, BioRaCe with US; G5, Reciproc; and G6, Reciproc with US. Irrigation was performed with saline solution. After biomechanical preparation, microbiological samples were performed with sterilized paper points, which were diluted and spread on BHI agar; after 48 h, the colony forming units (CFU/mL) were counted for each sample. RESULTS: Groups using ultrasonic agitation presented a greater antibacterial effect than the other ones, even using saline solution as irrigant. The ProTaper Universal system showed the best antibacterial activity of the tested systems (median of 0 CFU/mL with and without surfactant or ultrasonic activation [PUI]). Even with PUI, Reciproc (median of 2.5 CFU/mL with PUI and 5 without it) could not reduce as many colonies as ProTaper Universal without US. The BioRaCe system had greater bacterial reduction when using US (median of 0 CFU/mL with PUI and 30 without it). CONCLUSIONS: US promoted greater reduction in the number of bacteria in the flattened root canals prepared with nickel-titanium mechanized systems. Regarding the instruments used, the ProTaper Universal system was the most effective in reducing the bacterial number.

Antimicrobial Effect and Surface Tension of Some Chelating Solutions with Added Surfactants.

This study assessed the antimicrobial efficacy and surface tension of established irrigating solutions with a new experimental chelating solution in infected dentin tubes. Twenty-five specimens were randomly assigned to each of the irrigating solutions. Twenty specimens were used as negative and positive controls. After 21 days of contamination with E. faecalis, the irrigating solutions MTAD, QMiX and Tetraclean NA were delivered into each infected root canal. The solutions were removed and dentin samples were withdrawn from the root canals with sterile low-speed round burs with increasing ISO diameters. The dentin powder samples obtained with each bur were immediately collected in separate test tubes containing 3 mL of BHI broth. After that, 100 µL from each test tube was cultured on blood agar. The grown colonies were counted and recorded as colony-forming units (CFU). The surface tension of the irrigants was measured using a Cahn DCA-322 Dynamic Contact Angle Analyzer. A Kruskal Wallis nonparametric ANOVA and a Friedman test were used (p<0.05). Tetraclean NA showed lower surface tension and CFU values than MTAD and QMiX. Better antibacterial action and low surface tension were observed for Tetraclean NA, probably due to the improved penetration into the root canal and dentinal tubes.

Effect of ultrasonic streaming on intra-dentinal disinfection and penetration of calcium hydroxide paste in endodontic treatment.

OBJECTIVE: The antimicrobial effect of ultrasonic agitation of calcium hydroxide (CH) pastes in infected bovine dentin and their penetrability were evaluated using confocal laser scanning microscopy (CLSM) and microbiological culture. MATERIAL AND METHODS: Fifty-two bovine teeth were infected with Enterococcus faecalis using a new contamination protocol; then they received CH paste and were divided into groups with or without ultrasound. Ultrasonic agitation was conducted for 1 min with a plain point insert. After 15 d, the CLSM analyzed the viable and dead bacteria with Live and Dead assay. The dentinal wall debris was collected by burs, and the colony forming units (CFU/mL) were counted. The penetrability of the paste inside dentinal tubules was tested using the B-rodamine dye. RESULTS: The calcium hydroxide paste showed better results with the use of ultrasonic agitation (p<0.05). CONCLUSION: The ultrasonic agitation of CH paste increased its antimicrobial action and was responsible for intradentinal penetration with the fulfilment of the tubules.

Impact of irrigation protocols with some chelators and mechanical agitation on intratubular decontamination

Abstract: This study evaluated main canal and intratubular decontamination using different irrigation solutions followed by adjunctive agitation steps for infected root canals. Sixty-eight lower incisors were contaminated with Enterococcus faecalis and allocated to groups according to canal treatment (n = 10): G1, NaOCl followed by ethylenediaminetetraacetic acid (EDTA); G2, a mixture of NaOCl with hydroxyethylidene bisphosphonate (HEBP); and G3, NaOCl followed by EDTA-T (EDTA with sodium lauryl ether sulfate). All three groups of teeth were agitated with passive ultrasonic irrigation (PUI) using saline solution, whereby G4, G5, and G6 were prepared as above, and agitation was performed using an XP-Endo Finisher instrument. Microbiological samples were collected from the root canals with paper points at three times: before and after chemomechanical preparation and after agitation. The colony-forming units (CFU)/mL count was determined, and bacterial intratubular viability was analyzed via confocal laser scanning microscopy using Live/Dead staining. Statistical analysis was performed using a Kruskal-Wallis test followed by Dunn tests. A Friedman test was applied for colony-counting data (α = 0.05). CFU/mL counting indicated equally effective decontamination in the experimental groups (p > 0.05). According to microscopy images, the use of irrigation solutions followed by agitation with the XP-Endo Finisher yielded better results. Moreover, NaOCl+EDTA-T followed by XP-Endo Finisher resulted in significantly lower viability than in the PUI-activated groups (p < 0.05). The cervical and medium thirds of the specimens presented similar results. Overall, NaOCl+EDTA-T exhibited the best intratubular antibacterial activity, mainly for canals that were subsequently agitated using XP-Endo Finisher.

Consequences of chemical residue formation during potentiation of final irrigation - in vitro study

Seeking to increase the efficiency of endodontic irrigation, the association of different solutions as final irrigant has been investigated, such as sodium hypochlorite with chlorhexidine. The literature shows that the combination of these substances leads to the formation of a brownish precipitate, but does not reveal measurements of the intensity of this precipitate and its consequences. Aim: The present study aimed to evaluate the change in dentin color and the obliteration of the dentinal tubules after the association of sodium hypochlorite (NaOCl) with chlorhexidine (CHX) in the final irrigation. Methods: Fifty sterile human lower premolars were prepared with a ProDesign R 35.05 files and divided into 6 groups. Four different NaOCl concentrations (0.5%; 1%, 2.5% and 5.25%) associated with 2% CHX were tested, in addition to 2 control groups, using only 2.5% NaOCl and 2% CHX, respectively. After the final irrigation protocol, the dentin color change was evaluated by spectrophotometry immediately and after 24 hours, and the dentinal tubule obliteration was assessed by scanning electron microscopy. Results: It was possible to verify that regardless the NaOCl concentration used when associated with CHX, a chemical residue was formed, with consequent dentin pigmentation and tubular obstruction. There was a trend towards increased dentin pigmentation and tubular obstruction due to the deposition of the chemical residue formed by this association. Conclusion: It can be concluded that all concentrations of NaOCl associated with CHX caused color changes and tubular obstruction, being proportional to the concentration of NaOCl used

Influence of EDTA and its Association with Benzalkonium Chloride on Enterococcus faecalis Adhesion to Dentin / Influencia del EDTA y su Asociación con Cloruro de Benzalconio en la Adhesión de Enterococcus faecalis a la Dentina

ABSTRACT: The aim of this in vitro study was to investigate the influence of ethylenediaminetetraacetic acid (EDTA) associated with the benzalkonium chloride (BAK) on the adhesion and formation of Enterococcus faecalis biofilms attached to coated dentin. Discs standard bovine dentin blocks were treated with the coating materials evaluated: Saline solution (control), 17 % EDTA, 17 % EDTA associated with 1 % BAK for 5 minutes and subsequently washed with saline solution. Afterwards, biofilms of E. faecalis (ATCC 29212) were grown on the surface of coated dentin blocks for time intervals of 1 hour and 7 days (n = 20) and were subsequently washed with phosphate-buffered saline (PBS). Bacterial viability and total biovolume were analyzed by confocal laser scanning microscopy (CLSM) using the Live/Dead technique. Nonparametric Kruskal-Wallis followed by Dunn tests were used to determine statistical differences (a = 5 %). The 17 % EDTA + 1 % BAK group showed significantly lower biovolume and bacterial viability values at the end of 1 hour (p < 0.05). After 7 days of contamination, the 17 % EDTA and 17 % EDTA + 1 % BAK groups showed similar results that differed statistically from those of the control group (p < 0.05). The saline solution group showed higher values. The use of BAK associated with EDTA on dentin blocks surfaces before exposure to contamination was able to interfere in the adhesion of E. faecalis to dentin. Also, dentin treatment by BAK associated with a chelating agent influences the secondary biofilm formation, which could have important effects on the long-term success of root canal treatment.

RESUMEN: El objetivo del estudio consistió en investigar in vitro, la influencia del ácido etilendiamino-tetraacético (EDTA) con cloruro de benzalconio (BAK) en la adhesión y formación de biopelículas de Enterococcus faecalis a la dentina. Discos de dentina bovina fueron tratadas con solución salina (control), 17 % de EDTA, 17% de EDTA asociado con 1 % de BAK durante 5 minutos y lavadas con solución salina. Las biopelículas de E. faecalis (ATCC 29212) se cultivaron sobre los discos de dentina durante intervalos de tiempo de 1 hora y 7 días (n = 20), lavados con solución salina tamponada con fosfato (PBS). La viabilidad bacteriana y el biovolumen total se analizaron mediante microscopía de barrido por láser (CLSM) utilizando la técnica Live / Dead. Se realizó prueba no paramétrica de Kruskal-Wallis, seguida por Dunn con una diferencia estadística (a = 5 %). El grupo de 17 % EDTA + 1 % BAK mostró valores significativamente menores de biovolumen y viabilidad bacteriana al final de 1 hora (p < 0,05). Después de 7 días de contaminación, los grupos de 17 % EDTA y 17 % EDTA + 1 % BAK mostraron resultados similares que diferían estadísticamente del grupo control (p < 0,05). La solución salina mostró valores más altos. La asociación de BAK con EDTA antes de la contaminación interfirió en la adhesión de E. faecalis. Además, el tratamiento de la dentina por BAK asociado con EDTA influye en la formación de biopelículas secundarias, lo que podría tener efectos importantes sobre el éxito a largo plazo del tratamiento del conducto radicular.

Analysis of the reaction of subcutaneous tissues in rats and the antimicrobial activity of calcium hydroxide paste used in association with different substances.

The aim of this study was to evaluate the subcutaneous tissue response in rats and the antimicrobial activity of intracanal calcium hydroxide dressings mixed with different substances against E. faecalis. Fifty four rats were divided into three experimental groups according to the vehicle in the calcium hydroxide treatment: 0.4% chlorohexidine in propylene glycol (PG),Casearia sylvestris Sw in PG and calcium hydroxide+PG (control group). The pastes were placed into polyethylene tubes and implanted into the subcutaneous tissue. After 7, 14 and 30 days, the samples were processed and histologically evaluated (hematoxylin and eosin). The tissue surface in contact with the material was analyzed, and the quantitative analysis determined the volume density occupied by the inflammatory infiltrate (giant cells, polymorphonuclear cells and mononuclear cells), fibroblasts, collagen fibers and blood vessels. For the antimicrobial analysis, 20 dentin blocks infected with E. faecalis were treated with calcium hydroxide pastes in different vehicles; 0.4% chlorhexidine in PG, PG, extract from Casearia sylvestris Sw in PG and a positive control (infection and without medication) for 7 days. The efficiency of the pastes was evaluated by the live/dead technique and confocal microscopy. The results showed that 0.4% chlorhexidine induced a higher inflammatory response than the other groups. The Casearia sylvestris Sw extract showed satisfactory results in relation to the intensity of the inflammatory response. In the microbiological test, there were no statistical differences between the evaluated intracanal dressings and the percentage of bacterial viability was between 33 and 42%. The control group showed an 86% viability. Antimicrobial components such as chlorhexidine or Casearia sylvestris Sw did not improve the antimicrobial activity against E. faecalis in comparison to the calcium hydroxide+PG treatment. In addition, the incorporation of chlorhexidine in the calcium hydroxide paste promoted the highest inflammatory response.

A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy.

OBJECTIVES: To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM). MATERIAL AND METHODS: Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn's tests were used to evaluate the live and dead cells rates, and the scores obtained. RESULTS: The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05). CONCLUSION: The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.