Effects of heat in the properties of NaOCl alone and mixed with etidronate and alkaline tetrasodium EDTA.

AIM: To assess the free available chlorine concentration (FAC), organic tissue dissolution and smear layer removal capacity of sodium hypochlorite (NaOCl) alone and when mixtured with etidronate (HEDP) and tetrasodium EDTA (Na4 EDTA), and heated to different temperatures. METHODOLOGY: Mixtures at 1 : 1 ratio of 5% NaOCl with distilled water (considered NaOCl alone), 18% HEDP or 10% Na4 EDTA were heated to 25 °C, 37 °C, 48 °C and 60 °C. The FAC in the mixtures was assessed at 5, 10, 20, 30, 60 and 120 min. Samples of bovine muscle tissue (n = 10) were prepared with similar size and weighed before and after 5, 10 and 15 min of immersion in the mixtures heated to the different temperatures to verify organic matter dissolution. The intergroup results were compared statistically using one-way analysis of variance (anova) and intragroup by two-way analysis of variance (anova), both followed by Tukey's multiple-comparison test (α < 0.01). Bovine dentine blocks (n = 10) were analysed by scanning electron microscopy before and after immersion in the mixtures, and the time taken to remove the smear layer from the surfaces of the samples was determined. The Friedman test was used to compare the scores of the same group (α < 0.01), and the Kruskal-Wallis test with Dunn's post hoc was used to compare the different groups (α < 0.01). Saline solution was used as a control in the experiments of tissue dissolution and smear layer removal, RESULTS: Heating NaOCl alone did not affect its FAC. The higher the temperature of the mixtures with the chelators, the lower the FAC. Organic tissue dissolution was improved by increases in temperature of NaOCl alone and its mixture with HEDP (P < 0.01); however, the mixture with Na4 EDTA had no improvement (P > 0.01). Smear layer removal by NaOCl alone was enhanced by heating resulting in lower scores in some samples and became more rapid in the mixtures with the chelators. The saline solution did not promote tissue dissolution nor smear layer removal (P > 0.01). CONCLUSION: In this laboratory study, heating NaOCl alone or when mixed with HEDP improved its capacity to dissolve organic matter and remove the smear layer. However, the mixture with HEDP required frequent refreshment to retain these effects when heated. Due to the acceleration in the reaction between the irrigants, very rapid reductions in the free available chlorine in the mixtures with Na4 EDTA heated to the different temperatures occurred.

DNA methylation profiles of immune response-related genes in apical periodontitis.

AIM: To investigate the DNA methylation profiles of immune response-related genes in apical periodontitis (AP) lesions. METHODOLOGY: The methylation profiles on the cytosine-phosphate-guanine (CpG) regions of 22 gene promoters involved in inflammation and autoimmunity were assessed in 60 human AP lesions and 24 healthy periodontal ligaments (controls) using a pathway-specific real-time polymerase chain reaction array (EpiTect® Methyl Signature PCR Array Human Inflammatory Response). Differentially methylated genes were subsequently assessed for their mRNA expression. Data analyses (One-way anova, Tukey's multiple comparisons tests and Mann-Whitney tests) were performed using GraphPad Prism 6 software. P values ≤ 0.05 were considered statistically significant. RESULTS: Significant DNA hypermethylation was observed for CXCL3 and FADD gene promoters in AP lesions when compared to control tissues (P < 0.001) and among other genes (P < 0.05). In contrast, IL12B and IL4R were associated with significant hypomethylation in comparison to other genes (P < 0.05). IL12B, IL4R, CXCL3 and FADD had differential mRNA expression in AP lesions and controls (P < 0.001). CONCLUSIONS: Differential methylation profiles of immune response-related genes, such as FADD, CXCL3, IL12B and IL4R, may have an influence on individual AP susceptibility and patient treatment outcomes, through their potential contributions to altered expression of disease-relevant genes. Methylation and/or genetic variations in additional genes may also contribute to the dynamics of AP development and should be considered in future studies.

Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine.

AIM: To investigate the effects of combinations of several irrigants on the roughness and wettability of dentine, adhesion of Enterococcus faecalis and Candida albicans and adsorption of chlorhexidine (CHX) to the dentine. METHODOLOGY: Bovine dentine samples were prepared and their surface roughness standardized. The samples were distributed in groups (n = 10) and subjected to one of the following irrigation protocols: G1 - saline solution; G2 - sodium hypochlorite (NaOCl); G3 - NaOCl + ethylenediaminetetraacetic acid (EDTA); G4 - NaOCl + peracetic acid (PAA); G5 - NaOCl + 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP); G6 - NaOCl + EDTA + CHX; G7 - NaOCl + PAA + CHX; G8 - NaOCl + HEDP + CHX; and G9 - mixture of NaOCl + HEDP. After treatments, roughness and wettability were measured. In order to evaluate the adhesion of microorganisms to dentine, new dentine samples were prepared and after 2 h of contact with the microorganisms, were analysed using a confocal laser scanning microscope and the number of microorganisms adhering to the surfaces were determined. Absorption spectra were collected by attenuated total reflectance of Fourier-transform infrared spectroscopy before and after immersion of other dentine samples in each solution of G6, G7 and G8 and in a solution of 2% CHX at various time intervals. The areas of the band associated with CHX with the peak at 1492 cm-1 were calculated between 1479 and 1500 cm-1 of the spectral range. The data obtained in all experiments were subjected to one-way ANOVA (α < 0.05). The values of the CHX band were also subjected to one-way repeated measures ANOVA (α < 0.05). RESULTS: Saline solution, NaOCl, HEDP and CHX did not alter the roughness of the dentine (P > 0.05), whilst EDTA and PAA did (P < 0.05). Dentine surface wettability increased after the use of all irrigants compared to saline solution (P < 0.05), with HEDP causing the greatest increases (P < 0.05). The adhesion of E. faecalis was favoured on surfaces treated with only saline solution and NaOCl, and on samples that had decalcifying agents as the final irrigant (P < 0.05). The adhesion of C. albicans was highest on surfaces treated with only saline solution and on surfaces that had NaOCl used as the last irrigant (P < 0.05). The use of CHX as a final irrigant reduced the adhesion of both microorganisms. The roughness and wettability did not influence the adhesion of the microorganisms tested. The adsorption of CHX to the dentine was significant after 1 min of immersion of the mineralized samples in the irrigant (P < 0.05), and the use of chelating agents prior to CHX potentiated this adsorption. CONCLUSIONS: The irrigation solutions had a variable effect on the properties of dentine, on the adhesion of E. faecalis and C. albicans and the adsorption of CHX to the dentine surface.

Analysis of the effects of several decalcifying agents alone and in combination with sodium hypochlorite on the chemical composition of dentine.

AIM: To investigate the effects of several decalcifying agents alone and in combination with sodium hypochlorite (NaOCl) on the organic and inorganic components of dentine using attenuated total reflectance in Fourier transform infrared spectroscopy (ATR-FTIR). METHODOLOGY: Dentine slices from bovine teeth were submitted to (n = 5) the following: 0.9% saline, 9% and 18% etidronic acid (HEDP), 5% and 10% tetrasodium EDTA (EDTANa4 ), 17% trisodium EDTA (EDTAHNa3 ), and 0.5% and 2.0% peracetic acid (PAA) for 0.5-10 min; and to the combinations: G1 - mixture 5% NaOCl + 18% HEDP (5 and 10 min); G2 - mixture 5% NaOCl + 10% EDTANa4 (5 and 10 min); G2 - 2.5% NaOCl (5 min) + 17% EDTAHNa3 (1 min); G3 - 2.5% NaOCl (5 min) + 0.5% PAA (1 min); G4 - 2.5% NaOCl (5 min) + 9% HEDP (5 min). Specimens of G2, G3 and G4 received final flushes with 2.5% NaOCl for 0.5-10 min. Amide III/phosphate and carbonate/phosphate ratios of the spectra collected from the dentine specimens before and after immersion in the solutions were determined. Data were submitted to one-way repeated measures and one-way anova. RESULTS: For the same decalcifying agent, the higher the concentration and immersion time the greater the removal of phosphate, exposure of collagen matrix and consequently the increases in amide III/phosphate ratio. However, significant differences were found only between the two concentrations of PAA (P < 0.05). PAA caused greater increases in this ratio, followed by EDTAHNa3 , EDTANa4 and HEDP, and this order was retained in the combinations with NaOCl. This ratio was significantly reduced in G1 (P < 0.05) and not altered in G2 (P > 0.05). Due to collagen degradation, the amide III/phosphate ratio reduced significantly after the use of NaOCl in G3, G4 and G5 (P < 0.05). NaOCl required approximately 0.5 s to deproteinate the collagen matrix exposed after phosphate removal by EDTAHNa3 and PAA. The carbonate of dentine was removed more rapidly than phosphate by all decalcifying agents alone and in G3, G4 and G5. In the combinations with NaOCl, the last irrigant used defined the dentine amide III/phosphate and carbonate/phosphate ratios. CONCLUSIONS: HEDP and EDTANa4 caused minor whilst EDTAHNa3 and PAA caused greater demineralization of dentine; both effects were time and concentration dependent. NaOCl degraded the dentine organic matrix more rapidly when it was exposed. Combinations of NaOCl and decalcifying agents can be used to create dentine surfaces with varying compositions for interaction with endodontic sealers.

Mixture of alkaline tetrasodium EDTA with sodium hypochlorite promotes in vitro smear layer removal and organic matter dissolution during biomechanical preparation.

AIM: The aim of this study was to determine the following: (i) the quantity of free chlorine in mixtures of equal proportions of sodium hypochlorite (NaOCl) with trisodium ethylenediaminetetraacetic acid (EDTAHNa3 ) and alkaline tetrasodium ethylenediaminetetraacetic acid (EDTANa4 ); (ii) organic matter dissolution; and (iii) the time necessary to remove the smear layer by these irrigants alone and when mixed. METHODOLOGY: The solutions were mixed in a 1 : 1 ratio and then iodometrically titrated over time to determine the quantity of free available chlorine. The capability of organic matter dissolution by the solutions alone and the mixtures of irrigants was analysed by weighing bovine muscle tissue specimens before and after submission to the following groups (n = 10): G1 - 0.9% saline solution (control), G2 - 2.5% NaOCl, G3 - 17% EDTAHNa3 , G4 - 10% EDTANa4 , G5 - 20% EDTANa4 , G6 - 5% NaOCl + 17% EDTAHNa3 , G7 - 5% NaOCl + 10% EDTANa4 and G8 - 5% NaOCl + 20% EDTANa4 . The times necessary for smear layer removal were determinated on discs of bovine dentine with a standardized smear layer produced with SiC papers using a scanning electron microscope that did not require the samples to be sputter coated. The dentine discs were submitted to the same experimental groups previously described (n = 10) over several time periods, and the photomicrographs acquired were scored for the presence of smear layer. The parametric data of tissue dissolution were analysed using two-way anova and one-way anova with Tukey's post hoc tests (α < 0.05), whilst nonparametric data of smear layer removal were analysed by Friedman test (α < 0.05) and the Kruskal-Wallis test with Dunn's post hoc (α < 0.05). RESULTS: EDTAHNa3 caused an almost complete and immediate loss of free available chlorine from NaOCl, whilst EDTANa4 promoted a slow and concentrat-ion-dependent decline. The organic matter was not dissolved in the control group, EDTA groups or the mixture of NaOCl + 17% EDTAHNa3 group (P > 0.05). NaOCl alone and the associations of NaOCl + EDTANa4 dissolved tissue at all periods analysed (P < 0.05). The smear layer was not removed in the control and NaOCl groups (P > 0.05). The smear layer was removed at 1 min in the NaOCl + 17% EDTAHNa3 group (P < 0.05); 2 min in 17% EDTAHNa3 group (P < 0.05); and 5 min in 10% EDTANa4 , 20% EDTANa4 , 5% NaOCl + 10% EDTANa4 and 5% NaOCl + 20% EDTANa4 groups (P < 0.05). CONCLUSIONS: Alkaline EDTANa4 was slower in removing the smear layer than EDTAHNa3 , but when mixed with NaOCl during biomechanical canal preparation promoted organic matter dissolution and smear layer removal simultaneously. However, the mixing of NaOCl and EDTANa4 should be performed immediately before use to prevent the reduction of free available chlorine.

Etidronate causes minimal changes in the ability of sodium hypochlorite to dissolve organic matter.

AIM: To evaluate the effect of individual and combined use of sodium hypochlorite (NaOCl), etidronate (HEDP) and ethylenediaminetetraacetic acid (EDTA) in tissue dissolution. METHODOLOGY: Sixty fragments of bovine muscle tissue were prepared and their weights determined on a precision scale. The samples were then distributed in the following groups (n = 10): G1 - saline solution (control); G2 - 17% EDTA; G3 - 18% HEDP; G4 - 2.5% NaOCl; G5 - mixture of 5% NaOCl + 17% EDTA; and G6 - mixture of 5% NaOCl + 18% HEDP. The specimens in each group were immersed in the solutions for 5, 10 and 15 min and reweighted at each time period. Analysis of variance (anova) and Tukey's multiple-comparison tests (α<0.05) were applied to identify the intragroup and intergroup differences. RESULTS: G1, G2, G3 and G5 did not dissolve the organic matter. G4 and G6 significantly reduced the weights of specimens at all periods. Amongst the groups, the difference in ability to dissolve organic matter was greater and significant in the following order G4 = G6 > G5 = G3 = G2 = G1 after 5 min of immersion and G4 > G6 > G5 = G3 = G2 = G1 after 10 and 15 min of immersion. CONCLUSION: The only solution capable of dissolving organic matter was NaOCl. In the mixtures analysed, this ability was arrested by EDTA; however, it was minimally affected by the HEDP, proving that this combination, if used during the biomechanical preparation, is able to dissolve of organic matter.

Effects of sodium hypochlorite associated with EDTA and etidronate on apical root transportation.

AIM: To evaluate the influence of sodium hypochlorite associated with EDTA and etidronate on apical root transportation. METHODOLOGY: Forty-five roots of human mandibular molars with curvatures of 15-25° were embedded in acrylic resin to allow standardized angulation of the initial and final radiographs. The pre-instrumentation radiographs of the mesiobuccal canal of each root were taken using a radiograph digital sensor with a size 15 K-file in the canal. The canals were prepared with the ProTaper Universal system (Dentsply Maillefer, Ballaigues, Switzerland), using one of the following irrigation regimens during the instrumentation (n = 15): G1 - irrigation with 20 mL of saline solution (control); G2 - alternating irrigation with 2.5% hypochlorite solution (NaOCl) (15 mL); and 17% ethylenediaminetetraacetic acid (EDTA) (5 mL). During instrumentation, the canal was filled with NaOCl and then between each exchange of instrument filled with EDTA for 1 min, and G3 - irrigation with 20 mL of 5% NaOCl and 18% etidronate solution (HEBP) mixed in equal parts. The postinstrumentation radiographs were made with a F3 instrument in the canal. The images were magnified and superposed with Adobe Photoshop software (Adobe Systems, Mountain View, CA, USA). Apical transportation was determined with AutoCAD 2012 software (Autodesk Inc., San Rafael, CA, USA) by measuring the distance in millimetres between the tips of the instruments. The results were subjected to the nonparametric statistical Kruskal-Wallis test (α < 0.05). RESULTS: The median transportation and interquartile range values were 0.00 ± 0.05 for G1, 0.08 ± 0.23 for G2 and 0.13 ± 0.14 for G3. Comparison between groups showed that apical transportation in G3 was significantly greater than in G1 (P < 0.05). CONCLUSION: The use of NaOCl associated with etidronate increased apical transportation in the canals of extracted teeth.