Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

AIM: The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. METHODOLOGY: Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. RESULTS: Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. CONCLUSIONS: The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

Efficacy of mixed diclofenac solutions against root canal biofilms.

The objective of this research was to evaluate the efficacy of diclofenac sodium solutions, with or without cetrimide (CTR) added, against polymicrobial root canal biofilms grown in dentin specimens. The study groups were: (1) 5% diclofenac sodium (DCS); (2) 2.5% DCS; (3) 2.5% DCS + 0. 2% CTR; (4) 2.5% DCS + 0.4% CTR and (5) 0.9% saline solution (SS) as the control. After 5 min of solution contact with the biofilms, the antimicrobial activity was evaluated by means of the adenosine triphosphate (ATP) assay as well as confocal laser scanning microscopy (CLSM). Microbial quantification was indicated as the percentage reduction of relative light units (RLUs) for the ATP assay, the Log10 total biovolume and the viability percentage (green cells) for CLSM. Solutions of 2.5% DCS + 0.4% CTR and 5% DCS showed the highest antimicrobial efficacy. Cetrimide increased the antibiofilm activity of diclofenac sodium against endodontic biofilms.

Biological and chemical properties of 2-in-1 calcium-chelating and antibacterial root canal irrigants.

OBJECTIVES: To evaluate the capacity of canal wall smear layer removal, precipitation caused by irrigant interaction, antibacterial activity and cytotoxicity of three 2-in-1 root canal irrigating solutions. METHODS: Forty single-rooted teeth were mechanically instrumented and irrigated with QMix, SmearOFF, Irritrol or 0.9% saline. Each tooth was evaluated for smear layer removal using scanning electron microscopy. Precipitation after interaction of the irrigating solutions with sodium hypochlorite (NaOCl) was evaluated with 1H nuclear magnetic resonance and mass spectroscopy. Confocal laser scanning microscopy was used to evaluate the antimicrobial activity of the irrigants against Enterococcus faecalis biofilms. Neutral red and clonogenic assays were performed on Chinese hamster V79 cells to evaluate the short-term and long-term cytotoxicity of the irrigants. RESULTS: There was no significant difference between QMix and SmearOFF in eliminating smear layers from the coronal-third and middle-third of the canal spaces. In the apical-third, SmearOFF removed smear layers effectively. Irritrol incompletely removed smear layers from all the canal-thirds. When mixed with NaOCl, precipitation was evident only with Irritrol. QMix demonstrated a higher E. faecalis cell death percentage and a smaller biovolume. SmearOFF exhibited a larger decrease in biovolume compared with Irritrol, although Irritrol had a higher death percentage. Irritrol was more cytotoxic than the other irrigants on a short-term interval. In terms of long-term cytotoxicity, both Irritrol and QMix were cytotoxic. CONCLUSION: QMix and SmearOFF performed better in smear layer removal and antimicrobial activity. QMix and Irritrol were cytotoxic when compared to SmearOFF. Irritrol was associated with precipitation after interacting with NaOCl. CLINICAL SIGNIFICANCE: Evaluation of the smear layer removal capability, antibacterial activity and cytotoxicity of 2-in-1 root canal irrigants is necessary to ensure that they are safe to use during root canal treatment.

Effectiveness of conservative instrumentation in root canal disinfection.

OBJECTIVES: The impact of conservative instrumentation on the disinfection of root canals with different curvatures has not yet been determined. This ex vivo study aimed to evaluate and compare the effect of conservative instrumentation with TruNatomy (TN) and Rotate and a conventional rotary system, ProTaper Gold (PTG), on root canal disinfection during chemomechanical preparation of straight and curved canals. MATERIALS AND METHODS: Ninety mandibular molars with straight (n = 45) and curved (n = 45) mesiobuccal root canals were contaminated with polymicrobial clinical samples. Teeth were divided into three subgroups (n = 14) according to the file systems and the curvature. Canals were instrumented with TN, Rotate, and PTG, respectively. Sodium hypochlorite and EDTA were used as irrigants. Intracanal samples were taken before (S1) and after (S2) instrumentation. Six uninfected teeth were used as negative controls. The bacterial reduction between S1 and S2 was measured by ATP assay, flow cytometry, and culture methods. Kruskal-Wallis and ANOVA tests were followed by the Duncan post hoc test (p < 0.05). RESULTS: Bacterial reduction percentages were similar for the three file systems in straight canals (p > 0.05). However, PTG showed a lower reduction percentage of intact membrane cells in flow cytometry than TN and Rotate (p = 0.036). For the curved canals, no significant differences were obtained (p > 0.05). CONCLUSION: Conservative instrumentation of straight and curved canals using TN and Rotate files resulted in similar bacterial reduction compared to PTG. CLINICAL RELEVANCE: The disinfection efficacy of conservative instrumentation is similar to conventional instrumentation in straight and curved root canals.

Microbiome in paired root apices and periapical lesions and its association with clinical signs in persistent apical periodontitis using next-generation sequencing.

AIM: To assess and compare the microbiome of paired root apices and periapical lesions from cases with failed endodontic treatment and to associate the microbiome and bacterial metabolic pathways in both sites with asymptomatic apical periodontitis (AAP) and symptomatic apical periodontitis (SAP), using next-generation sequencing (NGS). METHODOLOGY: Matched root apices and periapical lesions of patients with failed root canal treatments were surgically extracted. Specimens were cryopulverized, bacterial DNA was extracted and the V3-V4 hypervariable regions of the 16 S rRNA gene were amplified and sequenced using the Illumina Miseq platform. Diversity and community composition were studied in the paired samples, as well as in AAP and SAP cases. Diversity indices were compared in each case by means of the Wilcoxon matched-pairs signed rank and Mann-Whitney U tests. Differences in the community composition were explored with multivariate statistical analysis and Linear discriminant analysis Effect Size (LEfSe). Bacterial functional study was performed through the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. RESULTS: Twenty-one paired apices and lesions were successfully sequenced and analysed, identifying a total of 21 phyla and 600 genera. A higher alpha-diversity was observed in the periapical lesions, although no global differences in the community composition between the two sites were found (p = .87), the most prevalent genera being Fusobacterium, Porphyromonas and Streptococcus. Prevotella, Clostridiales_vadinBB60_group, Bosea, Phreatobacter, Afipia and Xanthobacteriaceae_unclassified were enriched in SAP samples, while Pseudopropionibacterium, Campylobacter and Peptoniphilus were significantly more abundant in AAP cases (p < .05). Metabolic pathways involved in the amino acid metabolism or degradation and flagellum assembly were more abundant in SAP samples, whereas glucose metabolism-related pathways were associated with AAP. CONCLUSIONS: The bacterial community composition was similar in the apices and periapical lesions. The microbiome was different in AAP and SAP samples, gram-negative bacteria showing higher relative abundances in SAP cases. An association was observed between amino acid degradation and flagellum assembly pathways, and the development of tenderness to percussion or palpation.

Evaluation of the removal of 2-Hydroxyisocaproic acid from the root canal and its effect on the bond strength of MTA.

This study aimed to evaluate the removal of 2-hydroxyisocaproic acid (HICA) from the root canal system and its effect on the bond strength of the mineral trioxide aggregate (MTA). 126 single-rooted teeth were divided into 3 experimental groups (n = 36) that were treated with double antibiotic paste (DAP), HICA and calcium hydroxide (CH) and one control group (n = 18). In the first part, 18 teeth from each experimental group (n = 54) were examined to remove HICA from the root canal. In the second part, 72 teeth (3 experimental groups (n = 54) and one control group (n = 18)) were used for the evaluation of the push-out bond strength of MTA. There was no statistically significant difference between HICA, DAP and CH residues (p > 0.05). HICA group showed significantly less push-out bond strength (p < 0.05). DAP, HICA and CH could not be removed entirely from the root canal. HICA significantly reduced the bond strength of the MTA.

Antimicrobial potential of new diclofenac hydrogels for disinfection in regenerative endodontics: An in vitro and ex vivo study.

AIM: There is a need to explore new alternatives for root canal disinfection in regenerative endodontics, since the current strategies are far from ideal. Currently, the potential use of diclofenac (DC) is being investigated for controlling root canal infections. The objective was to evaluate the antimicrobial efficacy of novel DC-based hydrogels (DCHs) against polymicrobial biofilms grown in radicular dentine and root canals and to compare results with triantibiotic (TAH) and diantibiotic (DAH) hydrogels, and calcium hydroxide (Ca[OH]2 ). METHODOLOGY: The in vitro antimicrobial activity of intracanal medicaments was evaluated against 3-week-old polymicrobial root canal biofilms grown on human radicular dentine. Dentine samples were obtained and randomly divided into the study groups (n = 4/group): (1) 1 mg/ml TAH; (2) 1 mg/ml DAH; (3) 5% diclofenac (DCH); (4) 2.5% DCH; (5) 1.25% DCH; (6) 1 mg/ml DAH + 5% DCH; (7) Ca(OH)2 paste; (8) positive control. The microbial viability, in terms of percentage of intact cell membranes, was assessed after 7 days by confocal scanning laser microscopy (CSLM). The ex vivo efficacy of intracanal medications was evaluated in root canals infected with a polymicrobial suspension. Intracanal microbiological samples at baseline (S1) and 7 days post-treatment (S2) were taken; microbial quantification and cell viability were assessed by quantitative polymerase chain reaction (qPCR) and flow cytometry (FC). The mean Log10 of bacterial DNA copies in root canal samples before (S1) and the Log10 reduction of DNA copies S1-S2 in qPCR were recorded. The absolute value of total cells stained, and the percentage reduction of intact membrane cells after treatment (S1-S2), were analysed by FC. Global comparison was done using the Kruskal-Wallis test, whilst the Mann-Whitney U test was used for pair-by-pair comparison. RESULTS: Confocal scanning laser microscopy analysis indicated that the greatest effectiveness was obtained with 5% DCH, showing significant differences with respect to the other groups (p < .001). In root canals, the highest Log10 DNA reduction S1-S2 was obtained with 5% DCH and TAH, with no differences between them. The results of FC showed that only 5% DCH proved significantly superior to the other treatments. CONCLUSIONS: Sodium DC hydrogels demonstrate antimicrobial efficacy against endodontic biofilms.

Development of a chlorhexidine delivery system based on dental reline acrylic resins.

The high recurrence rate of common denture stomatitis after antifungal treatment is still concerning. This condition is caused by low patient compliance and incomplete local elimination of the main etiological factor - Candida albicans, often associated with other microorganisms, such as Streptococcus species. Impregnating denture materials with antimicrobials for local delivery is a strategy that can overcome the side effects and improve the efficacy of conventional treatments (topical and/or systemic). In this work, we describe the development of three hard autopolymerizing reline acrylic resins (Kooliner, Ufi Gel Hard, and Probase Cold) loaded with different percentages of chlorhexidine (CHX). The novel formulations were characterized based on their antimicrobial activity, mechanical, morphological and surface properties, in-vitro drug release profiles, and cytotoxicity. The addition of CHX in all resins did not change their chemical and mechanical structure. Among all the tested formulations, Probase Cold loaded with 5 wt% CHX showed the most promising results in terms of antimicrobial activity and lack of serious detrimental mechanical, morphological, surface, and biological properties.

Effect of the Controlled Delivery of Chelating Agents on the Pulp Tissue Dissolution Ability of Fresh Sodium Hypochlorite Solutions.

INTRODUCTION: The purpose of this study was to evaluate pulp tissue dissolution ability of sodium hypochlorite (NaOCl) when mixed with tetrasodic etidronate (Na4HEBP) and disodic ethylenediaminetetraacetate (Na2EDTA) under controlled flow. METHODS: Bovine pulp tissue was extracted from the lower incisors of 10 bovine jaws. Pulp specimens were standardized in size (1 × 3 × 10 mm), blotted dry, and weighed (initial weight [T0]: mean (SD) = 31.98 (1.18) mg). Specimens from the same jaw were randomly assigned to 2 control (n = 3 each) and 4 experimental groups (n = 10 each): NC (negative control/distilled water), PC (positive control/6%NaOCl), G1 (3%NaOCl), G2 (3%NaOCl-17% Na2EDTA), G3 (3%NaOCl-18% Na4HEBP), and G4 (3%NaOCl-9% Na4HEBP). Distilled water and NaOCl were provided using a delivery pump under a continuous controlled rate (1 mL/min). A second pump alternately delivered either Na2EDTA or Na4HEBP at the same rate with a 30-second programmed interval. Percentage of tissue weight loss was calculated at 2, 5, and 10 minutes (T2, T5, and T10) and compared among groups with analysis of variance. Free available chlorine and pH were controlled at T0 and T10. RESULTS: No tissue remained in PC at T5. No dissolution occurred in NC. There were no significant differences in the percentage of weight loss among experimental groups at any point of time. Some remnant tissue was found in G3 (1.4% ± 2.4) and G4 (1.6% ± 2.3) at T10, whereas nothing was left in G1 and G2. CONCLUSIONS: The controlled delivery of Na2EDTA and Na4HEBP did not alter tissue dissolution ability of NaOCl when fresh solutions were mixed in the root canal.

Present status and future directions - irrigants and irrigation methods.

Irrigation is considered the primary means of cleaning and disinfection of the root canal system. The purpose of this review was to set the framework for the obstacles that irrigation needs to overcome, to critically appraise currently used irrigants and irrigation methods, to highlight knowledge gaps and methodological limitations in the available studies and to provide directions for future developments. Organization of bacteria in biofilms located in anatomic intricacies of the root canal system and the difficulty to eliminate them is the main challenge for irrigants. Sodium hypochlorite remains the primary irrigant of choice, but it needs to be supplemented by a chelator. Delivery of the irrigants using a syringe and needle and activation by an ultrasonic file are the most popular irrigation methods. There is no evidence that any adjunct irrigation method, including ultrasonic activation, can improve the long-term outcome of root canal treatment beyond what can be achieved by instrumentation and syringe irrigation. It is necessary to redefine the research priorities in this field and investigate in greater depth the penetration of the irrigants, their effect on the biofilm and the long-term treatment outcome. New studies must also focus on clinically relevant comparisons, avoid methodological flaws and have sufficiently large sample sizes to reach reliable conclusions. Future multidisciplinary efforts combining the knowledge from basic sciences such as Chemistry, Microbiology and Fluid Dynamics may lead to more effective antimicrobials and improved activation methods to bring them closer to the residual biofilm in the root canal system.

A critical analysis of research methods and experimental models to study irrigants and irrigation systems.

Irrigation plays an essential role in root canal treatment. The purpose of this narrative review was to critically appraise the experimental methods and models used to study irrigants and irrigation systems and to provide directions for future research. Studies on the antimicrobial effect of irrigants should use mature multispecies biofilms grown on dentine or inside root canals and should combine at least two complementary evaluation methods. Dissolution of pulp tissue remnants should be examined in the presence of dentine and, preferably, inside human root canals. Micro-computed tomography is currently the method of choice for the assessment of accumulated dentine debris and their removal. A combination of experiments in transparent root canals and numerical modeling is needed to address irrigant penetration. Finally, models to evaluate irrigant extrusion through the apical foramen should simulate the periapical tissues and provide quantitative data on the amount of extruded irrigant. Mimicking the in vivo conditions as close as possible and standardization of the specimens and experimental protocols are universal requirements irrespective of the surrogate endpoint studied. Obsolete and unrealistic models must be abandoned in favour of more appropriate and valid ones that have more direct application and translation to clinical Endodontics.

Antibiofilm Activity of Diclofenac and Antibiotic Solutions in Endodontic Therapy.

INTRODUCTION: The aim of this study was to compare the antibiofilm effects of a triple antibiotic solution (TAS); a double antibiotic solution (DAS); and 5%, 2.5%, and 1.25% diclofenac solutions (DCSs) against Enteroccocus faecalis biofilm. METHODS: Eighty-four sterile radicular dentin blocks were used as biofilm substrate for 3 weeks. The study groups were as follows: (1) 1 mg/mL TAS (minocycline, metronidazole, and ciprofloxacin), (2) 1 mg/mL DAS (metronidazole and ciprofloxacin), (3) 5% DCS, (4) 2.5% DCS, (5) 1.25% DCS, and (6) 0.9% saline solution. The antimicrobial activity was evaluated by bacterial count determinations and confocal laser scanning microscopy. The contact time for the antimicrobial tests was 5 minutes. Bacterial counts were expressed as the reduction percentage of colony-forming units; for the confocal laser scanning microscopic evaluation, the log10 total biovolume and percentage of green population (live cells) were calculated. RESULTS: The colony-forming unit reduction percentage ranged between 62.98 and 98.62, respectively, for TAS and 5% DCS. The DCS showed a concentration-dependent effect.For the confocal laser scanning microscopy, the log10 total biovolume in all groups was very similar and showed a scarce (1.39-1.02) but significant reduction with respect to the control; 5% and 2.5% DCSs gave the lowest viable cell percentage. The TAS and DAS groups showed intermediate values without significant differences between them. CONCLUSIONS: DCSs at 5% and 2.5% have greater antimicrobial effects than TAS and DAS and may be considered a valid alternative for controlling the infection of teeth with apical periodontitis.

Antibacterial and antibiofilm activity over time of GuttaFlow Bioseal and AH Plus.

The sealers' antibacterial and antibiofilm activities against Enterococcus faecalis were evaluated by direct contact test (DCT) and confocal laser scanning microscopy (CLSM), respectively, after 1 day, and 1 and 4 weeks of aging. Cell viability was determined by adenosin triphosphate (ATP) assay after DCT. The parameters evaluated for the antibiofilm property were total biovolume and percentage of green cells in E.faecalis biofilms. The data from the bioluminescence ATP assay as well as the total biovolume and green percentage were analyzed by non-parametric tests, Kruskal-Wallis for global comparison and Kolmogorov-Smirnov for each two variables. Results of the DCT and CLSM for all parameters evaluated show that the antimicrobial activity of AH Plus decreased over time, whereas GuttaFlow Bioseal had an opposite property, increasing its antibacterial activity as the material aged.

Dentine tubule disinfection by different irrigation protocols.

The purpose of this study was to test the antimicrobial activity and the smear layer removal of different irrigation protocols-sodium hypochlorite (NaOCl), NaOCl followed by ethylenediaminetetraacetic acid (EDTA), and NaOCl combined with etidronic acid (HEBP)-against infected dentine tubules during root canal preparation. Single rooted premolars contaminated with Enterococcus faecalis were chemomechanically prepared. Depending on the irrigation protocols, the roots were divided into the following groups: (1) distilled water during and after instrumentation; (2) 2.5% NaOCl during and after instrumentation; (3) 2.5% NaOCl/9% HEBP during and after instrumentation; and (4) 2.5% NaOCl during instrumentation followed by 17% EDTA after instrumentation. The percentage of dead cells and the biovolume in infected dentine tubules were measured using confocal laser scanning microscopy and the live/dead technique. Smear layer removal on root canal wall surfaces was observed by means of scanning electron microscopy. Results were compared through parametric tests (p < .05). The groups NaOCl, NaOCl/HEBP, and NaOCl+EDTA exerted the highest antimicrobial activity (p > .05), followed by the group irrigated with water. All the irrigation protocols-including water-significantly reduced the bacteria biovolume. No dentine tubules free of smear layer were found in the positive control or the 2.5% NaOCl group. With NaOCl/HEBP and NaOCl+EDTA, respectively, 90.41% ± 7.33 and 76.54% ± 15.30 of dentine tubules were free of smear layer (p = .01). NaOCl/HEBP and NaOCl+EDTA exerted an important antimicrobial activity against bacteria inside dentine tubules, lowering the bacteria biovolume and eliminating a high amount of the smear layer, particularly in the NaOCl/HEBP group.

The Relationship of Surface Characteristics and Antimicrobial Performance of Pulp Capping Materials.

INTRODUCTION: Pulp capping materials need to be able to protect the pulp but also bond to the overlying restorative materials. Light-curable pulp capping materials bond better to restorative materials and are easier to place than most water-based cements. The aim of this study was to characterize new light-curable tricalcium silicate-based pulp capping materials and compare their surface and antimicrobial properties with clinically available Theracal (Bisco, Schaumburg, IL) and Biodentine (Septodont, Saint-Maur-des-Fossés, France). METHODS: The surface characteristics of 3 light-curable pulp capping materials based on a resin and filled with tricalcium silicate and tantalum oxide radiopacifier and Theracal and Biodentine were assessed by scanning electron microscopy, X-ray diffraction, and contact angle measurement. The radiopacity was measured following ISO 6876 standards. The antimicrobial activity was determined by the direct contact test and the antibiofilm activity by the adenosine triphosphate assay and the confocal laser scanning Live/Dead assay (Invitrogen, Eugene, OR) using a polymicrobial culture. RESULTS: The surface characteristics of the materials varied with the unfilled resin and Biodentine exhibiting a hydrophobic surface. Biodentine showed significantly higher antimicrobial properties in the direct contact test, but this property was absent in the antibiofilm activity tests. The resins filled with tricalcium silicate and Theracal showed higher antimicrobial activity than Biodentine in the adenosine triphosphate and live/dead assays. CONCLUSIONS: The surface characteristics of a material affect its antimicrobial properties. The experimental resin-modified materials exhibited comparable antimicrobial properties with other light-curable pulp capping agents. Further long-term studies on the materials' antimicrobial activity are required to assess whether they can result in better clinical outcomes.

Investigation of Acrylic Resin Disinfection Using Chemicals and Ultrasound.

PURPOSE: Dental prosthetic and orthodontic appliances are transported from the clinic to the laboratory for additions and repairs. These appliances, containing microbes from the oral flora, are a high risk for cross-contamination. The aim of this study was to evaluate the effect of chemical and ultrasound disinfection against two in vitro biofilms and an in vivo formed biofilm grown on unprepared and polished polymethyl methacrylate (PMMA) surfaces. MATERIALS AND METHODS: Rough and polished self-curing PMMA surfaces were infected with strains of both Candida albicans and Streptococcus oralis. After incubation, the samples were treated with different disinfection methods, including ultrasound treatment for both 15 and 30 seconds, and immersion in glutaraldehyde and alcohol-based chemical disinfectants (MD520 and Minuten, respectively). The disinfecting efficacy was assessed by colony forming units (CFU) analysis and by scanning electron microscopy (SEM). Furthermore the adequacy of bacterial elimination of application of 30-second ultrasound and MD520 was assessed on PMMA retrieved from ten volunteers by CFU analyses. ANOVA with p = 0.05 followed by the Tukey post hoc test and the Student t-test was used to analyze the data. RESULTS: The ultrasound treatment for 30 seconds, MD520, and Minuten were the most effective disinfectant methods as they reduced the microbial counts compared to the control (p < 0.05) as shown in the in vitro analyses. S. oralis adhered more to rough acrylic resin surfaces (p < 0.05). Ultrasound treatment was the most effective way to reduce microbial counts on PMMA exposed to oral flora (p = 0.043). CONCLUSION: Ultrasound treatment for 30 seconds was effective against C. albicans, S. oralis, and the oral flora as shown by testing microbial growth on agar plates and SEM.

Antibiofilm Activity of Sodium Hypochlorite and Alkaline Tetrasodium EDTA Solutions.

INTRODUCTION: The aim of this study was to determine the antimicrobial activity of mixed alkaline tetrasodium EDTA (EDTANa4)/sodium hypochlorite (NaOCl) solutions with and without the addition of cetrimide (CTR) against Enterococcus faecalis biofilms. METHODS: The antimicrobial solutions were evaluated on a 3-week biofilm of E. faecalis grown on radicular dentin blocks. The irrigating solutions were 2.5% NaOCl group, 20% EDTANa4 group, 10% EDTANa4 group, 2.5% NaOCl/10% EDTANa4 group, 2.5% NaOCl/5% EDTANa4 group, 2.5% NaOCl/10% EDTANa4/0.2% CTR group, 2.5% NaOCl/5% EDTANa4/0.2% CTR group, and 0.9% saline solution group. Cell viability was determined by adenosine triphosphate assay, and culture techniques were used to determine colony-forming unit (CFU) counts. In groups with positive growth, the Pearson linear correlation coefficient r (-1 ≤ r ≤ 1) between relative light units (RLUs) and log10 (CFUs + 1) was calculated. The results of the bioluminescence adenosine triphosphate assay and CFUs of the groups were analyzed by means of analysis of variance and the Duncan test. RESULTS: There was Pearson correlation between RLUs and log10 (CFUs + 1). A similar reduction of RLUs in all NaOCl groups was seen, giving statistical differences with respect to the control. In the NaOCl groups there was no bacterial growth. The lowest antimicrobial efficacy was found for the EDTANa4 groups. CONCLUSIONS: Mixed alkaline EDTANa4/NaOCl solutions with and without the addition of CTR do not interfere with the antimicrobial activity of NaOCl.

Antimicrobial and biological activity of leachate from light curable pulp capping materials.

OBJECTIVES: Characterization of a number of pulp capping materials and assessment of the leachate for elemental composition, antimicrobial activity and cell proliferation and expression. METHODOLOGY: Three experimental light curable pulp-capping materials, Theracal and Biodentine were characterized by scanning electron microscopy, energy dispersive spectroscopy and X-ray diffraction. The elemental composition of the leachate formed after 24h was assessed by inductively coupled plasma (ICP). The antimicrobial activity of the leachate was determined by the minimum inhibitory concentration (MIC) against multispecies suspensions of Streptococcus mutans ATCC 25175, Streptococcus gordonii ATCC 33478 and Streptococcus sobrinus ATCC 33399. Cell proliferation and cell metabolic function over the material leachate was assessed by an indirect contact test using 3-(4,5 dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The hydration behavior of the test materials varied with Biodentine being the most reactive and releasing the highest amount of calcium ions in solution. All materials tested except the unfilled resin exhibited depletion of phosphate ions from the solution indicating interaction of the materials with the media. Regardless the different material characteristics, there was a similar antimicrobial activity and cellular activity. All the materials exhibited no antimicrobial activity and were initially cytotoxic with cell metabolic function improving after 3days. CONCLUSIONS: The development of light curable tricalcium silicate-based pulp capping materials is important to improve the bonding to the final resin restoration. Testing of both antimicrobial activity and biological behavior is critical for material development. The experimental light curable materials exhibited promising biological properties but require further development to enhance the antimicrobial characteristics.

Influence of Smear Layer on the Antimicrobial Activity of a Sodium Hypochlorite/Etidronic Acid Irrigating Solution in Infected Dentin.

INTRODUCTION: The aim of this study was to evaluate the influence of the smear layer on the antimicrobial activity of a 2.5% sodium hypochlorite (NaOCl)/9% etidronic acid (HEBP) irrigating solution against bacteria growing inside dentin tubules. METHODS: Dentin tubules were infected with Enterococcus faecalis by centrifugation. After 5 days of incubation, the smear layer had formed in half of the samples, which were then treated with 2.5% NaOCl either alone or combined with 9% HEBP for 3 minutes. The percentage of dead cells in infected dentinal tubules was measured using confocal laser scanning microscopy and the live/dead technique. The smear layer on the surface of the root canal wall was also observed by scanning electron microscopy. Results of the percentage of dead cells were compared using parametric tests after subjecting data to the normalized Anscombe transformation. The level of significance was P < .05. RESULTS: In the absence of the smear layer, 2.5% NaOCl alone and combined with 9% HEBP showed high antimicrobial activity without significant differences between the 2. The smear layer reduced the antimicrobial activity of 2.5% NaOCl significantly, whereas the solution with HEBP was not affected. No dentin tubules free of the smear layer were obtained in the 2.5% NaOCl group. In the case of 2.5% NaOCl/9% HEBP, 95.40% ± 3.63% of dentin tubules were cleaned. CONCLUSIONS: The presence of the smear layer reduced the antimicrobial activity of 2.5% NaOCl. The combination of 2.5% NaOCl/9% HEBP exerted antimicrobial activity that was not reduced by the smear layer.