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.

Influencia de la dentina en el efecto antibacteriano de 2 concentraciones de hipoclorito de sodio en el crecimiento de Enterococcus faecalis ATCC 29212 / Influence of the dentin on the antibacterial effect of 2 concentrations of sodium hypochlorite in the growth of Enterococcus faecalis ATCC 29212

Este estudio in vitro evaluó la influencia de la dentina sobre el efecto antibacteriano contra Enterococcus faecalis ATCC 29212 de dos concentraciones de Hipoclorito de Sodio (NaOCl) 2,5 % y 5 %. Se empleó polvo de dentina a partir de dientes humanos (84 µg/ml) y la supervivencia de la bacteria se evaluó realizando recuento de unidades formadoras de colonias (UFC) a los 10, 30 y 60 segundos. Los datos se analizaron con la prueba estadística ANOVA factorial no encontrándose diferencias estadísticamente significativas entre los grupos con dentina y sin dentina. En conclusión, la dentina en este estudio no influyó en el efecto antibacteriano del Hipoclorito de Sodio en ninguna concentración, ni en los tiempos.

This in vitro study evaluated the influence of dentin on the antibacterial effect against Enterococcus faecalis ATCC 29212 of two concentrations of Sodium Hypochlorite NaOCl 2.5 % and 5 %. Dentin powder was used from human teeth (84 mg/ml) and the survival of the bacteria was evaluated by counting colony forming units (CFU) at 10, 30 and 60 seconds. The data were analyzed with the statistical ANOVA factorial test, finding no statistically significant differences between the groups with and without dentin. In conclusion, the dentin in this study had no inhibitory effect on antibacterial activity of Sodium Hypochlorite and any concentration, nor over time.

Efectividad bactericida del diamino fluoruro de plata a diferente concentración sobre estreptococos cariogénicos en muestras de saliva y dentina de escolares: un estudio in vitro / Bactericidal effectveness of silver diamine fluoride in different concentrations on cariogenic Streptococcus in salivary and dentinal samples of schoolchildren: an in vitro study

La OMS y la FDI han publicado que entre el 60 y 90% de los escolares padecen caries. En nuestro país, el Sistema de Vigilancia Epidemiológica de Patologías Orales (SIVEPAB) 2012, reporta un 85% de caries a nivel nacional en población pediátrica. Los agentes anticariogénicos como el diamino y el fluoruro de plata son un tratamiento alentador, este agente puede actuar como bactericida o bacteriostático en función de su concentración y su capacidad para inhibir el crecimiento de estreptococos del grupo viridans, y por ende, de la caries. Problema: ¿Cuál es la efectividad bactericida del diamino fluoruro de plata (Saforide®) a diferente concentración sobre la microbiota cariogénica de escolares? Objetivo: Determinar la eficacia bactericida del diamino fluoruro de plata (DFP) a diferentes concentraciones en el crecimiento bacteriano de Streptococcus mitis, S. mutans y S. salivarius en muestras de saliva y dentina en escolares. Material y métodos: Se llevó a cabo un estudio experimental con una variable independiente, el efecto bactericida del diamino fluoruro de plata y se tomó el halo de inhibición como la dependiente. Se utilizaron medidas descriptivas como prueba de comparación y análisis de varianza usando post-hoc Tukey≠ con una confianza del 95%, y análisis de datos exploratorios. Resultados: Se analizaron 100 muestras, de las cuales 48.3% correspondió a S. mutans, 41.4% a S. salivarius y 10.3% a S. mitis, se obtuvo una mayor zona de inhibición para las tres bacterias al 38% mostrando una diferencia estadísticamente significativa 12% (p < 0.05). También se observó un efecto bacteriostático al 12%, no así para el 38%, donde se encontró un efecto bactericida Conclusión: Nuestros resultados sugieren que al 38% de la concentración hay un claro efecto bactericida en el grupo de estreptococos viridans y el 12% no se recomienda para la detención de caries debido al efecto bacteriostático (AU)

WHO and FDI have ruled that 60-90% of schoolchildren are affected by caries. In our country, the System of Epidemiological Surveillance of Oral Pathologies (SIVEPAB) (SIVEPAB) 2012. Report a rate of 85% of caries nationally in pediatric population. Anticariogenic diamino agents such as silver fluoride are an encouraging decrease in treatment for these high rates of tooth decay in our country, this agent can act as bactericidal or bacteriostatic based on their concentration and their ability to inhibit endogenous metalloproteinase (MMP-2, 8, 9). Problem: What will be the bactericidal effectiveness of silver diamine fluoride different concentration on cariogenic Streptococci saliva samples taken from school and dentin? Objective: Determine the bactericidal effectiveness Silver diamine fluoride (SDF) to different concentration on bacterial growth of Streptococcus mitis, S. mutans, and S. salivarius in saliva samples and dentin in school. Material and methods: An experimental study was conducted as an independent variable the bactericidal effect of silver diamine fluoride was taken as dependent inhibition halo. Descriptive measures were used as a comparison test and analysis of variance using Post-hoc Tukey with 95% confidence, and exploratory data analysis. Results: One hundred samples, of which 48.3% corresponded to S. mutans, 41.4% to S. salivarius and 10.3% to S. mitis, were analyzed, we obtained a larger zone of inhibition for all three organisms at 38% showing a statistically significant difference from 12% (p < 0.05). It was also observed that the 12% sample bacteriostatic effect, not to the concentration of 38% was found a bactericidal effect. Conclusion: Our results suggest that 38% concentration has a bactericidal effect on Streptococcus viridans group and 12% showed not recommended for the arrest or detention of dentine caries bacteriostatic effect (AU)

Effect of Sweeteners on Root Dentine Demineralization Using a Microcosm Biofilm Model / Efecto de los Edulcorantes en la Desmineralización de la Dentina Radicular Utilizando un Modelo de Biofilm Microcosmo

ABSTRACT: The aim of the present study was to evaluate the effect of commercial sweeteners on root dentin demineralization using a microcosm biofilm model. Bovine dentin specimens with pre-determined surface hardness were randomized into six groups according to the studied sweeteners: sucralose, stevia, saccharin, aspartame. Sucrose was used as a positive control and an untreated group as a negative control. The specimens were submitted to biofilm development from one saliva donor and the cariogenic challenge occurred on subsequent five days, twice a day. At the end, the percentage of surface hardness loss (%SHL) and biomass was determined and submitted to ANOVA followed by Tukey's test. Sucrose presented the highest rate of demineralization, however, all sweeteners tested lead to a statistically higher root demineralization compared to the negative control (p <0.05). Sucrose caused greater demineralization in root dentin, however, the sweeteners were also able to induce it under this biofilm model.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto de los edulcorantes comerciales en la desmineralización de la dentina radicular utilizando un modelo de biofilm microcosmo. Se asignaron al azar muestras de dentina bovina con una dureza de la superficie predeterminada de acuerdo con los edulcorantes estudiados: sucralosa, estevia, sacarina, aspartame. La sacarosa se utilizó como control positivo y un grupo no tratado como control negativo. Las muestras se enviaron al desarrollo de biopelículas de un donante de saliva y el desafío cariogénico se produjo en los siguientes cinco días, dos veces al día. Al final, se determinó el porcentaje de pérdida de dureza de la superficie (% PDS) y biomasa y se aplicó un estudio estadístico de ANOVA seguido de la prueba de Tukey. La sacarosa presentó la mayor tasa de desmineralización; sin embargo, todos los endulzantes probados condujeron a una desmineralización de la raíz estadísticamente mayor en comparación con el control negativo (p<0,05). La sacarosa causó una mayor desmineralización en la dentina de raíz, sin embargo, los edulcorantes también fueron capaces de inducirla bajo este modelo de biofilm.

Comparison between static and semi-dynamic models for microcosm biofilm formation on dentin

Abstract Microcosm biofilm has been applied to induce carious lesions in dentin. However, no study has been done to compare the impact of the type of model for providing nutrients to microcosm biofilm formation on dentin. Objective This study compared the performance of two kinds of models (static and semi-dynamic) on the biofilm formation and the development of dentin carious lesions. Material and Methods In both models, biofilm was produced using inoculum from pooled human saliva mixed with McBain saliva for the first 8 h (5% CO2 and 37°C). Afterwards, for the static model, the samples were placed in 24-wells microplate containing McBain saliva with 0.2% sucrose, which was replaced at 24 h. In the semi-dynamic model, the samples were submitted to artificial mouth system with continuous flow of McBain saliva with 0.2% sucrose (0.15 ml/min, 37°C) for 10 h a day (for the other 14 h, no flow was applied, similarly to the static model). After 5 days, biofilm viability was measured by fluorescence and dentin demineralization by transverse microradiography. Results Biofilm viability was significantly lower for the static compared with semi-dynamic model, while dentin demineralization was significantly higher for the first one (p<0.05). The static model was able to produce a higher number of typical subsurface lesions compared with the semi-dynamic model (p<0.05). Conclusions The type of model (static and semi-dynamic) applied in the microcosm biofilm may have influence on it's viability and the severity/profile of dentin carious lesions.

Efficacy of removal of cariogenic bacteria and carious dentin by ablation using different modes of Er: YAG lasers

The primary objective of this in vitro study was to evaluate the efficiency of removal of cariogenic bacteria and carious dentin by ablation using two lasers: fluorescence-feedback controlled (FFC) Er:YAG laser and different pulses of Er:YAG laser based on variable square pulse technology (VSPt). The secondary objective was to measure the temperature during laser ablation of carious tissue. Seventy-two extracted human molars were used in this study. Sixty teeth with carious dentin were randomly divided into four experimental groups according to the treatment for caries removal: group 1: 400 µs (FFC group); group 2: super short pulse (SSP group, 50 µs pulse); group 3: medium short pulse (MSP group, 100 µs pulse); group 4: short pulse (SP group, 300 µs pulse) and one positive control group with no treatment. Twelve teeth without carious lesion were used as a negative control group. After caries removal, swabs were taken with cotton pellets and real-time PCR analysis was performed. During caries ablation, a thermal infrared camera was used to measure the temperature changes. In all experimental groups, specimens were free of bacterial contamination after the treatment. In the SSP, MSP and SP groups, temperatures measured during caries ablation were significantly higher compared to temperatures in the FFC group (P<0.001). In this in vitro study, laser treatment for removal of carious dentin and cariogenic bacteria was an efficient treatment modality without causing excessive temperatures that might adversely affect pulp vitality.

Doxycycline-containing glass ionomer cement for arresting residual caries: an in vitro study and a pilot trial

Abstract In a previous study, we demonstrated that the incorporation of doxycycline hyclate (DOX) into resin-modified glass ionomer cement (RMGIC) inhibited important cariogenic microorganisms, without modifying its biological and mechanical characteristics. In this study, we keep focused on the effect of that experimental material as a potential therapy for arresting residual caries by analyzing other in vitro properties and conducting a pilot clinical trial assessing the in vivo effect of DOX-containing RMGIC on residual mutans streptococci after partial carious removal in primary molars. Specimens of the groups RMGIC (control); RMGIC + 1.5% DOX; RMGIC + 3% DOX; and RMGIC + 4.5% DOX were made to evaluate the effect of DOX incorporation on surface microhardness and fluoride release of RMGIC and against biofilm of Streptococcus mutans. Clinical intervention consisted of partial caries removal comparing RMGIC and RMGIC + 4.5% DOX as lining materials. After 3 months, clinical and microbiologic evaluations were performed. Data were submitted to ANOVA/Tukey or Wilcoxon/Mann-Whitney set as α=0.05. Fluoride release and surface microhardness was not influenced by the incorporation of DOX (p>0.05). There was a significant reduction of S. mutans biofilm over the material surface with the increase of DOX concentration. After clinical trial, the remaining dentin was hard and dry. Additionally, mutans streptococci were completely eliminated after 3 months of treatment with RMGIC + 4.5% DOX. The incorporation of DOX provided better antibiofilm effect, without jeopardizing fluoride release and surface microhardness of RMGIC. This combination also improved the in vivo shortterm microbiological effect of RMGIC after partial caries removal.

Estudio histológico descriptivo de la colonización de bacterias en los túbulos dentinarios de dientes extraídos con necrosis pulpar / A descriptive histological study of bacterial colonization in the dentinal tubules of extracted teeth with pulp necrosis

Antecedentes: uno de los objetivos del tratamiento endodónticoconsiste en lograr la eliminación de los microorganismos residentes enlos conductos radiculares. Sin embargo, los microorganismos presentesen la necrosis pulpar se adaptan a las condiciones de los conductosnecróticos penetrando en los túbulos dentinarios, lo que complica elpronóstico del tratamiento. Objetivo: El propósito de esta investigaciónfue describir histológicamente las zonas de formación y distribución dela biopelícula tanto en los conductos como en los túbulos dentinarios dedientes extraídos con patología pulpo-periapical. Material y métodos:Se estudiaron 34 muestras de dientes extraídos con lesiones periapicales.Ninguno de los especímenes tenía tratamiento de conductos previo, nilesión endoperiodontal, ni fractura longitudinal o fractura de la raíz.Los dientes fueron descalcifi cados en ácido fórmico al 5% en formolamortiguado durante siete semanas. Se realizó el procedimiento histoló-gico de rutina para inclusión de las muestras en parafi na. Se obtuvieroncortes seriados longitudinales del conducto pulpar para someterlos atinción con hematoxilina y eosina, tinción de ácido peryódico de Schiff ,metenamina de plata y de Gram & Taylor Brown-Brenn para identifi carlos túbulos dentinarios, la presencia de hongos y bacterias. Resultados:De los 544 cortes estudiados, 75% (405) tuvieron colonizaciónmicrobiana. No se encontraron evidencias de la presencia de hongos.Con respecto a la profundidad de penetración de los microorganismosen los túbulos se identifi caron 194 cortes (35.6%) con presencia debacterias en 150 μm y 211 muestras (38.7%) en los que la penetraciónfue más allá de 500 μm...

Background: one of the main goals of endodontic treatment is toachieve the elimination of resident microorganisms in the root canal.However, the microorganisms involved in the pulp necrosis adapt to theconditions of necrotic canals, penetrating the dentinal tubules, whichcomplicates treatment. Objective: The purpose of this research was tohistologically describe the areas of formation and distribution of biofi lmin both the canals and the dentinal tubules of teeth extracted with pulpand periapical pathology. Material and methods: 34 samples of teethwith periapical lesions were studied. None of the specimens had priorcanal treatment, endoperiodontal injury, fracture nor longitudinal rootfracture. Teeth were decalcifi ed with 5% formic acid and buff ered withformalin for 7 weeks. Histological routine procedure for includingsamples in paraffi n was conducted. Longitudinal serial sections wereobtained of the pulp canal space for submission to staining withhematoxylin and eosin, peryodic acid Schiff , methenamine silver, andGram & Taylor Brown-Brenn, to identify dentinal tubules and thepresence of fungi and bacteria. Results: Of the 544 histological sectionsunder study 75% (405) showed microbial colonization. No evidence offungi was found. 194 histological sections (35.6%) had microorganismspenetrating the dentinal tubules to a depth of 150 microns, and 211histological sections (38.7%) had microorganisms penetrating thedentinal tubules for more than 500 μm...

Effects of different irrigation regimes in physico-chemical properties of dentin and consequences of changes in the adhesion of microorganisms and AH Plus sealer / Efeitos de diferentes regimes de irrigação nas propriedades físicoquímicas da dentina e consequências das alterações na adesão de microrganismos e do cimento endodôntico AH Plus

Besides of the desired effects, the chemical solutions used to assist the endodontic instruments in the cleanliness and disinfection of the root canal system can also cause changes in the physicochemical properties of dentin, and consequently affect the adhesion of endodontic sealers and microorganisms to the root canal walls. However, the effects of new irrigators and irrigation protocols remain unknown. The objectives of this thesis were to verify the alterations in the properties of some irrigants when used combined in mixtures, to define the time necessary for the smear layer removal by a new irrigant, to determine the organic matter dissolution capacity and the effects in the physicochemical properties of dentin of some irrigation solutions and protocols, and to evaluate the adhesion of microorganisms and AH Plus sealer to dentin after its submission to different irrigation sequences. In all experiments with dentin, the samples used were obtained from bovine teeth. In the analysis performed in this thesis, the following solutions were tested isolated and combined in different irrigation protocols: saline solution (control), sodium hypochlorite (NaOCl), trisodium (EDTAHNa3), alkaline ethylenediaminetetraacetic acid tetrasodium (EDTANa4), chlorhexidine (CHX), peracetic acid (PAA), and etidronic acid (HEDP). The EDTAHNa3 and EDTANa4 were tested in relation to their effects on the free chlorine content of NaOCl. The solutions were mixed in a 1:1 ratio and the iodometric titration of the mixtures performed in different time intervals. The time necessary for smear layer removal from dentin samples by solutions of EDTAHNa3 and different concentrations of EDTANa4 isolated and mixed with NaOCl was determined with the aid of the scanning electron microscope (SEM). The capacity of NaOCl to dissolve organic matter was determined by weighting fragments of bovine muscle before and after immersion in solutions of 1%, 2.5%, and 5% of NaOCl in different periods of time. Also, the effects of EDTAHNa3, EDTANa4 and HEDP on the organic matter dissolution by NaOCl were evaluated. The alterations produced by all solutions isolated and some irrigation protocols in the organic and inorganic components of the dentin surface were analysed by the attenuated total reflectance of Fourier transform infrared spectroscopy (ATR-FTIR) technique. Absorbance spectra were collected from the dentin surface before and after immersion of samples in the irrigants and the ratios of the amide III/phosphate and carbonate/phosphate bands were calculated. To quantify the adhesion of CHX to mineralized dentin and to dentin demineralized by different irrigation protocols, the areas of the band associated with CHX with the peak in 1492 cm−1 were determined in spectra obtained by ATR-FTIR. The effects of different irrigation protocols in the roughness and wettability of dentin surface were measured with a benchtop roughness tester and the sessile drop technique, respectively. For the assays of microorganisms' adhesion, samples were prepared and treated the same way and with the same irrigation protocols used in the roughness and wettability tests. Following, Candida albicans and Enterococcus faecalis were maintained in contact with the dentin for 2 hours and the samples were analyzed on the confocal laser scanning microscope (CLSM). Tests of push-out were performed to determine the impact of different irrigation protocols on the dentin bonding strength of AH Plus sealer over time. Canals of bovine incisors teeth were instrumented, irrigated and following obturated using only the sealer AH Plus. Half of the samples were submitted to pushout assessment 7 days after the obturation and the other half 20 months later. The results of the experiments showed that the EDTAHNa3 caused an almost complete and immediate loss of free available chlorine from NaOCl, whilst EDTANa4 promoted a slow and concentration-dependent decline. The smear layer was removed only by decalcifying solutions and in about 1 min by the 17% EDTAHNa3 and 5 min by the EDTANa4, both isolated or mixed with NaOCl. The increase in NaOCl concentration and contact time with the samples intensified the dissolution of organic matter. The mixtures of NaOCl with EDTANa4 and HEDP were able to dissolve the fragments of bovine muscle over-time, however, the EDTAHNa3 strongly affected the NaOCl dissolution capacity when they were mixed. The results of ATR-FTIR experiments showed that the increase in the NaOCl concentration intensified the deproteination of the dentin collagen with a reduction in the amide III/phosphate ratio. For the same decalcifying agent, the higher the concentration and immersion time the greater the removal of phosphate, exposure of the collagen matrix and consequently the increases in amide III/phosphate ratio. The PAA caused greater increases in amide III/phosphate ratio, followed by EDTAHNa3, EDTANa4 and HEDP and this order was maintained in the protocols in which NaOCl was used before the decalcifying agents. NaOCl required approximately 0.5 min to deproteinate the collagen matrix exposed after phosphate removal by EDTAHNa3 and PAA. The carbonate/phosphate ratio decreased after 30 s of samples immersion in solutions of NaOCl at 1%, 2.5% and 5% with no more alterations over time. The carbonate of the dentine was removed faster than phosphate by all decalcifying agents employed alone and in the irrigation protocols in which the use of the NaOCl was followed by the use of the EDTAHNa3, PAA and HEDP. For irrigation protocols that associate NaOCl with chelating solutions, the last irrigant used defined the final dentine amide III/phosphate and carbonate/phosphate ratios. For the ATR-FTIR analysis of CHX adhesion, the results showed that the adsorption of this irrigant to the dentin was potentiated when chelating agents were used prior to the CHX. In relation to the experiments of surface roughness, the saline solution, NaOCl, HEDP and CHX did not alter the roughness of the dentin, but EDTAHNa3 and PAA increased it. The wettability of the surface increased after the use of all irrigants, being the HEDP to cause the greater increases. In the assays of microorganisms' adhesion, the smear layer and collagen exposed by the chelating agents favored the adhesion of E. faecalis. The C. albicans adhesion was major in surfaces with smear layer and more mineral. The use of CHX as the final irrigant reduced the adhesion of both microorganisms. The wettability did not influence the microorganisms' adhesion, while increases in roughness seems to potentiate the adherence of E. faecalis. The experiments of bond strength of AH Plus to the dentin showed that the irrigation with NaOCl and mixture of NaOCl + EDTANa4 produced the lowest push-out bond strength values in 7 days compared to NaOCl + EDTAHNa3, NaOCl + EDTAHNa3 + NaOCl, NaOCl + EDTAHNa3 + CHX and the mixture of NaOCl + HEDP. After 20 months the lowest values were obtained in the groups irrigated with NaOCl and NaOCl + EDTAHNa3. The groups of NaOCl + EDTAHNa3 + NaOCl, mixture NaOCl + HEDP, and mixture NaOCl + EDTANa4 presented values of push-out bond strength in 20 months similar to the values in 7 days. It was possible to conclude that the irrigation solutions tested in this study have different effects in the organic and inorganic matter and some of them can affect the action of each other when mixed. Independent of being used isolated or combined in irrigation protocols, these irrigants cause modifications in the dentin physicochemical properties that influence the adhesion of AH Plus sealer in short and long term and the microorganisms' adherence to the surface in cases of recontaminations.(AU)

Além dos efeitos desejados, as soluções químicas utilizadas para auxiliar os instrumentos endodônticos na limpeza e desinfecção do sistema radiculares podem causar alterações nas propriedades físico-químicas da dentina e consequentemente afetar a adesão de cimentos endodônticos e microrganismos às paredes do canal radicular. Contudo, os efeitos de novos irrigantes e protocolos de irrigação ainda são desconhecidos. Os objetivos desta tese foram verificar as alterações nas propriedades de alguns irrigantes quando utilizados combinados em misturas, definir o tempo necessário para a remoção da camada de smear layer por um novo irrigante, determinar a capacidade de dissolução de matéria orgânica e os efeitos de algumas soluções e protocolos de irrigação nas propriedades físico-químicas de dentina e avaliar a adesão de microrganismos e cimento AH Plus à dentina após a submissão desta a diferentes sequências de irrigação. Em todos os experimentos com dentina as amostras utilizadas foram obtidas a partir de dentes bovinos. Nas análise realizadas nesta tese as seguintes soluções foram testadas isoladas e combinadas em diferentes protocolos de irrigação: solução salina (controle), hipoclorito de sódio (NaOCl), ácido etilenodiaminotetraacético trisódico (EDTAHNa3), ácido etilenodiaminotetracético tetrassódico alcalino (EDTANa4), clorexidina (CHX), ácido peracético (PAA) e ácido etidrônico (HEDP). O EDTAHNa3 e o EDTANa4 foram testados em relação aos seus efeitos sobre o teor de cloro livre do NaOCl. As soluções foram misturadas em uma proporção de 1:1 e a titulação iodométrica das misturas realizada em diferentes intervalos de tempo. O tempo necessário para a remoção da smear layer de amostras de dentina pela solução de EDTAHNa3 a 17% e diferentes concentrações de EDTANa4 isoladas e misturadas com NaOCl foi determinado com o auxílio do microscópio eletrônico de varredura (SEM). A capacidade de dissolução de matéria orgânica pelo NaOCl foi determinada pesando fragmentos de músculo bovino antes e depois da imersão em soluções de 1%, 2,5% e 5% de NaOCl em diferentes períodos de tempo. Além disso, os efeitos do EDTAHNa3, EDTANa4 e HEDP na dissolução de matéria orgânica pelo NaOCl foram avaliados. As alterações produzidas por todas as soluções isoladas e alguns protocolos de irrigação nos componentes orgânicos e inorgânicos da superfície da dentina foram analisadas pela técnica de reflexão total atenuada em espectroscopia no infravermelho por transformação de Fourier (ATRFTIR). Espectros de absorbância foram coletados da superfície da dentina antes e após a imersão das amostras nos irrigantes, e foram calculadas as razões das bandas de amida III/fosfato e carbonato/fosfato. Para quantificar a adesão da CHX à dentina mineralizada e à dentina desmineralizada por diferentes protocolos de irrigação, foram determinadas as áreas da banda associada a CHX com pico em 1492 cm−1 em espectros obtidos por ATR-FTIR. Os efeitos de diferentes protocolos de irrigação na rugosidade e molhabilidade da superfície da dentina foram medidos com um rugosímetro de bancada e a técnica de gota séssil, respectivamente. Para os ensaios de adesão de microrganismos, amostras foram preparadas e tratadas da mesma maneira e com os mesmos protocolos de irrigação utilizados nos testes de rugosidade e molhabilidade. Em seguida, Candida albicans e Enterococcus faecalis foram mantidos em contato com a dentina por 2 horas e as amostras foram analisadas no microscópio confocal de varredura laser (CLSM). Testes de push-out foram realizados para determinar o impacto de diferentes protocolos de irrigação na resistência de união à dentina do cimento AH Plus ao longo do tempo. Canais de dentes incisivos de bovinos foram instrumentados, irrigados e em seguida obturados utilizando apenas o cimento AH Plus. Metade das amostras foi submetida a avaliação de push-out 7 dias após a obturação e a outra metade após 20 meses. Os resultados dos experimentos mostraram que o EDTAHNa3 causou uma perda quase completa e imediata do cloro livre do NaOCl, enquanto o EDTANa4 promoveu um declínio lento e concentração dependente. A smear layer foi removida apenas por soluções descalcificantes e em cerca de 1 min pelo EDTAHNa3 a 17% e em 5 min pelo EDTANa4, tanto isolados ou misturados com o NaOCl. O aumento da concentração de NaOCl e do tempo de contato com os fragmentos de músculo bovino intensificou a dissolução da matéria orgânica. As misturas de NaOCl com EDTANa4 e HEDP foram capazes de dissolver as amostras de músculo ao longo do tempo, no entanto, o EDTAHNa3 afetou fortemente a capacidade de dissolução do NaOCl quando eles foram misturados. Os resultados dos experimentos com ATR-FTIR mostraram que o aumento da concentração do NaOCl intensificou a desproteinização do colágeno da dentina com redução da relação amida III/fosfato. Para o mesmo agente de descalcificação, quanto maior a concentração e o tempo de imersão, maior a remoção de fosfato, exposição da matriz de colágeno e consequentemente o aumento da proporção amida III/fosfato. O PAA causou os maiores aumentos na relação amida III/fosfato, seguido de EDTAHNa3, EDTANa4 e HEDP e esta ordem foi mantida nos protocolos em que o NaOCl foi usado antes dos agentes descalcificantes. O NaOCl necessitou aproximadamente 0,5 min para desproteinizar a matriz de colágeno exposta após a remoção de fosfato pelo EDTAHNa3 e o PAA. A relação carbonato/fosfato diminuiu após 30 s de imersão das amostras em soluções de NaOCl a 1%, 2,5% e 5%, sem mais alterações ao longo do tempo. O carbonato da dentina foi removido mais rápido do que o fosfato por todos os agentes descalcificantes empregados sozinhos e nos protocolos de irrigação em que o uso do NaOCl foi seguido pelo uso do EDTAHNa3, PAA e HEDP. Para os protocolos de irrigação que associam o NaOCl com soluções quelantes, o último irrigante utilizado definiu as proporções finais de amida II/fosfato e carbonato/fosfato da dentina. Para as análises da adesão da CHX em ATR-FTIR, os resultados mostraram que a adsorção deste irrigante à dentina foi potencializada quando agentes quelantes foram utilizados antes da CHX. Em relação aos experimentos de rugosidade da superfície, a solução salina, o NaOCl, o HEDP e a CHX não alteraram a rugosidade da dentina, mas o EDTAHNa3 e o PAA a aumentaram. A molhabilidade da superfície aumentou após o uso de todos os irrigantes, sendo que o HEDP causou os maiores aumentos. Nos ensaios de adesão dos microrganismos, a smear layer e o colágeno exposto pelos agentes quelantes favoreceram a adesão de E. faecalis. A adesão da C. albicans foi maior em superfícies com smear layer ou mais mineral. O uso de CHX como irrigante final reduziu a adesão de ambos os microrganismos. A molhabilidade não influenciou a adesão dos microrganismos, enquanto o aumento da rugosidade parece potencializar a adesão do E. faecalis. Os experimentos de resistência de união do AH Plus à dentina mostraram que a irrigação com NaOCl e a mistura de NaOCl + EDTANa4 produziram valores de resistência de união em 7 dias mais baixos em comparação com NaOCl + EDTAHNa3, NaOCl + EDTAHNa3 + NaOCl, NaOCl + EDTAHNa3 + CHX e a mistura de NaOCl + HEDP. Após 20 meses, os valores mais baixos foram obtidos nos grupos irrigados com NaOCl e NaOCl + EDTAHNa3. Os grupos de NaOCl + EDTAHNa3 + NaOCl, mistura de NaOCl + HEDP e mistura de NaOCl + EDTANa4 apresentaram valores de força de união por push-out em 20 meses semelhante aos valores em 7 dias. Foi possível concluir que as soluções de irrigação testadas neste estudo têm diferentes efeitos na matéria orgânica e inorgânica e elas podem afetar as ações umas das outras quando misturadas. Independentemente de serem utilizadas isoladas ou combinadas em protocolos de irrigação, os irrigantes causam modificações nas propriedades físico-químicas dentinárias que influenciam na adesão do cimento AH Plus a curto e longo prazo e na adesão de microrganismos à superfície em casos de recontaminação.(AU)

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

ABSTRACT 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.

Exopolysaccharide dispelled by calcium hydroxide with volatile vehicles related to bactericidal effect for root canal medication

ABSTRACT Objective: Enterococcus faecalis is the dominant microbial species responsible for persistent apical periodontitis with ability to deeply penetrate into the dentin. Exopolysaccharides (EPS) contribute to the pathogenicity and antibiotic resistance of E. faecalis. Our aim was to investigate the antimicrobial activity of calcium hydroxide (CH), camphorated parachlorophenol (CMCP), and chlorhexidine (CHX) against E. faecalis in dentinal tubules. Material and Methods: Decoronated single-canal human teeth and semicylindrical dentin blocks were incubated with E. faecalis for 3 weeks. Samples were randomly assigned to six medication groups for 1 week (n=10 per group): CH + 40% glycerin-water solution (1:1, wt/vol); CMCP; 2% CHX; CH + CMCP (1:1, wt/vol); CH + CMCP (2:3, wt/vol); and saline. Bacterial samples were collected and assayed for colony-forming units. After dentin blocks were split longitudinally, confocal laser scanning microscopy was used to assess the proportion of viable bacteria and EPS production in dentin. Results: CMCP exhibited the best antimicrobial activity, while CH was the least sensitive against E. faecalis (p<0.05). CHX showed similar antimicrobial properties to CH + CMCP (1:1, wt/vol) (p>0.05). CH combined with CMCP inhibited EPS synthesis by E. faecalis, which sensitized biofilms to antibacterial substances. Moreover, increasing concentrations of CMCP decreased EPS matrix formation, which effectively sensitized biofilms to disinfection agents. Conclusion: The EPS matrix dispelled by CH paste with CMCP may be related to its bactericidal effect; the visualization and analysis of EPS formation and microbial colonization in dentin may be a useful approach to verify medicaments for antimicrobial therapy.

In vivo model for microbial invasion of tooth root dentinal tubules

ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0) to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.

Antimicrobial activity and substantivity of Uncaria tomentosa in infected root canal dentin

Abstract The aim of this study was to analyze the antimicrobial activity and substantivity of Uncaria tomentosa Willd DC (cat's claw, CC) in root dentin contaminated with Enterococcus faecalis. Forty-eight human premolars were contaminated with E. faecalis (ATCC 29212) and randomly divided into four groups according to the irrigant used during chemomechanical preparation (CMP): CC group: 2% CC gel; CHX group: 2% chlorhexidine digluconate gel (CHX); NaOCl group: 5.25% sodium hypochlorite (NaOCl); and SS group: sterile saline (SS). Microbiological samples were collected before (S1) and after (S2) CMP and after 7 days (S3). Colony-forming units (CFU/mL) at the different sampling times and comparisons among the groups were statistically analyzed by Wilcoxon and Kruskal-Wallis tests (p < 0.05). Significant bacterial reduction was achieved in all groups after CMP (p < 0.05). Results show no significant difference between S3 and S2 (p > 0.05) in the CC and CHX groups. Bacterial load was higher in S3 than in S2 samples (p < 0.05) in the NaOCl and SS groups. Our results suggest antibacterial effect of 2% CC gel against E. faecalis in infected dentin, in addition to antibacterial substantivity of 2% CC and 2% CHX up to 7 days.

Enamel and dentine demineralization by a combination of starch and sucrose in a biofilm - caries model

Abstract Sucrose is the most cariogenic dietary carbohydrate and starch is considered non-cariogenic for enamel and moderately cariogenic for dentine. However, the cariogenicity of the combination of starch and sucrose remains unclear. The aim of this study was to evaluate the effect of this combination on Streptococcus mutans biofilm composition and enamel and dentine demineralization. Biofilms of S. mutans UA159 were grown on saliva-coated enamel and dentine slabs in culture medium containing 10% saliva. They were exposed (8 times/day) to one of the following treatments: 0.9% NaCl (negative control), 1% starch, 10% sucrose, or 1% starch and 10% sucrose (starch + sucrose). To simulate the effect of human salivary amylase on the starch metabolization, the biofilms were pretreated with saliva before each treatment and saliva was also added to the culture medium. Acidogenicity of the biofilm was estimated by evaluating (2 times/day) the culture medium pH. After 4 (dentine) or 5 (enamel) days of growth, biofilms (n = 9) were individually collected, and the biomass, viable microorganism count, and polysaccharide content were quantified. Dentine and enamel demineralization was assessed by determining the percentage of surface hardness loss. Biofilms exposed to starch + sucrose were more acidogenic and caused higher demineralization (p < 0.0001) on either enamel or dentine than those exposed to each carbohydrate alone. The findings suggest that starch increases the cariogenic potential of sucrose.

Clinical effect of photodynamic therapy on primary carious dentin after partial caries removal

Abstract This study was conducted to assess the clinical effect of photodynamic therapy (PDT) in the decontamination of the deep dentin of deciduous molars submitted to partial removal of carious tissue. After cavity preparation, dentin samples were taken from the pulp wall of nineteen deciduous molars before and after PDT application. Remaining dentin was treated with 0.01% methylene blue dye followed by irradiation with an InGaAlP diode laser (λ - 660 nm; 40 mW; 120 J/cm2; 120 s). Dentin samples were microbiologically assessed for the enumeration of total microorganisms, Lactobacillus spp. and mutans streptococci. There was no significant difference in the number of colony-forming units (CFU) for any of the microorganisms assessed (p > 0.05). Photodynamic therapy, using 0.01% methylene blue dye at a dosimetry of 120 J/cm2 would not be a viable clinical alternative to reduce bacterial contamination in deep dentin.

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.

Influence of antimicrobial solutions in the decontamination and adhesion of glass-fiber posts to root canals

AbstractObjective This study evaluated the effect of root canal disinfectants on the elimination of bacteria from the root canals, as well as their effect on glass-fiber posts bond strength.Material and Methods Fifty-three endodontically treated root canals had post spaces of 11 mm in length prepared and contaminated with E. faecalis. For CFU/ml analysis, eight teeth were contaminated for 1 h or 30 days (n=4). Teeth were decontaminated with 5% NaOCl, 2% CHX, or distilled water. As control, no decontamination was conducted. After decontamination, sterile paper points were used to collect samples, and CFU/ml were counted. For push-out, three groups were evaluated (n=15): irrigation with 2.5% NaOCl, 2% CHX, or sterile distilled water. A bonding agent was applied to root canal dentin, and a glass-fiber post was cemented with a dual-cured cement. After 24 h, 1-mm-thick slices of the middle portion of root canals were obtained and submitted to the push-out evaluation. Three specimens of each group were evaluated in scanning electron microscopy (SEM). Data were analyzed with one-way ANOVA and Dunnett’s T3 test (α=0.05).Results The number of CFU/ml increased from 1 h to 30 days of contamination in control and sterile distilled water groups. Decontamination with NaOCl was effective only when teeth were contaminated for 1 h. CHX was effective at both contamination times. NaOCl did not influence the bond strength (p>0.05). Higher values were observed with CHX (p<0.05). SEM showed formation of resin tags in all groups.Conclusion CHX showed better results for the irrigation of contaminated root canals both in reducing the bacterial contamination and in improving the glass-fiber post bonding.

Papacarie: A Chemomechanical Caries Removal Agent.

Chemomechanical caries removal (CMCR) is a noninvasive technique eliminating infected dentine via a chemical agent. Th e objective of CMCR is to eliminate the outer layer or infected dentin, leaving the aff ected layer or partly demineralized dentin, which can be remineralized and repaired. As this process based on minimally invasive dentistry, it not only removes infected tissues, also preserves healthy dental structure, avoiding pulp irritation and patient discomfort. Th is is a method of caries removal based on dissolution. Instead of drilling, this method uses a chemical agent assisted by an a traumatic mechanical force to remove soft carious structure. Th e chemomechanical method for caries removal is most outstanding among other alternative methods. Th is paper reviews one of the chemomechanical removal agent, Papacarie.

In vitro effectiveness of Brazilian brown propolis against Enterococcus faecalis

The aim of this study was to evaluate the in vitro antimicrobial activity of Brazilian brown propolis as an intracanal medication againstEnterococcus faecalis. Thirty dentin discs prepared from intact freshly extracted bovine maxillary central incisors were infected withE. faecalis for 21 days. The specimens were distributed into six groups according to the medicament used as follows: G1- calcium hydroxide paste; G2- Carbowax 400 (control group); G3- 20% brown propolis paste; G4- 40% brown propolis paste; G5- 20% brown propolis paste + calcium hydroxide paste; and G6- 40% brown propolis paste + calcium hydroxide paste. The experimental pastes were placed into the canal lumen and left for 14 days. After each period, irrigation was performed with sterile saline to remove the medicament, and the canals were dried with sterile paper points. The dentin chips were removed from the canals with sequential sterile round burs at low speed and were immediately collected in separate test tubes containing BHI broth. The tubes were incubated at 37°C, and microbial growth was analyzed by spectrophotometry after 15 days. All the experimental medications significantly reduced the number of viable bacteria. The G4 and G5 pastes were more effective than the G1 paste, with 35.8%, 41%, and 21.3% antibacterial activity, respectively. Brazilian brown propolis shows antibacterial capacity againstE. faecalis.

Effect of cariogenic challenge on the stability of dentin bonds

Objective: The oral environment is subject to biofilm accumulation and cariogenic challenge, and few studies exist on the effect of these factors on the bond strength of adhesive systems. The aim of this study was to test if the exposure of adhesive interfaces to cariogenic challenge under biofilm accumulation could promote higher degradation than the exposure to biofilm accumulation alone. Material And Methods: Five molars were ground until exposure of medium dentin and then restored (Single Bond 2 and Z250 3M ESPE). The tooth/resin sets were cut to obtain beam-shaped specimens, which were distributed according to the aging conditions (n=20): water for 24 h (control); biofilm under cariogenic challenge for 3, 5 or 10 days; biofilm without cariogenic challenge for 10 days; and water for 3 months. Microcosm biofilms were formed from human saliva and grown in a saliva analogue medium, supplemented or not with sucrose to promote cariogenic challenge. Specimens were tested for microtensile bond strength, and failure modes were classified using light microscopy. Bond strength data were analyzed using ANOVA and failure modes were analyzed using ANOVA on ranks (α=0.05). Results: No significant differences in bond strength were detected among the aging methods (P=0.248). The aging period was associated with an increase in the frequency of adhesive failures for the groups aged for 10 days or longer (P<0.001). Conclusion: Aging leads to a higher prevalence of interfacial adhesive failures, although this effect is not associated with cariogenic challenge or reduction in bond strengths. .