Green synthesis, characterization and antimicrobial evaluation of silver nanoparticles for an intracanal dressing

Abstract Green chemistry has been applied in different areas due to the growing demands for renewable processes and one of them is nanotechnology. The aim of this study was to characterize a formulation containing silver nanoparticles (AgNPs) produced by a green synthesis and to evaluate its antimicrobial activity. The formulation will be used as an intracanal dressing exploiting the AgNPs' antimicrobial properties, which are crucial to prevent infections and bacterial reinfections that can compromise endodontic treatments. In the green synthesis, silver nitrate was employed as the precursor salt, maltose as a reducing agent, and gelatin as a stabilizing agent. The formulation was prepared mixing 50 % of a liquid containing the AgNPs and 50 % of hydroxyethylcellulose gel at 1.5 % with proper evaluation of the process inherent parameters. Techniques such as molecular absorption spectrometry and dynamic light scattering were used in characterization step. The antimicrobial activity of the AgNPs against Escherichia coli ATCC 25922, Enterococcus faecalis NCTC 775, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 and Streptococcus mutans ATCC 25175 was verified according to National Comittee for Clinical Laboratory Standards (NCCLS) by determining minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The obtained results indicated the formulation containing AgNPs produced by a green synthesis was properly characterized by the selected techniques. Furthermore, the formulation assessment proved that it is suitable for the proposal as well as it has potential to be used as an intracanal dressing since presented antimicrobial activity against all bacterial strains evaluated.

Resumo A química verde tem sido aplicada em diferentes áreas devido à crescente demanda por processos renováveis e uma delas é a nanotecnologia. O objetivo deste estudo foi caracterizar uma formulação contendo nanopartículas de prata (AgNPs) produzidas por meio de síntese verde e avaliar sua atividade antimicrobiana. A formulação será usada como curativo intracanal explorando as propriedades antimicrobianas das AgNPs que são cruciais para prevenir infecções e reinfecções bacterianas que podem comprometer os tratamentos endodônticos. Na síntese verde, nitrato de prata foi empregado como sal precursor, maltose como agente redutor e gelatina como agente estabilizador. A formulação foi preparada misturando-se 50% do líquido contendo as AgNPs e 50% de gel de hidroxietilcelulose a 1,5% com avaliação adequada dos parâmetros inerentes ao processo. Técnicas como espectrometria de absorção molecular e espalhamento dinâmico de luz foram usadas na etapa de caracterização. A atividade antimicrobiana das AgNPs contra Escherichia coli ATCC 25922, Enterococcus faecalis NCTC 775, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 e Streptococcus mutans ATCC 25175 foi verificada de acordo com o National Comittee for Clinical Laboratory Standards (NCCLS), determinando-se a concentração inibitória mínima (MIC) e a concentração bactericida mínima (MBC). Os resultados obtidos indicaram que a formulação contendo AgNPs produzidas por meio de síntese verde foi devidamente caracterizada pelas técnicas selecionadas. Além disso, a avaliação da formulação provou que ela é adequada para a proposta, bem como tem potencial para ser utilizada como curativo intracanal já que apresentou atividade antimicrobiana contra todas as cepas bacterianas avaliadas.

Influence of solvents on the bond strength of resin sealer to intraradicular dentin after retreatment

Abstract This study evaluated the removal of filling material with ProTaper Universal Rotary Retreatment system (PTR) combined with solvents and the influence of solvents on the bond strength (PBS) of sealer to intraradicular dentin after canal reobturation. Roots were endodontically treated and distributed to five groups (n = 12). The control group was not retreated. In the four experimental groups, canals were retreated with PTR alone or in combination with xylol, orange oil, and eucalyptol. After filling material removal, two specimens of each group were analysed by SEM and µCT to verify the presence of filling remnants on root canal walls. The other roots were reobturated and sectioned in 1-mm-thick dentin slices that were subjected to the push-out test. Data were analysed by two-way ANOVA and Tukey’s test (α = 0.05). SEM and µCT analysis revealed that all retreatment techniques left filling remnants on canal walls. The control group (3.47 ± 1.21) presented significantly higher (p < 0.05) PBS than the experimental groups. The groups retreated with PTR alone (2.59 ± 0.99) or combined with xylol (2.54 ± 0.77) and orange oil (2.32 ± 0.93) presented similar bond strength (p > 0.05), and differed significantly from the group with eucalyptol (1.89 ± 0.63). The solvents reduced the PBS of the sealer to dentin and no retreatment technique promoted complete removal of filling material.

New Methodology to Evaluate Bond Strength of Root-End Filling Materials

This study evaluated the bond strength of root-end filling materials to root-end cavities using a new methodology. Twenty maxillary central incisors were subjected to biomechanical preparation (#80 hand file) and sectioned transversally 2 mm short of the apex and 4 mm coronally to this point. The root cylinders were embedded in acrylic resin and positioned at 45° to the horizontal plane for preparation of root-end cavities with a diamond ultrasonic retrotip. Two groups (n=10) were formed according to the root-end filling material: MTA and Super EBA. A gutta-percha cone (#80) was tug-backed at the limit between the canal and the root-end cavity. The cavity was filled and the gutta-percha cone was removed after complete setting of the sealer. The specimens were placed in an Instron machine with the root-end filling turned downwards. The push-out shaft was inserted in the space previously occupied by the gutta-percha cone and run at a crosshead speed of 1.0 mm/min for pushing out the root-end filling material. Data were analyzed by ANOVA (α=5%). Super EBA (6.03±1.31) presented higher bond strength (MPa) than MTA (1.81±0.45) (p>0.05). There was a predominance of cohesive failures for Super EBA and mixed for MTA. The protocol of specimen preparation is effective and introduces a specific methodology for assessing bond strength of root-end filling materials to dentin. Among the materials, Super EBA presented the highest bond strength.

Este estudo avaliou a resistência de união de materiais retrobturadores a cavidades retrógradas, usando uma nova metodologia. Vinte incisivos centrais superiores foram submetidos ao preparo biomecânico (lima manual #80) e seccionadas transversalmente a 2 mm do ápice e a 4 mm do primeiro corte. Os cilindros de raízes foram incluídos em resina acrílica e posicionado a 45° em relação ao plano horizontal para confecção das retrocavidades com ponta ultrassônica diamantada. Dois grupos (n = 10) foram formados de acordo com os cimentos utilizados: MTA e Super EBA. Um cone de guta-percha (#80) foi travado no limite da retrocavidade com o canal radicular. A retrocavidade foi preenchida e, após o tempo de endurecimento do cimento utilizado, o cone de guta-percha foi removido. Os espécimes foram fixados na máquina universal de ensaios Instron com a retrobturação voltada para baixo. A haste empregada para pressionar o cimento retrobturador no teste de push-out foi inserida no espaço anteriormente ocupado pela guta-percha e acionada com velocidade de cruzeta de 1,0 mm/min, para extrusão do material retroburador. Os dados foram analisados por meio do teste ANOVA (α=5%). O cimento Super EBA (6,03 ± 1,31) apresentou maior resistência de união (MPa) comparado ao cimento MTA (1,81 ± 0,45) (p> 0,05). Houve predomínio de falhas coesivas para o cimento Super EBA e falhas mistas para o MTA. O protocolo de preparação das amostras é eficaz e apresenta uma metodologia específica para avaliação da resistência de união de materiais retrobturadores à dentina. Dentre os materiais testados, o cimento Super EBA apresentou a maior resistência de união.

Effects of various irrigation/aspiration protocols on cleaning of flattened root canals

This study aimed to evaluate, by means of scanning electron microscopy (SEM), the cleaning of flattened root canals, varying irrigation/aspiration protocols during biomechanical preparation. Thirdy human mandibular incisors were distributed into three groups (n = 10) according to the aspiration/irrigation protocols: conventional, conventional + brush, and apical negative pressure irrigation. Irrigation procedure was performed with 5 mL of 1% NaOCl at each change of instrument; final irrigation was conducted with 17% EDTA for 5 min. After biomechanical preparation, the roots were sectioned and prepared for SEM analysis. The images obtained were evaluated under 35× and 1,000× magnification by three calibrated examiners, following a double-blind design. All data were analyzed using Kruskal–Wallis and Dunn’s post hoctests (α = 0.05). Canals wherein the conventional method and apical negative pressure irrigation were employed revealed less debris, statistically different from the conventional + brush group (p < 0.05). Regarding the presence of smears, apical negative pressure irrigation was more effective in cleaning, showing lowest scores (p < 0.05), compared with the other tested protocols. Comparing each root canal third revealed that the apical portion was difficult to clean as all the tested protocols showed similar high scores (p > 0.05), both for the presence of debris and smear layer. In conclusion, although none of the studied irrigation/aspiration protocols have completely cleaned flattened root canals, apical negative pressure irrigation was more effective in smear layer removal, whereas the conventional + brush protocol was the least effective in removing the debris and smear layer.

Quantitative Assessment of Root Canal Roughness with Calcium-Based Hypochlorite Irrigants by 3D CLSM

Chemical solutions play important roles in endodontic treatment and promote ultrastructural changes in dentin surface. The aim of this study was to quantify root canal roughness at different concentrations of calcium hypochlorite (Ca(OCl)2) and sodium hypochlorite (NaOCl) by confocal laser scanning microscopy (CLSM). Fifty-two human mandibular premolars were sectioned and randomly organized into thirteen groups (n=8): saline (control); 1%, 2.5% and 5% NaOCl; 1%, 2.5% and 5% Ca(OCl)2; the hypochlorite groups were further divided into with or without EDTA. The chlorine concentrations of the different solutions were measured by iodine titration (%). The superficial roughness (Sa) was quantified by CLSM. Ca(OCl)2 presented substantial decrease in chlorine concentration that differed from the package indication, but without compromising the dentin ultrastructure changes. There were no significant differences in dentin roughness between Ca(OCl)2 or NaOCl at all studied concentrations. The combination with EDTA provided similar roughness values among the solutions (p>0.05). The 5% Ca(OCl)2 and NaOCl solutions significantly increased dentin roughness and did not differ from the EDTA association (p>0.05). Ca(OCl)2 promoted similar dentin roughness as the NaOCl at the same concentrations and combined with EDTA. It may be concluded that Ca(OCl)2 modified the root canal dentin roughness similarly to NaOCl, at the same concentrations and EDTA combinations used in this study. Ca(OCl)2 and NaOCl, both at 5%, significantly altered dentin roughness, overcoming EDTA association, thus Ca(OCl)2 concentrations ranging from 1% to 2.5% may be suitable solutions for root canal irrigation protocols.

Soluções químicas são fundamentais para o tratamento endodôntico; entretanto, promovem alterações ultraestruturais na superfície dentinária. O objetivo deste estudo foi quantificar a rugosidade da dentina radicular com diferentes concentrações de hipoclorito de cálcio (Ca(OCl)2) e hipoclorito de sódio (NaOCl) utilizando microscopia confocal à laser (CLSM). Foram utilizados 52 premolares humanos inferiores e aleatoriamente divididos em treze grupos (n=8): Soro fisiológico (controle); NaOCl a 1%, 2,5% and 5%; Ca(OCl)2 a 1%, 2,5% and 5%; os grupos de hipoclorito foram subdivididos pela associação ou não ao ácido etilenodiaminotetracético (EDTA). A concentração de cloro ativo foi avaliada para diferentes soluções utilizando titulação iodométrica (%). A rugosidade superficial (Sa) foi quantificada por CLSM. Ca(OCl)2 apresentou perda substancial de cloro ativo e que foi distinta da condição descrita pelo fabricante, sem entretanto comprometer as alterações no substrato dentinário. Não houve diferenças significantes na rugosidade dentinária produzida pelos Ca(OCl)2 e NaOCl em todas as concentrações estudadas e associação com EDTA. A associação ao EDTA produziu rugosidade semelhante entre as soluções (p>0.05). O Ca(OCl)2 e NaOCl na concentração de 5% aumentaram significativamente a rugosidade dentinária e não apresentaram diferenças dos valores obtidos com a associação de EDTA (p>0.05). O Ca(OCl)2 alterou a rugosidade da dentina radicular de forma semelhante ao NaOCl, nas concentrações e associações utilizadas neste estudo. Como a concentração de 5% de Ca(OCl)2 e NaOCl, apresentou maior rugosidade dentinária, independente da associação ao EDTA, pode-se concluir que Ca(OCl)2 nas concentrações de 1% e 2,5% pode ser considerado uma solução adequada para a irrigação de canais radiculares.

Development of Intracanal Formulation Containing Silver Nanoparticles

This study aimed to synthetize, characterize and evaluate the antimicrobial properties of silver nanoparticles to be used in the development of a root intracanal formulation. Silver nanoparticles (AgNPs) were obtained by reduction of silver nitrate with sodium borohydride and characterized by UV-Visible spectrophotometry, scanning electron microscopy (SEM) and dynamic light scattering (DLS). The antimicrobial activity of nanoparticle formulation was evaluated by determinations of the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against different bacterial species by the microdilution method, according to recommendations of the Clinical and Laboratory Standards Institute (CLSI). Three potential vehicles, hydroxyethylcellulose, Carbomer and polyethylene glycol were tested as carriers for formulations containing AgNPs. The efficiency of the synthesis method chosen to produce AgNPs was demonstrated by four characterization techniques. The nanoparticles showed antibacterial activity against all species tested. Incorporation of AgNPs into all experimental vehicles produced stable formulations but the one in hydroxyethylcellulose presented better physical proprieties. The results indicate that silver nanoparticles are potential antiseptic agents to be used in root canals and incorporation in adequate vehicles may favor a broader application.

O presente estudo teve como objetivo sintetizar, caracterizar e avaliar as propriedades antimicrobianas de nanopartículas de prata visando o desenvolvimento de uma formulação intracanal. As nanopartículas de prata (AGNPS) foram obtidas pela redução de nitrato de prata com borohidreto de sódio e caracterizados por espectrofotometria UV-Visível, microscopia eletrônica de varredura (MEV) e espalhamento de dinâmico de luz (DLS). A atividade antimicrobiana da formulação de nanopartículas foi avaliada por meio das determinações da concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) contra diferentes espécies de bactérias pelo método de microdiluição, de acordo com recomendações do Clinical and Laboratory standards Institute (CLSI). Três potenciais veículos, hidroxietilcelulose, carbómero e polietileno glicol foram testados como veículos para as formulações de AGNPS. A eficiência do método de síntese escolhido para produzir AGNPS foi demonstrada por quatro técnicas de caracterização. As nanopartículas apresentaram atividade antibacteriana contra todas as espécies bacterianas testadas. A incorporação de AGNPS em todos os veículos experimentais produziram formulações estáveis, porém, quando utilizado a hidroxietilcelulose foram obtidos melhores propriedades físicas. Os resultados indicam que as nanopartículas de prata são potenciais agentes anti-sépticos para serem usados na terapia endodôntica e a incorporação em veículos adequados pode favorecer uma aplicação mais ampla.

Physicochemical properties of endodontic sealers of different bases

OBJECTIVE: To assess the setting time (ST), flow (FL), radiopacity (RD), solubility (SB) and dimensional change following setting (DC) of different sealers (AH Plus®, Polifil, Apexit Plus®, Sealapex®, Endométhasone® and Endofill®) according to American National Standards Institute/American Dental Association (ANSI/ADA) Specification 57. MATERIAL AND METHODS: Five samples of each material were used for each test. For ST, cast rings were filled with sealers and tested with a Gilmore needle. For FL, the sealer was placed on a glass plate. After 180 s, another plate with 20 g and a load of 100 g were applied on the material, and the diameters of the discs formed were measured. In RD, circular molds were filled with the sealers, radiographed and analyzed using Digora software. For SB, circular molds were filled with the sealers, a nylon thread was placed inside the material and another glass plate was positioned on the set, pressed and stored at 37°C. Samples were weighed, placed in water, dried and reweighed. The water used for SB was analyzed by atomic absorption spectrometry. For DC, circular molds were filled with the sealers, covered by glass plates and stored at 37°C. Samples were measured and stored in water for 30 days. After this period, they were dryed and measured again. RESULTS: Regarding ST, AH Plus®, Apexit® and Endofil® sealers are in accordance with ANSI/ADA standards. Endométhasone's manufacturer did not mention the ST; Polifil is an experimental sealer and Sealapex® did not set. Considering RD, SB and DC, all sealers were in accordance with ANSI/ADA. The spectrometric analysis showed that a significant amount of K+ and Zn2+ ions was released from Apexit Plus® and Endofill®, respectively. CONCLUSION: Except for DC, all other physicochemical properties of the tested sealers conformed to ANSI/ADA requirements.

Radiopacity evaluation of Portland and MTA-based cements by digital radiographic system

OBJECTIVE: The aim of the present study was to evaluate the radiopacity of Portland and MTA-based cements using the Digora TM digital radiographic system. MATERIAL AND METHODS: The performed tests followed specification number 57 from the American National Standard Institute/American Dental Association (2000) for endodontic sealing materials. The materials were placed in 5 acrylic plates, especially designed for this experiment, along with a graduated aluminum stepwedge varying from 1 to 10 mm in thickness. The set was radiographed at a 30 cm focus-object distance and with 0.2 s exposure time. After the radiographs were taken, the optical laser readings of radiographs were performed by Digora TM system. Five radiographic density readings were performed for each studied material and for each step of the aluminum scale. RESULTS: White ProRoot MTA (155.99±8.04), gray ProRoot MTA (155.96±16.30) and MTA BIO (143.13±16.94) presented higher radiopacity values (p<0.05), while white non-structural Portland (119.76±22.34), gray Portland (109.71±4.90) and white structural Portland (99.59±12.88) presented lower radiopacity values (p<0.05). CONCLUSIONS: It was concluded that MTA-based cements were the only materials presenting radiopacity within the ANSI/ADA specifications.

Influence of apical enlargement in cleaning and extrusion in canals with mild and moderate curvatures

Optical microscopy and morphometric analysis were used in this study to evaluate, in vitro, the cleaning of the apical region in root canals with mild or moderate curvatures subjected to biomechanical preparation with a rotary system, as well as to assess the amount of extruded material to the periapical area. Lateral incisors (n = 32), 16 with curvature angles smaller or equal to 10º (GI) and 16 between 11º and 25º angles (GII) were submitted to Hero 642 rotary instrumentation with different surgical diameters: (A) 30.02 and (B) 45.02. Irrigation was performed at each change of instrument with 5 mL of ultrapure Milli-Q water and the extruded material through the apical foramen was collected. Root cross-sections were subjected to histological analysis by optical microscopy (×40) and the images were evaluated morphometrically using the Image Tool software. Quantification of the extruded material was performed by weighing after liquid evaporation. ANOVA showed no statistically significant differences (p>0.05) among the groups with respect to the procedures used to clean the apical region. Considering the amount of extruded material, the Tukey's HSD showed that canals with mild curvature prepared with the 45.02 surgical diameter showed significantly higher values (p<0.05) that those of the other groups, which were similar between themselves (p>0.05). In conclusion, the effect of cleaning the apical region did not differ in the groups, considering root curvature and the surgical diameter of instruments used for apical preparation. The amount of extruded material was greater in canals with mild curvature that were prepared with the 45.02 surgical instrument diameter.

Este estudo avaliou, in vitro, por meio de microscopia óptica e morfometria, a limpeza da região apical em canais radiculares com curvatura leve e moderada submetidos ao preparo biomecânico com sistema rotatório, bem como avaliou a quantidade de material extruído para a área apical. Incisivos laterais (n = 32), sendo 16 com ângulo de curvatura igual ou inferior a 10º (GI) e 16 entre 11º e 25º (GII), foram submetidos à instrumentação rotatória com o sistema Hero 642 com diferentes diâmetros cirúrgicos: (A) 30,02 e (B) 45,02. A irrigação, a cada troca de instrumento, foi realizada com água ultra-pura de Milli Q (5 mL), e o material extruído através do forame apical foi coletado. As raízes foram submetidas a análise histológica sob microscopia óptica (40×) e as imagens foram submetidas à análise morfométrica utilizando o software Image Tool. A quantificação do material extruído foi realizada por pesagem, após a evaporação do líquido. A análise de variância não mostrou diferença estatística significante em relação à limpeza da região apical nos diferentes grupos testados (p>0,05). Considerando a quantidade de material extruído, o teste de T6ukey mostrou que os canais com curvatura leve preparados com o diâmetro cirúrgico 45,02 apresentaram os maiores valores, sendo estatisticamente diferente dos demais grupos (p<0,05) que foram semelhantes entre si (p>0,05). Pode-se concluir que a limpeza da região apical não diferiu entre os diferentes grupos, considerando a curvatura radicular e o diâmetro cirúrgico. A quantidade de material extruído foi maior nos canais preparados com curvatura leve que foram preparados com diâmetro cirúrgico 45,02.

Evaluation of physico-chemical properties of Portland cements and MTA

The purpose of this study was to evaluate the hydrogenionic potential and electrical conductivity of Portland cements and MTA, as well as the amount of arsenic and calcium released from these materials. In Teflon molds, samples of each material were agitated and added to plastic flasks containing distilled water for 3, 24, 72 and 168 h. The results were analyzed with a Kruskal-Wallis non-parametric test for global comparisons and a Dunn-Tukey test for pairwise comparisons. The results revealed no significant differences in the pH of the materials (p > 0.05). The electrical conductivity of the cements were not statistically different (p > 0.05). White non-structural cement and MTA BIO released the largest amount of calcium ions into solution (p < 0.05), while arsenic release was insignificant in all of the materials (p > 0.05). The results indicated that the physico-chemical properties of Portland cements and MTA were similar. Furthermore, all materials produced an alkaline environment and can be considered safe for clinical use because arsenic was not released. The electrical conductivity and the amount of calcium ions released into solution increased over time.

Cleaning of flattened root canals with different irrigating solutions and nickel-titanium rotary instrumentation

The aim of this study was to evaluate the cleaning capacity of Profile .04 files combined with different irrigating solutions in flattened root canals using histological, morphometrical and SEM analyses. Eighty human mandibular incisors were prepared with Profile .04 instruments and randomly divided into 4 groups according to the irrigating solutions used (n=20): G1: distilled water (control); G2: 1 percent sodium hypochlorite (NaOCl); G3: 1 percent NaOCl alternated with 17 percent EDTA, and G4: 1 percent NaOCl with RCPrep cream. Ten teeth of each group were evaluated with an optical microscope to determine the percentage of root canal debris. The remaining teeth were evaluated under scanning electron microscopy (SEM). Data were analyzed statistically by ANOVA and Tukey's test (á=0.01).There was a significant difference (p<0.001) among the groups regarding the percentage of debris left in the canals (distilled water: 18.82 ± 5.55; 1 percent NaOCl: 6.29 ± 5.55; 1 percent NaOCl + 17 percent EDTA: 12.47 ± 6.92; 1 percent NaOCl + RCPrep: 7.82 ± 1.91). The SEM analysis showed the best results for 1 percent NaOCl + 17 percent EDTA on smear layer removal. It may be concluded that the combination of Profile .04 rotary instrumentation and the tested solutions was not able to totally remove debris and smear layer from flattened root canals.

O objetivo deste estudo foi avaliar a capacidade de limpeza dos instrumentos Profile .04 associados a diferentes soluções irrigantes, em canais radiculares achatados, por meio de análises histológica, morfométrica e MEV. Oitenta incisivos inferiores humanos foram submetidos ao preparo biomecânico com instrumentos Profile .04 e aleatoriamente distribuídos em 4 grupos de acordo com as soluções irrigantes usadas (n=20): G1 - água destilada (controle); G2 - NaOCl 1 por cento; G3 - NaOCl 1 por cento alternado com EDTA 17 por cento e G4 - NaOCl 1 por cento com RCPrep creme. Dez dentes de cada grupo foram avaliados em microscopia óptica para determinação da porcentagem de debris do canal radicular. Os demais dentes foram avaliados em microscopia eletrônica de varredura. A análise estatística mostrou diferença significante (p<0,001) entre os grupos ao considerar a porcentagem de debris remanescente nos canais (água destilada: 18,82 ± 5,55; NaOCl: 6,29 ± 5,55; NaOCl + EDTA: 12,47 ± 6,92; NaOCl + RCPrep: 7,82 ± 1,91). A análise em MEV evidenciou melhores resultados na remoção da camada de smear com o NaOCl 1 por cento alternado com EDTA 17 por cento. Pode-se concluir que a associação entre a instrumentação rotatória com Profile .04 e as soluções testadas não foi capaz de remover totalmente debris e camada de smear de canais radiculares achatados.