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This study aimed to evaluate the effects of flavonoids incorporated into poly(N-vinylcaprolactam) (PNVCL) hydrogel on cell viability and mineralization markers of odontoblast-like cells. MDPC-23 cells were exposed to ampelopsin (AMP), isoquercitrin (ISO), rutin (RUT) and control calcium hydroxide (CH) for evaluation of cell viability, total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition by colorimetric assays. Based on an initial screening, AMP and CH were loaded into PNVCL hydrogels and had their cytotoxicity and effect on mineralization markers determined. Cell viability was above 70% when MDPC-23 cells were treated with AMP, ISO and RUT. AMP showed the highest ALP activity and mineralized nodule deposition. Extracts of PNVCL+AMP and PNVCL+CH in culture medium (at the dilutions of 1/16 and 1/32) did not affect cell viability and stimulated ALP activity and mineralized nodules' deposition, which were statistically higher than the control in osteogenic medium. In conclusion, AMP and AMP-loaded PNVCL hydrogels were cytocompatible and able to induce bio-mineralization markers in odontoblast-cells.

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OBJECTIVE: This study aimed to evaluate the cytotoxicity and synergistic effect of epigallocatechin gallate (EGCG) and fosfomycin (FOSFO) on biofilms of oral bacteria associated with endodontic infections. METHODOLOGY: This study determined minimum inhibitory and bactericidal concentration (MIC/MBC) and fractionated inhibitory concentration (FIC) of EGCG and FOSFO against Enterococcus faecalis, Actinomyces israelii, Streptococcus mutans, and Fusobacterium nucleatum. Monospecies and multispecies biofilms with those bacteria formed in polystyrene microplates and in radicular dentin blocks of bovine teeth were treated with the compounds and control chlorhexidine (CHX) and evaluated by bacterial counts and microscopy analysis. Toxicity effect of the compounds was determined on fibroblasts culture by methyl tetrazolium assays. RESULTS: The combination of EGCG + FOSFO demonstrated synergism against all bacterial species, with an FIC index ranging from 0.35 to 0.5. At the MIC/FIC concentrations, EGCG, FOSFO, and EGCG+FOSFO were not toxic to fibroblasts. EGCG+FOSFO significantly reduced monospecies biofilms of E. faecalis and A. israelli, whereas S. mutans and F. nucleatum biofilms were eliminated by all compounds. Scanning electron microscopy of multispecies biofilms treated with EGCG, EGCG+FOSFO, and CHX at 100x MIC showed evident biofilm disorganization and substantial reduction of extracellular matrix. Confocal microscopy observed a significant reduction of multispecies biofilms formed in dentin tubules with 84.85%, 78.49%, and 50.6% of dead cells for EGCG+FOSFO, EGCG, and CHX at 100x MIC, respectively. CONCLUSION: EGCG and fosfomycin showed a synergistic effect against biofilms of oral pathogens related to root canal infections without causing cytotoxicity.

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BACKGROUND: Early childhood caries (ECC) is the most common chronic disease in young children and a public health problem worldwide. It is characterized by the presence of atypical and fast progressive caries lesions. The aggressive form of ECC, severe early childhood caries (S-ECC), can lead to the destruction of the whole crown of most of the deciduous teeth and cause pain and sepsis, affecting the child's quality of life. Although the multifactorial etiology of ECC is known, including social, environmental, behavioral, and genetic determinants, there is a consensus that this disease is driven by an imbalance between the oral microbiome and host, or dysbiosis, mediated by high sugar consumption and poor oral hygiene. Knowledge of the microbiome in healthy and caries status is crucial for risk monitoring, prevention, and development of therapies to revert dysbiosis and restore oral health. Molecular biology tools, including next-generation sequencing methods and proteomic approaches, have led to the discovery of new species and microbial biomarkers that could reveal potential risk profiles for the development of ECC and new targets for anti-caries therapies. This narrative review summarized some general aspects of ECC, such as definition, epidemiology, and etiology, the influence of oral microbiota in the development and progression of ECC based on the current evidence from genomics, transcriptomic, proteomic, and metabolomic studies and the effect of antimicrobial intervention on oral microbiota associated with ECC. CONCLUSION: The evaluation of genetic and proteomic markers represents a promising approach to predict the risk of ECC before its clinical manifestation and plan efficient therapeutic interventions for ECC in its initial stages, avoiding irreversible dental cavitation.

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Abstract Objective This study aimed to evaluate the cytotoxicity and synergistic effect of epigallocatechin gallate (EGCG) and fosfomycin (FOSFO) on biofilms of oral bacteria associated with endodontic infections. Methodology This study determined minimum inhibitory and bactericidal concentration (MIC/MBC) and fractionated inhibitory concentration (FIC) of EGCG and FOSFO against Enterococcus faecalis, Actinomyces israelii, Streptococcus mutans, and Fusobacterium nucleatum. Monospecies and multispecies biofilms with those bacteria formed in polystyrene microplates and in radicular dentin blocks of bovine teeth were treated with the compounds and control chlorhexidine (CHX) and evaluated by bacterial counts and microscopy analysis. Toxicity effect of the compounds was determined on fibroblasts culture by methyl tetrazolium assays. Results The combination of EGCG + FOSFO demonstrated synergism against all bacterial species, with an FIC index ranging from 0.35 to 0.5. At the MIC/FIC concentrations, EGCG, FOSFO, and EGCG+FOSFO were not toxic to fibroblasts. EGCG+FOSFO significantly reduced monospecies biofilms of E. faecalis and A. israelli, whereas S. mutans and F. nucleatum biofilms were eliminated by all compounds. Scanning electron microscopy of multispecies biofilms treated with EGCG, EGCG+FOSFO, and CHX at 100x MIC showed evident biofilm disorganization and substantial reduction of extracellular matrix. Confocal microscopy observed a significant reduction of multispecies biofilms formed in dentin tubules with 84.85%, 78.49%, and 50.6% of dead cells for EGCG+FOSFO, EGCG, and CHX at 100x MIC, respectively. Conclusion EGCG and fosfomycin showed a synergistic effect against biofilms of oral pathogens related to root canal infections without causing cytotoxicity.

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This study aimed to evaluate the cytotoxicity and microbiological properties of poly (N-vinylcaprolactam)-PNVCL hydrogels containing flavonoids as intracanal medication for endodontic therapy. Antimicrobial activity of ampelopsin (AMP), isoquercitrin and rutin was determined against Enterococcus faecalis, Actinomyces israelii, Lactobacillus casei, Streptococcus mutans, and Fusobacterium nucleatum by the microdilution method. After synthesis and characterization by rheology, PNVCL hydrogels were loaded with AMP and controls calcium hydroxide (CH) and chlorhexidine (CHX), and determined the compounds release profile. PNVCL+AMP, PNVCL+CH, PNVCL+CHX were evaluated on multi-species biofilms and analyzed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). Cytotoxicity was determined after fibroblasts exposure to serial dilutions of AMP and PNVCL hydrogel extracts. AMP was effective against all of the bacteria tested, especially against S. mutans, A. israelli and F. nucleatum. SEM and CLSM analysis showed that PNVCL + AMP caused a significant decrease and disorganization of multi-species biofilms and reduction of intracanal viable cells, superior to the other groups. AMP affected fibroblast viability at concentrations above 0.125 mg/mL, and extracts of PNVCL+AMP showed low cytotoxicity. In conclusion, PNVCL containing AMP demonstrated cytocompatibility and potent effect against multi-species biofilms and could be potential intracanal medication for endodontic purposes.

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Ainda não foi encontrado um medicamento capaz de desinfetar os canais radiculares e permitir a recuperação celular e a regeneração tecidual em dentes permanentes jovens com comprometimento endodôntico. Dois importantes flavonóis detectados no vinho tinto, morina (MO) e miricetina (MY), são atualmente estudados por suas amplas propriedades biológicas, incluindo atividade antimicrobiana. No entanto, o desenvolvimento de sistemas de liberação controlada pode ser útil para a liberação desses flavonóis para fins de terapia endodôntica. Este estudo avaliou a citocompatibilidade e os efeitos antimicrobianos/antibiofilme de MO e MY, isolados ou incorporados em hidrogéis termorreversíveis de quitosana-poloxamer-ß-glicerofosfato de sódio (CPG), além dos efeitos de MO e MY, isolados e combinados sobre a viabilidade, atividade de ALP e produção de nódulos de mineralização em células MDPC-23. A atividade antimicrobiana dos compostos foi avaliada em Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum em condições planctônicas, em biofilmes dual-espécies e multiespécies e analisadas por contagem bacteriana e microscopia de varredura. Os hidrogéis CPG foram caracterizados por reometria de fluxo e oscilatória, temperatura de gelificação, perfil de textura e análise de bioadesão em espécimes de dentina. MO, MY e controles (hidróxido de cálcio ­ CH e clorexidina ­ CHX) foram incorporados em hidrogéis de CPG e o efeito do antibiofilme sobre biofilmes multiespécies formados em amostras de dentina radicular foi avaliado por microscopia confocal. O efeito de toxicidade dos compostos isolados ou incorporados em hidrogéis de CGP foi determinado em cultura de fibroblastos por ensaios de resazurina. Os dados foram analisados estatisticamente pelos testes ANOVA e Tukey considerando p < 0,05. A combinação de MO e MY foi sinérgica ou aditiva contra bactérias endodônticas testadas a partir de concentrações de 0,03 mg/mL MO + 0,06 mg/mL MY e não foram tóxicas para fibroblastos até 0,125mg/mL. MO + MY apresentou melhor efeito sobre biofilmes dual-espécies e multiespécies considerando suas menores concentrações quando comparados com os flavonóis isolados. Os hidrogéis CPG foram caracterizados como termorreversíveis e com propriedades mecânicas e bioadesivas adequadas. Hidrogéis de CPG carregados com MO+MY, CH e CHX apresentaram efeitos inibitórios semelhantes quando aplicados em biofilmes multiespécies formados no interior dos túbulos dentinários radiculares por 48h e seus extratos apresentaram citotoxicidade acima de 50% de diluição. As células semelhantes a odontoblastos (MDPC-23) foram expostas a diferentes concentrações de MO, MY, isoladamente ou em combinação e CH como controle positivo por 24h e 48h, e troca contínua de meio osteogênico por 8 dias e 14 dias. As combinações de MO+MY ou CH também foram incorporadas em hidrogéis de quitosana-poloxamer-ß-glicerofosfato e seus extratos em meio de cultura celular foram coletados após 48h e 7 dias. Viabilidade celular, atividade de fosfatase alcalina (ALP) e ensaios de deposição de nódulos mineralizados (MN) foram realizados pelo método de resazurina, ensaios de monofosfato de timolftaleína e coloração com vermelho de alizarina, respectivamente. Todos os compostos não causaram citotoxicidade nas concentrações testadas em 24h e 8 dias e 0,5 mg/mL MO e MY isolados reduziram a viabilidade celular em 48h. A atividade de ALP e a deposição de MN foram aumentadas para a combinação MO+MY e CH em células MDPC-23. Extratos de hidrogel de 7 dias contendo ou não MO+MY não foram citotóxicos até diluição de 25% em 48h e em baixas concentrações estimularam a atividade de ALP e deposição de MN aos 8 e 14 dias de avaliação. Em conclusão, a combinação de morina e miricetina incorporada ou não em hidrogéis de CPG apresentou efeito antibiofilme sobre patógenos orais e baixa toxicidade sobre fibroblastos. Morina e miricetina em baixas concentrações, isoladas, em combinação ou em hidrogéis CPG não foram citotóxicas e foram eficazes na indução de marcadores de mineralização em células semelhantes a odontoblastos(AU)

A drug capable of disinfecting the root canals and allow cell recovery and tissue regeneration in permanent young teeth with endodontic problems has not been found yet. Two important flavonols detected in red wine, morin (MO) and myricetin (MY), are currently studied for their wide biological properties including antimicrobial activity. However, the development of controlled release systems could be useful for the delivery of these flavonols for endodontic therapies. This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of MO and MY, alone or incorporated in thermoreversible chitosanpoloxamer hydrogels containing sodium ß-glycerophosphate (CPG), in addition to the effects of isolated and combined morin and myricetin flavonols on viability, ALP activity and production of mineralization nodules in MDPC-23 cells. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on dual-species and multispecies biofilms and analyzed by bacterial counts and scanning microscopy. CPG hydrogels were characterized by flow and oscillatory rheometry, gelation temperature, texture profile and bioadhesion analysis in dentin specimens. MO, MY and controls (calcium hydroxide ­ CH and chlorhexidine ­ CHX) were incorporated in CPG hydrogels and antibiofilm effect on multispecies biofilms formed in radicular dentin samples were evaluated by confocal microscopy. Cytotoxicity of the compounds alone or incorporated in CGP hydrogels was determined on fibroblasts culture by resazurin assays. Data were statistically analyzed by ANOVA and Tukey considering p < 0.05. The combination of MO and MY had synergistic or additive against oral bacteria tested starting at concentrations of 0.03 mg/mL MO + 0.06 mg/mL MY and they were not toxic to fibroblasts up to 0.125mg/mL. MO + MY had better effect on dual-species and multispecies biofilms considering their lower concentrations when compared with the flavonols alone. CPG hydrogels were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. CPG hydrogels loaded with MO+MY, CH and CHX have similar inhibitory effects when applied on multispecies biofilms formed inside root dentin tubules for 48h and their extracts were cytotoxicity above 50% dilution. Furthermore, the effects of morin, myricetin, alone or in combination or incorporated in chitosan-based hydrogels on cytotoxicity and expression of mineralization markers in odontoblast-like cells. The MDPC-23 cells were exposed to different concentrations of morin (MO), myricetin (MY), alone or in combination and calcium hydroxide (CH) as a positive control for 24h and 48h, and continuous osteogenic medium changing for 8 days and 14 days. The combinations of MO+MY or CH were also incorporated in chitosan-poloxamer-ß- glycerophosphate hydrogels and their extracts in cell culture media were collected after 48h and 7 days. Cell viability, alkaline phosphatase (ALP) activity and assays mineralized nodules (MN) deposition were performed using resazurin method, thymolphthalein monophosphate assays and alizarin red staining, respectively. Data were statistically analyzed considering p< 0.05. All compounds were non-toxic at the concentrations tested at 24h and 8 days and 0.5 mg/mL MO and MY alone reduced cell viability at 48h. ALP activity and deposition of MN were increased for MO+MY combination and CH in MDPC-23 cells. 7 days hydrogel extracts containing or not MO+MY were not cytotoxic up to 25% dilution at 48h and at low concentrations stimulated ALP activity and MN deposition at 8 and 14 days of evaluation. In conclusion, the combination of morin and myricetin incorporated or not in CPG hydrogels presented antibiofilm effect on oral pathogens and low cytotoxicity on fibroblasts. Morin myricetin at low concentrations, alone, in combination or in CPG hydrogels were not cytotoxic and were effective in inducing mineralization markers in odontoblast-like cells(AU)

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This clinical study compared the effectiveness of two rotary systems: HyFlex CM (Coltene-Whaledent, Altstetten, Switzerland) and ProTaper Next (Dentsply Sirona, Ballaigues, Switzerland) on the removal of cultivable bacteria and endotoxins from primarily infected root canals. This study was designed as a randomized single-blinded, 2-arm, clinical trial. Twenty-four primarily infected root canals were selected and randomly divided into 2 groups: HyFlex CM (n = 12); and ProTaper Next (n = 12). Samples were collected before and after the biomechanical preparation and inoculated in specific flasks. Irrigation was performed using 2.5% sodium hypochlorite. A kinetic turbidimetric lysate assay of limulus amoebocytes was used to quantify endotoxins. Microbiological culture technique was used to determine the count of bacterial colony forming units (CFU/mL). Data collected were statistically analyzed using SigmaPlot 12.0 for Windows. The Two-Way ANOVA statistical test was performed and the level of significance was 5%. In the samples before the biomechanical preparation, cultivable bacteria and endotoxins were evidenced in 100% of the cases. The culture analysis revealed that there was no statistically significant difference in the bacterial reduction between the two instrumentation systems. Endotoxins were present in 100% of the canals after instrumentation and there was no statistical difference between the two systems in endotoxin reduction. Thus, it was concluded that both instrumentation systems were effective in reducing root canal bacteria and endotoxins with primary endodontic infection and that there was no statistical difference between them. However, no system was able to eliminate 100% of the bacteria and their by-products.

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O objetivo do presente estudo foi avaliar a efetividade na redução bacteriana de um protocolo de irrigação final de canais radiculares com infecção primária utilizando o sistema Easy Clean. Vinte e quatro canais radiculares com infecções endodônticas primárias crônicas foram analisados. As amostras foram coletadas com 3 pontas de papel estéreis em meio de transporte microbiológico antes da instrumentação (S1), após instrumentação rotatória com dois sistemas diferentes (S2), e após o protocolo final de irrigação utilizando o Easy Clean para agitação das soluções de irrigação (S3). Técnica de cultura foi utilizada para determinar a contagem de unidades formadoras de colônia (UFC/mL). Os resultados foram analisados estatisticamente pelo teste Mann-Whitney ao nível de significância de 5% para comparação entre as variáveis. Bactérias foram detectadas em 100% dos casos analisados, mostrando em S1 uma média de 2,0x106 UFC/mL, em S2 2,3x104 UFC/mL e em S3 6,6x103 UFC/mL. Em conclusão, embora não tenha sido estatisticamente significante, houve uma redução no número de bactérias após o uso da Easy Clean. Portanto, pode-se sugerir que o protocolo final de irrigação com o auxílio do Easy Clean auxilia na redução de bactérias(AU)

The objective of the present study was to evaluate the effectiveness in the bacterial reduction of a final irrigation protocol of root canals with primary infection using the Easy Clean system. Twenty-four root canals with chronic primary infection were analyzed. Samples were collected with 3 sterile paper points in microbiological transport medium before instrumentation (S1), after rotary instrumentation with two different systems (S2), and after the final irrigation protocol using the Easy Clean system for agitation irrigating solutions (S3). Culture technique was used to determine the count of colony forming units (CFU/mL). The results were statiscally analyzed by the MannWhitney test at a significance level of 5% for comparison among the variables. Bacteria were detected in 100% of analyzed cases, showing in S1a mean of 2.0x106 CFU/mL, in S2 2.3x104 CFU/mL, and in S3 6.6x103 CFU/mL. Statistically significant differences were found between the rotary systems Protaper Next and HyFlex CM in the reduction of the bacterial load, but not in the analysis before and after Easy Clean. In conclusion, although not statistically significant, there was a reduction in the number of bacteria after using Easy Clean. Therefore, it could be suggested that the final irrigation protocol with the aid Easy Clean assists in the reduction of bacteria(AU)

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