Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Pro-inflammatory mediators expression by pulp cells following tooth whitening on restored enamel surface

Abstract This paper aimed to assess the influence of adhesive restoration interface on the diffusion of hydrogen peroxide (H2O2), indirect toxicity, and pro-inflammatory mediators expression by odontoblast-like cells, after in-office tooth whitening. Dental cavities prepared in bovine enamel/dentin discs were adhesively restored and subjected or not to hydrolytic degradation (HD). A whitening gel with 35% H2O2 (WG) was applied for 45 min onto restored and non-restored specimens adapted to artificial pulp chambers giving rise to the groups: SD- intact discs (control); SD/HP- whitened intact discs; RT/HP- restored and whitened discs; and RT/HD/HP- restored and whitened discs subjected to HD. The extracts (culture medium + WG components diffused through enamel/dentin/restoration interface) were collected and applied to odontoblast-like MDPC-23 cells. The study evaluated the amount of H2O2 in the extracts, as well as the cell viability (CV), cell morphology (CM), and gene expression of inflammatory mediators (TNF-α and COX-2) by the pulp cells exposed to the extracts (ANOVA and Tukey tests; 5% significance). All whitened groups presented lower CV than SD (control; p<0.05). The highest CV reduction and gene expression of TNF-α and COX-2 was observed in the RT/HD/HP group in comparison with SD/HP and RT/HP (control; p<0.05). CM alterations occurred in all whitened groups. The intensity of these cell side effects was directly related with the amount of H2O2 in the extracts. We concluded that adhesive restoration of dental cavity increases the H2O2 diffusion after in-office whitening, enhancing the indirect toxicity of this therapy and trigger pro-inflammatory overexpression by MDPC-23 cells.

Resumo Este trabalho teve como objetivo avaliar a influência da interface de uma restauração adesiva na difusão do peróxido de hidrogênio (H2O2), toxicidade indireta e expressão de mediadores pró-inflamatórios por células odontoblastóides, após clareamento dental em consultório. Cavidades dentárias preparadas em discos de esmalte / dentina foram restauradas com adesivo e submetidas ou não à degradação hidrolítica (HD). Um gel clareador com 35% H2O2 (WG) foi aplicado por 45 min em discos restaurados e não restaurados adaptados às câmaras pulpares artificiais dando origem aos grupos: SD- discos intactos (controle); SD / HP - Discos intactos clareados; RT / HP - discos restaurados e clareados; e RT / HD / HP - discos restaurados, clareados e submetidos a HD. Os extratos (meio de cultura + componentes WG difundidos através da interface esmalte/dentina/restauração) foram coletados e aplicados em células odontoblastóides MDPC-23. Foi avaliada a quantidade de H2O2 nos extratos, bem como a viabilidade (CV), morfologia (CM) e expressão gênica de mediadores inflamatórios (TNF-α e COX-2) pelas células pulpares expostas aos extratos (ANOVA e testes de Tukey; 5% de significância). Todos os grupos clareados apresentaram menor CV do que SD (controle; p <0,05). A maior redução CV e expressão gênica de TNF-α e COX-2 foi observada no grupo RT / HD / HP em comparação com SD / HP e RT / HP (controle; p <0,05). Alterações na CM ocorreram em todos os grupos clareados. A intensidade desses efeitos celulares teve relação direta com a quantidade de H2O2 nos extratos. Concluímos que a presença de uma cavidade contendo restauração adesiva aumenta a difusão de H2O2 após o clareamento em consultório, o que, por sua vez, aumenta a toxicidade indireta dessa terapia e desencadeia a expressão de mediadores pró-inflamatórios pelas células pulpares MDPC-23.

Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.

Bond strength of universal adhesive applied to dry and wet dentin: one-year in vitro evaluation

Aim: This study evaluated the influence of dentin wettability on the immediate and extended microtensile bond strength (mTBS) of a universal adhesive system used in the etch-and-rinse strategy. Methods: Twenty human third molars were selected and divided into four groups according to the adhesive system and dentin wettability. The mTBS values of each group were registered 24 h and one year after adhesive system application and resin composite block build-up (n=30). Data were analyzed by the t-test (p<0.05). Results: When both adhesive systems were compared, there was no statistically significant difference when they were applied following wet bonding (p>0.05). However, the dry bonding reduced µTBS values of the Adper Single Bond 2 adhesive (p<0.05). Regarding storage time, both groups presented similar µTBS values at 24 h and one year (p>0.05). Conclusions: Therefore, the Scotchbond Universal Adhesive can be applied to dry or wet dentin without compromising the etch-and-rinse bonding quality and the durability of the restorations

Bond Strength and Cytotoxicity of a Universal Adhesive According to the Hybridization Strategies to Dentin

Abstract This study evaluated application protocol (etch-and-rinse/ER and self-etching/SE) and dentin wettability (wet and dry) on microtensile bond strength (μTBS) and transdentinal cytotoxicity of ScotchbondTM Universal (SU) adhesive system. The μTBS values and fracture mode were registered 24 h after adhesive system application and resin composite block build-up (n=5). For analysis of transdentinal cytotoxicity, odontoblast-like MDPC-23 cells were seeded on pulpal surface of dentin discs (0.4 mm thick) adapted to artificial pulp chambers (n=8). The adhesive system was applied to occlusal surface, followed by 24-h incubation time. Cell viability (Alamar Blue) and morphology (SEM) were assessed. Adper Single Bond 2 and Clearfil SE Bond were used as positive controls of the ER and SE application protocols, respectively. No treatment was performed on negative control (NC) group. Data were analyzed by ANOVA and Tukey's tests (α=5%). Higher μTBS values were found for ER mode in comparison with SE protocol (p<0.05). Dentin wettability had no effect on bond strength of SU in both the ER and SE techniques (p>0.05). Most fractures involved hybrid layer and/or adhesive layer. Neither variable prevented the intense toxic effects of adhesive systems on MDPC-23 cultured cells, since intense reduction in cell viability (±88%) and severe alterations in cell morphology were observed for all groups compared to NC, with no differences among them (p>0.05). Therefore, it was concluded that application of SU following the ER protocol had better adhesive performance. However, this adhesive system featured intense transdentinal cytotoxicity to pulp cells, regardless of application protocol and dentin wettability.

Resumo Este estudo avaliou o protocolo de aplicação (convencional/ER e autocondicionante/SE) e o grau de umidade da dentina (úmida e seca) sobre a resistência de união à microtração (μTBS) e a citotoxicidade transdentinária do sistema adesivo ScotchbondTM Universal (SU). Os valores de μTBS e o modo de fratura foram registrados 24 h após aplicação do sistema adesivo e restauração com resina composta pela técnica incremental. Para avaliação da citotoxicidade transdentinária, células odontoblastóides MDPC-23 foram semeadas na face pulpar de discos de dentina (0,4 mm de espessura) adaptados a câmaras pulpares artificiais (n = 8). O sistema adesivo foi aplicado na superfície oclusal, seguido de incubação por 24 h. A viabilidade e morfologia celular foram avaliadas pelo teste de Alamar Blue e MEV, respectivamente. Adper Single Bond 2 e Clearfil SE Bond foram utilizados como controle positivo do protocolo de aplicação ER e SE, respectivamente. Nenhum tratamento foi realizado no grupo controle negativo (NC). Os dados foram analisados pelos testes de ANOVA e Tukey (α = 5%). Maiores valores de μTBS foram encontrados para o modo ER em comparação com o protocolo SE (p < 0,05). O grau de umidade da dentina não apresentou efeito na resistência de união do SU em ambos os protocolos ER e SE (p > 0.05). A maioria das fraturas envolveu a camada híbrida e / ou camada adesiva. Ambas as variáveis não preveniram o intenso efeito citotóxico dos sistemas adesivos sobre as células MDPC-23 em cultura, uma vez que redução intensa na viabilidade celular (± 88%) e alterações severas na morfologia celular foram observadas para todos os grupos quando comparados ao NC, sem diferenças entre eles (p > 0.05). Desta forma, foi concluído que a aplicação do SU seguindo o protocolo ER apresentou melhor performance adesiva. No entanto, esse sistema adesivo promoveu intensa citotoxicidade transdentinária sobre células pulpares, independente do protocolo de aplicação e grau de umidade dentinária.

Influência do gluconato de cálcio sobre a citotoxicidade trans-amelodentinária de um agente clareador com 20% de peróxido de hidrogênio / Influence of calcium gluconate on the trans-enamel and trans-dentinal cytotoxicity of a bleaching agent with 20% hydrogen peroxide

Objetivo: Avaliar a citotoxicidade de um agente clareador contendo 2% de gluconato de cálcio (GC) sobre células pulpares humanas (HDPCs). Materiais e Métodos: Discos de esmalte-dentina adaptados em câmaras pulpares artificiais (CPAs) foram posicionados em compartimentos de forma que a dentina permaneceu imersa em meio de cultura, enquanto que o esmalte foi submetido ao clareamento com géis a 20% de H2O2 contendo ou não GC, durante 1x 45, 1x15 ou 1x5 minutos. No controle positivo foi realizado clareamento com 35% de H2O2 aplicado por 1x 45 minutos, sendo que no controle negativo nenhum tratamento foi realizado sobre o esmalte. A viabilidade celular (teste do MTT) e a difusão trans-amelodentinária de H2O2 (violeta leuco- -cristal/peroxidase) foram avaliadas (ANOVA/Tukey α = 5%; n = 8). Resultados: Foi observada redução significativa na viabilidade celular em todos os grupos clareados quando comparados ao controle negativo (p < 0,05); no entanto, os grupos expostos aos géis contendo 20% de H2O2, com ou sem GC, apresentaram valores de viabilidade celular significativamente superiores ao controle positivo (p < 0,05). A redução da viabilidade celular e a difusão de H2O2 residual para os grupos clareados com 20% de H2O2 foi proporcional ao tempo de contato dos produtos com a superfície dental, sendo que a presença de GC resultou em minimização significativo do efeito tóxico/difusão de H2O2 para os protocolos 1x 15 e 1x 5 min (p < 0,05). Conclusão: A presença de 2% de GC nos géis com 20% de H2O2 resulta em redução da difusão de H2O2 residual pela estrutura dental e do efeito citotóxico sobre células pulpares humanas, quando o produto é aplicado por curtos períodos sobre a superfície dental.

Objective: To evaluate the cytotoxicity of a bleaching agent containing 2% calcium gluconate (CG) on human pulp cells (HDPCs). Materials and Methods: Enamel-dentin disks adapted in artificial pulp chambers (CPAs) were placed in compartments so that dentin remained immersed in culture medium, while the enamel was subjected to bleaching with 20% H2O2 gels containing or not CG for 1x 45, 1x15 or 1x5 minutes. In the positive control, bleaching was performed with 35% H2O2 applied for 1x 45 minutes, and in the negative control no treatment was performed on the enamel. Cell viability (MTT test) and transenamel and trans-dentinal diffusion of H2O2 (leuco-crystal violet / peroxidase) were evaluated (ANOVA / Tukey α = 5%, n = 8). Results: A significant reduction in cell viability was observed in all bleached groups when compared to the negative control (p <0.05); However, groups exposed to gels containing 20% H2O2, with or without CG, had significantly higher values of cell viability than the positive control (p <0.05). The reduction of cell viability and the diffusion of residual H2O2 to the bleached groups with 20% H2O2 was proportional to the contact time of the products with the dental surface, and the presence of CG resulted in a significant minimization of the toxic /diffusion effect of H2O2 For the 1x15 and 1x5min protocols (p <0.05). Conclusion: The presence of 2% GC in gels with 20% H2O2 results in reduction of residual H2O2 diffusion by dental structure and cytotoxic effect on human pulp cells when the product is applied for short periods on the dental surface.

Bioestimulação de células pulpares humanas expostas a sinvastatina / Biostimulation of human pulp cells exposed to simvastatin

O objetivo desse estudo foi avaliar o potencial bioativo da sinvastatina (SV), aplicada por diferentes períodos sobre células da polpa dental humana (HDPCs). Para isto, HDPCs em 80% de confluência (n=6) foram tratadas com meio osteogênico suplementado com 0,01 µM de SV pelos períodos de 24 h, 72 h ou continuamente por até 21 dias. No controle negativo, as células foram mantidas em meio osteogênico. A viabilidade celular (MTT) foi avaliada em períodos de 1, 3, 7, 14 e 21 dias, e a deposição de matriz mineralizada (alizarin red) após 14 e 21 dias de cultivo celular. Os dados foram submetidos aos testes ANOVA e Tukey (α=5%). Foi observado que nos períodos de 1, 3, 7 e 14 dias não houve diferença significativa na viabilidade das células submetidas aos tratamentos com SV em comparação ao controle (p<0,05); no entanto, redução tardia foi observada aos 21 dias para as células tratadas com SV por 72 h ou de modo contínuo (p<0,05). Em contrapartida, aumento na deposição de matriz mineralizada foi observado para o tratamento contínuo com SV aos 21 dias, quando comparado ao controle (p<0,05). Foi possível concluir que o tratamento contínuo de células pulpares humanas com 0,01µM de SV foi capaz de bioestimular a deposição de matriz mineralizada in vitro.

The objective of this study was to evaluate the bioactive potential of simvastatin (SV), applied during different periods on human dental pulp cells (HDPCs). For this, HDPCs at 80% confluency (n = 6) were treated with osteogenic medium supplemented with 0.01 µM SV for periods of 24 h, 72 h or continuously up to 21 days. In the negative control group, the cells were cultivated in osteogenic medium. The cell viability (MTT) was evaluated after 1, 3, 7, 14 and 21 days, and the mineralized matrix deposition (alizarin red) was assessed at 14 and 21 days of cell culture. Data were submitted to ANOVA and Tukey's test (α=5%). No significant difference in cell viability was observed at 1, 3, 7 and 14 days for the cells exposed to SV compared to negative control (p<0.05); however, significant reduction was observed at 21 days for cells treated with SV during 72 h or continuously (p<0.05). On the other hand, increase in mineralized matrix deposition at 21 days was observed for cells treated continuously with SV when compared to control (p<0.05). It was possible to conclude that the continuous treatment of human pulp cells with 0.01 µM of SV was able to biostimulate mineralized matrix deposition in vitro.

Antifungal activity of components used for decontamination of dental prostheses on the growth of Candida albicans / Atividade antifúngica de produtos utilizados na descontaminação de próteses dentárias sobre o crescimento de Candida albicans

Introduction: The effectiveness of antimicrobial solutions employed in dental prosthesis decontamination is still uncertain. Aim: To evaluate the antifungal activity of cleaners used in the decontamination of dental prostheses on the growth of Candida albicans. Material and method: The evaluated products were: Corega Tabs(r) (S1), Sodium Hypochlorite 1% (S2), Sodium Bicarbonate 1% (S3), Hydrogen Peroxide 1% (S4), Chlorhexidine Digluconate 0.12% - Periogard (r) (S5), Mouthrinse based on essential oils - Listerine(r) (S6), essential oil from Rosmarinus officinalis (rosemary) at concentrations of 1% (S7) and 2% (S8). The antifungal activity of the products was evaluated by agar diffusion technique and the determination of microbial death curve of samples of C. albicans (ATCC 90028) in concentration 1.5 × 106 CFU/mL. The tests were performed in triplicate and statistical analysis was made by ANOVA Two-Way and Tukey tests, with the confidence level of 95%. Result: The average of the zones of inhibition growth, in millimeters, obtained for the products were: 0.0 (S1), 44.7 (S2), 0.0 (S3), 21.6 (S4), 10.0 (S5), 6.1 (S6), 0.0 (S7) and 2.4 (S8). Considering the determination of microbial death curve, all products showed a statistical difference (p<0.01) from control (0.85% sodium chloride) and S3 groups. Fungal growth less than 2×104 CFU/mL and an accentuation of the microbial death curve were observed after 30 minutes, with exception for S3 and control groups. Conclusion: The studied compounds, with the exception of Sodium Bicarbonate, have antifungal effect against C. albicans, which contribute for dental prostheses hygiene. .

Introdução: A efetividade de soluções antimicrobianas empregadas na descontaminação de próteses ainda é incerta. Objetivo: Avaliar a atividade antifúngica de soluções empregadas na descontaminação de próteses sobre o crescimento de Candida albicans. Material e método: Foram avaliados os produtos: Corega Tabs Branqueador(r) (S1), Hipoclorito de Sódio 1% (S2), Bicarbonato de Sódio 1% (S3), Peróxido de Hidrogênio 1% (S4), Digluconato de Clorexidina 0,12% - Periogard(r) (S5), Enxaguatório bucal a base de óleos essenciais - Listerine(r) (S6), e óleo essencial de Rosmarinus officinalis (alecrim) nas concentrações 1% (S7) e 2% (S8). A atividade antifúngica foi avaliada por meio da técnica de difusão em ágar e da determinação da curva de morte microbiana de amostras de C. albicans (ATCC 90028) na concentração 1,5×106 UFC/mL. Os testes foram realizados em triplicata e a análise estatística se deu pelos testes ANOVA Two-Way e Tukey, sendo adotado nível de confiança de 95%. Resultado: A média dos halos de inibição do crescimento, em milímetros, obtidos para os produtos foram: 0,0 (S1); 44,7 (S2); 0,0 (S3); 21,6 (S4); 10,0 (S5); 6,1 (S6); 0,0 (S7) e 2,4 (S8). Para curva de morte microbiana, todos os produtos apresentaram diferença estatisticamente significante (p<0,05) do grupo controle (cloreto de sódio 0,85%) e do grupo S3. Verificou-se crescimento fúngico inferior a 2×104 UFC/mL e acentuação na curva de morte microbiana após 30 minutos de ação, a exceção do grupo S3. Conclusão: As substâncias analisadas, a exceção do Bicarbonato de Sódio, possuem ação antifúngica frente C. albicans, podendo contribuir para higienização de próteses. .

Atividade antifúngica do óleo essencial de Rosmarinus officinalis sobre a cinética do crescimento de Candida albicans e Candida tropicalis / Antifungal activity of Rosmarinus officinalis essential oil on growth kinetics of Candida albicans and Candida tropicalis

Objetivo: Avaliar a atividade antifúngica do óleo essencial de Rosmarinus officinalis sobre a cinética do crescimento de Candida albicans (ATCC289065) e C. tropicalis (ATCC40042). Material e Métodos: Em tubos de vidro estéreis foram adicionados 1,8mL de Caldo Sabouraud Dextrose, 0,2mL das suspensões fúngicas (1,5x10 elevado a 6UFC/mL) e 2mL da diluição do óleo essencial de R. officinalis nas concentrações 2% e 1%. Posteriormente, realizou-se a semeadura de 10µL das soluções testadas nos tempos zero, 30, 60 e 120 minutos. Após incubação a 37ºC por 24 horas, determinou-se o total de UFC/mL para cada amostra. A Solução de Hipoclorito de Sódio a 0,5% e o meio de cultura sem a adição de antimicrobianos funcionaram como controle. Resultados: O R. officinalis provocou, para C. albicans e C. tropicalis, acentuação na curva de morte microbiana nos intervalos 30 minutos (<3,5x10 elevado a 4 UFC/mL) e 120 minutos (<9x10 elevado a 4UFC/mL), respectivamente. Para C. albicans, observou-se diferença estatisticamente significante (p-valor<0,01) entre o controle de crescimento e o total de UFC/mL produzido pelos produtos testados. Para C. tropicalis, verificou-se diferença estatisticamente significante (p-valor<0,01) entre o efeito provocado pelo R. officinalis e o total de UFC/ml do controle de crescimento e do Hipoclorito de Sódio. Conclusão: O óleo essencial de R. officinalis apresentou ação antifúngica sobre o crescimento de C. albicans e C. tropicalis, destacando-se maior efeito diante do maior tempo de contato.

Objective: The aim of the study was to evaluate the antifungal activity of the essential oil of Rosmarinus officinalis (rosemary) on the growth kinetics of Candida albicans (ATCC289065) and C. tropicalis (ATCC40042). Methods: Into sterile glass tubes were inserted 1.8 mL of SabouraudDextrose broth, 0.2 mL of antifungal suspensions (1.5 x 10 the high 6 UFC/mL) and 2 mL of the dilution of R. officinalis essential oil at 2% and 1%. Posteriorly, 10ìL of the test solutions were sowed on plates at the times: zero, 30, 60 and 120 minutes. After incubation at 37 ° C for 24 hours, the counting of CFU/ mL for each sample was determined. Sodium hypochlorite solution at 0.5% and antimicrobial-free culture media were used as controls. Results: Upon C. albicans and C. tropicalis, R. officinalis intensified microbial death curve at 30 minutes (<3.5 x 10 the high 4 CFU/mL) and 120 minutes (<9 x 10 the high 4 CFU/mL), respectively. On C. albicans, statistically significant difference (p<0.01) was found between growth control and total CFU/mL promoted by the test products. Against C. tropicalis, a statistically significant difference (p<0.01) was verified between the effect caused by R. officinalis and the total CFU/mL of growth control and sodium hypochlorite. Conclusion: It was concluded that Rosmarinus officinalis essential oil showed antifungal activity against C. albicans and C. tropicalis, and better effects were observed in a longer contact time.