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.

Proliferation rate and expression of stem cells markers during expansion in primary culture of pulp cells

Abstract: The aim of the present study was to evaluate the proliferation rate and the expression of stem cells markers during expansion in primary culture of dental pulp stem cells (DPSCs), comparing different techniques (explant and enzymatic digestion), subject ages (up to 40 and over 40) and cell passages (#2, #5 and #8). DPSCs were isolated using either the enzymatic digestion (ED) or explant (EX) technique. The number of days needed for the cells to reach confluence was determined. Immunophenotyping was performed by immunofluorescence and flow cytometry analysis using antibodies specific for nestin, vimentin, CD44, CD146, Oct3/4 and CD34. Data were subjected to three-way analysis of variance (n = 6/group). The ANOVA tests were complemented by Tukey's or t-tests (p < 0.05). The variables "donor age" and "technique" were analyzed to define the optimal desirability value using a response optimization. DPSCs presented a high proliferation rate from passages 2 to 5 while cells from passage 8 proliferated at a slower rate. For all markers, no significant difference was observed among passages, irrespective of the technique used or the donor's age. The mean fraction of specific antibodies was 73.7% (± 11.5), 49.0% (± 18.7), 80.1% (± 8.0), 45.2% (± 13.7), 64.7% (± 5.3) and 2.0% (± 1.5) for CD44, OCT, vimentin, nestin, CD146 and CD34, respectively. The highest optimal desirability value was obtained using the ED technique and cells from younger patients (d = 0.92). However, it was concluded that neither the isolation technique nor the donor age or cell passage significantly interfered with the stem cell phenotype and proliferation rate during cell expansion.

Influence of bisphosphonates on the behavior of osteoblasts seeded onto titanium discs

Abstract Among other factors, types of bisphosphonates and treatment regimens seem to be strongly associated with the success or failure of installation of osseointegrated implants. This study investigated the influence of two bisphosphonates, sodium alendronate (SA) and zoledronic acid (ZA), on the metabolism of osteoblasts. Human osteoblasts (Saos-2) were seeded onto machined or acid-treated titanium discs previously placed on 24-well plates in complete culture medium. After 24 h, cells were exposed to bisphosphonates at 0.5, 1 or 5 µM for 24 h, 48 h or 7 days. The effects of SA and ZA on osteoblasts were assessed based on the adhesion of these cells to the titanium surfaces by direct fluorescence, cell viability, total protein and collagen synthesis. Alkaline phosphatase activity and mineral nodule deposition by these cells were also evaluated. Data were evaluated by ANOVA and Tukey tests (α=0.05). Decreased adhesion of cells to the titanium discs was observed when exposed to both bisphosphonates; however, this lack of cell adhesion was more evident for ZA-treated cells. In addition, the exposure of osteoblasts to ZA decreased the viability, ALP activity and mineral nodule deposition, which may be related to poor osseointegration after implant installation.

Resumo Entre outros fatores, os tipos de bisfosfonatos bem como os regimes de tratamento parecem estar diretamente associados com o sucesso ou falhas na instalação de implantes osseointegrados. Este estudo avaliou a influência de dois bisfosfonatos, o alendronato de sódio (AS) e o ácido zoledrônico (AZ), no metabolismo de osteoblastos. Osteoblastos humanos (Saos-2) foram cultivados sobre discos de titânio polidos ou submetidos a tratamento ácido superficial, previamente alocados em placas de 24 compartimentos, utilizando meio de cultura completo. Após 24 horas, as células foram expostas aos bisfosfonatos, nas concentrações de 0,5, 1 ou 5 µM, por 24 h, 48 h, ou 7 dias. Os efeitos do AZ e AZ sobre os osteoblastos foram determinados considerando a adesão destas células às superfícies de titânio, por meio de fluorescência direta, a viabilidade celular, produção de proteína total e síntese de colágeno. A atividade de fosfatase alcalina e a deposição de nódulos mineralizados também foram avaliadas. Os dados foram analisados por meio do teste ANOVA complementado por Tukey (α = 0.05). Menor adesão dos osteoblastos foi observada quando estas células foram expostas a ambos os bisfosfonatos, porém, esta falha na adesão foi mais evidente para as células tratadas com AZ. Além disso, a exposição dos osteoblastos ao AZ também resultou em diminuição da viabilidade, atividade de ALP e deposição de nódulos mineralizados, o que pode estar relacionado a uma pobre osseointegração após a instalação do implante.

Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel

Abstract Objective This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity. Methodology First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey's test (n=8. p<0.05). Results All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness. Conclusion Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.

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.

Análise da citotoxicidade de um gel clareador com 10% de peróxido de hidrogênio sobre células odontoblastóides de acordo com a espessura do substrato dental / Analysis of the cytotoxicity of a 10% hydrogen peroxide bleaching gel to odontoblast-like cells according to the thickness of dental substrate

Objetivo: Avaliar a citotoxicidade de um gel clareador contendo 10% de peróxido de hidrogênio (H2O2), aplicado sobre discos de esmalte/dentina simulando diferentes espessuras dentais. Material e método: Discos com 2,3; 3,5; e 4,0 mm de espessura foram obtidos para simular incisivos centrais inferiores, incisivos centrais superiores e segundos pré-molares superiores, respectivamente. Para cada espessura, o gel com 10% de H2O2 foi aplicado sobre o esmalte por 3x 15 min, 1x 15 min ou 1x 5 min. O protocolo 35% H2O2 3x 15 min foi empregado como controle positivo (CP), e nenhum tratamento foi realizado no controle negativo (CN). Células odontoblastóides MDPC-23 foram expostas por 1 h aos componentes da difusão trans-amelodentinária coletados imediatamente após o clareamento, sendo realizada análise da viabilidade celular, estresse oxidativo, deposição de nódulos de mineralização, bem como a quantificação da difusão de H2O2 pelos discos. Resultados: O gel com 10% de H2O2 não promoveu redução significativa da viabilidade celular em relação ao CN para todos os protocolos e espessuras testadas, resultando em valores de difusão de H2O2 significativamente inferiores ao CP. Apenas o protocolo 10% 3x 15 min aplicado sobre os discos simulando incisivos promoveu aumento no estresse oxidativo e reduziu a deposição de nódulos de mineralização em relação ao CN; porém, estes efeitos foram significativamente inferiores ao CP. Conclusão: De acordo com a metodologia usada neste estudo, foi possível concluir que, independente da espessura dental, a aplicação de um gel clareador com 10% de H2O2 por 5-45 min sobre o esmalte causa limitado efeito citotóxico sobre células pulpares.

Objective: To evaluate the cytotoxicity of a bleaching gel with 10% hydrogen peroxide (H2O2) applied onto enamel/dentin discs simulating different dental thicknesses. Material and methods: Discs with 2.3; 3.5; and 4.0 mm thickness were obtained to simulate low central incisors, upper central incisors and upper second pre-molars, respectively. For each thickness, the 10% H2O2 gel was applied for 3x 15 min, 1x 15 min or 1x 5 min. A gel with 35% H2O2 applied for 3x 15 min was used as positive control (PC) and no treatment was performed in negative control (NC). Odontoblast- like MDPC-23 cells were exposed for 1 h to the transenamel and trans-dentinal components collected immediately after bleaching. Cell viability, oxidative stress and mineralized nodule deposition were assessed as well as the quantification of H2O2 diffused through the discs. Results: The 10% H2O2 gel did not promote significant reduction on cell viability in comparison to NC for all tested protocols and thicknesses, resulting in H2O2 diffusion values significantly lower than PC. Only the protocol 10% H2O2 3x 15 min applied onto discs simulating incisors increased significantly the oxidative stress and reduced mineralized nodule deposition compared to NC; however, these effects were significantly lower than PC. Conclusion: According to the methodology employed in this laboratorial study, the application of a bleaching gel with 10% H2O2 for 5-45 min onto dental structure featured limited cytotoxicity to pulp cells, disregarding the enamel/dentin thicknesses.

In vitro and in vivo evaluations of glass-ionomer cement containing chlorhexidine for Atraumatic Restorative Treatment

Abstract Objectives: Addition of chlorhexidine has enhanced the antimicrobial effect of glass ionomer cement (GIC) indicated to Atraumatic Restorative Treatment (ART); however, the impact of this mixture on the properties of these materials and on the longevity of restorations must be investigated. The aim of this study was to evaluate the effects of incorporating chlorhexidine (CHX) in the in vitro biological and chemical-mechanical properties of GIC and in vivo clinical/ microbiological follow-up of the ART with GIC containing or not CHX. Material and Methods: For in vitro studies, groups were divided into GIC, GIC with 1.25% CHX, and GIC with 2.5% CHX. Antimicrobial activity of GIC was analyzed using agar diffusion and anti-biofilm assays. Cytotoxic effects, compressive tensile strength, microhardness and fluoride (F) release were also evaluated. A randomized controlled trial was conducted on 36 children that received ART either with GIC or GIC with CHX. Saliva and biofilm were collected for mutans streptococci (MS) counts and the survival rate of restorations was checked after 7 days, 3 months and one year after ART. ANOVA/Tukey or Kruskal-Wallis/ Mann-Whitney tests were performed for in vitro tests and in vivo microbiological analysis. The Kaplan-Meier method and Log rank tests were applied to estimate survival percentages of restorations (p<0.05). Results: Incorporation of 1.25% and 2.5% CHX improved the antimicrobial/anti-biofilm activity of GIC, without affecting F release and mechanical characteristics, but 2.5% CHX was cytotoxic. Survival rate of restorations using GIC with 1.25% CHX was similar to GIC. A significant reduction of MS levels was observed for KM+CHX group in children saliva and biofilm 7 days after treatment. Conclusions: The incorporation of 1.25% CHX increased the in vitro antimicrobial activity, without changing chemical-mechanical properties of GIC and odontoblast-like cell viability. This combination improved the in vivo short-term microbiological effect without affecting clinical performance of ART restorations.

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.

Repair of Bone Defects with Chitosan-Collagen Biomembrane and Scaffold Containing Calcium Aluminate Cement

Abstract Innovative biomaterials can provide a promising new direction for the treatment of bone defects, stimulating a proper repair process, with no damage to adjacent tissues. The purpose of this in vivo study was to evaluate the biocompatibility and the osteoinductive capacity of chitosan-collagen biomembrane and scaffold containing calcium aluminate cement. Eighteen New Zealand white rabbits (Oryctolagus cuniculus) were distributed according to the experimental times of analysis (7, 15 and 30 days). Four bone defects were created in the rabbits calvaria, which were individually filled with the biomembrane, scaffold, blood clot (negative control) and autologous bone (positive control). Histopathological analysis was performed using optical microscope at 32´, 64´, 125´ and 320´ magnifications. Cell response to inflammation and new bone tissue formation was quantified using a score system. The biomembrane group presented greater inflammatory response at 15 days, with significant difference to autologous bone group (p<0.05). There was no statistically significant difference for foreign body type reaction among groups (p>0.05). Concerning new bone formation, linear closure of the defect area was observed more evidently in the group with autologous bone. The scaffold group presented similar results compared with the autologous bone group at 30 days (p>0.05). Both tested biomaterials presented similar biocompatibility compared with the control groups. In addition, the biomembrane and scaffold presented similar osteoinductive capacity, stimulating bone repair process in the course of the experimental time intervals.

Resumo Biomateriais inovadores podem fornecer uma promissora nova direção para o tratamento de defeitos ósseos, estimulando um processo de reparo adequado, sem danos aos tecidos adjacentes. O objetivo deste estudo in vivo foi avaliar a biocompatibilidade e a capacidade osteoindutora de uma biomembrana e um scaffold compostos por colágeno e quitosana, contendo cimento de aluminato de cálcio. Dezoito coelhos (New Zealand White, Oryctolagus cuniculus) foram distribuídos de acordo com os períodos experimentais de análise (7, 15 e 30 dias). Quatro defeitos foram criados na calvaria dos coelhos, que foram individualmente preenchidos com a biomembrana, scaffold, coágulo (controle negativo) e osso autólogo (controle positivo). A avaliação histopatológica foi realizada em microscópio óptico em aumentos de 32´, 64´, 125´ e 320´. A resposta celular à inflamação e à formação de novo tecido ósseo foi quantificada utilizando um sistema de escore. O grupo da biomembrana apresentou maior resposta inflamatória no período de 15 dias, com diferença significativa para o grupo do osso autólogo (p<0,05). Não houve diferença estatística significante para a reação do tipo corpo estranho entre os grupos (p>0,05). Em relação à neoformação óssea, observou-se fechamento linear da área do defeito, que foi mais evidente no grupo em que se utilizou o osso autólogo. O grupo scaffold apresentou resultados semelhantes ao grupo do osso autólogo no período de 30 dias (p>0,05). Ambos os biomateriais testados apresentaram biocompatibilidade similar em comparação com os grupos controle. Além disso, a biomembrana e o scaffold apresentaram capacidade osteoindutora similar, estimulando o reparo ósseo ao longo dos intervalos de tempo experimentais.

Cytotoxicity Evaluation of Root Canal Sealers Using an In Vitro Experimental Model with Roots

Abstract The aim of the present study was to evaluate the cytotoxicity of root canal sealers under conditions closely resembling a clinical reality. A primary human fibroblast cell line was seeded in 24-well acrylic plates with Dulbecco’s modified Eagle’s medium supplemented with 10% serum fetal bovine (SFB) and incubated for 24 h. Root canals from premolars were filled and individually attached to nylon devices to be stabilized in the wells with the already seeded cells. Specimens were divided into groups as follows: Control: gutta-percha cones (GPC); AH Plus+GPC; Sealapex+GPC; MTA Fillapex+GPC and Endofill+GPC. After 24 and 48 h, cell viability and morphology were evaluated by MTT assay and scanning electron microscopy (SEM), respectively. Statistical analysis was performed by Mann-Whitney test, complemented by Kruskal Wallis test (p<0.05). Only Endofill presented cytotoxicity after 24 h. MTA Fillapex and Endofill reduced the production of succinic desidrogenase after 48 h. AH Plus was non-toxic at any time point. SEM showed that the AH Plus and MTA Fillapex groups presented fibroblasts with morphology close to the control group, while the Endofill group presented few cells with thin extensions cells. The present study showed that good results were present in AH Plus and Sealapex, but not the Endofill group after 48 h. The method used enabled evaluation of the cytotoxicity of the studied sealers that diffused through the root apex.

Resumo O objetivo do presente estudo foi avaliar a citotoxicidade dos cimentos dos canais radiculares em condições próximas à realidade clinica. Uma linhagem primária de fibrolastos humanos foi semeada em placas acrílicas de 24-poços com meio de cultura Dulbecco’s modified Eagle’s medium suplementado com 10% de soro fetal bovino e incubados por 24 h. Os canais radiculares de pré-molares foram obturados e individualmente adaptados aos dispositivos de nylon para serem estabilizados nos poços com as células já semeadas. Amostras foram dividas de acordo com os grupos: Controle: cones de gutta-percha (CGP); AH Plus+CGP; Sealapex+CGP; MTA Fillapex+CGP e Endofill+CGP. Após 24 e 48 h, a viabilidade e a morfologia celular foram avaliadas pelo ensaio de MTT e microscopia eletrônica de varredura (MEV), respectivamente. Análises estatísticas foram realizadas pelo teste de Mann-Whitney, complementadas por Kruskal Wallis (p<0,05). Apenas o Endofill apresentou citotoxicidade após 24 h. MTA Fillapex e Endofill reduziram a produção da enzima desidrogenase succinica após 48 h. AH Plus não apresentou toxicidade em nenhum momento. MEV mostrou que os grupos AH Plus e o MTA Fillapex apresentaram fibroblastos com morfologia próxima ao grupo controle, enquanto que o grupo do Endofill apresentou poucas células com finos prolongamentos. O presente estudo demonstrou que resultados satisfatórios foram apresentados nos grupos AH Plus e Sealapex, mas não para o Endofill após 48 h. O método utilizado permitiu avaliar a citotoxicidade dos cimentos que se difundem pelo ápice radicular.

Cytotoxicity of New Calcium Aluminate Cement (EndoBinder) Containing Different Radiopacifiers

Abstract This study aimed to evaluate the cytotoxicity of a calcium aluminate cement (EndoBinder) containing different radiopacifiers, Bi2O3, ZnO or ZrO2, compared with Mineral Trioxide Aggregate (MTA). According to ISO 10993-12:2012 (E) recommendations, 0.2 g of each cement were applied in transwell inserts and placed in 24-well culture plates containing 1 mL of culture medium (DMEM). After 24 h of incubation, the extracts (DMEM containing components released from the cements) were applied to immortalized odontoblast-like MDPC-23 cells. Cell viability (MTT test), alkaline phosphatase activity (ALP), total protein production and cell morphology (Scanning Electron Microscopy - SEM) were evaluated. The volume of 50 µL of extract was used to determine the chemical elements released by the cements using Energy Dispersive Spectroscopy (EDS). The following groups were established (n=6): NC - negative control (without treatment); EB - EndoBinder without radiopacifier; EBBO - EndoBinder+Bi2O3; EBZnO - EndoBinder+ZnO; EBZrO - EndoBinder+ZrO2 and WMTA - White MTA. Data were subjected to statistical analysis (Kruskal-Wallis test, level of significance=5%). Cells exposed to the different versions of EndoBinder presented small reduction in viability, total protein production and ALP activity, with values similar to the NC and WMTA groups (p>0.05). Different elements (C, O, Na, Al, P, Si, Cl, Bi, K) released by the cements were detected in the extracts. However, the cells had no significant changes in their morphology. EndoBinder and MTA did not affect negatively the metabolism of the odontoblastic-like cells, showing it to be cytocompatible, irrespective of the used radiopacifier.

Resumo Este estudo avaliou a citotoxicidade de um cimento de aluminato de cálcio (EndoBinder) contendo diferentes radiopacificadores, Bi2O3, ZnO ou ZrO2, comparativamente ao trióxido mineral agregado (MTA). Seguindo a norma ISO 10993-12:2012 (E), 0,2 g de cada cimento foi aplicada em insertos transwell, que foram colocados em placas de cultura de 24 wells contendo 1 mL de meio de cultura (DMEM). Após 24 h de incubação, os extratos (DMEM contendo componentes liberados dos cimentos) foram aplicados sobre células pulpares imortalizadas MDPC-23. Viabilidade celular (teste de MTT), atividade da fosfatase alcalina (ALP), produção de proteína total e a morfologia das células (Microscópio Eletrônico de Varredura - MEV) foram avaliadas. Um volume de 50 µL do extrato foi utilizado para determinar, através de Espectroscopia de Energia Dispersiva (EDS), os elementos químicos liberados pelos cimentos. Os seguintes grupos foram estabelecidos (n=6): NC - controle negativo (sem tratamento); EB - EndoBinder sem radiopacificador; EBBO - EndoBinder+Bi2O3; EBZnO - EndoBinder+ZnO; EBZrO - EndoBinder+ZrO2 e WMTA - MTA branco. Os dados foram submetidos à análise estatística (teste de Kruskal-Wallis, nível de significância=5%). Células expostas às diferentes versões de EndoBinder apresentaram pequena redução na viabilidade, produção de proteína total e atividade da ALP, com valores semelhantes aos grupos NC e WMTA (p>0,05). Diversos elementos (C, O, Na, Al, P, Si, Cl, Bi, K) liberados pelos cimentos foram detectados nos extratos. Entretanto, as células não apresentaram alterações significativas em sua morfologia. EndoBinder e MTA, não afetaram negativamente o metabolismo das células odontoblastóides, mostrando-se citocompatíveis, independente do radiopacificador utilizado.

Functional Differences In Gingival Fibroblasts Obtained from Young and Elderly Individuals

Abstract Fibroblasts participate in the wound repair process through proliferation and migration as well as the synthesis of factors growth and extracellular matrix molecules. However, cell aging and the individual himself can lead to reduction of cell functions and consequently, the ability of tissue repair. This study evaluated the activity of gingival fibroblasts from young (Y) and elderly (Y) patients and their responsiveness to tumor necrosis factor alpha (TNF-a). Gingival fibroblasts were isolated from six patients (3Y; and 3E) and seeded in complete culture medium (DMEM). For cell viability analysis, total protein production and collagen synthesis, fibroblasts were cultured in 96-well plates for 24, 48 or 72 h (n=36). Cell responses to TNF-a, was evaluated by application of this cytokine to cultured cells (100 ng/mL) for 24 h, followed by evaluation of reactive oxygen species (ROS), nitric oxide (NO) and CCL5 production (n=36). Data were analyzed by Kruskal-Wallis and the Mann-Whitney U tests (a = 0.05). Viability of E fibroblasts was higher than Y fibroblasts for 24 and 48 h, but these cells showed gradual reduction of viability over the course of time. For Y cells, reduced collagen synthesis was observed at 48 h. No difference was observed in ROS production for both cells after TNF-a exposure. However, both cultures showed increased production of NO and CCL5 in the presence of TNF-a. Functional differences and distinct responsiveness to TNF-a were observed according to patient's age.

Resumo Fibroblastos participam no processo de reparação de ferida através da proliferação e migração, bem como a síntese de fatores de crescimento e moléculas da matriz extracelular. No entanto, o envelhecimento celular e o próprio indivíduo podem levar à redução de funções celulares e, consequentemente, a capacidade de reparação de tecidos. Este estudo avaliou a atividade dos fibroblastos gengivais de pacientes jovens (J) e idosos (I) e sua capacidade de resposta frente ao fator de necrose tumoral alfa (TNF-a). Fibroblastos gengivais foram isolados de seis pacientes (3J e 3I) e semeados em meio de cultura completo (DMEM). Para a análise de viabilidade celular, a produção de proteína total e a síntese de colágeno, fibroblastos foram cultivados em placas de 96 poços, durante 24, 48 ou 72 h (n = 36). Respostas celulares frente ao TNF-a, foram avaliadas por aplicação desta citocina (100 ng/mL) nas células cultivadas durante 24 h, seguida por avaliação de espécies reativas de oxigênio (EROs), produção de óxido nítrico (NO) e produção CCL5 (n= 36). Os dados foram analisados por testes de Kruskal-Wallis e Mann-Whitney U (a = 0,05). A viabilidade de fibroblastos I foi mais elevada do que os fibroblastos J para 24 e 48 h, mas estas células mostraram uma redução gradual de viabilidade ao longo do tempo. Para as células de J, foi observada redução da síntese de colágeno em 48 h. Não foi observada diferença na produção de EROs para ambas as células após exposição ao TNF-a. No entanto, ambas as culturas apresentaram aumento da produção de NO e CCL5 na presença de TNF-a. Diferenças funcionais e alteração na capacidade de resposta ao TNF-a foram observadas de acordo com a idade do paciente.

Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells

Abstract The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells.

Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells

Abstract The present study aimed at evaluating the cytotoxic effects of a novel cement called CER on periodontal fibroblast-like cells of mice (MDPL-20), in comparison with different formulations of Mineral Trioxide Aggregate (MTA), by means of the cell viability test (MTT) and cell morphology analysis. Thirty-two round-shaped samples were fabricated with the following cements: white MTA, white and gray CER and experimental white MTA. The samples were immersed in serum-free culture medium for 24 hours or 7 days (n = 16). The extracts (culture medium + components released from the cements) were applied for 24 hours to previously cultured cells (40.000 cells/cm2) in the wells of 24-well plates. Cells seeded in complete culture medium were used as a negative control. Cell viability was assessed using the MTT assay. Two samples of each cement were used for cell morphology analysis by Scanning Electron Microscopy (SEM). The extracts obtained at the 7-day period presented higher cytotoxicity compared with the 24-hour period (p < 0.05). The gray CER obtained at 24 hours presented the highest cytotoxic effect, whereas the experimental white MTA presented the lowest, similar to the control (p > 0.05). However, at the 7-day period, the experimental white MTA presented no significant difference in comparison with the other cements (p > 0.05). At the 7-day period, CER cement presented cytotoxic effects on fibroblast-like cells, similar to different MTA formulations. However, the immersion period in the culture medium influenced the cytotoxicity of the cements, which was greater for CER cement at 24 hours.

Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid

Abstract Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

At-Home Bleaching: Color Alteration, Hydrogen Peroxide Diffusion and Cytotoxicity

This study evaluated the color change, cytotoxicity and hydrogen peroxide (HP) diffusion caused by different home bleaching protocols: 10% carbamide peroxide (CP) for 3 or 1.5 h, 6% hydrogen peroxide for 1.5 h or 45 min. To quantify the peroxide penetration, disks of bovine teeth were placed in artificial pulp chambers (APCs) containing acetate buffer, which was collected for evaluation in a spectrophotometer. For analysis of cytotoxicity, specimens were adapted in APCs containing culture medium, which subsequently was applied on MDPC-23 odontoblast-like cells for 1 h. Cellular metabolism was evaluated by methyl tetrazolium (MTT) assay and the color change of the specimens was analyzed using the CIE L * a * b * system. The data were submitted to ANOVA and Fisher test (α=5%). The treatment with 10% CP for 3 h was the most effective, and 6% HP for 45 min produced the lowest color change. The groups 10% CP for 1.5 h and 6% HP for 45 min had the lowest trans-enamel dentinal HP penetration, and the 6% HP for 1.5 h had the highest. None of the protocols affected cellular metabolism and morphology. In conclusion, reduced peroxide exposure time reduced the bleaching result; higher HP diffusion did not mean higher effectiveness.

Este estudo avaliou a alteração de cor, a citotoxicidade e a difusão de peroxido de hidrogênio ocorridos em diferentes protocolos clareadores caseiros: peróxido de carbamida (PC) 10% por 3 ou 1,5 h; peróxido de hidrogênio (PH) 6% por 1,5 h ou 45 min. Para a quantificação da penetração do peróxido, discos de dentes bovinos foram posicionados em câmaras pulpares artificiais (CPAs) contendo solução tampão de acetato, que foi coletada para avaliação em espectrofotômetro. Para análise da citotoxicidade, os espécimes foram adaptados nas CPAs contendo meio de cultura, que posteriormente foi aplicado sobre células odontoblastóides MDPC-23 por 1 h. O metabolismo celular foi avaliado pelo teste MTT e a alteração de cor dos espécimes foi analisada pelo sistema CIE L*a*b*. Os dados foram submetidos a ANOVA e teste de Fisher (α=5). O tratamento com PC10% por 3 horas foi o mais efetivo, enquanto que o tratamento com PH 6% por 45 min produziu a menor alteração cromática. Os grupos PC 10% por 1,5 h e PH 6% por 45 min causaram a menor penetração trans-amelodentinária do peróxido, e PH 6% por 1,5 h, a maior difusão. Nenhum tratamento alterou o metabolismo celular. A redução do tempo de exposição aos peróxidos comprometeu o resultado clareador; maior penetração de peroxido não significa maior efetividade clareadora.

Dose-responses of Stem Cells from Human Exfoliated Teeth to Infrared LED Irradiation

Despite several reports regarding tissue regeneration, including pulp repair induced by different light sources, only limited data have been reported concerning the effects of light-emitting diodes (LED) on stem cells from human exfoliated deciduous teeth (SHEDs). The aim of this study was to evaluate the effects of different energy densities of infrared LED on the cell viability, number of cells and mineralized tissue production by SHEDs. SHEDs were obtained from near-exfoliation primary teeth (n=3), seeded in plain DMEM (104 cells/cm2), and irradiated by a LED prototype (LEDTable 850 nm, 40 mW/cm2) delivering 0 (control), 2, 4, 8, 15 or 30 J/cm2 (n=9). Cell viability (MTT assay), cell proliferation (trypan blue assay), and mineralized nodule (MN) formation (alizarin red stain) were assessed 12 and 72 h post-irradiation. Data were subjected to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Cells irradiated with 2 or 4 J/cm2 exhibited higher metabolism at 72 h, and all energy densities provided increase in cell proliferation after 12 h. Regarding MN formation, the best results were observed at 72 h after SHED irradiation with 8 and 15 J/cm2. It was concluded that the cell viability, cell number and MN formation by pulp cells are enhanced after exposure to infrared LED irradiation. Overall, the greatest SHED biostimulation was obtained with 4 and 8 J/cm2.

Apesar de diversos estudos envolvendo regeneração tecidual, incluindo o reparo pulpar induzido por diferentes fontes de luz, dados limitados têm sido reportados a respeito dos efeitos da irradiação com diodos emissores de luz (LED) sobre células-tronco de dentes decíduos esfoliados (SHEDs). O objetivo do presente estudo foi avaliar os efeitos de diferentes doses de energia (DE) do LED infravermelho sobre a viabilidade celular, número de células viáveis e produção de nódulos mineralizados (NM) por SHEDs. As células foram obtidas a partir de dentes decíduos próximos ao período de esfoliação (n=3), semeadas em DMEM completo (10⁴ células/cm2) e irradiadas utilizando um protótipo de LED (LEDTable 850 nm, 40 mW/cm²⁾ com as doses de 0 (controle), 2, 4, 8, 15 ou 30 J/cm² (n=9). A viabilidade celular (MTT), o número de células viáveis (trypan blue assay) e a formação de NM (alizarin red stain) foram realizados 12 e 72 h após a irradiação. Os dados foram avaliados utilizando os testes Kruskal-Wallis e Mann-Whitney (α=0,05). As células irradiadas com 2 ou 4 J/cm² exibiram uma maior viabilidade em 72 h, e todas as DE aumentaram o número de células viáveis após 12 h. Para a formação de NM, os melhores resultados foram observados 72 h após a irradição das SHEDs, com as doses de 8 e 15 J/cm². Concluiu-se que a viabilidade celular, o número de células e a formação de NM por células pulpares são aumentados após exposição ao LED infravermelho. De um modo geral, a melhor bioestimulação celular (SHEDs) foi obtida com 4 e 8 J/cm².

Response of Human Pulps to Different In-Office Bleaching Techniques: Preliminary Findings

This study evaluated a whitening effect and the likely side effect (tooth sensitivity and pulp response) of human teeth subjected to different in-office bleaching (IOB) techniques and materials, mainly the presence of calcium in the IOB materials. A calcium-free (CF) and a calcium-containing (CC) 35% hydrogen peroxide (HP) gels were evaluated. The CF was refreshed every 15 minutes, three times (CF 3-15) or in a single 45-min application (CF 1-45) at one bleaching appointment. The CC was used only in a single 45-min application (CC 1-45). Each technique was applied in 5 mandibular incisors scheduled for extraction for different patients. In control group, no tooth bleaching was performed. The tooth colour (TC) and tooth sensitivity (TS) were recorded at baseline and after IOB. The teeth were extracted 2 days after the application of IOB and subjected to histological analysis. The data was submitted to appropriate statistical analysis (α=0.05). The changes of TC were similar between groups and statistically different from the control (p<0.05). However, TS of groups bleached with CF was statistically higher than that recorded for CC and the control (p<0.05). In CF 3-15 and CF 1-45 groups, the coronal pulp tissue exhibited partial necrosis associated with tertiary dentin deposition. In CC 1-45 group smaller area of necrosis occurred only in three bleached teeth in which tertiary dentin deposition was observed. The calcium-containing 35%HP gel could be preferable for in-office bleaching because it caused less tooth sensibility and pulp damage.

O objetivo do estudo foi avaliar o efeito clareador e seus efeitos adversos (sensibilidade e resposta pulpar) de dentes humanos submetidos a diferentes técnicas e materiais para o clareamento em consultório (CLCO), principalmente a presença de cálcio nos materiais para CLCO. Um agente clareador a base de peróxido de hidrogênio (PH) a 35% sem cálcio (SC) e com cálcio (CC) foram avaliados. O agente clareador SC foi usado em duas técnicas de aplicação: o gel clareador foi reaplicado a cada 15 minutos, três vezes (SC 3-15) ou 1 x 45-min por aplicação (SC 1-45) em uma sessão clínica. O agente clareador CC foi usado apenas em 1 x 45-min por aplicação (CC 1-45). Cada técnica foi aplicada em 5 incisivos inferiores indicados para extração de pacientes diferentes. No grupo controle, o clareamento não foi realizado. O efeito clareador (EC) e a sensibilidade dental (SD) foram registrados inicialmente e após o CLCO. Os dentes foram extraídos após 2 dias da aplicação do CLCO e foram submetidos ao análise histológica. Os dados foram submetidos a análise estatística apropriada (α=0.05). As mudanças foram semelhantes entre os grupos e significativamente diferentes do controle (p<0.05). Entretanto, a SD nos grupos clareados com SC foi estatisticamente maior do que a registrada nos grupos CC e do grupo controle (p<0.05). Nos grupos SC 3-15 e SC CF 1-45, o tecido pulpar da região coronária exibia necrose parcial associada a deposição de dentina terciária. No grupo CC 1-45, pequenas áreas de necrose ocorreram somente em 3 dentes clareados, nos quais deposição de dentina terciária também foi observada. O gel CC de HP a 35%HP gel poderia ser preferível para a realização de CLCO devido ao fato de causar menos danos ao tecido pulpar.

Dose-Response and Time-Course of a-Tocoferol Mediating the Cytoprotection Of Dental Pulp Cells Against Hydrogen Peroxide

This in vitro study evaluated the potential protective effect of vitamin E alpha-tocopherol (α-T) isomer against the toxicity of hydrogen peroxide (HP) applied on dental pulp cells. Odontoblast-like MDPC-23 cells were seeded on 96-well plates for 72 h, treated with different concentrations of α-T (1, 3, 5, and 10 mM) for different times (1, 4, 8, and 24 h) and then exposed or not to a 0.018% HP solution for 30 min. In positive and negative control groups, cells were exposed to HP or culture medium (DMEM containing 5% DMSO), respectively. Cell viability was assessed by the MTT assay and the absorbance numeric data, expressed as percentage values, were subjected to the statistical analysis by Kruskal-Wallis and Mann-Whitney tests (α=5%). Considering the cells in the negative control as having 100% of cell viability, all combinations of α-T concentrations and pretreatment times showed a protective effect against HP cytotoxicity. Significant reduction of cell viability (59%) was observed in the positive control compared with the negative control. The highest values of pulp cell viability were obtained after pretreatment with 1 and 3 mM α-T concentrations for 24 h followed by exposure to HP (126% and 97% of cell viability, respectively). Under the tested conditions, the most effective cell protection against the cytotoxic effects of HP was provided by the lowest concentrations of α-T (1 and 3 mM) applied for 24 h.

Neste estudo, foi avaliado o potencial protetor do isômero alfa-tocoferol da vitamina E (-T) contra a ação tóxica do peróxido de hidrogênio (PH) aplicado sobre células pulpares. Células odontoblastóides MDPC-23 foram semeadas em placas de 96 compartimentos por 72 h, tratadas com diferentes concentrações de α-T (1, 3, 5 e 10 mM) por diferentes períodos (1, 4, 8 e 24 h) e, então, expostas ou não a uma solução com 0,0018% de PH por 30 min. Nos controles positivo e negativo, as células foram expostas ao PH ou meio de cultura (DMEM contendo 5% de DMSO), respectivamente. A viabilidade celular foi avaliada pelo teste do MTT e os valores numéricos de absorbância, expressos em porcentagem, foram submetidos a análise estatística pelos testes de Kruskal-Wallis e Mann-Whitney (α=5%). Considerando as células do grupo controle negativo como apresentando 100% de viabilidade celular, todas as combinações de α-T, nas diferentes concentrações, e tempos de pré-tratamento demonstraram um efeito protetor contra a citotoxicidade do PH. Redução significativa da viabilidade (59%) foi observada para o grupo controle positivo comparado ao controle negativo. Os maiores valores de viabilidade celular foram obtidos após pré-tratamento com 1 e 3 mM de α-T por 24 h seguido de exposição ao PH (126% e 97% de viabilidade celular, respectivamente). Assim, de acordo com as condições experimentais, o efeito protetor mais efetivo contra os efeitos tóxicos do PH foi observado para as menores concentrações de α-T (1 e 3 mM) aplicado por 24 h.