Abstract This study evaluated the bioactive potential of a macro-porous chitosan scaffold incorporated with calcium hydroxide (CH-Ca) and functionalized with bioactive doses of simvastatin (SV) for bone tissue regeneration. Initially, the bioactive dose of SV in osteoblastic cells (SAOS-2) was determined. For the direct contact experiment, SAOS-2 cells were plated on scaffolds to assess cell viability and osteogenic differentiation. The second assay was performed at a distance using extracts from scaffolds incubated in culture medium to assess the effect of conditioned medium on viability and osteogenic differentiation. The initial screening showed that 1 μM SV presented the best biostimulating effects, and this dose was selected for incorporation into the CH-Ca and pure chitosan (CH) scaffolds. The cells remained viable throughout the direct contact experiment, with the greatest cell density in the CH-Ca and CH-Ca-SV scaffolds because of their higher porosity. The CH-Ca-SV scaffold showed the most intense bio-stimulating effect in assays in the presence and absence of osteogenic medium, leading to an increased deposition of mineralized matrix. There was an increase in the viability of cells exposed to the extracts for CH-Ca, CH-SV, and CH-Ca-SV during the one-day period. There was an increase in ALP activity in the CH-Ca and CH-Ca-SV; however, the CH-Ca-SV scaffold resulted in an intense increase in the deposition of mineralized nodules, approximately 56.4% at 7 days and 117% at 14 days, compared with CH (control). In conclusion, functionalization of the CH-Ca scaffold with SV promoted an increase in bioactivity, presenting a promising option for bone tissue regeneration.

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.

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.

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.

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.

Abstract The current study evaluated the effects of taxifolin treatments on the viability of osteoblast-like cells, and on the expression of early mineralization markers, as part of the ongoing search for new endodontic materials able to induce periapical healing without causing cytotoxicity. Saos-2 osteoblast-like cells were exposed to different concentrations of taxifolin (5 and 10 µM), applied as pretreatments either for 24h and 72h, or continuously throughout the experimental protocol. Cell viability using the methylthiazole tetrazolium (MTT) assay, alkaline phosphatase activity using thymolphthalein monophosphate assays, deposition of mineralized nodules using alizarin red staining, and expression of ALP and COL-1 by qPCR were determined after 6 and 13 days of treatment. The data were analyzed statistically (p<0.05). Taxifolin was not cytotoxic in the concentrations tested. Pretreatments with taxifolin for 24h and 72h at 10 µM stimulated ALP activity, and increased mineralized nodule deposition by Saos-2 cells. Continuous treatment with taxifolin was not effective in stimulating ALP activity and mineralization. ALP and COL-1 gene expression increased with taxifolin pretreatments, since the highest mRNA levels were observed after 72h of pretreatment with taxifolin at 10 µM on day 13. In conclusion, taxifolin was cytocompatible, and induced mineralization markers when applied for short periods in osteoblast-like cell culture.

Resumo O presente estudo avaliou o potencial odontogênico de células da polpa dental humana (HDPCs) em contato com scaffolds de quitosana contendo aluminato de cálcio (CHAlCa) associado ou não à baixas dosagens de sinvastatina (SV). Scaffolds de quitosana receberam uma suspensão de aluminato de cálcio e foram imersos em soluções contendo a droga. Foram estabelecidos três grupos experimentais: scaffolds de quitosana e aluminato de cálcio (CHAlCa - controle), scaffolds de quitosana-aluminato de cálcio com 0.5 µM SV (CHAlCa-SV0.5), e quitosana-aluminato de cálcio com 1.0 µM SV (CHAlCa-SV1.0). A morfologia e composição foram avaliados por MEV e EDS, respectivamente. Após 14 dias do cultivo das HDPCs sobre os scaffolds, foram avaliados a viabilidade celular, adesão e espalhamento, deposição de matriz mineralizada e expressão gênica de marcadores odontogênicos. Observou-se que as partículas de aluminato de cálcio foram incorporadas à matriz de quitosana, a qual exibiu poros regulares distribuídos por toda sua estrutura. As dosagens selecionadas de sinvastatina foram biocompatíveis com as HDPCs. A concentração de 1 µM de SV induziu intensa expressão de fenótipo odontoblástico pelas HDPCs, demonstrando aumento da deposição de matriz mineralizada e maior expressão de ALP, Col1A1 e DMP-1. Portanto, podemos concluir que a incorporação de aluminato de cálcio e sinvastatina em scaffolds de quitosana apresentou um efeito sinérgico nas HDPCs, favorecendo a diferenciação celular e deposição de matriz mineralizada.

Abstract The present study evaluated the odontogenic potential of human dental pulp cells (HDPCs) exposed to chitosan scaffolds containing calcium aluminate (CHAlCa) associated or not with low doses of simvastatin (SV). Chitosan scaffolds received a suspension of calcium aluminate (AlCa) and were then immersed into solutions containing SV. The following groups were established: chitosan-calcium-aluminate scaffolds (CHAlCa - Control), chitosan calcium-aluminate with 0.5 µM SV (CHAlCa-SV0.5), and chitosan calcium-aluminate with 1.0 µM SV (CHAlCa-SV1.0). The morphology and composition of the scaffolds were evaluated by SEM and EDS, respectively. After 14 days of HDPCs culture on scaffolds, cell viability, adhesion and spread, mineralized matrix deposition as well as gene expression of odontogenic markers were assessed. Calcium aluminate particles were incorporated into the chitosan matrix, which exhibited regular pores homogeneously distributed throughout its structure. The selected SV dosages were biocompatible with HDPCs. Chitosan-calcium-aluminate scaffolds with 1 µM SV induced the odontoblastic phenotype in the HDPCs, which showed enhanced mineralized matrix deposition and up-regulated ALP, Col1A1, and DMP-1 expression. Therefore, one can conclude that the incorporation of calcium aluminate and simvastatin in chitosan scaffolds had a synergistic effect on HDPCs, favoring odontogenic cell differentiation and mineralized matrix deposition.

Resumo O objetivo deste estudo foi o desenvolvimento de um recobrimento de vidro bioativo sobre a zircônia (Zr) para modular o fenótipo de fibroblastos gengivais. Para este propósito, partículas de Biosilicato® (6 mg/mL) em um veículo a base de água/álcool isopropílico (1:1) foram aplicadas sobre discos de zircônia seguido por tratamento térmico a 1100 °C por 20 min. A topografia de superfície (MEV), composição química (EDX), rugosidade de supefície (Ra; microscopia confocal), energia livre de superfície (goniômetro) e alteração de cor (Espectrofotometria UV-vis) foram avaliadas (n=6). A seguir, fibroblastos L929 foram semeados sobre discos de Zr e BS+Zr e a proliferação (Alamar Blue; n=6), morfologia (MEV; n=2), migração celular (wound healing; n=4) e a síntese de colágeno (Sirius Red; n=6) foram avaliados até 7 dias. Os dados foram analisados pelos testes ANOVA/Tukey (a=5%). Um recobrimento homogêneo consistindo de Si, Na, O e Ca foi detectado na superfície da Zr após o tratamento térmico com BS, o qual promoveu um aumento significante na rugosidade e energia livre de superfície (p<0,05). Nenhuma mudança nos parâmetros de cor foi observada (p>0,05). Células semeadas na superfície de Zr+BS apresentaram maior proliferação, expressão de colágeno e capacidade de migração em comparação com aquelas cultivadas sobre a superfície de Zr (p<0,05). Imagens de MEV revelaram que o espalhamento celular ocorreu mais rápido na presença de BS. Assim, conclui-se que o tratamento térmico da superfície da Zr com BS levou a deposição de um recobrimento bioativo, o qual induziu in vitro o espalhamento, proliferação e migração de fibroblastos gengivais e expressão de colágeno.

Abstract The objective of this study was the development of a bioactive glass coating on zirconia (Zr) to modulate the gingival fibroblast phenotype. For this purpose, Biosilicate® (BS) particles in a water/isopropyl alcohol (1:1) vehicle (6 mg/mL) were applied to zirconia discs followed by thermal treatment at 1100 °C for 20 min. The surface topography (SEM), chemical composition (EDX), surface roughness (Ra; confocal microscopy), surface free energy (goniometry), and color alteration (UV-vis spectrophotometry) were assessed (n=6). Thereafter, L929 fibroblasts were seeded onto Zr and Zr+BS discs, and cell proliferation (Alamar Blue; n=6), morphology (SEM; n=2), migration (wound healing; n=4), and collagen synthesis (Sirius Red; n=6) were evaluated up to 7 days. Data were analyzed by ANOVA/Tukey tests (a=5%). A homogeneous coating consisting of Si, Na, O, and Ca was detected on the Zr surface after thermal treatment with BS, which led to a significant increase in surface roughness and free energy (p<0.05). No change in color parameters was observed (p>0.05). Cells seeded on the Zr+BS surface featured increased proliferation, collagen expression, and migration capability in comparison with those cultured on plain Zr (p<0.05). SEM images revealed that cell spreading occurred faster in the presence of BS. Therefore, it was concluded that thermal treatment of the Zr surface with BS led to the deposition of a bioactive coating, which induced gingival fibroblast spread, proliferation, migration, and collagen expression in vitro.

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.

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.

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.

Abstract: The aim of this study was to evaluate the bioactivity and cytocompatibility of simvastatin (SV) applied to MDPC-23 odontoblast-like cells. For this purpose, MDPC-23 cells were seeded in 96-well plates and submitted to treatments with 0.01 or 0.1 μM of SV for 24 h, 72 h or continuously throughout the experimental protocol. The negative control group (NC) was maintained in DMEM. Cell viability (MTT), ALP activity (thymolphthalein monophosphate), and mineralized matrix deposition (alizarin red) were analyzed at several time points. The data were submitted to ANOVA and Tukey's test (α = 0.05). Although cell viability was observed in the groups treated with SV, these groups did not differ from the NC up to 7 days. There was a reduction in cell viability for the groups treated with 0.1 μM of SV for 72 h, and submitted to continuous mode after 14 days. A significant increase in ALP activity occurred in the group treated with 0.01 μM of SV for 24 h, compared with the NC; however, only the group treated with 0.1 μM of SV in continuous mode reduced the ALP activity, in comparison with the NC. After 14 days, only continuous treatment with 0.1 μM of SV did not differ from NC, whereas the other experimental groups showed increased mineralized matrix deposition. Thus, it was concluded that low concentrations of simvastatin were bioactive and cytocompatible when applied for short periods to cultured MDPC-23 odontoblast-like cells.

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.

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.

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.

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.

<p>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.</p>

<p>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.</p>

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.

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.