Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines

Abstract Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

Differentiation of odontoblast-like cells from human exfoliated deciduous teeth-derived stem cells by transforming growth factor β3 in combination with heparin in vitro / 实用医学杂志

Objective To investigate the ability of human exfoliated deciduous teeth-derived stem cells (SHED) to differentiate into odontoblast-like cells. Methods SHEDs were isolated by enzyme digestion method. The 3nd passage SHEDs were incubated with 25 ng/mL recombinant human TGF-β3 , or with TGF-β3 in combination with heparin. The DSPP expression was detected by Q-PCR and Western-blotting assay. Alizarin red staining, immunhistochemistry assay and alkaline phosphatase(AKP) activity assay were performed, respectively. Result The AKP activity was enhance by TGF-β3 in combination with heparin. Alizarin red staining was positive in TGF-β3-heparin groups, with the increase of DSPP expression at both mRNA and protein level. Conclusion TGF-β3 in combination with heparin can enhance the differentiation of human exfoliated deciduous teeth-derived stem cells into odontoblast-like cells.

Efeito protetor da vitamina E (α-Tocoferol) contra a atividade citotóxica do peróxido de hidrogênio / Protective effect of vitamin E (α-tocopherol) against the cytotoxicity of hydrogen peroxide

O clareamento dental de consultório, realizado com géis que apresentam elevada concentração de peróxido de hidrogênio (PH), pode causar danos intensos para as células da polpa dentária. Dentro deste contexto, a administração de agentes antioxidantes previamente ao clareamento dental, tem sido considerada uma terapia promissora, pois pode eliminar ou pelo menos minimizar os efeitos adversos deste procedimento clínico estético. Assim, o objetivo do presente estudo foi avaliar o possível efeito protetor da Vitamina E (alfa-Tocoferol / α-T) contra à ação citotóxica do PH sobre células odontoblastóides (MDPC-23). Para isto, células foram semeadas em placas de 96 wells durante 72 horas e então submetidas a diferentes tempos de pré-tratamento (1, 4, 8 e 24 horas) com variadas concentrações de α-T (1, 3, 5 e 10 mM). Após os períodos de pré-tratamento, as células foram expostas ou não ao PH (0,018%) durante 30 minutos. Nos grupos controle positivo e negativo, as células foram somente expostas a uma solução de PH (0,018%) ou meio de cultura, respectivamente. O metabolismo celular foi avaliado pelo teste de MTT. A absorbância foi transformada em porcentagem e analisada pelo Teste de Kruskal-Wallis, complementado por Mann-Whitney (α=0,05%). Todas as concentrações de αT e tempos de pré-tratamento propostos neste estudo apresentaram proteção das células contra os efeitos citotóxicos do PH. Porém, os melhores resultados foram obtidos com as concentrações de 1 e 3 mM (126% e 97%, respectivamente) por 24 horas em relação a 41% de metabolismo celular do grupo controle positivo (p<0,05). Portanto, as células pré-tratadas com α-T por 24 horas, em concentrações baixas (1 e 3 mM), apresentaram os melhores resultados de viabilidade celular, ou seja, maior efeito protetor frente à agressão direta do PH

The in-office tooth bleaching using gels with high concentrations of hydrogen peroxide (HP) may cause irreversible damage to pulp tissue. Therefore, the administration of antioxidazing agents previously to the tooth bleaching procedures has been considered as a promising therapy, mainly due the capacity of these agents to prevent, or at last reduce, the negative side-effects caused by toxic products used in this esthetic treatment. Therefore, the aim of the present in vitro study was to evaluate the protective activity of vitamin E (alfa-Tocoferol / α-T) against the toxic effects of HP applied to cultured odontoblast-like cells (MDPC-23). Cells were seeded in wells of 96-well plates for 72 hs and then pre-treated with different concentrations of α-T (1, 3, 5, and 10 mM) for variable periods (1, 4, 8, and 24 hs). Following, the cells were exposed to a HP solution (0,018%) for 30 min. In positive and negative control groups, the cells were exposed only to HP solution (0,018%) or culture medium, respectively. The cell metabolism was assessed by MTT assay and the absorbance numeric data, expressed as percentage values, were subjected to the statistical analysis of Kruskal-Wallis complemented by Mann-Whitney test (α=0.05%). All α-T concentrations and pre-treatments evaluated in this study showed cell protection against the cytotoxic effects of HP. However, considering the MDPC23 cells in the negative control group as presenting 100% metabolism, it was observed that the most relevant data occurred when the cells were pre-treated with α-T at 1 and 3 mM (126% and 97% of cell metabolism, respectively) for 24hs compared to the positive control group (41%) (p<0.05). Based upon the methodology used in the present investigation, it can be concluded that low concentrations of α-T (1 and 3 mM) applied for 24 hs to the cultured odontoblast-like MDPC-23 cells provide the best protective effects against the cytotoxicity caused by hydrogen peroxide

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

O objetivo foi avaliar o potencial odontogênico de células indiferenciadas da polpa (OD-21) por meio de indução química in vitro. As células foram divididas em grupos: controle (OD-21), induzido (OD-21 em meio suplementado/OD-21 + OM), e células odontoblastóides (MDPC-23). Após 3, 7, 10 e 14 dias, avaliou-se proliferação e viabilidade celular, proteína total e fosfatase alcalina (ALP), mineralização, imunolocalização da proteína da matriz dentinária 1 (DMP1), ALP e osteopontina (OPN), assim como a expressão dos genes ALP, OSTERIX (Osx), DMP1 e fator de transcrição RUNX2 por PCR em tempo real. Os dados foram avaliados pelo teste de Kruskal-Wallis seguido pelo teste de Mann-Whitney U (p<0.05). Houve diminuição na proliferação celular em OD-21 + OM, com viabilidade celular similar em todos os grupos, exceto aos sete dias. O conteúdo de proteína total foi maior no grupo OD-21 + OM em todos os períodos; o mesmo ocorreu com a atividade de ALP quando comparada com o grupo OD-21, além de apresentar resultados similares ao grupo MDPC-23. A mineralização foi maior em OD-21 + OM quando comparada com o controle negativo. A imunolocalização demonstrou expressão de DMP1 e ALP em MDPC-23 e OD-21 + OM, enquanto que todos os grupos foram positivos para OPN. A expressão gênica de DMP1 e ALP foi maior nas culturas de MDPC-23, enquanto que a de RUNX2 foi menor para estas células e maior no controle negativo. A expressão de OSTERIX foi menor em OD-21 + OM quando comparada aos outros grupos. Sugere-se que as células indiferenciadas da polpa da linhagem OD-21 apresentam potencial odontogênico e poderiam ser usadas para a engenharia tecidual.

Citotoxidade direta e transdentinária de sistemas adesivos com diferentes graus de hidrofilia sobre células odontoblastóides / Direct and transdentinal cytotoxicity of adhesive systems with different degrees of hydrophilicity on odontoblast-like cells

O objetivo geral desse trabalho, dividido em dois experimentos (capítulos 1 e 2), foi avaliar a citotoxicidade de sistemas adesivos experimentais (SAEs), com diferentes graus de hidrofilia, e do etanol como solução de solvatação da dentina, sobre células odontoblastóides. No capítulo 1, discos de papel filtro esterilizados foram impregnados com 10 uL de cada SAE (n=22): R1, R2, R3, R4 e R5 (em ordem crescente de hidrofilia), seguido de fotoativação. Os discos foram individualmente imersos em meio de cultura DMEM para obtenção de extratos (DMEM + componentes liberados dos SAEs), os quais foram posteriormente aplicados sobre células MDPC-­23 em cultura. Discos não impregnados (R0) serviram como controle. Foram avaliados o metabolismo celular (teste de MTT), a expressão de proteína total (PT) e a atividade de fosfatase alcalina (FA), além do tipo de morte celular (citometria de fluxo) e grau de conversão monomérica (FTIR) dos SAEs. Os dados de cada variável resposta do estudo foram analisados por testes de Kruskal-­Wallis e Mann-­Whitney (α=0,05). Considerando R0 como 100%, foi observada redução do metabolismo celular de 36,4%, 3,1%, 0,2%, 21,5% e 65,7%, respectivamente para R1, R2, R3, R4 e R5. Apenas R1 e R5 diferiram estatisticamente do controle. Para PT, R1, R4 e R5 tiveram expressão estatisticamente inferior ao controle, enquanto que a atividade de FA foi significativamente reduzida por R1 e R5. Esses mesmos SAEs juntamente com R4 induziram as maiores porcentagens de morte...

The overall aim of this study, divided into two experiments, was to evaluate the cytotoxicity of experimental adhesive systems (EAS) with different hydrophilicity, and ethanol as a dentin solvation solution, on odontoblast-­like cells. In the first experiment, sterilized filter paper discs were impregnated with 10 uL of each EAS: R1, R2, R3, R4 and R5 (in increasing rank of hydrophilicity), followed by light activation. The paper discs were individually immersed in DMEM culture medium for obtaining the extracts (DMEM + released components of EAS), which were applied on MDPC-23 cells in culture. Non-­impregnated paper discs (R0) were used as control. Cell metabolism (MTT assay), total protein expression (TP) and alkaline phosphatase activity (ALP) were assessed, in addition to the type of cell death (flow cytometry) and the degree of monomer conversion (FTIR). Data for each response variable were submitted to Kruskal-­Wallis and Mann-­Whitney tests (α=0.05). Compared to the control (100%), cell metabolism was decreased by 36.4%, 3.1%, 0.2%, 21.5% and 65.7% for R1, R2, R3, R4 and R5, respectively. However, only R1 and R5 differed from the control. R1, R4 and R5 decreased the expression of TP compared to the control, whereas only R1 and R5 significantly reduced the activity of ALP. The later EAS plus R4 caused the highest percentages of cell death by necrosis. A higher percentage of monomer conversion was detected as a function of the hydrophilicity. According to the experimental conditions, it could be...