Effect of preheating on cytotoxicity and physicochemical properties of light-cured calcium-based cements.

The aim of this study was to evaluate the degree of conversion, cytotoxicity, solubility and pH of photopolymerizable calciumbased cements submitted to preheating. The degree of conversion was analyzed by Fourier transform infrared, cytotoxicity by the MTT test and solubility through loss of mass. The data were subjected to statistical tests (ANOVA / Tukey's, p<0.05). The photopolymerizable materials showed a low degree of conversion, regardless of preheating. All materials caused a reduction in cell viability at 24 hours and 7 days, with the Dycal (control) being more cytotoxic. Heat had a positive effect on Biocal at 7 days. Dycal is the most soluble material. Heat had no effect on the solubility or pH of the polymerizable materials. It is concluded that photopolymerizable calcium-based cements have a low degree of conversion and are soluble, which results in mild to moderate cytotoxicity.

O objetivo do presente estudo foi avaliar o grau de conversão, citotoxicidade, solubilidade e pH de cimentos à base de cálcio fotopolimerizáveis submetidos a pré-aquecimento. O grau de conversão foi analisado por espectroscopia no infravermelho com transformada de Fourier, a citotoxicidade pelo teste de MTT e a solubilidade através da perda de massa. Os dados foram submetidos a testes estatísticos (ANOVA/Tukey, p<0,05). Os materiais fotopolimerizáveis apresentaram baixo grau de conversão, independente do pré-aquecimento. Todos os materiais causaram redução da viabilidade celular nas análises de 24 horas e 7 dias, sendo que o Dycal (controle) apresentouse mais citotóxico e o calor apresentou efeito positivo sobre o Biocal na análise de 7 dias. O Dycal é o material mais solúvel e o calor não causou efeito na solubilidade e pH dos materiais polimerizáveis. Assim, conclui-se que os cimentos à base de cálcio fotopolimerizáveis apresentam baixo grau de conversão e são solúveis, que resulta em citotoxicidade suave e moderada.

Effect of preheating on cytotoxicity and physicochemical properties of light-cured calcium-based cements / Efeito do pré-aquecimento na citotoxicidade e propriedades físico-químicas de cimentos à base de cálcio fotopolimerizáveis

ABSTRACT The aim of this study was to evaluate the degree of conversion, cytotoxicity, solubility and pH of photopolymerizable calciumbased cements submitted to preheating. The degree of conversion was analyzed by Fourier transform infrared, cytotoxicity by the MTT test and solubility through loss of mass. The data were subjected to statistical tests (ANOVA / Tukey's, p<0.05). The photopolymerizable materials showed a low degree of conversion, regardless of preheating. All materials caused a reduction in cell viability at 24 hours and 7 days, with the Dycal (control) being more cytotoxic. Heat had a positive effect on Biocal at 7 days. Dycal is the most soluble material. Heat had no effect on the solubility or pH of the polymerizable materials. It is concluded that photopolymerizable calcium-based cements have a low degree of conversion and are soluble, which results in mild to moderate cytotoxicity.

RESUMO O objetivo do presente estudo foi avaliar o grau de conversão, citotoxicidade, solubilidade e pH de cimentos à base de cálcio fotopolimerizáveis submetidos a pré-aquecimento. O grau de conversão foi analisado por espectroscopia no infravermelho com transformada de Fourier, a citotoxicidade pelo teste de MTT e a solubilidade através da perda de massa. Os dados foram submetidos a testes estatísticos (ANOVA/Tukey, p<0,05). Os materiais fotopolimerizáveis apresentaram baixo grau de conversão, independente do pré-aquecimento. Todos os materiais causaram redução da viabilidade celular nas análises de 24 horas e 7 dias, sendo que o Dycal (controle) apresentouse mais citotóxico e o calor apresentou efeito positivo sobre o Biocal na análise de 7 dias. O Dycal é o material mais solúvel e o calor não causou efeito na solubilidade e pH dos materiais polimerizáveis. Assim, conclui-se que os cimentos à base de cálcio fotopolimerizáveis apresentam baixo grau de conversão e são solúveis, que resulta em citotoxicidade suave e moderada.

Cytotoxicity and bioactivity of various pulpotomy materials on stem cells from human exfoliated primary teeth.

AIMS: To investigate the cytotoxicity and bioactivity of several pulpotomy materials: Biodentine (Septodont, Saint-Maur-des-Fosses, France) MTA (Angelus, Londrina, PR, Brazil), Theracal LC (Bisco Inc., Schamburg, IL, USA) and IRM (Dentsply DeTrey GmbH, Konstanz, Germany), after contact with stem cells isolated from human exfoliated primary teeth (SHEDs). METHODOLOGY: SHEDs were cultured in the presence of the eluates of various pulpotomy materials for 24, 48 and 72 h. Cell viability was determined by mitochondrial dehydrogenase enzymatic (MTT) assay. Apoptosis and changes in cell phenotype were evaluated by flow cytometry. Also, an in vitro scratch wound-healing assay was used to determine their effects on cell migration. To assess cell morphology and attachment to the different pulpotomy materials, SHEDs were directly seeded onto the material surfaces and analysed by scanning electron microscopy (SEM). Finally, the deposition of a calcified matrix in presence of these materials was verified by Alizarin Red staining. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). RESULTS: Cell viability in the presence of Biodentine eluates was significantly higher to that obtained using complete medium alone (control; P < 0.01) and was also significantly higher than using MTA Angelus from 48 h of incubation (P < 0.01). However, Theracal LC and IRM were associated with low rates of cell viability (P < 0.001). Similar results were obtained in an apoptosis assay. In addition, SHEDs maintained their mesenchymal phenotype in all conditions although their capacity to migrate was higher in the presence of Biodentine. SEM studies revealed a suitable proliferation rate, cell spreading and attachment, especially when using Biodentine and MTA Angelus discs. Finally, Biodentine eluates significantly induced calcified matrix deposition from 7 days of culture (P < 0.01). CONCLUSIONS: Biodentine exhibited better cytocompatibility and bioactivity than MTA Angelus, Theracal LC and IRM.

Evaluation of the genotoxicity and mutagenicity of Ca3SiO5-based cement.

Ca3SiO5 is new cement based on the composition of Portland that has been developed to have superior physicochemical and biological properties. In a clinical evaluation, the cement did not appear to have cytotoxic properties and allowed for the proliferation of pulp cells and gingival fibroblasts. However, no previous studies have evaluated the genotoxicity or the mutagenicity of Ca3SiO5in vivo. Therefore, the goal of this study is to evaluate the genotoxic and mutagenic potential of Ca3SiO5-based cement in vivo. Twenty-four male Wistar rats were divided into 3 groups (n = 8). Group A rats received subcutaneous implantation of Ca3SiO5 in the dorsum. Group B rats received a single dose of cyclophosphamide (positive control). Group C rats received subcutaneous implantation of empty tubes in the dorsum (negative control). After 24 hours, all animals were euthanized and the bone marrow of the femurs was collected for use in the comet assay and the micronucleus test. The comet assay revealed that the Ca3SiO5 group had a tail intensity of 23.57 ± 7.70%, the cyclophosphamide group had a tail intensity of 27.43 ± 7.40%, and the negative control group had a tail intensity of 24.75 ± 5.55%. The average number of micronuclei was 6.25 (standard deviation, SD = 3.53) in the Ca3SiO5 group, 9.75 (SD = 2.49) in the cyclophosphamide group, and 0.75 (SD = 1.03) in the negative control group. There was an increase in the micronuclei frequency in the Ca3SiO5 group compared to that of the negative control group (p < 0.05). Our data showed that exposure to the Ca3SiO5-based cement resulted in an increase in the frequency of micronuclei, but no genotoxicity was detected according to the comet assay.

Evaluation of the genotoxicity and mutagenicity of Ca3SiO5-based cement

Abstract Ca3SiO5 is new cement based on the composition of Portland that has been developed to have superior physicochemical and biological properties. In a clinical evaluation, the cement did not appear to have cytotoxic properties and allowed for the proliferation of pulp cells and gingival fibroblasts. However, no previous studies have evaluated the genotoxicity or the mutagenicity of Ca3SiO5in vivo. Therefore, the goal of this study is to evaluate the genotoxic and mutagenic potential of Ca3SiO5-based cement in vivo. Twenty-four male Wistar rats were divided into 3 groups (n = 8). Group A rats received subcutaneous implantation of Ca3SiO5 in the dorsum. Group B rats received a single dose of cyclophosphamide (positive control). Group C rats received subcutaneous implantation of empty tubes in the dorsum (negative control). After 24 hours, all animals were euthanized and the bone marrow of the femurs was collected for use in the comet assay and the micronucleus test. The comet assay revealed that the Ca3SiO5 group had a tail intensity of 23.57 ± 7.70%, the cyclophosphamide group had a tail intensity of 27.43 ± 7.40%, and the negative control group had a tail intensity of 24.75 ± 5.55%. The average number of micronuclei was 6.25 (standard deviation, SD = 3.53) in the Ca3SiO5 group, 9.75 (SD = 2.49) in the cyclophosphamide group, and 0.75 (SD = 1.03) in the negative control group. There was an increase in the micronuclei frequency in the Ca3SiO5 group compared to that of the negative control group (p < 0.05). Our data showed that exposure to the Ca3SiO5-based cement resulted in an increase in the frequency of micronuclei, but no genotoxicity was detected according to the comet assay.

Development of calcium phosphate/sulfate biphasic cement for vital pulp therapy.

OBJECTIVES: Bioactive calcium phosphate cement (CPC) has been used widely to repair bone defects because of its excellent biocompatibility and bioactivity. However, the poor handling properties, low initial mechanical strength, and long setting time of CPC limit its application in vital pulp therapy (VPT). The aim of this study was to synthesize biphasic calcium phosphate/sulfate cements and evaluate the feasibility of applying these cements in VPT. METHODS: The physical, chemical, and mechanical properties of CPC were improved by mixing the cement with various amounts of α-calcium sulfate hemihydrate (CSH). The hydration products and crystalline phases of the materials were characterized using scanning electron microscopy and X-ray diffraction analysis. In addition, the physical properties, such as the setting time, compressive strength, viscosity, and pH were determined. Water-soluble tetrazolium salt-1 and lactase dehydrogenase were used to evaluate cell viability and cytotoxicity. RESULTS: The developed CPC (CPC/CSH cement), which contains 50wt% CSH cement, exhibited no obvious temperature increase or pH change during setting when it was used as a paste. The initial setting time of the CPC/CSH biphasic cement was substantially shorter than that of CPC, and the initial mechanical strength was 23.7±5.6MPa. The CPC/CSH cement exhibited higher viscosity than CPC and, thus, featured acceptable handling properties. X-ray diffraction analysis revealed that the relative peak intensity for hydroxyapatite increased, and the intensity for calcium sulfate dehydrate decreased as the amount of CPC was increased. The cell viability and cytotoxicity test results indicated that the CPC/CSH cement did not harm dental pulp cells. SIGNIFICANCE: The developed CPC/CSH biphasic cement exhibits substantial potential for application in VPT.

In vitro cytotoxicity evaluation of a novel root repair material.

INTRODUCTION: This study examined the effect of a new bioactive dentin substitute material (Biodentine) on the viability of human gingival fibroblasts. METHODS: Biodentine, White ProRoot mineral trioxide aggregate (MTA), and glass ionomer cement were evaluated. Human gingival fibroblasts were incubated for 1, 3, and 7 days both in the extracts from immersion of set materials in culture medium and directly on the surface of the set materials immersed in culture medium. Fibroblasts cultured in Dulbecco modified Eagle medium were used as a control group. Cytotoxicity was evaluated by flow cytometry, and the adhesion of human gingival fibroblasts to the surface of the set materials was assessed by using scanning electron microscopy. The data of cell cytotoxicity were analyzed statistically by using a one-way analysis of variance test at a significance level of P< .05. RESULTS: Cells exposed to extracts from Biodentine and MTA showed the highest viabilities at all extract concentrations, whereas cells exposed to glass ionomer cement extracts displayed the lowest viabilities (P< .05). There was no significant difference in cell viabilities between Biodentine and MTA during the entire experimental period (P> .05). Human gingival fibroblasts in contact with Biodentine and MTA attached to and spread over the material surface after an overnight culture and increased in numbers after 3 and 7 days of culture. CONCLUSIONS: Biodentine caused gingival fibroblast reaction similar to that by MTA. Both materials were less cytotoxic than glass ionomer cement.

Novel experimental cements for use on the dentin-pulp complex.

This aim of this study was to evaluate the physicochemical and biological properties of novel experimental cements (Hybrid, Paste and Resin) based on synergistic combinations of existing materials, including pH, diametral tensile strength (DTS) and cytotoxicity comparing them with mineral trioxide aggregate (MTA - Angelus®) and a glass ionomer cement (GIC) developed at our laboratory. For the physicochemical and biological tests, specimens with standard dimensions were produced. pH measurements were performed with digital pH meter at the following time intervals: 3, 24, 48 and 72 h. For the DTS test, cylindrical specimens were subjected to compressive load until fracture. The MTT assay was performed for cytotoxicity evaluation. Data were analyzed by ANOVA and Tukey's test (α=0.05). Paste group showed pH values similar to MTA, and Hybrid group presented pH values similar to GIC (p>0.05). The tested materials showed pH values ranging from alkaline to near neutrality at the evaluated times. MTA and GIC showed similar DTS values. The lowest and highest DTS values were seen in the Paste and Resin groups, respectively (p<0.05). Cell viability for MTA and experimental Hybrid, Paste and Resin groups was 49%, 93%, 90% and 86%, respectively, when compared with the control group. The photo-cured experimental resin cement showed similar or superior performance compared with the current commercial or other tested experimental materials.

Chlorhexidine, ethanol, lipopolysaccharide and nicotine do not enhance the cytotoxicity of a calcium hydroxide pulp capping material.

AIM: To determine whether cells pre-stressed by known cytotoxic or inflammatory agents are more susceptible to the deleterious effects of a calcium hydroxide formulation used in pulp capping. METHODOLOGY: Adult human dermal fibroblasts were treated for 48 h with 0.001% chlorhexidine, 0.2% ethanol, 5 µg mL(-1) Escherichia coli lipopolysaccharide (LPS) or 0.05 mmol L(-1) nicotine. Cells were subsequently treated with the soluble materials extracted from Dycal pellets for an additional 24 h. Controls included cells cultured in medium only and cells exposed to Dycal only. Cytotoxicity was measured using colorimetric MTT, WST and secreted lactate dehydrogenase assays. In addition, mitotic activity was evaluated using a colorimetric histone H3 phosphorylation assay. Data were statistically analysed using anova with Tukey's multiple comparison post-test and significance at P ≤ 0.05. RESULTS: For all assays, measured values for cells treated with chlorhexidine, ethanol, LPS or nicotine plus the soluble materials extracted from Dycal pellets were significantly lower compared to control (P < 0.05) for all comparisons between experimental conditions. However, between treatments and for comparisons of treatments with Dycal, there were no differences observed for any assay. CONCLUSIONS: Calcium hydroxide in a formulation used in dental clinical procedures is highly cytotoxic to cultured cells, as evidenced by several cellular assays. However, other known toxic agents, including chlorhexidine, ethanol, bacterial LPS and nicotine, do not appear to function synergistically to increase the deleterious cellular effects of the calcium hydroxide in an in vitro model of cytotoxicity.

Cytotoxicity comparison of three current direct pulp-capping agents with a new bioceramic root repair putty.

INTRODUCTION: The purpose of this in vitro study was to compare the cytotoxicity of white mineral trioxide aggregate cement (AMTA, MTA-Angelus), Brasseler Endosequence Root Repair Putty (ERRM), Dycal, and Ultra-blend Plus (UBP) by using human dermal fibroblasts and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. METHODS: Cultured adult human dermal fibroblasts were exposed to multiple concentrations of material elutes. The test material samples were immersed and incubated in the culture medium for 2, 5, or 8 days at 37°C. The cytotoxic effects were recorded by using an MTT-based colorimetric assay. Positive and negative controls were used. The results were statistically examined by one-way analysis of variance and Tukey post tests. RESULTS: The cell viability of cultures exposed to all dilutions of AMTA, ERRM, and UBP was statistically similar to the negative control at 2 and 5 days. Only the Dycal-exposed specimens exhibited a statistically significant increase in cytotoxicity at the 2 initial evaluation periods. After exposure to the 8-day elutes, the respective percentage of cell survivability was 91% (Brasseler), 88% (MTA-Angelus), 76% (Ultra-blend Plus), and 37% (Dycal). CONCLUSIONS: From the data in this in vitro study, AMTA, ERRM, and UBP had statistically similar adult human dermal fibroblast cytotoxicity levels. Relative to the negative control, only Dycal was shown to have a statistically significant cytotoxic effect to adult human dermal fibroblasts at all tested intervals.

Novel experimental cements for use on the dentin-pulp complex

This aim of this study was to evaluate the physicochemical and biological properties of novel experimental cements (Hybrid, Paste and Resin) based on synergistic combinations of existing materials, including pH, diametral tensile strength (DTS) and cytotoxicity comparing them with mineral trioxide aggregate (MTA - Angelus®) and a glass ionomer cement (GIC) developed at our laboratory. For the physicochemical and biological tests, specimens with standard dimensions were produced. pH measurements were performed with digital pH meter at the following time intervals: 3, 24, 48 and 72 h. For the DTS test, cylindrical specimens were subjected to compressive load until fracture. The MTT assay was performed for cytotoxicity evaluation. Data were analyzed by ANOVA and Tukey's test (α=0.05). Paste group showed pH values similar to MTA, and Hybrid group presented pH values similar to GIC (p>0.05). The tested materials showed pH values ranging from alkaline to near neutrality at the evaluated times. MTA and GIC showed similar DTS values. The lowest and highest DTS values were seen in the Paste and Resin groups, respectively (p<0.05). Cell viability for MTA and experimental Hybrid, Paste and Resin groups was 49%, 93%, 90% and 86%, respectively, when compared with the control group. The photo-cured experimental resin cement showed similar or superior performance compared with the current commercial or other tested experimental materials.

O objetivo deste estudo foi avaliar propriedades físico-químicas e biológicas de novos cimentos experimentais (Híbrido, Pasta e Resinoso) baseado na combinação sinérgica de materiais existentes, incluindo pH, resistência à tração diametral (RTD) e citotoxidade, comparando-os ao MTA (Angelus®) e a um cimento de ionômero de vidro (CIV) desenvolvido em nosso laboratório. Para a realização dos testes físico-mecânico e biológico, foram confeccionados espécimes com dimensões padrão. O teste de pH foi realizado por meio de pH-metro digital nos tempos: 3, 24, 48 e 72 h. Para o teste de RTD, espécimes cilíndricos foram submetidos a carga compressiva até sua fratura. Para avaliação da citotoxidade, utilizou-se o teste MTT. Os dados foram analisados utilizando ANOVA e teste de Tukey (α=0,05). O grupo Pasta apresentou valores de pH semelhantes ao MTA, assim como o grupo Híbrido seguiu os parâmetros do CIV (p>0,05). Todos os materiais apresentaram valores de pH alcalinos ou próximosà neutralidade nos tempos avaliados. MTA e CIV apresentaram valores de RTD similares. Os menores e maiores valores observados foram do grupo Pasta e Resinoso, respectivamente (p<0,05). A viabilidade celular para os grupos MTA, Híbrido, Pasta, Resinoso, quando comparados ao grupo controle foi de: 49, 93, 90 e 86%, respectivamente. O cimento experimental Resinoso apresentou desempenho similar ou superior aos materiais comerciais e experimentais avaliados.

Insulin-like growth factor 1 and transforming growth factor-ß stimulate cystine/glutamate exchange activity in dental pulp cells.

INTRODUCTION: The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-ß (TGF-ß) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-ß were through the enhancement of an endogenous antioxidant mechanism. METHODS: We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-ß, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-). RESULTS: We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-ß were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-ß on cystine uptake. Both growth factors stimulated system xc-mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line. CONCLUSIONS: The results suggest that IGF-1 and TGF-ß are protective through the stimulation of system xc-mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells.

TEGDMA reduces mineralization in dental pulp cells.

Direct application of dentin bonding agents onto the exposed pulp has been advocated, but in vivo studies indicate a lack of reparative dentin formation. Our objective was to investigate the role of triethylene glycol dimethacrylate (TEGDMA), a commonly used compound in dentin bonding agents, as a potential inhibitor of mineralization. Human pulp cells were exposed to different concentrations of TEGDMA, and expression of the mineralization-related genes collagen I, alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, and dentin sialophosphoprotein was analyzed. Gene expression studies by real-time polymerase chain-reaction revealed a concentration- and time-dependent decrease of mineralization markers. A subtoxic TEGDMA concentration (0.3 mM) reduced expression levels by 5 to 20% after 4 hrs and by 50% after 12 hrs. Furthermore, alkaline phosphatase activity and calcium deposition were significantly lower in dental pulp cells treated with TEGDMA over 14 days. These findings indicate that even low TEGDMA concentrations might inhibit mineralization induced by dental pulp cells, thus impairing reparative dentin formation after pulp capping with dentin bonding agents.