Abstract
Objectives
The aims of this study were to evaluate the cytotoxicity of four orthodontic primers, including two hydrophilic and two hydrophobic materials, and to investigate the role of the reactive oxygen species (ROS) in induced cell damage. Moreover, the effects of the anti-oxidant N-acetyl cysteine (NAC) on primers toxicity was analyzed.
Methods
Human gingival fibroblasts (HGF) were exposed to different concentrations of primers (0–0.25 mg/ml) in the presence or absence of NAC, and the cytotoxicity was assessed by the MTT assay, while cell death was quantified by flow cytometry after propidium iodide staining. The increase in the induced ROS levels was detected by flow cytometry measuring the fluorescence of the oxidation-sensitive dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA).
Results
All materials decreased cell viability in a dose-related manner after a 24 h exposure period. Cytotoxicity of orthodontic primers based on concentrations which caused a 50% decrease in cell viability (TC 50 ) in HGF was ranked as follows (median values): Eagle Fluorsure (0.078 mg/ml) > Transbond XT (0.081 mg/ml) > Transbond MIP (0.128 mg/ml) > Ortho solo (0.130 mg/ml). Moreover, in HGF cells, all materials induced a dose-dependent increase in ROS levels compared to untreated cells. Incubation of HGF with NAC significantly reduced ROS production and decreased the cell damage and cytotoxicity caused by all materials tested ( p < 0.001).
Significance
Our results suggested that hydrophilic primers were less cytotoxic than hydrophobic materials. Moreover, we demonstrated a major role of ROS in the induction of cell death since the antioxidant N-acetyl cysteine was able to prevent cell damage induced by all materials tested.
1
Introduction
In orthodontic clinical practice the role of primers is to provide an adhesive interface between tooth and composite materials . Similar to traditional dental bonding, orthodontic primers (OPs) are a mixture of monomers diluted in a solvent such as water, acetone, or ethanol . Dental monomers, including triethylene glycol dimethacrylate (TEGDMA), 2-hydroxyethyl methacrylate (HEMA), bisphenol A diglycidyl dimethacrylate (Bis-GMA), and/or other components might be released from adhesives and composites even after polymerization . These leachables from resin based-materials (RBM) are a likely cause of cell damage and cellular stress via the formation of ROS . It has been reported that dental monomers reduced the levels of the natural radical scavenger glutathione (GSH), which protects cell structures from damage caused by oxidative stress . Depletion of the intracellular GSH pool may increase the intracellular ROS levels leading to cell death through necrosis or apoptosis . ROS levels, if not counteracted by cellular antioxidants, cause acute injury and damage of important biomolecules including cellular proteins, lipids and DNA leading to cell death . Previous investigations showed toxic effects of orthodontic and dentin bonding agents (DBAs) in various cell lines . The cytotoxicity of these materials might be caused by an impairment of the cellular pro- and anti-oxidant redox balance resulting in an increase in intracellular ROS levels .
In recent years, research focused on cellular mechanisms leading to cell death or survival in order to find molecules such as N-acetyl cysteine (NAC), which are able to reduce adverse effects and to increase the useful effects of dental materials . NAC, a cysteine derivative, has been shown to inhibit cell death caused by a variety of compounds including dental monomers through its anti-oxidant activity , NFkB activation or cell differentiation induction . We recently demonstrated that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation . Hence, a chemical reaction between the sulfhydryl base of NAC and the beta-carbon of the double bond in methyl methacrylate (MMA)-based materials is most likely to occur resulting in the formation of an MMA/NAC adduct .
Therefore, the purpose of our study was to compare the cytotoxicity induced by different OPs, including two recently developed hydrophilic OPs, Transbond MIP and Ortho Solo, as well as two hydrophobic, Transbond XT and Eagle Fluorsure. The cytotoxic effects of the primers were estimated by evaluating cell viability using the MTT assay, the production of ROS, and the number of surviving cells after PI-staining. Moreover, we tested the hypothesis that NAC was able to counteract the OPs toxicity and induced cell death.
2
Materials and methods
2.1
Materials, chemicals and cells
N-acetyl cysteine (NAC), 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), cell culture medium and supplements were purchased from Sigma Chemical Co. (Milan, Italy); 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, USA).
Human primary gingival fibroblasts (HGF) were obtained from four healthy patients after informed consent . The protocol was reviewed and approved by the Institutional Review Board (University of Napoli “Federico II”). HGFs were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mm glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, at 37 °C in a humified atmosphere of 5% CO 2 in air.
2.2
Primers and sample preparation
The orthodontic primers Transbond XT and Transbond MIP (3 M, Monrovia, CA, USA), Eagle Fluorsure (American Orthodontics, Sheboygan, USA), and Ortho Solo (Ormco, Glendora, CA, USA) were tested in this study. The materials were dissolved in pure ethanol (100 mg/ml) at room temperature, and then stock solutions were prepared in culture medium at a concentration of 10 mg/ml in order to produce different final concentrations . Cells cultured in the presence of 0.2% ethanol alone served as controls in all experiments.
2.3
Cytotoxicity of primers
Cytotoxic concentrations of primers were identified by MTT as previously reported . HGF were seeded in a 96-well tissue culture dish at 10,000 cells/well for 24 h at 37 °C. Then, the medium was removed and cell monolayers were exposed to various concentrations of primers (0–0.25 mg/ml) for 24 h in presence or absence of 10 mM NAC for 24 h. Next, the medium was replaced by 100 μL/well MTT (0.5 mg/ml) in phosphate-buffered saline (PBS), and the cells were incubated at 37 °C for 1 h in a 5% CO 2 atmosphere. The MTT solution was replaced by 100 μL/well of DMSO, and gently swirled for 10 min. The optical density in each well was immediately measured using a spectrophotometer (Sunrise, TECAN, Männedorf, Zurich, Switzerland) at a wavelength of 540 nm. Each experiment was performed four times in quadruplicate. The optical density readings obtained from 16 individual cell cultures treated with each primer concentration were expressed as the percentage of untreated cells (100%). The concentrations of the primers (mg/ml) which caused fifty percent cell death (TC 50 values) were calculated with a computer-associated, curve-fitting program. Differences between median TC 50 values were statistically analyzed using the Mann–Whitney U -test ( p = 0.05).
2.4
Measurement of reactive oxygen species (ROS) production
The generation of ROS was measured using an oxidation-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA). Intracellular esterase activity results in the formation of DCFH, a non-fluorescent compound which emits fluorescence when it is oxidized to DCF-DA . HGF (1 × 10 5 cells) were incubated with different concentrations of primers (0–0.25 mg/ml) in the presence or absence of 10 mM NAC for 1 h at 37 °C in a humidified atmosphere of 5% CO 2 in air. In order to measure ROS production induced by tested materials, cells were stained with 10 μM of DCFH-DA for 30 min at 37 °C, detached with trypsin/EDTA, washed, re-suspended in PBS, and then immediately analyzed by flow cytometry. We used a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) to measure the generation of ROS by the fluorescence intensity (FL-1, 530 nm) of 20,000 cells. Mean fluorescence intensity was obtained by histogram statistics using WinMDI 2.8. Fluorescence intensities obtained from at least four independent experiments were normalized to untreated control cultures (1.0), and differences between median values were statistically analyzed using the Mann–Whitney U test for pairwise comparisons among groups at the 0.05 level of significance.
2.5
Cell death detection
HGF cells were exposed to primers (0–0.20 mg/ml) for 24 h in the presence or absence of 10 mM NAC. After treatment, floating and adherent cells were collected by centrifugation, then washed and resuspended in PBS. Untreated and treated cells were stained with PI, and immediately analyzed by flow cytometry (FACScan, Becton–Dickinson, San Jose, CA, USA). Viable cells (no staining), and necrotic cells (PI) were detected and quantified as a percentage of the entire cell population . Analysis of the data was performed by means of the WinMDI 2.8 program. The percentage of dead cells in treated cell cultures and untreated controls were calculated in triplicate in three independent experiments ( n = 9). Differences between median values were statistically analyzed using the Mann–Whitney U test for pairwise comparisons among groups at the 0.05 level of significance.
2
Materials and methods
2.1
Materials, chemicals and cells
N-acetyl cysteine (NAC), 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT), propidium iodide (PI), cell culture medium and supplements were purchased from Sigma Chemical Co. (Milan, Italy); 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, USA).
Human primary gingival fibroblasts (HGF) were obtained from four healthy patients after informed consent . The protocol was reviewed and approved by the Institutional Review Board (University of Napoli “Federico II”). HGFs were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mm glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, at 37 °C in a humified atmosphere of 5% CO 2 in air.
2.2
Primers and sample preparation
The orthodontic primers Transbond XT and Transbond MIP (3 M, Monrovia, CA, USA), Eagle Fluorsure (American Orthodontics, Sheboygan, USA), and Ortho Solo (Ormco, Glendora, CA, USA) were tested in this study. The materials were dissolved in pure ethanol (100 mg/ml) at room temperature, and then stock solutions were prepared in culture medium at a concentration of 10 mg/ml in order to produce different final concentrations . Cells cultured in the presence of 0.2% ethanol alone served as controls in all experiments.
2.3
Cytotoxicity of primers
Cytotoxic concentrations of primers were identified by MTT as previously reported . HGF were seeded in a 96-well tissue culture dish at 10,000 cells/well for 24 h at 37 °C. Then, the medium was removed and cell monolayers were exposed to various concentrations of primers (0–0.25 mg/ml) for 24 h in presence or absence of 10 mM NAC for 24 h. Next, the medium was replaced by 100 μL/well MTT (0.5 mg/ml) in phosphate-buffered saline (PBS), and the cells were incubated at 37 °C for 1 h in a 5% CO 2 atmosphere. The MTT solution was replaced by 100 μL/well of DMSO, and gently swirled for 10 min. The optical density in each well was immediately measured using a spectrophotometer (Sunrise, TECAN, Männedorf, Zurich, Switzerland) at a wavelength of 540 nm. Each experiment was performed four times in quadruplicate. The optical density readings obtained from 16 individual cell cultures treated with each primer concentration were expressed as the percentage of untreated cells (100%). The concentrations of the primers (mg/ml) which caused fifty percent cell death (TC 50 values) were calculated with a computer-associated, curve-fitting program. Differences between median TC 50 values were statistically analyzed using the Mann–Whitney U -test ( p = 0.05).
2.4
Measurement of reactive oxygen species (ROS) production
The generation of ROS was measured using an oxidation-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA). Intracellular esterase activity results in the formation of DCFH, a non-fluorescent compound which emits fluorescence when it is oxidized to DCF-DA . HGF (1 × 10 5 cells) were incubated with different concentrations of primers (0–0.25 mg/ml) in the presence or absence of 10 mM NAC for 1 h at 37 °C in a humidified atmosphere of 5% CO 2 in air. In order to measure ROS production induced by tested materials, cells were stained with 10 μM of DCFH-DA for 30 min at 37 °C, detached with trypsin/EDTA, washed, re-suspended in PBS, and then immediately analyzed by flow cytometry. We used a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) to measure the generation of ROS by the fluorescence intensity (FL-1, 530 nm) of 20,000 cells. Mean fluorescence intensity was obtained by histogram statistics using WinMDI 2.8. Fluorescence intensities obtained from at least four independent experiments were normalized to untreated control cultures (1.0), and differences between median values were statistically analyzed using the Mann–Whitney U test for pairwise comparisons among groups at the 0.05 level of significance.
2.5
Cell death detection
HGF cells were exposed to primers (0–0.20 mg/ml) for 24 h in the presence or absence of 10 mM NAC. After treatment, floating and adherent cells were collected by centrifugation, then washed and resuspended in PBS. Untreated and treated cells were stained with PI, and immediately analyzed by flow cytometry (FACScan, Becton–Dickinson, San Jose, CA, USA). Viable cells (no staining), and necrotic cells (PI) were detected and quantified as a percentage of the entire cell population . Analysis of the data was performed by means of the WinMDI 2.8 program. The percentage of dead cells in treated cell cultures and untreated controls were calculated in triplicate in three independent experiments ( n = 9). Differences between median values were statistically analyzed using the Mann–Whitney U test for pairwise comparisons among groups at the 0.05 level of significance.
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