Highlights
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Approaches to improve the hybrid layer and bond strength have been developed.
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5% DMSO is able to inactivate human gelatinases MMP-2 and MMP-9.
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Evaluation the possible toxic effect of DMSO against the pulp tissue.
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Low concentrations of DMSO did not evoke any substantial cytopathic effect.
Abstract
Objectives
To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells.
Methods
Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm 2 ) for 24 h. Eight replicates ( n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates ( n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal–Wallis and Mann–Whitney’s tests ( p < 0.05).
Results
Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO.
Significance: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.
1
Introduction
Long-term stability of resin–dentin bonds in a hostile environment like the oral cavity is still challenging . Constituents of the resin–dentin bond such as the hybrid layer, the dentin adhesive layer, and collagen fibrils inadvertently left unprotected within the hybridized dentin , are susceptible to protease-induced degradation . Dentinal endogenous proteases, matrix metalloproteinases (MMPs) and cysteine cathepsins (cathepsin K and B) play a significant role in resin–dentin interface collagen degradation .
Different approaches to preserve hybrid layer integrity and bond strength durability have been developed . Chlorhexidine (CHX) is a nonspecific MMP inhibitor that also inhibits dentin cathepsin K and B . The use of CHX as a primer is effective in slowing the degradation of hybrid layers produced in vitro and in vivo . Although CHX has a protective effect in hybrid layers, adhesive interfaces produced in vitro after the application of CHX are stable for up to 2 years while the analysis of resin–dentin bonds produced in vivo indicates that the longevity of CHX effect may be reduced over time . It may occur because the binding of CHX to dentin is only electrostatic and there is no chemical interaction between CHX molecules and collagen fibrils. This electrostatic bond may be not strong enough to prevent CHX from being leached out of the interface , which makes it interesting to investigate other potential enzyme inhibitors that would interact with collagen in a more stable manner.
It has been recently reported that 5% dimethyl sulfoxide (DMSO) is able to inactivate human gelatinases MMP-2 and MMP-9 . Additionally, when used as a dentin primer, DMSO prevented resin–dentin bond degradation after 12 months of aging . DMSO [(CH 3 ) 2 SO] is a colorless liquid classified as an organosulfur complex that is capable of dissolving both polar and nonpolar compounds because it is a polar aprotic solvent. It is also miscible in several organic solvents, including those used in adhesive dentistry, and in water. The polar nature of DMSO combined with its capacity to accept hydrogen bonds and its relative small and compact structure (MW = 78.13 g/mol) are responsible for its unique capability to penetrate living tissues . These properties also result in the ability of DMSO to associate with water, proteins, carbohydrates, nucleic acid, ionic substances, and other constituents of living systems. DMSO has the ability to compete with water molecules in the interpeptide hydrogen bonds in collagen matrix resulting in the dissociation of extracellular collagen matrix .
Because the idea of using DMSO as a MMP inhibitor and as a resin–dentin bond stability promoter involves its application on dentin, it is important to evaluate the possible toxic effect of this compound against the pulp tissue. Odontoblasts are the first cells to be in contact with substances diffused through the dentinal tubules; therefore, the aim of this study was to evaluate the direct cytotoxicity of DMSO against odontoblast-like cells.
2
Materials and methods
2.1
Culture of odontoblast-like cells MDPC-23
Immortalized odontoblast-like MDPC-23 cells were seeded at a density of 5 × 10 4 cells/cm 2 into 24-well culture plates (COSTAR 3595 – Corning Incorporated, Corning, NY, USA) in 1 mL of DMEM (Dulbecco’s Modified Eagle’s Medium; Sigma–Aldrich Corp., St. Louis, MO, USA) supplemented with 10% FBS (Fetal Bovine Serum; Cultilab, Campinas, SP, Brazil) containing l -glutamine (2 mmol/L), penicillin (100 IU/mL), and streptomycin (2 mmol/L) (Gibco, Grand Island, NY, USA). The cells were kept in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere (Isotemp Fisher Scientific, Pittsburgh, PA, USA).
2.2
DMSO solutions
After incubation for 48 h, the culture medium (DMEM) of each cell was aspirated and replaced by 1 mL of a solution containing the following concentrations of DMSO (Dimehtylsulfoxide, DMSO; Mallinckrodt Baker Inc, Phillipsburg, NJ, USA): 0.05 mM (0.0004%); 0.1 mM (0.0008%); 0.3 mM (0.0024%); 0.5 mM (0.004%); or 1 mM (0.008%) mixed in plain DMEM. The control group was represented by DMSO-free DMEM. The cell were kept in contact with the solutions for 24 h and maintained in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere during that period of time.
2.3
Cell viability
After placing the cells in direct contact with the DMSO solutions or DMEM (control) for 24 h, the cells were incubated with 5 mg/mL MTT for 4 h. Mitochondrial dehydrogenase enzymes in living cells convert the yellow water-soluble tetrazolium salt 3-(4,5-dimethylthia-zol-2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich Corp., St. Louis, MO, USA) into violet formazan crystals stored in the cytoplasm of cells. Then, the culture medium with the MTT solution was aspirated and replaced by 400 μL of acidified isopropanol solution (0.04 N HCl) in each well to dissolve and extract the violet formazan crystals. After agitation and confirmation of the homogeneity of the solutions, three 100 μL aliquots of each well were transferred to a 96-well plate (Corning Incorporated, Corning, NY, USA). Cell viability was evaluated by spectrophotometry as being proportional to the absorbance measured at 570 nm wavelength using a microplate reader (Thermo Plate, Nanshan District, Shenzhen, Gandong, China). The average of the values obtained from the three aliquots was calculated as a single value for each sample for statistical analysis. For this analysis, 12 replicates were performed per group in triplicate and results were presented as percentage of the control defined as 100% of cell viability.
2.4
Viable cell counts
Viable cells were counted by excluding those stained with Trypan Blue dye. After 24-h contact of MDPC-23 cells with the DMSO solutions or fresh DMEM (control), trypan blue test was performed as previously described by Basso et al. . In brief, the DMSO solutions and DMEM (control) in contact with the cells were aspirated and replaced by 0.12% trypsin (Invitrogen, Carlsbad, CA, USA) that was kept in contact with the cells for 10 min to detach them from the acrylic. Then 50 μL aliquots of this cell suspension were added to 50 μL of 0.04% trypan blue dye (Sigma–Aldrich Corp., St. Louis, MO, USA) and left undisturbed for 2 min. Ten microliters of the solution were taken to a hemocytometer and examined with an inverted light microscope (Nikon Eclipse TS 100, Nikon Corporation, Tokyo, Japan) to determine the number of total cells and nonviable ( i.e. trypan blue stained) cells. The number of viable cells was calculated by subtracting the number of nonviable cells from the number of total cells. Twelve replicates were done for each group and the analysis was done in triplicate.
2.5
Identification of cell death by necrosis (flow cytometry)
Flow cytometry was employed to analyze necrotic cell death using propidium iodide (PI) staining. The cells were analyzed by fluorescence-activated cell sorting (FACS) in a flow cytometer (FACSCanto; BD Biosciences, San Jose, CA, USA) equipped with argon laser and Cell Quest software (BD Biosciences). MDPC-23 cells (3 × 10 4 cells/cm 2 ) were cultured in DMEM in 24-well plates for 48 h, and then the culture medium was replaced by the DMSO solutions or fresh DMEM (control). After 24-h incubation, the cells were harvested with trypsin, centrifuged at 2300 rcf for 2 min to remove the supernatant and resuspended in 300 μL of ligation buffer containing 10 mM HEPES pH 7.4, 150 mMNaCl, 5 mMKCl, 1 mM MgCl 2 and 1.8 mM CaCl 2 . Acquisition of cells stained positively for necrosis was made immediately after the addition of 3 μg/mL PI (Sigma–Aldrich Corp., St. Louis, MO, USA) at a concentration of 100 μg of reagent per mL of buffering solution. The samples were acquired in the FL-2 (PI) channels of the flow cytometer. Data for statistical analysis were obtained as the percentage of cells stained in relation of the total number of MDPC-23 cells identified by the flow cytometer. Eight samples per group were analyzed and the method was run in duplicate.
2.6
Cell adhesion (crystal violet staining)
The solutions in contact with the cells were aspirated after 24 h and the cells were fixed with 2.5% (v/v) glutaraldehyde (Sigma–Aldrich Corp., St Louis, MO, USA) for 60 min. Then, cells were dyed with 0.1% crystal violet for 15 min followed by two repeated rinsing with deionized water. Cell adherence was analyzed in a light microscope (Olympus BX51, Miami, FL, USA) connected to a computer. Images (Olympus C5060, Miami, FL, USA) were taken from four randomly selected areas of each well with 10× magnification. Images were evaluated using an imaging software (Image J 1.45S, Wayne Rasband, National Institutes of Health, USA). Data ( n = 12 per group) were presented as % of the control (defined as 100%) and performed in triplicate.
2.7
Quantification of total protein
Total protein (TP) production was evaluated as previously described by Basso et al. and was performed simultaneously with the MTT assay. After 24 h incubation in contact with the cells, the solutions were aspirated and the cells were washed three times with 1 mL PBS at 37 °C. One mL of 0.1% sodium lauryl sulfate in water (Sigma–Aldrich Corp., St. Louis, MO, USA) was added to each well and maintained for 40 min at room temperature to produce cell lysis. The samples were homogenized and an aliquot of 1 mL of each well was transferred to Falcon tubes while the blank tube received 1 mL of deionized water. One mL of Lowry reagent solution (Sigma–Aldrich Corp., St. Louis, MO, USA) was added to all samples and the tubes were agitated for 10 s in a tube shaker (Phoenix AP 56, Araraquara, SP, Brazil). After 20 min at room temperature, 500 μL of Folin-Ciocalteau’s phenol reagent solution (Sigma–Aldrich Corp., St. Louis, MO, USA) were added to each sample and homogenized. After 30 min, three 100-μL aliquots of each tube were transferred to a 96-well culture plate and the absorbance of the test was measured at 655 nm wavelength with the ELISA plate reader (Thermo Plate). The average of the three values was calculated for statistical analysis. The absorbance of the control was set as 100% and the results were presented as percentage of the control. Twelve samples were performed for TP quantification and the analysis was run in triplicate.
2.8
Mineralized nodule formation (Alizarin Red)
Only for this protocol, DMSO solutions were prepared using DMEM enriched with ascorbic acid, β-glycerophosphate (Sigma–Aldrich Corp., St Louis, MO, USA) and FBS. The solutions (1 mL) were replenished every three days and the cells were maintained in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere. Then, on the seventh day, the solutions were aspirated and the cells were fixed with 70% ethanol for 60 min. The fixed cells were stained with 1% Alizarin red (Sigma–Aldrich Corp., St Louis, MO, EUA) diluted in deionized water for 20 min under agitation. The remaining dye was carefully aspirated and the cells were washed with deionized water for three consecutive times. 10% cetylpyridinium chloride in PBS was added to the wells (500 μL) and left under agitation for 15 min. Three 100-μL aliquots from each well were transferred to wells of a 96-well culture plate and the absorbance was read in a microplate reader (Thermo Plate) at 562 nm wavelength . The analysis was performed in triplicate ( n = 12) and the results presented as absolute values.
2.9
Statistical analyses
Data from cell number, adhesion, viability and total protein production were normally distributed and presented homoscedasticity. Therefore, data were submitted to one-way ANOVA (“concentration of DMSO”) complemented by Tukey’s test for pairwise comparisons. Data from mineralized nodules formation and cell death by necrosis did not adhere to the normal curve and were analyzed by Kruskal–Wallis and Mann–Whitney tests. Additionally, Spearman’ or Pearson’s tests were applied to correlate the concentration of DMSO with the response variables of the study. For all statistical tests a p < 0.05 was taken as statistically significant.
2
Materials and methods
2.1
Culture of odontoblast-like cells MDPC-23
Immortalized odontoblast-like MDPC-23 cells were seeded at a density of 5 × 10 4 cells/cm 2 into 24-well culture plates (COSTAR 3595 – Corning Incorporated, Corning, NY, USA) in 1 mL of DMEM (Dulbecco’s Modified Eagle’s Medium; Sigma–Aldrich Corp., St. Louis, MO, USA) supplemented with 10% FBS (Fetal Bovine Serum; Cultilab, Campinas, SP, Brazil) containing l -glutamine (2 mmol/L), penicillin (100 IU/mL), and streptomycin (2 mmol/L) (Gibco, Grand Island, NY, USA). The cells were kept in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere (Isotemp Fisher Scientific, Pittsburgh, PA, USA).
2.2
DMSO solutions
After incubation for 48 h, the culture medium (DMEM) of each cell was aspirated and replaced by 1 mL of a solution containing the following concentrations of DMSO (Dimehtylsulfoxide, DMSO; Mallinckrodt Baker Inc, Phillipsburg, NJ, USA): 0.05 mM (0.0004%); 0.1 mM (0.0008%); 0.3 mM (0.0024%); 0.5 mM (0.004%); or 1 mM (0.008%) mixed in plain DMEM. The control group was represented by DMSO-free DMEM. The cell were kept in contact with the solutions for 24 h and maintained in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere during that period of time.
2.3
Cell viability
After placing the cells in direct contact with the DMSO solutions or DMEM (control) for 24 h, the cells were incubated with 5 mg/mL MTT for 4 h. Mitochondrial dehydrogenase enzymes in living cells convert the yellow water-soluble tetrazolium salt 3-(4,5-dimethylthia-zol-2,5-diphenyltetrazolium bromide (MTT; Sigma–Aldrich Corp., St. Louis, MO, USA) into violet formazan crystals stored in the cytoplasm of cells. Then, the culture medium with the MTT solution was aspirated and replaced by 400 μL of acidified isopropanol solution (0.04 N HCl) in each well to dissolve and extract the violet formazan crystals. After agitation and confirmation of the homogeneity of the solutions, three 100 μL aliquots of each well were transferred to a 96-well plate (Corning Incorporated, Corning, NY, USA). Cell viability was evaluated by spectrophotometry as being proportional to the absorbance measured at 570 nm wavelength using a microplate reader (Thermo Plate, Nanshan District, Shenzhen, Gandong, China). The average of the values obtained from the three aliquots was calculated as a single value for each sample for statistical analysis. For this analysis, 12 replicates were performed per group in triplicate and results were presented as percentage of the control defined as 100% of cell viability.
2.4
Viable cell counts
Viable cells were counted by excluding those stained with Trypan Blue dye. After 24-h contact of MDPC-23 cells with the DMSO solutions or fresh DMEM (control), trypan blue test was performed as previously described by Basso et al. . In brief, the DMSO solutions and DMEM (control) in contact with the cells were aspirated and replaced by 0.12% trypsin (Invitrogen, Carlsbad, CA, USA) that was kept in contact with the cells for 10 min to detach them from the acrylic. Then 50 μL aliquots of this cell suspension were added to 50 μL of 0.04% trypan blue dye (Sigma–Aldrich Corp., St. Louis, MO, USA) and left undisturbed for 2 min. Ten microliters of the solution were taken to a hemocytometer and examined with an inverted light microscope (Nikon Eclipse TS 100, Nikon Corporation, Tokyo, Japan) to determine the number of total cells and nonviable ( i.e. trypan blue stained) cells. The number of viable cells was calculated by subtracting the number of nonviable cells from the number of total cells. Twelve replicates were done for each group and the analysis was done in triplicate.
2.5
Identification of cell death by necrosis (flow cytometry)
Flow cytometry was employed to analyze necrotic cell death using propidium iodide (PI) staining. The cells were analyzed by fluorescence-activated cell sorting (FACS) in a flow cytometer (FACSCanto; BD Biosciences, San Jose, CA, USA) equipped with argon laser and Cell Quest software (BD Biosciences). MDPC-23 cells (3 × 10 4 cells/cm 2 ) were cultured in DMEM in 24-well plates for 48 h, and then the culture medium was replaced by the DMSO solutions or fresh DMEM (control). After 24-h incubation, the cells were harvested with trypsin, centrifuged at 2300 rcf for 2 min to remove the supernatant and resuspended in 300 μL of ligation buffer containing 10 mM HEPES pH 7.4, 150 mMNaCl, 5 mMKCl, 1 mM MgCl 2 and 1.8 mM CaCl 2 . Acquisition of cells stained positively for necrosis was made immediately after the addition of 3 μg/mL PI (Sigma–Aldrich Corp., St. Louis, MO, USA) at a concentration of 100 μg of reagent per mL of buffering solution. The samples were acquired in the FL-2 (PI) channels of the flow cytometer. Data for statistical analysis were obtained as the percentage of cells stained in relation of the total number of MDPC-23 cells identified by the flow cytometer. Eight samples per group were analyzed and the method was run in duplicate.
2.6
Cell adhesion (crystal violet staining)
The solutions in contact with the cells were aspirated after 24 h and the cells were fixed with 2.5% (v/v) glutaraldehyde (Sigma–Aldrich Corp., St Louis, MO, USA) for 60 min. Then, cells were dyed with 0.1% crystal violet for 15 min followed by two repeated rinsing with deionized water. Cell adherence was analyzed in a light microscope (Olympus BX51, Miami, FL, USA) connected to a computer. Images (Olympus C5060, Miami, FL, USA) were taken from four randomly selected areas of each well with 10× magnification. Images were evaluated using an imaging software (Image J 1.45S, Wayne Rasband, National Institutes of Health, USA). Data ( n = 12 per group) were presented as % of the control (defined as 100%) and performed in triplicate.
2.7
Quantification of total protein
Total protein (TP) production was evaluated as previously described by Basso et al. and was performed simultaneously with the MTT assay. After 24 h incubation in contact with the cells, the solutions were aspirated and the cells were washed three times with 1 mL PBS at 37 °C. One mL of 0.1% sodium lauryl sulfate in water (Sigma–Aldrich Corp., St. Louis, MO, USA) was added to each well and maintained for 40 min at room temperature to produce cell lysis. The samples were homogenized and an aliquot of 1 mL of each well was transferred to Falcon tubes while the blank tube received 1 mL of deionized water. One mL of Lowry reagent solution (Sigma–Aldrich Corp., St. Louis, MO, USA) was added to all samples and the tubes were agitated for 10 s in a tube shaker (Phoenix AP 56, Araraquara, SP, Brazil). After 20 min at room temperature, 500 μL of Folin-Ciocalteau’s phenol reagent solution (Sigma–Aldrich Corp., St. Louis, MO, USA) were added to each sample and homogenized. After 30 min, three 100-μL aliquots of each tube were transferred to a 96-well culture plate and the absorbance of the test was measured at 655 nm wavelength with the ELISA plate reader (Thermo Plate). The average of the three values was calculated for statistical analysis. The absorbance of the control was set as 100% and the results were presented as percentage of the control. Twelve samples were performed for TP quantification and the analysis was run in triplicate.
2.8
Mineralized nodule formation (Alizarin Red)
Only for this protocol, DMSO solutions were prepared using DMEM enriched with ascorbic acid, β-glycerophosphate (Sigma–Aldrich Corp., St Louis, MO, USA) and FBS. The solutions (1 mL) were replenished every three days and the cells were maintained in a humidified incubator at 37 °C under a 5% CO 2 and 95% air atmosphere. Then, on the seventh day, the solutions were aspirated and the cells were fixed with 70% ethanol for 60 min. The fixed cells were stained with 1% Alizarin red (Sigma–Aldrich Corp., St Louis, MO, EUA) diluted in deionized water for 20 min under agitation. The remaining dye was carefully aspirated and the cells were washed with deionized water for three consecutive times. 10% cetylpyridinium chloride in PBS was added to the wells (500 μL) and left under agitation for 15 min. Three 100-μL aliquots from each well were transferred to wells of a 96-well culture plate and the absorbance was read in a microplate reader (Thermo Plate) at 562 nm wavelength . The analysis was performed in triplicate ( n = 12) and the results presented as absolute values.
2.9
Statistical analyses
Data from cell number, adhesion, viability and total protein production were normally distributed and presented homoscedasticity. Therefore, data were submitted to one-way ANOVA (“concentration of DMSO”) complemented by Tukey’s test for pairwise comparisons. Data from mineralized nodules formation and cell death by necrosis did not adhere to the normal curve and were analyzed by Kruskal–Wallis and Mann–Whitney tests. Additionally, Spearman’ or Pearson’s tests were applied to correlate the concentration of DMSO with the response variables of the study. For all statistical tests a p < 0.05 was taken as statistically significant.
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