Abstract
Objectives
The aim of the study was to evaluate the biological effects of water eluents from polycarbonate based esthetic orthodontic brackets.
Methods
The composite polycarbonate brackets tested were Silkon Plus (SL, fiber-glass-reinforced), Elan ME (EL, ceramic particle-reinforced) and Elegance (EG, fiber-glass-reinforced). An unfilled polyoxymethylene bracket (Brilliant, BR) was used as control. The brackets’ composition was analyzed by ATR-FTIR spectrometry. The cytotoxicity and estrogenicity of the eluents obtained after 3 months storage of the brackets in water (37 °C) were investigated in murine fibroblasts (NIH 3T3), breast (MCF-7) and cervical cancer (CCl-2/Hela) cell lines.
Results
SL and EG were based on aromatic-polycarbonate matrix, whereas EL consisted of an aromatic polycarbonate-polyethylene terepthalate copolymer. A significant induction of cell death and a concurrent decrease in cell proliferation was noted in the EG eluent-treated cells. Moreover, EG eluent significantly reduced the levels of the estrogen signaling associated gene pS2, specifically in MCF7 cells, suggesting that cell death induced by this material is associated with downregulation of estrogen signaling pathways. Even though oxidative stress mechanisms were equally activated by all eluents, the EG eluents induced expression of apoptosis inducing factor (AIF) and reduced Bcl-xL protein levels.
Significance
Some polycarbonate-based composite brackets when exposed to water release substances than activate mitochondrial apoptosis.
1
Introduction
Polycarbonate resins have been increasingly used as dental biomaterials due to their biocompatibility, exceptional esthetics and tailored mechanical attributes . An interesting and growing application of polycarbonate resins was the production of esthetic orthodontic brackets. Early attempts to produce orthodontic brackets from unfilled polycarbonates were unsuccessful, due to excessive in-service distortion, discoloration and staining . To improve water resistance, new glass-particle or glass-fiber reinforced materials were introduced with metallic-strengthened slots, in an attempt to diminish the undesirable features.
Bisphenol-A (BPA) is the main raw material used in the production of the aromatic polycarbonate (ArPCB) matrix of many plastic esthetic brackets. The benzene rings and the quaternary carbon atoms of the BPA structure create a bulk, stiff chain which offers rigidity, strength and less susceptibility to biodegradation in comparison with aliphatic polycarbonates . Moreover, ArPCBs offer temperature and impact resistance, excellent optical properties, large plastic deformations without cracking and easy molding and thermoforming capacity, making this material attractive for component manufacturing.
The widespread use of polycarbonate-based orthodontic brackets has caused concerns on the possible biological and systemic health side-effects of the eluents of these materials released intraorally by physical and chemical processes. Brackets made of ArPCBs have demonstrated sensitivity to water plasticization and water cracking, resulting in water degradation and release of traceable amounts of BPA in the oral cavity of patients and in aqueous environments after long term immersion . Cytotoxic responses of plastic bracket eluents in human gingival fibroblasts have been presented so far, leading to reduced viability, plasma membrane damage, DNA fragmentation and increased cell death .
BPA and BPA derivatives, increase the levels of reactive oxygen species that are known mediators of signaling cascades under physiological conditions. Elevated levels of such compounds can disrupt the cellular redox equilibrium, causing oxidative DNA damage and apoptosis in mammalian cells. BPA specifically, has been previously shown to activate multiple cytotoxic mechanisms and induce DNA damage by activating oxidative stress, p53 and other cell cycle proteins , mitochondrial and endoplasmic reticulum proteins and mTOR pathways . The role of BPA in the canonical apoptotic pathways has been poorly examined and there is limited data associating its role in mitochondrial cell death of T cell lines and germ cells after UV irradiation and hydroquinone treatment . At the same time, epidemiological and genetic studies have shown that BPA is an environmental estrogenic compound that can exert proliferative responses and more specifically can induce hormonal-related effects including altered peripubertal mammary gland development in mice ; early puberty in females and feminization in males; higher risk for breast cancer in females and prostate cancer in males ; induction of calcium influx, which leads to prolactin release and associated behavioral effects ; development of hyperglycemia and insulin tolerance ; elevation of oxidative stress mediators and upregulation of the cAMP response element-binding factor, which inhibits apoptosis .
Due to the significant use of polycarbonate particle- and fiber-reinforced esthetic brackets in the orthodontic practice and the implication of their constituents with contrasting biological activities, the present study was designed aiming to investigate possible cytotoxic and estrogenic effects of eluents from three types of polycarbonate brackets on NIH 3T3 fibroblasts, Hela cells and the estrogen receptive MCF7 cell lines. The hypothesis tested was that there are no statistically significant differences in the performance of the brackets.
2
Materials and methods
2.1
Brackets
The brackets used in this study are listed in Table 1 . According to the manufacturers’ product information sheets, EL, EG and SL are all polycarbonate composite brackets reinforced with filler-particles (EL) or glass-fibers (EG, SL). The polycarbonate-free bracket (BR), composed of unfilled polyoxymethylene, was used as a control, whereas triple distilled water was used as the immersion medium for brackets in all the experiments performed.
| Product (code) | Composition a | Slot type | Manufacturer |
|---|---|---|---|
| Brilliant (BR) | Unfilled polyoxymethylene | Plastic | Forestadent GmbH, Pforzheim, Germany |
| Elan ME (EL) | Ceramic particle-reinforced polycarbonate | Metallic | GAC, Central Islip, NY, USA |
| Elegance (EG) | Fiber-glass-reinforced polycarbonate | Metallic | Dentaurum GmbH, Ispringen, Germany |
| Silkon Plus (SL) | Fiber-glass-reinforced polycarbonate | Plastic | American Orthodontics, Sheboygan, WI, USA |
2.2
Composition
The molecular composition of the plastic brackets was investigated by micro-attenuated reflection Fourier transform infrared spectroscopy (micro-ATR FTIR). The wings of each bracket were pressed against the diamond reflective element of a micro-ATR accessory (Golden-Gate MKII; Specac) attached to an FTIR spectrometer (Spectrum GX; Perkin-Elmer) and spectra were recorded under the following conditions: 4000–600 cm −1 range, 4 cm −1 resolution, 40 scans acquisition, 2 mm diameter sampling area, single-reflection diamond ATR element, 2 μm estimated depth of analysis. Spectra were subjected to baseline and ATR correction, employing Spectrum v 5.1 (Perkin-Elmer) software.
2.3
In vitro aging
Five brackets of each brand (upper incisors) were immersed in sterile silicone-sealed glass-beakers with 200 ml of triple-distilled water and kept at 37 °C for 3 months. The brackets were then removed, the eluents were diluted (1:10) and used for all the following procedures. A sealed glass-beaker with blank water was used as control.
2.4
Cell culture
The NIH 3T3, CCL-2 (Hela) and MCF-7 cell lines were obtained from ATCC-LGC (Wesel). Briefly, NIH 3T3 were cultured in DMEM whereas Hela and MCF-7 were grown in Eagle’s Minimum Essential Medium supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. They were maintained in a humidified atmosphere of 5% CO 2 at 37 °C. Cells were used at a confluency of approximately 80% for all experiments.
2.5
LDH cytotoxicity assay
Quantitation of LDH activity released from damaged cells after addition of the eluents was performed using the Cytotoxicity Detection Kit (Roche) according to the manufacturer’s instructions. Supernatants from cells cultured for 24 h in the presence of the eluents were incubated with the substrate mixture and assessed for LDH release. The LDH released in the supernatants reduced the tetrazolium salt ING to formazan, by a coupled enzymatic reaction. Formazan dye quantification was measured using an ELISA plate reader at 500 nm. The increased LDH activity directly correlated with the cell death increase in cell cultures. Three samples were used per experiment in three sets of independent experiments.
2.6
WST proliferation assay
The WST-1 reagent (Roche) was used to measure the metabolic activity of viable cells. Cells were incubated with the eluents for 20 h and WST was added in the cells for another 4 h. The formazan formed was quantified at 450 nm using an ELISA plate reader and the absorbance was directly correlated with viable cell numbers. Three samples were used per experiment in three sets of independent experiments.
2.7
Hoechst staining
Cell death of NIH 3T3 cells, 24 h after incubation with EG, was visualized by staining cell nuclei with the DNA-binding fluorochrome Hoechst 33258 (Molecular Probes) according to the manufacturer’s instructions. Fragmented or condensed nuclei were indicative of apoptotic cells. Staining was replicated twice per experiment in two independent experiments.
2.8
Western blot analysis
Total protein extracts from cells treated with EG eluent at various time points were prepared by scraping the cells in cold-lysis buffer containing 50 mM Tris–HCl (pH: 7.4), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1% Triton-X and a cocktail of complete protease inhibitors (Roche). Aliquots of 50 μg total protein extracts were boiled in a buffer containing 6% SDS, 40% glycerol, 125 mM DTT and 3% bromophenol blue, then resolved on 10–12% polyacrylamide gels under denaturing conditions and finally transferred onto nitrocellulose membranes. Blots were probed with antibodies against caspase 8 (Santa Cruz, 1:1000), caspase 3 (Santa Cruz, 1:2000) and apoptosis inducing factor-AIF (Santa Cruz, 1:500) at 4 °C overnight. Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgGs (Cell Signaling, 1:1000 up to 1:2000). Antibody binding was detected using the ECL Plus detection system (Amersham Pharmacia). To normalize for protein content, membranes were stripped and reprobed with anti-β-tubulin antibody (Santa Cruz, 1:2000). Western blot analysis was replicated twice per set in two independent sets of experiments.
2.9
NOS activity
NOS activity of EG eluent-treated cells was measured using the NOS detection kit (Cell Technology). Briefly, cells were cultured in black clear bottom 96 well plates, washed with phosphate buffer (PBS, pH: 7.5) and incubated with diaminofluorescein-2 diacetate (DAF-2DA) substrate diluted 1:200 for various time points (3 h, 6 h and 24 h). Fluorescence was measured using a fluorescence plate reader (488 nm excitation, 515 nm emission, Gemini XPS; Molecular Devices). Duplicate samples per test were evaluated in a set of two independent tests.
2.10
Regulation of estrogenic signaling
MCF7 cells, that expresses estrogen receptors and the human pS2 gene that was originally identified as transcriptionally induced by estrogen in these cells , Hela cells that can also be estrogen receptive due to expression of low levels of estrogen receptors and NIH 3T3 cells lacking estrogen receptors, were used in the study. Total RNA was isolated from cells incubated with the different eluents for 12 h and then semi-quantitative PCR was performed to detect the mRNA of the pS2 gene in all cell lines. Total RNA was extracted with TRIzol (InVitrogen) according to the manufacturer’s instructions, whereas for semi-quantitative RT-PCR, DNase-treated (Promega) RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Promega) and random hexamers (InVitrogen). Primers were used for the detection of human pS2, mouse pS2, human β-actin (forward: 5′-ACA-CTG-TGC-CCA-TCT-ACG-AGG-3′ and reverse: 5′-AGG-GGC-CGG-ACT-CGT-CAT-ACT-3′) and mouse β-actin (forward: 5′-CAT-CAC-TAT-TGG-CAA-CGA-GC-3′ and reverse: 5′-ACG-CAG-CTC-AGT-AAC-AGT-CC-3′) were amplified as a loading control. Densitometric analysis was performed using Image Quant 5.2 (Storm Scanner 600; Molecular Dynamics) and relative band intensities were determined. Three samples were used per experiment in three sets of independent experiments.
2.11
Statistical analysis
For all tests means and standard errors were calculated. For RT-PCR analyses, measurements of band intensities were expressed as pixel intensity per unit area. Values were normalized using the β-actin values and were compared using one-way ANOVA plus Bonferroni t -test for pairwise comparisons. For cell viability, one-way ANOVA on Ranks followed by Bonferroni t -test for pairwise comparisons was used. For all comparisons a 95% significance level was used ( a : 0.05). Statistical analyses were performed with Sigma Stat 2.0 software (Jandel).
2
Materials and methods
2.1
Brackets
The brackets used in this study are listed in Table 1 . According to the manufacturers’ product information sheets, EL, EG and SL are all polycarbonate composite brackets reinforced with filler-particles (EL) or glass-fibers (EG, SL). The polycarbonate-free bracket (BR), composed of unfilled polyoxymethylene, was used as a control, whereas triple distilled water was used as the immersion medium for brackets in all the experiments performed.
| Product (code) | Composition a | Slot type | Manufacturer |
|---|---|---|---|
| Brilliant (BR) | Unfilled polyoxymethylene | Plastic | Forestadent GmbH, Pforzheim, Germany |
| Elan ME (EL) | Ceramic particle-reinforced polycarbonate | Metallic | GAC, Central Islip, NY, USA |
| Elegance (EG) | Fiber-glass-reinforced polycarbonate | Metallic | Dentaurum GmbH, Ispringen, Germany |
| Silkon Plus (SL) | Fiber-glass-reinforced polycarbonate | Plastic | American Orthodontics, Sheboygan, WI, USA |
2.2
Composition
The molecular composition of the plastic brackets was investigated by micro-attenuated reflection Fourier transform infrared spectroscopy (micro-ATR FTIR). The wings of each bracket were pressed against the diamond reflective element of a micro-ATR accessory (Golden-Gate MKII; Specac) attached to an FTIR spectrometer (Spectrum GX; Perkin-Elmer) and spectra were recorded under the following conditions: 4000–600 cm −1 range, 4 cm −1 resolution, 40 scans acquisition, 2 mm diameter sampling area, single-reflection diamond ATR element, 2 μm estimated depth of analysis. Spectra were subjected to baseline and ATR correction, employing Spectrum v 5.1 (Perkin-Elmer) software.
2.3
In vitro aging
Five brackets of each brand (upper incisors) were immersed in sterile silicone-sealed glass-beakers with 200 ml of triple-distilled water and kept at 37 °C for 3 months. The brackets were then removed, the eluents were diluted (1:10) and used for all the following procedures. A sealed glass-beaker with blank water was used as control.
2.4
Cell culture
The NIH 3T3, CCL-2 (Hela) and MCF-7 cell lines were obtained from ATCC-LGC (Wesel). Briefly, NIH 3T3 were cultured in DMEM whereas Hela and MCF-7 were grown in Eagle’s Minimum Essential Medium supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin. They were maintained in a humidified atmosphere of 5% CO 2 at 37 °C. Cells were used at a confluency of approximately 80% for all experiments.
2.5
LDH cytotoxicity assay
Quantitation of LDH activity released from damaged cells after addition of the eluents was performed using the Cytotoxicity Detection Kit (Roche) according to the manufacturer’s instructions. Supernatants from cells cultured for 24 h in the presence of the eluents were incubated with the substrate mixture and assessed for LDH release. The LDH released in the supernatants reduced the tetrazolium salt ING to formazan, by a coupled enzymatic reaction. Formazan dye quantification was measured using an ELISA plate reader at 500 nm. The increased LDH activity directly correlated with the cell death increase in cell cultures. Three samples were used per experiment in three sets of independent experiments.
2.6
WST proliferation assay
The WST-1 reagent (Roche) was used to measure the metabolic activity of viable cells. Cells were incubated with the eluents for 20 h and WST was added in the cells for another 4 h. The formazan formed was quantified at 450 nm using an ELISA plate reader and the absorbance was directly correlated with viable cell numbers. Three samples were used per experiment in three sets of independent experiments.
2.7
Hoechst staining
Cell death of NIH 3T3 cells, 24 h after incubation with EG, was visualized by staining cell nuclei with the DNA-binding fluorochrome Hoechst 33258 (Molecular Probes) according to the manufacturer’s instructions. Fragmented or condensed nuclei were indicative of apoptotic cells. Staining was replicated twice per experiment in two independent experiments.
2.8
Western blot analysis
Total protein extracts from cells treated with EG eluent at various time points were prepared by scraping the cells in cold-lysis buffer containing 50 mM Tris–HCl (pH: 7.4), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1% Triton-X and a cocktail of complete protease inhibitors (Roche). Aliquots of 50 μg total protein extracts were boiled in a buffer containing 6% SDS, 40% glycerol, 125 mM DTT and 3% bromophenol blue, then resolved on 10–12% polyacrylamide gels under denaturing conditions and finally transferred onto nitrocellulose membranes. Blots were probed with antibodies against caspase 8 (Santa Cruz, 1:1000), caspase 3 (Santa Cruz, 1:2000) and apoptosis inducing factor-AIF (Santa Cruz, 1:500) at 4 °C overnight. Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgGs (Cell Signaling, 1:1000 up to 1:2000). Antibody binding was detected using the ECL Plus detection system (Amersham Pharmacia). To normalize for protein content, membranes were stripped and reprobed with anti-β-tubulin antibody (Santa Cruz, 1:2000). Western blot analysis was replicated twice per set in two independent sets of experiments.
2.9
NOS activity
NOS activity of EG eluent-treated cells was measured using the NOS detection kit (Cell Technology). Briefly, cells were cultured in black clear bottom 96 well plates, washed with phosphate buffer (PBS, pH: 7.5) and incubated with diaminofluorescein-2 diacetate (DAF-2DA) substrate diluted 1:200 for various time points (3 h, 6 h and 24 h). Fluorescence was measured using a fluorescence plate reader (488 nm excitation, 515 nm emission, Gemini XPS; Molecular Devices). Duplicate samples per test were evaluated in a set of two independent tests.
2.10
Regulation of estrogenic signaling
MCF7 cells, that expresses estrogen receptors and the human pS2 gene that was originally identified as transcriptionally induced by estrogen in these cells , Hela cells that can also be estrogen receptive due to expression of low levels of estrogen receptors and NIH 3T3 cells lacking estrogen receptors, were used in the study. Total RNA was isolated from cells incubated with the different eluents for 12 h and then semi-quantitative PCR was performed to detect the mRNA of the pS2 gene in all cell lines. Total RNA was extracted with TRIzol (InVitrogen) according to the manufacturer’s instructions, whereas for semi-quantitative RT-PCR, DNase-treated (Promega) RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Promega) and random hexamers (InVitrogen). Primers were used for the detection of human pS2, mouse pS2, human β-actin (forward: 5′-ACA-CTG-TGC-CCA-TCT-ACG-AGG-3′ and reverse: 5′-AGG-GGC-CGG-ACT-CGT-CAT-ACT-3′) and mouse β-actin (forward: 5′-CAT-CAC-TAT-TGG-CAA-CGA-GC-3′ and reverse: 5′-ACG-CAG-CTC-AGT-AAC-AGT-CC-3′) were amplified as a loading control. Densitometric analysis was performed using Image Quant 5.2 (Storm Scanner 600; Molecular Dynamics) and relative band intensities were determined. Three samples were used per experiment in three sets of independent experiments.
2.11
Statistical analysis
For all tests means and standard errors were calculated. For RT-PCR analyses, measurements of band intensities were expressed as pixel intensity per unit area. Values were normalized using the β-actin values and were compared using one-way ANOVA plus Bonferroni t -test for pairwise comparisons. For cell viability, one-way ANOVA on Ranks followed by Bonferroni t -test for pairwise comparisons was used. For all comparisons a 95% significance level was used ( a : 0.05). Statistical analyses were performed with Sigma Stat 2.0 software (Jandel).
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