Highlights
- •
Orthodontic bonding indices an increase in BPA concentration immediately after the 1st post-bonding rinse.
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Reduced BPA concentration after the 2nd post-bonding rinse.
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BPA concentrations measured after 2nd post-bonding were at the pre-bonding levels.
Abstract
Objective
To assess the in vivo amount of BPA released from a visible light-cured orthodontic adhesive, immediately after bracket bonding.
Methods
20 orthodontic patients were recruited after obtaining informed consent. All patients received 24 orthodontic brackets in both dental arches. In Group A (11 patients), 25 ml of tap water were used for mouth rinsing, whereas in Group B (9 patients) a simulated mouth rinse formulation was used: a mixture of 20 ml de-ionized water plus 5 ml absolute ethanol. Rinsing solutions were collected before, immediately after placing the orthodontic appliances and after washing out the oral cavity and were then stored in glass tubes. Rinsing was performed in a single phase for 60 s with the entire volume of each liquid. The BPA analysis was performed by gas chromatography–mass spectrometry.
Results
An increase in BPA concentration immediately after the 1st post-bonding rinse was observed, for both rinsing media, which was reduced after the 2nd post-bonding rinse. Water exhibited higher levels of BPA concentration than water/ethanol after 1st and 2nd post-bonding rinses. Two-way mixed Repeated Measures ANOVA showed that the primary null hypothesis declaring mean BPA concentration to be equal across rinsing medium and rinsing status was rejected ( p -value <0.001). The main effects of the rinsing medium and status, as well as their interaction were found to be statistically significant ( p -values 0.048, <0.001 and 0.011 respectively).
Significance
A significant pattern of increase of BPA concentration, followed by a decrease that reached the initial values was observed. The amount of BPA was relatively low and far below the reference limits of tolerable daily intake.
1
Introduction
Resin-based dental materials may induce several undesirable side effects in the oral environment, including localized and systemic reactions, mainly due to release of reactive species like residual monomers, catalysts, oxidation byproducts etc. . In orthodontics, adhesives have received limited attention relative to other orthodontic materials, as apparent in the pertinent literature .
The majority of orthodontic resinous materials are derived from Bisphenol-A (BPA). The BPA configuration assembles a bulk, stiff chain that provides low susceptibility to biodegradation as well as significant strength and rigidity in BPA-derived dimethacrylate polymers based on monomers like Bisphenol-A glycidyl dimethacrylate (BisGMA), its ethoxylated analogue (BisEDMA), Bispenol-A dimethacrylate (BisDMA) and urethane-modified BisGMA . Although BPA is not used as a raw material in dental resin composites, it is likely to be present as an impurity from the chemical synthesis procedure .
The unique biologic effects of BPA arise at ranges within the levels of the detection threshold for a majority of analytical techniques and show a non-monotonic curve pattern on tissues, characterized by intense reactivity at low levels and no response at very high ones . This model of action originates from natural human hormones, such as 17β-estradiol, which can generate effects at concentrations markedly lower than those required to block the specific receptors. BPA and BPA derivatives, increase the levels of reactive oxygen species , which 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.
More specifically, BPA has already been shown to activate multiple cytotoxic mechanisms and induce DNA damage . The role of BPA in the canonical apoptotic pathways has been inadequately appraised 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 . Moreover, epidemiological and genetic studies have shown that BPA is an environmental estrogenic compound that can exert proliferative responses and more specifically may induce hormonal-related effects . Although the majority of the evidence on the effect of BPA derives from in vitro or animal studies, recent research with human tissues confirmed intense redox activity and cross-linking of the DNA in human spermatozoa . In addition, epidemiologic assays have demonstrated augmented incidence of infertility treatment and an increase in the number of abnormal sperm heads among female and male workers respectively in the plastics industry .
In orthodontics, BPA dimethacrylate derivatives are mostly used for bonding brackets (bonding resins and resin composites as main adhesives) and lingual retainers, whereas BPA-polycarbonates are used for manufacturing plastic brackets. In vitro studies have documented the release of BPA from polycarbonate brackets , orthodontic adhesives and resin composites that are frequently used for bonding lingual retainers . For traditional and flowable resin composites used as lingual retainers, BPA release was confirmed in vivo as well , with the highest values in saliva measured immediately after polymerization.
The aim of the present study was to assess the likelihood of in vivo release of BPA from a visible light-cured orthodontic adhesive, immediately after bracket bonding, between two groups of patients using different mouth-rinsing solutions. The primary null hypothesis was that the mean BPA concentration would not vary between study groups.
2
Materials and methods
2.1
Patients and setting
Orthodontic patients were recruited from patients undergoing orthodontic treatment in the 251 Air Force General Hospital. The study design was approved by the ethics and research committee of the institution (Approval Number: F.076/AD.20714) and was accomplished in accordance with the guidelines of the Declaration of Helsinki. All patients signed an informed consent before commencement of the study.
Recruitment of eligible patients began in September 2013 and ended in June 2014. Volunteers with resin fillings, sealants or other resin restorations were excluded from the study. In addition, volunteers with occupation related to chronic and severe BPA exposure or in any work environment associated with plastics were excluded as well.
2.2
Clinical procedures
A total of 20 patients were finally considered eligible for inclusion. All patients received fixed orthodontic appliances in the upper and lower jaw (1st molar to 1st molar of the contralateral side). For standardization purposes, one orthodontist performed all bracket bonding procedures. Metallic brackets were used in all patients (In-Ovation R, Dentsply GAC, Milford, DE, USA). The materials used for the bonding procedure are listed in Table 1 . The bonding procedure was performed as follows: The buccal surfaces of the teeth were cleaned with a fruoride-free prophylaxis pumice, rinsed with water and then acid-etched with the gel etchant for 15 s. Following 5 s water rinsing and 5 s air-drying, the primer was applied on the acid-etched surface in a thin layer. Then, the orthodontic adhesive was applied onto the bracket base, the bracket was pressed against the primed enamel, flash resin was carefully removed with a sharp explorer and finally light-cured by using a LED curing unit emitting 1400 mW/cm 2 light intensity at 395–480 nm range (Valo, Ultradent Products, Inc, S. Jordan, UT, USA). Light-curing was performed by irradiating the occlusal and gingival margins of the adhesive, for 5 s each.
| Product | Composition (wt% range) | Manufacturer |
|---|---|---|
| Scotchbond Etchant | Water (55–65), Phosphoric acid (30–40), Synthetic amorphous silica (5–10) | 3M Unitek, Monrovia CA, USA |
| Transbond MIP Primer | Resin: Bisphenol-A diglycidyl ether dimethacrylate (15–25). 2-Hydroxyethyl methacrylate (10–20) 2-Hydroxy-1,3-dimethacryloxypropane (5–15) Copolymer of itaconic and acrylic acid (5–15) Diurethane dimethacrylate (1–10) Solvents: Ethyl alcohol (30–40) Water (1–10) |
3M Unitek, Monrovia CA, USA |
| Transbond XT Adhesive | Resin: Bisphenol-A diglycidyl ether dimethacrylate (10–20) Bisphenol-A bis(2-hydroxyethyl ether) dimethacrylate (5–10) Fillers: Silane treated quartz (70–80), Silane treated silica (<2), Catalysts: Diphenyl iodonium hexafluorophosphate (<0.2) |
3M Unitek, Monrovia CA, USA |
The patients were randomly classified in two groups (A = 11, B = 9). Simple, computer-based, randomization was implemented. To evaluate the levels of BPA release, rinsing solutions were collected from each patient in the same appointment, at three different periods: (a) before bracket bonding, (b) immediately after bracket bonding (first rinse) and (c) immediately after the first rinse (second rinse). In Group A, 25 ml of tap water were used for mouth rinsing, whereas in Group B a mixture of 20 ml de-ionized water plus 5 ml absolute ethanol were used. Rinsing was performed in a single phase for 60 s with the entire volume of each liquid. Only glassware was used in the process involving sample collection and storage to prevent background contamination of BPA. All samples were then refrigerated at 4 °C until analysis.
2.3
BPA determination
2.3.1
BPA extraction
BPA was recovered from samples by employing solid phase extraction (SPE) cartridges (OASIS HLB, 6 cm 3 /200 mg, 30 μm particle size, Waters Corp., Milford, MA, USA). The cartridges were placed on a vacuum manifold and conditioned sequentially with acetone, methanol and Milli-Q water (Merc Millipore, Billerica, MA, USA). The sample was percolated through the cartridges at a flow rate 5 ml/min. Then, the cartridges were dried under nitrogen and BPA was eluted with acetone. The eluates were evaporated up to 0.5 ml volume in a rotary evaporator and then up to dryness under a mild stream of nitrogen. The extracted compound was submitted to derivatization by adding 100 μl of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Fluka, Buchs, Switzerland) at 70 °C for 30 min. Surrogate standard deuterated Bisphenol A (BPA-d16, Aldrich, Dorset, UK) was added in all samples before extraction as well as in standard solutions used for calibration.
2.3.2
Analytical determination of BPA
The BPA analysis was performed by gas chromatography–mass spectrometry employing a gas-chromatograph (Trace GC Ultra, Thermo Finnigan Electron Corporation, Waltham, MA, USA) coupled with an ion trap mass spectrometer (Polaris Q, Thermo Finnigan) and an autosampler (AI 3000, Thermo Finnigan). A 5% diphenyl–95% dimethylpolysiloxane capillary column of 30 m length, 0.25 mm internal diameter and 0.25 μm film thickness (Rtx–5MS Crossbond, Thames Restek Ltd., Bucks, UK) was used with He carrier gas at a flow rate of 1.5 ml/min. The column temperature program was set as follows: Initial T = 80 °C for 1 min, increase up to T = 150 °C at 20 °C/min rate, further increase up to T = 280 °C at 10 °C/min rate, which was maintained for 2 min. The temperature of the injector was 280 °C, the ion source 200 °C and the transfer line 300 °C, respectively.
Mass spectra were obtained using electron impact ionization (70 eV). The identification of BPA and BPA-d16 was based on relative retention times, the presence of target ions ( m / z 357.2 and 358.2 for BPA and 368.3 and 369.3 for BPA-d16) and their relative abundance. BPA was quantified by the relative response factor to the surrogate internal standard BPA-d16. The m / z 357.2 and 368.3 were used for quantitation of BPB and BPA16, based on relative response factor. A linear fit with high correlation coefficient (0.998) was obtained for the working standards. The instrumental repeatability was 2.3% and the detection limit was 0.10 ng/ml. The recovery of BPA ranged from 97 to 104%.
2.4
Statistical analysis
Descriptive statistics (mean and standard deviation) of the BPA concentration by rinsing medium and status were calculated. The rinsing medium (water, water/ethanol) and the rinsing status (before bracket bonding, after the 1st rinse and following the 2nd rinse) divided the dataset into six subgroups. The primary null hypothesis was that the mean BPA concentration did not vary between these six subgroups. This null hypothesis was tested by using two-way mixed repeated measures analysis of variance (2-way mixed RM ANOVA) with the BPA concentration as the dependent variable. The fixed portion of the model included rinsing medium, rinsing status and their interaction. The random portion of the model was the rinsing medium. Post-hoc comparisons were performed by Sidak’s correction. The level of statistical significance was set to 95% ( α = 0.05). All analyses were conducted using Stata 13.0/SE software (StataCorp LP, College Station, TX, USA).
2
Materials and methods
2.1
Patients and setting
Orthodontic patients were recruited from patients undergoing orthodontic treatment in the 251 Air Force General Hospital. The study design was approved by the ethics and research committee of the institution (Approval Number: F.076/AD.20714) and was accomplished in accordance with the guidelines of the Declaration of Helsinki. All patients signed an informed consent before commencement of the study.
Recruitment of eligible patients began in September 2013 and ended in June 2014. Volunteers with resin fillings, sealants or other resin restorations were excluded from the study. In addition, volunteers with occupation related to chronic and severe BPA exposure or in any work environment associated with plastics were excluded as well.
2.2
Clinical procedures
A total of 20 patients were finally considered eligible for inclusion. All patients received fixed orthodontic appliances in the upper and lower jaw (1st molar to 1st molar of the contralateral side). For standardization purposes, one orthodontist performed all bracket bonding procedures. Metallic brackets were used in all patients (In-Ovation R, Dentsply GAC, Milford, DE, USA). The materials used for the bonding procedure are listed in Table 1 . The bonding procedure was performed as follows: The buccal surfaces of the teeth were cleaned with a fruoride-free prophylaxis pumice, rinsed with water and then acid-etched with the gel etchant for 15 s. Following 5 s water rinsing and 5 s air-drying, the primer was applied on the acid-etched surface in a thin layer. Then, the orthodontic adhesive was applied onto the bracket base, the bracket was pressed against the primed enamel, flash resin was carefully removed with a sharp explorer and finally light-cured by using a LED curing unit emitting 1400 mW/cm 2 light intensity at 395–480 nm range (Valo, Ultradent Products, Inc, S. Jordan, UT, USA). Light-curing was performed by irradiating the occlusal and gingival margins of the adhesive, for 5 s each.
| Product | Composition (wt% range) | Manufacturer |
|---|---|---|
| Scotchbond Etchant | Water (55–65), Phosphoric acid (30–40), Synthetic amorphous silica (5–10) | 3M Unitek, Monrovia CA, USA |
| Transbond MIP Primer | Resin: Bisphenol-A diglycidyl ether dimethacrylate (15–25). 2-Hydroxyethyl methacrylate (10–20) 2-Hydroxy-1,3-dimethacryloxypropane (5–15) Copolymer of itaconic and acrylic acid (5–15) Diurethane dimethacrylate (1–10) Solvents: Ethyl alcohol (30–40) Water (1–10) |
3M Unitek, Monrovia CA, USA |
| Transbond XT Adhesive | Resin: Bisphenol-A diglycidyl ether dimethacrylate (10–20) Bisphenol-A bis(2-hydroxyethyl ether) dimethacrylate (5–10) Fillers: Silane treated quartz (70–80), Silane treated silica (<2), Catalysts: Diphenyl iodonium hexafluorophosphate (<0.2) |
3M Unitek, Monrovia CA, USA |
The patients were randomly classified in two groups (A = 11, B = 9). Simple, computer-based, randomization was implemented. To evaluate the levels of BPA release, rinsing solutions were collected from each patient in the same appointment, at three different periods: (a) before bracket bonding, (b) immediately after bracket bonding (first rinse) and (c) immediately after the first rinse (second rinse). In Group A, 25 ml of tap water were used for mouth rinsing, whereas in Group B a mixture of 20 ml de-ionized water plus 5 ml absolute ethanol were used. Rinsing was performed in a single phase for 60 s with the entire volume of each liquid. Only glassware was used in the process involving sample collection and storage to prevent background contamination of BPA. All samples were then refrigerated at 4 °C until analysis.
2.3
BPA determination
2.3.1
BPA extraction
BPA was recovered from samples by employing solid phase extraction (SPE) cartridges (OASIS HLB, 6 cm 3 /200 mg, 30 μm particle size, Waters Corp., Milford, MA, USA). The cartridges were placed on a vacuum manifold and conditioned sequentially with acetone, methanol and Milli-Q water (Merc Millipore, Billerica, MA, USA). The sample was percolated through the cartridges at a flow rate 5 ml/min. Then, the cartridges were dried under nitrogen and BPA was eluted with acetone. The eluates were evaporated up to 0.5 ml volume in a rotary evaporator and then up to dryness under a mild stream of nitrogen. The extracted compound was submitted to derivatization by adding 100 μl of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA, Fluka, Buchs, Switzerland) at 70 °C for 30 min. Surrogate standard deuterated Bisphenol A (BPA-d16, Aldrich, Dorset, UK) was added in all samples before extraction as well as in standard solutions used for calibration.
2.3.2
Analytical determination of BPA
The BPA analysis was performed by gas chromatography–mass spectrometry employing a gas-chromatograph (Trace GC Ultra, Thermo Finnigan Electron Corporation, Waltham, MA, USA) coupled with an ion trap mass spectrometer (Polaris Q, Thermo Finnigan) and an autosampler (AI 3000, Thermo Finnigan). A 5% diphenyl–95% dimethylpolysiloxane capillary column of 30 m length, 0.25 mm internal diameter and 0.25 μm film thickness (Rtx–5MS Crossbond, Thames Restek Ltd., Bucks, UK) was used with He carrier gas at a flow rate of 1.5 ml/min. The column temperature program was set as follows: Initial T = 80 °C for 1 min, increase up to T = 150 °C at 20 °C/min rate, further increase up to T = 280 °C at 10 °C/min rate, which was maintained for 2 min. The temperature of the injector was 280 °C, the ion source 200 °C and the transfer line 300 °C, respectively.
Mass spectra were obtained using electron impact ionization (70 eV). The identification of BPA and BPA-d16 was based on relative retention times, the presence of target ions ( m / z 357.2 and 358.2 for BPA and 368.3 and 369.3 for BPA-d16) and their relative abundance. BPA was quantified by the relative response factor to the surrogate internal standard BPA-d16. The m / z 357.2 and 368.3 were used for quantitation of BPB and BPA16, based on relative response factor. A linear fit with high correlation coefficient (0.998) was obtained for the working standards. The instrumental repeatability was 2.3% and the detection limit was 0.10 ng/ml. The recovery of BPA ranged from 97 to 104%.
2.4
Statistical analysis
Descriptive statistics (mean and standard deviation) of the BPA concentration by rinsing medium and status were calculated. The rinsing medium (water, water/ethanol) and the rinsing status (before bracket bonding, after the 1st rinse and following the 2nd rinse) divided the dataset into six subgroups. The primary null hypothesis was that the mean BPA concentration did not vary between these six subgroups. This null hypothesis was tested by using two-way mixed repeated measures analysis of variance (2-way mixed RM ANOVA) with the BPA concentration as the dependent variable. The fixed portion of the model included rinsing medium, rinsing status and their interaction. The random portion of the model was the rinsing medium. Post-hoc comparisons were performed by Sidak’s correction. The level of statistical significance was set to 95% ( α = 0.05). All analyses were conducted using Stata 13.0/SE software (StataCorp LP, College Station, TX, USA).
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