Introduction
The objectives of this study were to quantify in vitro the Bisphenol A (BPA) release from 5 orthodontic composites and to assess in vivo the BPA level in patients’ saliva and urine after bracket bonding with an orthodontic adhesive system.
Methods
For the in-vitro portion of this study, 5 orthodontic composites were evaluated: Eagle Spectrum (American Orthodontics, Sheboygan, Wis), Enlight (Ormco, Orange, Calif), Light Bond (Reliance Orthodontic Products, Itasca, Ill), Mono Lok II (Rocky Mountain Orthodontics, Denver, Colo), and Transbond XT (3M Unitek, Monrovia, Calif). Simulating intraoral conditions, the specimens were immersed in a water/ethanol solution, and the BPA (ng.g −1 ) liberation was measured after 30 minutes, 24 hours, 1 day, 1 week, and 1 month by the gas chromatography system coupled with mass spectrometry. Twenty patients indicated for fixed orthodontic treatment participated in the in-vivo study. Saliva samples were collected before bracket bonding and then 30 minutes, 24 hours, 1 day, 1 week, and 1 month after bonding the brackets. Urine samples were collected before bonding and then at 1 day, 1 week, and 1 month after bonding. The results were analyzed statistically using analysis of variance and Tukey posttest, with a significance level of 5%.
Results
All composites evaluated in vitro released small amounts of BPA. Enlight composite showed the greatest release, at 1 month. Regarding the in-vivo study, the mean BPA level in saliva increased significantly only at 30 minutes after bonding in comparison with measurements recorded before bonding.
Conclusions
All orthodontic composites released BPA in vitro. Enlight and Light Bond had, respectively, the highest and lowest BPA releases in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion.
Highlights
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Five orthodontic composites released BPA in vitro.
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BPA levels peaked 30 days after bonding.
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Enlight released the most BPA, and Light Bond released the least.
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Detected BPA levels were lower than the reference dose for daily ingestion.
During orthodontic treatment with fixed appliances, bracket bonding with composite materials based on bisphenol A glycidyl methacrylate is of great importance for treatment outcome. It is the main monomer used in resins and orthodontic adhesives, and bisphenol A (2,2′-bis [4-hydroxyphenyl] propane; BPA) is the main component of this monomer.
BPA is a synthetic chemical substance widely used for production of epoxy resin and polycarbonate plastic used to manufacture various products, including bottles, food packaging, baby bottles, toys, detergents, pesticides, cars, and dental resinous materials, such as composites and pit-and-fissure sealants. Due to the increase in the number of products based on epoxy resins and polycarbonate plastics, human exposure to BPA has increased rapidly.
BPA has been extensively studied as one of the most common environmental endocrine disruptors, having an estrogenic action from competitive binding of estrogen-like polymer molecules to natural hormone receptors. The environment—water, air, and soil—can be a route of exposure to BPA, but foods are the primary source of human contact. The daily human consumption of BPA is less than 1 μg.kg −1 , and greater doses may lead to destructive adverse effects on the endocrine system, especially during fetal development. Although medical research has demonstrated the harmful effects of BPA-releasing materials to the body, dental research in this field is limited. The presence of BPA in human saliva, urine, and blood after use of resinous restorative materials and pit-and-fissure sealants has been demonstrated. Kang et al evaluated the release of BPA from a composite resin used to bond orthodontic lingual retainers, but no study has quantified the amount of BPA released either in vitro after different experimental periods or in vivo in the saliva and urine of patients after bonding of orthodontic brackets.
Based on these findings, the objectives of this study were to quantify in vitro the release of BPA from 5 contemporary orthodontic composites, and to assess in vivo the levels of BPA in the saliva and urine of patients after bracket bonding with an orthodontic adhesive system.
Material and methods
In the in-vitro experiment, the release of BPA was evaluated from 5 orthodontic composites commonly used for bracket bonding. The materials and their compositions, manufacturers, and batch numbers are listed in Table I .
| Transbond XT | Mono Lok II | Light bond | Eagle Spectrum | Enlight | |
|---|---|---|---|---|---|
| Composite | Silica, Bis-GMA, silano, N-dimethyl benzocaine, hexa-fluoride phosphate | Composite based on Bis-GMA with small parts of glass and photosensitive catalysis system | Fused silica, urethane dimethacrylate, triethyleneglycol dimethacrylate | Not given by the manufacturer | Uncured methacrylate ester monomers, silica, activators, conservatives |
| Adhesive composite | Triethylene glycol dimethacrylate Bis-GMA | Fluid resin in Bis-GMA base and a photosensitive catalysis system | Biphenyl dimethacrylate, hydroxyethyl methacrylate, acetone | Not given by the manufacturer | Ethyl alcohol, dimethacrylate resins, barium aluminoborosilicate, glass fumed silica (silicon dioxide), sodium hexafluorosilicate |
| Manufacturer | 3M Unitek, Monrovia, Calif | Rocky Mountain Orthodontics, Denver, Colo | Reliance, Orthodontic Products, Itasca, Ill | American Orthodontics, Sheboygan, Wis | Ormco, Orange, Calif |
| Batch number | 1323200750 | K2601-1 | 131812 | B 41282 | 4571779 |
Four disc-shaped (5 mm diameter × 3 mm thick) samples of each resin were prepared using a synthetic fluorine-containing resin matrix and photoactivated for 60 seconds with a halogen light-curing unit with 450 mW/cm 2 light intensity (XL 3000; DMC, Plantation, Fla). Each sample was weighed and immersed in a separate flask containing 4 mL of an ethanol/water solution (75:25 v/v) at 37°C. Aliquots of 1 mL of the solutions with the samples were collected 30 minutes, 24 hours, 1 week, and 1 month after immersion. After addition of 20 μL of an internal standard working solution (BPA-d16; 1 μg.mL −1 ), 100 μL aliquots of the ethanol/water solutions were dried under a vacuum system at 45°C, and the samples were derivatized. Derivatization consisted of the addition of 50 μL of the reagent BSTFA + TMCS to the dry residue, vortexing for 30 seconds, and immersion in a thermostatized bath at 37°C for 30 minutes. After that, 1 μL of the derivatized solution was injected into the chromatography system for analysis.
The ethics committee of the University of São Paulo, Ribeirão Preto, São Paulo, Brazil, reviewed and approved our research (protocol 34805914.9.0000.5419). The subjects enrolled in the study were 20 patients of both sexes aged 12 to 18 years (mean age, 12.3 years) who needed fixed orthodontic treatment and fulfilled the following inclusion criteria: complete permanent dentition; no caries, periodontal disease, restorations (composite resin, amalgam, or glass ionomer), or pit-and-fissure sealants; good general health; and nonsmokers. In addition, during the experiment, the patients did not receive any restorations to minimize biases or confounding factors that could influence the results. A patient was excluded from the study if bracket rebonding was necessary. For strict adherence to the research protocol, the patients were instructed not to use plastic utensils, such disposable glasses, cutlery, or dishes.
The 20 patients were planned to receive complete fixed orthodontic appliances with metallic brackets from the left second premolar to the right second premolar in both arches, totaling 20 teeth. Before placement of the orthodontic appliances, 1 mL of nonstimulated saliva and 5 mL of urine were collected in glass tubes for analysis of preexisting BPA (quantified in ng.g −1 ). Next, rubber cup pumice dental prophylaxis was performed, and the enamel was etched with phosphoric acid for 15 seconds, rinsed with air/water spray, and dried with a mild air blow. A calibrated and experienced orthodontist (L.G.M.) performed bracket bonding using the Transbond XT light-cure orthodontic adhesive system (3M Unitek, Monrovia, Calif). After application of Transbond XT Primer, Transbond XT resin was applied on the bracket base, bonded to the teeth, and photoactivated for 20 seconds with a halogen light-curing unit at 450 mW/cm 2 light intensity. Then 1 mL of nonstimulated saliva and 5 mL of urine were collected from each patient 30 minutes, 24 hours, 1 week, and 1 month after bracket bonding.
Urine samples were subjected to an enzymatic treatment consisting of mixing 1 mL of urine with 1 mL of sodium acetate buffer (0.1 mol L -1 , pH 5, containing 0.1% [w/v] ascorbic acid) and 20 μL of the diluted enzyme solution (in 0.2% saline solution [w/v] freshly prepared and cooled; 10,000 units.mL -1 for glucuronidase and 937 units.mL -1 for sulfatase). The mixture was maintained at 37°C for 4 hours. Next, 20 μL of the internal standard (1 μg.mL -1 ) was added for liquid-liquid extraction. Two 1-mL aliquots of the extraction solvent (MTBE) was added, and the samples were vortexed for 30 seconds and centrifuged at 15°C for 5 minutes at 5000 rpm. The supernatant was collected and dried under vacuum, and the residue was derivatized as previously described.
The saliva samples were treated in the same way as the urine samples, except that enzymatic treatment was not necessary. A 40-μL aliquot of the internal standard (1 μg.mL -1 ) was added to 1 mL of saliva, and liquid-liquid extraction and derivatization were performed as previously described.
The analysis of BPA in the samples (in-vitro and in-vivo experiments) was performed in a gas chromatograph mass spectrometer (GCMS-QP2010 Plus; Shimadzu, Tokyo, Japan). Chromatographic separations were achieved on an NST-05MS (5% diphenyl, 95% dimethylpolysiloxane) analytical column (30 mm × 0.25 mm × 0.25 μm). The injector, ion source, and interface temperatures were set at 280°C, 230°C, and 250°C, respectively, and the solvent cut time was 3 minutes. The samples were injected in the split mode (1:3), and the injection volume was 1 μL. The column temperature was held at 180°C for 3 minutes, followed by an increase at a 20°C per minute rate to 240°C, which was maintained for 4 minutes, and a further increase at a 20°C per minute rate to 300°C, which was maintained for 2 minutes. Helium at a constant flow rate of 1 mL per minute −1 was used as the carrier gas, and the electron impact (70 eV) ionization mode was used. The data were acquired by Selected Ion Monitoring mode, in which a fragment was used for quantification and another fragment was used to confirm both BPA and internal standard ( Table II ).
| Quantification ion (m/z) | Confirmation ion (m/z) | Retention time (min) | |
|---|---|---|---|
| BPA | 357 | 372 | 8.27 |
| BPA-d16 (PI) | 368 | 386 | 8.19 |
The data from the in-vitro and in-vivo (urine and saliva in ng.g −1 ) experiments were examined for normal distribution (Shapiro-Wilk test, P >0.05) and homogeneity of variance (Levene test, P >0.05). Data were expressed as means and standard deviations, and the differences between time points (baseline, 30 minutes, 24 hours, 1 week, and 1 month) were verified by analysis of variance and Tukey post hoc tests. Data were analyzed using Bioestat statistical software (version 5.3; Mamirauá Institute, Belém, PA, Brazil), and the significance level was set at 5%.
Results
Means and standard deviations of BPA release from the orthodontic composites in vitro are presented in Table III . BPA was released from all materials at all time points, and this release increased with time. Enlight and Light Bond had, respectively, the highest and the lowest amounts of BPA release ( P <0.05) at all time points.
| Time point | Transbond XT | Mono-Lok II | Eagle Spectrum | Enlight | Light bond |
|---|---|---|---|---|---|
| 30 minutes after bracket bonding | 28.0 ± 1.0 | 9.4 ± 0.3 a | 38.4 ± 1.9 | 48.1 ± 2.0 | 10.0 ± 0.4 a |
| 24 hours after bracket bonding | 54.6 ± 2.1 | 81.8 ± 4.4 | 167.1 ± 3.8 | 252.4 ± 3.9 | 21.6 ± 2.1 |
| 1 week after bracket bonding | 142.9 ± 8.8 | 71.5 ± 3.2 | 295.0 ± 8.8 | 415.2 ± 29.3 | 31.0 ± 0.7 |
| 1 month after bracket bonding | 324.1 ± 17.5 | 146.6 ± 20.2 | 607.6 ± 47.2 | 1020.1 ± 50.4 | 65.0 ± 0.6 |
Descriptive results of BPA levels in saliva are presented in Table IV . The mean BPA level 30 minutes after bracket bonding was significantly higher ( P <0.05) than at baseline and all other time points. The mean BPA levels at 24 hours, 1 week, and 1 month after bracket bonding were statistically similar to each other and not significantly ( P >0.05) different from the values obtained before bonding.
| Time point | BPA concentration (ng.g −1 ) | ||
|---|---|---|---|
| Minimum | Maximum | Mean (SD) ∗ | |
| Before bracket bonding | 0.49 | 0.73 | 0.56 (0.06) a |
| 30 minutes after bracket bonding | 0.74 | 1.82 | 1.04 (0.28) b |
| 24 hours after bracket bonding | 0.50 | 1.34 | 0.64 (0.21) a |
| 1 week after bracket bonding | 0.50 | 1.52 | 0.76 (0.33) a |
| 1 month after bracket bonding | 0.49 | 1.23 | 0.61 (0.16) a |
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