Release of leachable products from resinous compounds in the saliva of children with anterior open bite treated with spur

Abstract To evaluate the release of bisphenol-A glycidyl methacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), bisphenol A (BPA), and phthalates of the composite resin used in the bonding of spurs applied in the treatment of children with anterior open bite and its effects on human keratinocytes. Methodology Saliva samples of 22 children were collected before spur attachment (baseline) and 30 minutes (min) and 24 hours (h) after spur bonding. Analysis was performed using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (HPLC–MS/MS) and gas chromatography coupled to mass spectrometry (GC–MS). Standardized resin increments were added to three different dilutions of the cell culture medium. Keratinocytes (HaCaT) were cultivated in the conditioned media and evaluated for cell viability (MTT) and cell scratch assay. Results The levels of BisGMA (1.74±0.27 μg/mL), TEGDMA (2.29±0.36 μg/mL), and BPA (3.264±0.88 μg/L) in the saliva after 30 min, in comparison to baseline (0±0 μg/mL, 0±0 μg/mL, and 1.15±0.21 μg/L, respectively), presented higher numbers. After 24 h, the levels of the monomers were similar to the baseline. Phthalates showed no significant difference among groups. HaCat cells showed increased viability and reduced cell migration over time after exposure to methacrylate-based resin composites. Conclusion Resin composites, used to attach spurs in children with anterior open bite during orthodontic treatment, release monomers after polymerization and can influence the behavior of human keratinocytes, even at very low concentrations. Orthodontists should be aware of the risks of the resinous compounds release and preventive procedures should be held to reduce patient exposure.


Introduction
Methacrylate-based resin composites have been commonly used in bonding accessories in orthodontic treatment. 1 The base monomer of the organic matrix is bisphenol-A glycidyl methacrylate (BisGMA), which, due to its high viscosity, is mixed with other dimethacrylates, such as triethylene glycol dimethacrylate (TEGDMA) and/or other monomers 2 and additives, such as phthalates. 3 Dental materials are susceptible to degradation when applied clinically and, as a consequence, may leach into the oral environment. 2, [4][5][6][7][8] The release of composite resin monomers is potentially hazardous [8][9][10][11] with systemic 11 and local effects on the oral mucosa, gum, and dental pulp. 8,12 One of the products resulting from the decomposition of BisGMA is bisphenol-A (BPA), which is considered a xenoestrogen and can simulate the function of estrogen. 13 In addition, exposure to BPA may lead to early sexual maturation in children, 14 infertility 14 increased risk of breast and prostate cancer, 15 and changes in immune functions. 16 Another leachable product found in dental materials is the phthalate, 3 which is synthetic chemical ester of phthalic acid applied in the production of plastic materials. 17 Exposure to phthalates is a risk because they are endocrine disruptors and can cause sexual changes in children. 17 Studies demonstrated the release of resinous compounds in human saliva, urine, and blood after their use in restorative procedures, pit-andfissure sealants, and orthodontics. 5,10,18,19 Despite being similar, the chemical composition and clinical applications of these compounds directly influence the amount of leachable products release. 18  Three participants missed the appointments and the study remained with 22 participants that agreed to participate, including eight males and 14 females, with a mean age of 8.95±1.45 years (Figure 1). The consent forms were collected and signed by the legal guardians and the participants signed the assent forms.
Saliva collection was performed before the device was placed (baseline) and within 30 minutes (min) and 24 hours (h) after the device was bonded, following Tou, et al. 21 (2022

Saliva collection
To collect unstimulated saliva, volunteers were advised to sit comfortably with their heads slightly tilted down, allowing saliva to accumulate in their mouth, and then collect it in a glass vial. The collected saliva of each participant was immediately stored on ice and then at −80°C until the moment of analysis. A

In vitro experiment
To mimic the oral microenvironment in the condition of spur attachment, cell culture tests were performed to evaluate cell viability (3-[4,5-(dimethylthiazol-2-yl  The value of P<0.05 was considered statistically significant.
BPA leached after spur attachment, without significant differences in phthalate levels between the baseline and 24 h groups (Table 2).
We noted no significant differences in the levels of phthalates (DiBP and DBP) among the different time points (Table 2) quantifying products from resinous dental materials because of its high efficiency in the evaluation of these compounds. 9,12,19 However, the operational process and the techniques used for the extraction of sample compounds are widely variable, 9,18,23,26 which may generate different findings among the studies. In addition, the amount and size of samples may vary in studies, including in vitro and in vivo analysis, so standardization of the samples may be challenging. 2,4,8,27 The difference in the commercial brands used may also lead to differences in the number of components released by the materials and their genotoxic and cytotoxic potential. 4,10,19 Our study was based on the experimental protocol of Moreira,et al. 18 (2017) and Gomes, et al. 23 (2020) to reduce the risk of error, thus increasing the capability of comparison among other studies.
Methacrylate-based dental materials are susceptible to degradation when applied clinically, so byproducts may be released into the oral environment. 8 Compounds applied for bonding orthodontic accessories in the treatment of adults and pre-adolescent patients are among the degradable materials. 11,18 We observed a significant pattern of increase in BisGMA, TEGDMA, and BPA concentrations after spur attachment in children, followed by a decrease that reached the initial values.
An in vitro study quantified the elution of compounds from resin-based dental composites over one year and verified that BisGMA, HEMA, and UDMA were able to continuously elute from the materials up to 52 weeks after initial immersion. 28 In accordance, other studies also identified component release within the first minutes after polymerization. 19 Polydorou, et al. 4 (2007) identified the presence in vitro of BisGMA and TEGDMA at 24 h and 7 days after polymerization.
The clinical application of each compound is also a factor that can interfere with the quantity of released byproducts. 5,8,11,[29][30][31][32] More fluid resinous materials present a higher percentage of leachable products released. 8,30 Another important clinical factor to be    4,5,27,29,32 and that most of the oligomer and monomer components are released within the first three to six hours after polymerization depending on the environment, with a rate of 80-100% release within the first 24 hours. 27 Moreira, et al. 18 (2017) identified significant BPA release in the saliva 30 minutes after light curing and did not find significant water. 34 Consequently, BPA has spread through our environment, making daily human exposure to BPA very intense. In this study, the baseline and 24 h after the spur attachment with resinous material groups presented BPA levels different from zero, with increased levels 30 minutes after bonding. These initial and 24 h after BPA values might be explained due to the patients' environmental exposure to BPA from other possible sources. BPA exposure and average daily release were analyzed by previous studies in dental composite resins for dental restorations, glass ionomers, sealants, root canal sealers, and adhesives systems for orthodontic purposes. 5,8,11,[29][30][31][32] The average daily release of BPA from dental composite and resin glass ionomers ranged from 0.58±0.06 ng/g/day to 7.87±1.33 ng/g/day and 0 ng/g/day to 0.48±0.27 ng/g/day, respectively, in artificial saliva on the first day of an in vitro experiment, which is dependent on polymerization condition. 29 In addition, BPA released from composite materials (one fissure sealant, two adhesives, and one root canal sealer) varied from 1.1±0.2 pg BPA/mg material to 21.4±2.3 pg BPA/mg material according to the type and amount of material and light-curing system used. 5 Bagley, et al. 11 (2021) demonstrated that the highest total BPA exposure was estimated for a dental restoration application compared to dental sealants and orthodontic adhesives, with the dental restoration application showing an average BPA exposure of 323.8 ng/treatment. TEGDMA may also arise from dental-cured products. 8,30,31 TEGDMA lightcure dependent release from sealant samples ranged from 26.6 ppm to 84.98 ppm 31 and TEGDMA in-vitro release from orthodontic adhesives was 31.7 µg/ mL. 8,30 Our results showed that 30 minutes after the spur bonding with the orthodontic adhesive system, the BPA levels increased almost three times compared to baseline levels and presented approximately 76% reduction after 24 hours.
Phthalates have been linked to health problems, such as early puberty in women, 35  can influence their concentration levels. The main sources of DiBP and DBP are not limited to food packages, cosmetics, and personal care products, but also solvents, plastic materials, and even dust particles that can be aspirated and ingested. 17, 39 The patients from this study did not have access to items that could be sources of phthalates in the sample collection. In our study, although not significant, DiBP and DBP were detected in all periods of salivary samples collected before and after the spur bonding. Similar events have been reported in other in vitro studies. 36 The concern with the release of resin composite byproducts is that these components are toxic to tissues and cells. 10,25,40 Cell culture studies demonstrated that BisGMA release simulated estrogen function in the body 13,41 and that BPA was able to induce migration, proliferation, and estrogenic activity in MCF-7 breast cancer cells. 41,42 TEGDMA exhibited excellent viscosity and copolymerization behavior and revealed considerable cell cytotoxic potency. 40 We identified changes in cellular behavior in HaCat cells, showing an increase in cell migration in cultures exposed to methacrylates.
The resin-based products may also be cytotoxic to human gingival fibroblasts and keratinocytes and might interfere with cell proliferation and migration. 10,25,40,43 The cytotoxicity of monomer release may occur in a dose-dependent manner and is dependent on the followability of the resin. 44,45 Theilig, et al. 25 (2000)

Conflict of interest
The authors declare no conflict of interest.

Data availability statement
The datasets generated and analyzed during this study are available in the SciELO Data repository,