Physicochemical properties and biological effects of quaternary ammonium methacrylates in an experimental adhesive resin for bonding orthodontic brackets

Abstract Fixed orthodontic appliances may lead to biofilm accumulation around them that may increase caries risk. This study aimed to evaluate the influence of quaternary ammonium methacrylates (QAMs) on the physicochemical properties, cytotoxicity, and antibacterial activity of adhesive resins for orthodontic purposes. Methodology: A base resin was prepared with a comonomer blend and photoinitiator/co-initiator system. Two different QAMs were added to the base adhesive: dimethylaminododecyl methacrylate at 5 wt.% (DMADDM) or dimethylaminohexadecyl methacrylate (DMAHDM) at 10 wt.%. The base adhesive, without QAMs, (GC) and the commercial Transbond™ XT Primer 3M (GT) were used as control. The resins were tested immediately and after six months of aging in the water regarding the antibacterial activity and shear bond strength (SBS). The antibacterial activity was tested against Streptococcus mutans via metabolic activity assay (MTT test). The groups were also tested for the degree of conversion (DC) and cytotoxicity against keratinocytes. Results: The resins containing QAM showed antibacterial activity compared to the commercial material by immediately reducing the metabolic activity by about 60%. However, the antibacterial activity decreased after aging (p<0.05). None of the groups presented any differences for SBS (p>0.05) and DC (p>0.05). The incorporation of DMADDM and DMAHDM significantly reduced the keratinocyte viability compared to the GT and GC groups (p<0.05). Conclusion: Both adhesives with QAMs showed a significant reduction in bacterial metabolic activity, but this effect decreased after water aging. Lower cell viability was observed for the group with the longer alkyl chain-QAM, without significant differences for the bonding ability and degree of conversion. The addition of QAMs in adhesives may affect the keratinocytes viability, and the aging effects maybe decrease the bacterial activity of QAM-doped materials.


Introduction
Fixed orthodontic treatment leads to significant caries risk due to biofilm accumulation around the appliance components and the challenges to achieve a reliable oral hygiene. 1-5 During orthodontic bonding procedures, the excess of adhesive is invariably left on the tooth surface at the bracket-enamel interface. This adhesive excess may be a site for rapid attachment and growth of oral microorganisms such as Streptococcus mutans. 6  Antibacterial materials could be a promising alternative to biofilm control in orthodontics patients. 8,9 The effectiveness of different antibacterial materials added to adhesives has been evaluated in vitro, 8,[10][11][12][13] in situ, 10 and in vivo, 9 showing positive results to decrease biofilm viability. However, the duration of the antibacterial effect of resins remains uncertain.
Among the antibacterial agents that have been tested, the incorporation of methacrylate monomers derived from quaternary ammonium salts has increasing attention. Quaternary ammonium methacrylates (QAM) present antimicrobial mechanisms by disturbing the electrical balance of bacterial membrane through their positively charged molecules with the negatively charged compounds on the bacteria surface. This event leads to bacterial membrane disruption and cell death. [13][14][15][16] Besides providing antibacterial activity, the incorporation of antimicrobial agents should not alter dental material physical properties. 15,16 For orthodontic purposes, the shear bond strength (SBS) between the adhesive and the bracket should not be affected.
However, the literature is divergent regarding the most appropriate QAM and its concentration to be added in adhesive resins for bonding orthodontic brackets. 11,12,17 Previously, the physical properties and antimicrobial activity of two QAMdimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM)were tested at two different concentrations (5 wt.% or 10 wt.%). 18,19 The longer the carbon chain of the QAM and the higher its concentration, the higher the antimicrobial capacity achieved. However, the physical properties of the resin materials were jeopardized by increasing QAM concentration. DMADDM at 5 wt.%, in turn, led to the lowest polymer degradation after water storage among groups 19 while maintaining the antimicrobial property. 18 However, there is no evaluation of SBS nor cytotoxicity of these adhesives.
Our study aimed at evaluating the influence of quaternary ammonium methacrylates (QAM) in the physicochemical properties, cytotoxicity, and antibacterial activity of adhesive resins for orthodontic purposes. For this purpose, we evaluated the addition of DMADDM at 5 wt.% or DMAHDM at 10 wt.% in the base resin considering both the immediate and the long-term antibacterial property, along with SBS. We also analyzed the degree of conversion and cytotoxicity against human keratinocytes. The null hypothesis is that QAM addition in the experimental adhesive will not affect physicochemical properties tested and will not promote antibacterial activity and cytotoxic effect.

Synthesis of antibacterial monomers
Menschutkin reaction was used to synthesize the antimicrobial monomers, as reported 14,20 and previously published. 18,19 A tertiary amine and an organo-halide were added to this reaction, in equal amounts (60 mmol), into a round bottom flask coupled to a condenser with 20 mL of ethanol and were refluxed for 24 h. The pure monomer was obtained when the solvent was evaporated in a rotatory evaporator until dryness and so it did not need purification. For each monomer, a different organo-halide was used, since the tertiary amine was always 2-(dimethylamino)ethyl methacrylate (DMAEMA).

All groups had an increased value of SBS between T0
and T1 (p<0.05). Table 1  This approach may assist in preventing caries lesions in non-cooperative patients. 11,12,25 In our study, the addition of QAM did not change the physicochemical properties evaluated, and antibacterial activity was   13,15 There were no significant differences when the group of 5 wt.% of DMADDM was compared with 10 wt.% of DMAHDM, which differs from our previous study. 18 Although there was no statistically significant difference between G16C/10 and the control groups in T0, the percentage difference was high (around 60 % compared to GT), and there was no statistical difference among both adhesives containing QAM. In our study, we used MTT assay to evaluate the antibacterial activity. In this test, the reduction of tetrazolium salts to formazan is associated with microorganism metabolic activity. A higher absorbance in the MTT assay suggests a higher formazan concentration and higher metabolic activity. However, this test does not measure cell viability, such as colonyforming units assay. As an initial test to compare the antibacterial activity immediately and over time, it is interesting to use MTT because the pathogenicity of S. mutans is related to its metabolic activity and acid production. 26 Further studies to evaluate the developed materials via colony-forming unit tests would assist in understanding the antibacterial effects. 25 After water aging, the effect was lower, and no difference was observed among groups. According to Rego, et al. 18 (2017) the composites containing QAM showed antibacterial activity after brushing simulation.
However, there was an increase in viable biofilm after brushing and polishing, suggesting that the antibacterial effects of composites with QAM decrease over time. 18 The effect of water in the chemical and structure of the polymer network is essential to understand the possible behavior of dental materials in the oral environment. 27 Besides the aging performed in our study, the effect of degradation was previously demonstrated to reduce the surface hardness, increase sorption and solubility, and hygroscopic expansion. 19 The leaching part of the QAM from the cured adhesive surface may explain the weakened effect of antibacterial activity after water-aging in our study. 13 Even with methacrylate groups, part of the QAM may not have copolymerized the resin and was lost during the storage.
There is no standard method for assessing the SBS of brackets to enamel since the loads applied in clinical settings on brackets are dynamic and complex. 28 A mixture of shear and tensile loads are applied to brackets in vivo, and they cannot be perfectly simulated in vitro. The forces during chewing are abrupt and impose a high risk of damage to the enamel when compared with the force applied during the SBS test. 28,29 However, this is the most widely used assay to assess bonding effectivenesss of orthodontics. 8 In our study, the SBS results showed no differences among groups, indicating that the addition of the QAM did not affect the mechanical property of the material, corroborating with previous studies. 28,30,31 In the immediate SBS, there was no difference among groups. Interestingly, all adhesives showed higher SBS outcomes after six months. The rationale for that may be delayed in the polymerization reaction when the specimens were stored in warm distilled water over time. 32 The addition of DMADDM at 5 wt.% also resulted in less degradation for the composite in a previous study. 19 Besides the non-difference observed for the SBS among groups in the immediate or in the long-term analysis, the DC of the orthodontic resins also did not change with the addition of QAM. This Cytotoxicity of the adhesives against keratinocytes was evaluated because these cells are present in the oral mucosa. Viable cells were identified using the SRB dye test. Compared to the MTT assay, which is another colorimetric test and an ISO-indicated method. The SRB test has better predictive power by marking viable cell proteins and not relying on cell metabolism for MTT assay. In our study, the cell viability was lower than ISO requires (inferior to 70 % is considered a cytotoxic material when the MTT test is applied), indicating that, even with copolymerizable QAM, probably some of these components may leach from the resin matrix.