Abstract
Objective
Investigate antimicrobial properties and surface texture of model composites with different concentration and alkyl chain length of quaternary ammonium monomers (QAS).
Methods
Monomers derived from QAS salts with alkyl chain lengths of 12 carbons ((dimethylaminododecyl methacrylate) DMADDM) and 16 carbons (dimethylaminohexadecyl methacrylate—DMAHDM) were obtained from the reactions of their respective organo-halides with the tertiary amine 2-(dimethylamino)ethyl methacrylate (DMAEMA). DMADDM and DMAHDM were incorporated into model composite in concentrations of 5 or 10%, resulting the following groups: G12.5 (DMADDM 5%), G12.10 (DMADDM 10%), G16.5 (DMAHDM 5%), G16.10 (DMAHDM 10%) and GC (control). Biofilm viability, lactic acid production and surface roughness were analysed 24 h after samples preparation (initial), repeated after toothbrush abrasion and after polishing simulation. Data were submitted to ANOVA and Tukey’s test (p ≤ 0.05).
Results
The longer the molecular chain size of QAS and the higher its concentration (G16.10), the lower was the viability and the production of lactic acid by the biofilm. No differences were detected in initial roughness’ measurements among groups. However, after abrasion, there was an increase of biofilm viability and lactic acid production. Composites containing QAS presented rougher surfaces compared to the CG. After polishing, biofilm viability and surface roughness were statistically similar for all groups. Nevertheless, DMAHDM at 10% showed reduction in lactic acid production.
Significance
Chain length and concentration of QAS influenced biofilm development and production of lactic acid. Longer chains and higher concentrations of QAS promoted better antimicrobial properties. Changes in surface texture caused by abrasion, decreased antibiofilm properties.
1
Introduction
Secondary caries stand out as one of the main reason for failure in the use of dental composite restorations . The biofilm formed at restorations can, through the metabolism of carbohydrate and the formation of acids in the biofilm accumulated at the interface of restorations cause secundary caries . Furthermore, the unreacted monomers from the resin matrix stimulate bacterial growth .
The development of recurrent caries is a multifactorial issue. Features related both to the patient and to the restorative procedure will dictate the propensity of secondary caries formation. Opdam et al. observed that the risk of development of secondary caries is higher in patients with medium and high decay rate, and the greater the number of involved faces, the higher is the probability of failure (about 30–40% higher) . The formation of secondary caries is also related to the marginal dental substrate and the cavity’s depth, so that margins in dentine and deeper lesions are more prone to recurrent caries . Its histopathology, however, is not different from primary caries, which is a localized process of both demineralization of dentin and enamel caused by biofilm .
One alternative to control biofilm growth and consequent development of a new cavity is the incorporation of methacrylic monomers derived of quaternary ammonium salts (QAM) to the organic matrix of resin composites . These monomers cause a disturbance in the electrical balance of the bacterial membrane through the contact of their positively charged molecules with the negatively charged cell, causing rupture of its membrane and cell death . According to some studies, quaternary ammonium methacrylates also exhibit low cytotoxicity to cells of the oral cavity about twenty times lower than the BIS-GMA, a monomer widely used in dental composites available on the market .
Among the monomers derived from quaternary ammonium salts dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM) stand out . They are generally incorporated in concentrations of 5% and 10% by mass in adhesives and resin composites . The molecules of DMADDM and DMAHDM have an amine core, a methacrylate termination and a long chain of hydrocarbons (12 alkyl chain to DMADDM and 16 to DMAHDM). The amine nucleus is responsible for the positive charge of the molecule and consequent antimicrobial action; the methacrylate termination ensures the incorporation of the monomer to the polymer chain, allowing inhibitory action in the long term . Regarding the hydrocarbon chain, studies show the relationship between chain length and bactericidal action, so that longer chains promote increased hydrophobicity, leading to greater penetration into the bacterial membrane and consequent inhibition of biofilm .
Besides promising, the studies available in the literature do not simulate clinical conditions, such as finishing and polishing or toothbrush abrasion of composites containing QAM, which may affect the antibiofilm properties of these materials. Therefore, the objective of this study was to determine the effect of concentration and chain length of the quaternary ammonium methacrylates on (a) viability of induced Streptococcus mutans , (b) the formation of lactic acid on composites and (c) surface roughness of novel resin composites after toothbrush abrasion and polishing.
The study hypothesis was that the higher the concentration and chain length of quaternary ammonium, the lower the viability of the biofilm induced on the composite; the lower the formation of lactic acid by S. mutans on composites; the lower the surface roughness of the resin after toothbrush abrasion.
2
Materials and methods
2.1
Synthesis of antimicrobial monomers
The antimicrobial monomers were synthesized using the Menschutkin reaction, as previously reported . In this addition reaction, a tertiary amine and an organo-halide were added, 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. After that, the solvent was evaporated to dryness (rotary evaporator) to afford the pure monomer, which did not require any purification. For each monomer, a different organo-halide was used ( Scheme 1 ), since the tertiary amine was always 2-(dimethylamino)ethyl methacrylate (DMAEMA).
To characterize the reaction products, Nuclear Magnetic Resonance (NMR) Spectroscopy and High-Resolution Mass Spectrometry (HRMS) were used. 1 H and 13 C NMR spectra were recorded on an Avance 200 MHz spectrometer (Bruker, Billerica, MA, USA), using CDCl 3 as the solvent. Standard Bruker software was used throughout and chemical shifts were given in ppm (δ scale) and coupling constants ( J ) were given in hertz (Hz). High-resolution mass spectra were obtained on a Bruker microTOF II mass spectrometer using ESI.
2.1.1
Dimethylaminododecyl methacrylate (DMADDM)
Yield 24.07 g (98.8%); 1 H NMR (CDCl 3 ) δ 6.11 (s, 1H), 5.64 (s, 1H), 4.64 (br s, 2H), 4.11 (br s, 2H), 3.72–3.50 (m, 2H), 3.47 (s, 6H), 1.92 (s, 3H), 1.81–1.63 (m, 2H), 1.30–1.17 (m, 18H), 0.84 (t, J = 6.6 Hz, 3H); 13 C NMR (CDCl 3 ) δ 166.4, 135.3, 127.4, 65.6, 62.4, 58.3, 52.0, 32.0, 29.6, 29.5, 29.5, 29.4, 29.3, 26.4, 23.0, 22.7, 18.3, 14.2; HRMS-ESI: m / z [M−Br] + calculated for C 20 H 40 NO 2 Br: 326.3059; found: 326.3062.
2.1.2
Dimethylaminohexadecyl methacrylate (DMAHDM)
Yield 27.52 g (99.3%); 1 H NMR (CDCl 3 ) δ 6.12 (s, 1H), 5.65 (s, 1H), 4.64 (br s, 2H), 4.13 (br s, 2H), 3.70–3.55 (m, 2H), 3.49 (s, 6H), 1.93 (s, 3H), 1.90–1.61 (m, 2H), 1.37–1.20 (m, 26H), 0.85 (t, J = 5.7 Hz, 3H); 13 C NMR (CDCl 3 ) δ 166.4, 135.3, 127.4, 65.6, 62.3, 58.3, 52.0, 32.0, 29.7, 29.6, 29.5, 29.4, 29.3, 26.4, 23.0, 22.7, 18.3, 14.2; HRMS-ESI: m / z [M−Br] + calculated for C 24 H 48 NO 2 Br: 382.3685; found: 382.3691.
2.2
Formulation of composites with antimicrobial action
For each material, Bis-GMA and TEGDMA (Esstech Inc., USA, batch TSMPOO4397) were used in a 1:1 ratio by weight, 1% camphorquinone (Esstech Inc., USA; batch TSNP004397) and 1% of amine EDMAB (Sigma–Aldrich, USA, batch MKBB3614). Furthermore, different concentrations (5% or 10%) of the antibacterial monomers listed above were also incorporated into the resin matrix. Inorganic filler particles were added in order to obtain a composite containing 50% mass load. Regarding the inorganic content, 15% of 40 nm pre-silanized spherical silica particles (Aerosil R 812, batch 3523102) and 85% of 0.7 μm pre-silanized borosilicate barium glass particles (V-258-4107, batch 699-17, Esstech Inc.) were incorporated into the composite. Thus, four resin composites were formulated, containing different antimicrobial agents in different concentrations, and one control group without antibacterial action ( Table 1 ).
| Groups | Antibiofilm monomer |
|---|---|
| Group 12.5 | DMADDM 5% |
| Group 12.10 | DMADDM 10% |
| Group 16.5 | DMAHDM 5% |
| Group 16.10 | DMAHDM 10% |
| Group C | Control |
2.3
Specimens preparation
The experimental composites were bulk-filled in a metal mold with dimensions of 8 mm diameter and 2 mm thickness. A metal weight of 65 g was placed over a mylar strip and a glass slide for 30 s to plan the sample and remove the excess of resin. Subsequently, the samples were photoactivated with a LED light source (LED Demi, Kerr, Wisconsin, USA) for 40 s on each side, at an irradiance of 800 mW/cm 2 checked with a radiometer (LED Radiometer, Demetron SDS Kerr, Wisconsin, EUA) at the end of preparation of each specimen. Ten specimens were prepared (n = 10) for each experimental group to assess surface roughness, gloss retention, and of biofilm growth inhibition. At the end of this step, one side of the specimen was standardized so the readings and aging procedures could be performed.
2.4
Induction of biofilm
The mature biofilm used was composed exclusively of S. mutans . This microorganism was selected because it is one of the main microorganisms involved in caries initiation and progression.
Specimens were exposed to ultraviolet light in a laminar flow cabinet for 30 min to be sterilized. After this step, put in wells of sterile culture cells plates.
Isolates of “American Type Culture Collection” (ATCC 25175, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil) were used for the induction of S. mutans biofilm on the specimens. S. mutans aliquots are stored in liquid nitrogen (approximately −180 °C). For each experiment, a single aliquot was thawed.
Aliquots of cell suspension with 2 × 10 5 cells/ml in a culture medium supplemented with 2% sucrose were inoculated onto the specimens and kept for 48 h at 37 °C in microaerophilic conditions.
2.5
Analysis of metabolic activity (lactate concentration)
After a 48 h incubation period, the specimens were rinsed with cysteine peptone water (CPW) to remove loose cells and transferred to new sterile plates containing 1.5 ml of buffered peptone water solution (BPW) supplemented with 0.2% sucrose (v/v). To allow the acid formation, the plates were incubated anaerobically for 3 h at 37 °C. After this period, the lactate concentration in the BPW solution was determined using standard enzymatic method of lactate dehydrogenase .
2.6
Analysis of bacterial viability
A colorimetric analysis of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) was used, which measures the reduction of MTT to form crystals by metabolically active and viable biofilm cells.
The specimens were inserted in Falcon tubes containing 1 ml of sterile phosphate buffered saline (PBS) and vortexed for 1 min for 3 times to promote detachment of the biofilm from the surface of the specimens. The PBS containing the biofilm was aspirated and transferred to microtubes and then taken to the centrifuge. After centrifugation, the isolated biofilm on the bottom of the microtube was retained and the remaining PBS was aspirated and discarted. 100 μl of MTT was added in each microtube and incubated in microaerophilic conditions at 37° C for 1 h. After this period, 100 μl of dimethylsulfoxide (DMSO) were added in each microtube and kept for 20 min under stirring and sheltered from the light. Finally, the content of each microtube was transferred to a 96 well plate to be read in a microplate reader at absorbance of 540 nm.
2.7
Surface roughness
The surface roughness was measured using a contact profilometer (SJ-201—Mitutoyo, Japan) using a constant speed of 1.0 mm/s at the applied force of 6 mN. The tip covered a distance of 2 mm and 6 readings were done by specimen, calculating an average to obtain a value for each specimen, given in μm. The parameter used was the Ra (arithmetic mean of peaks and valleys of a surface).
2.8
Toothbrush abrasion
A toothbrush simulator (MEV2, Odeme, São Paulo, Brazil) was used for abrasion on the surface of the specimens. Toothbrushes (Oral-B Indicator ® Plus, soft bristles) were coupled to the machine in order to present intimate contact with the surface of the resins. Brushing simulation was done by applying a vertical load of 2.5 N, with horizontal movements. The ratio of distilled water/dentifrice (Colgate Total 12 Clean Mint) used was 1:1. For each specimen, 3 ml of the mixture water/dentifrice (“slurry”) were initially added and 1 ml of the mixture was refilled every 1000 cycles. Subsequently, the samples were removed from the machine, washed with tap water and taken to ultrasonic bath for 10 min to remove any remainder of the slurry.
2.9
Polishing of specimens
The specimens were polished (Aropol-e, AROTEC, Brazil) with 600 and 4000 grid sandpaper at 150 rpm for 60 s under water irrigation.
2.10
Statistical analysis
The results will be submitted to two-way analysis of variance (ANOVA) with repeated measures and Tukey’s test (95%).
2
Materials and methods
2.1
Synthesis of antimicrobial monomers
The antimicrobial monomers were synthesized using the Menschutkin reaction, as previously reported . In this addition reaction, a tertiary amine and an organo-halide were added, 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. After that, the solvent was evaporated to dryness (rotary evaporator) to afford the pure monomer, which did not require any purification. For each monomer, a different organo-halide was used ( Scheme 1 ), since the tertiary amine was always 2-(dimethylamino)ethyl methacrylate (DMAEMA).
To characterize the reaction products, Nuclear Magnetic Resonance (NMR) Spectroscopy and High-Resolution Mass Spectrometry (HRMS) were used. 1 H and 13 C NMR spectra were recorded on an Avance 200 MHz spectrometer (Bruker, Billerica, MA, USA), using CDCl 3 as the solvent. Standard Bruker software was used throughout and chemical shifts were given in ppm (δ scale) and coupling constants ( J ) were given in hertz (Hz). High-resolution mass spectra were obtained on a Bruker microTOF II mass spectrometer using ESI.
2.1.1
Dimethylaminododecyl methacrylate (DMADDM)
Yield 24.07 g (98.8%); 1 H NMR (CDCl 3 ) δ 6.11 (s, 1H), 5.64 (s, 1H), 4.64 (br s, 2H), 4.11 (br s, 2H), 3.72–3.50 (m, 2H), 3.47 (s, 6H), 1.92 (s, 3H), 1.81–1.63 (m, 2H), 1.30–1.17 (m, 18H), 0.84 (t, J = 6.6 Hz, 3H); 13 C NMR (CDCl 3 ) δ 166.4, 135.3, 127.4, 65.6, 62.4, 58.3, 52.0, 32.0, 29.6, 29.5, 29.5, 29.4, 29.3, 26.4, 23.0, 22.7, 18.3, 14.2; HRMS-ESI: m / z [M−Br] + calculated for C 20 H 40 NO 2 Br: 326.3059; found: 326.3062.
2.1.2
Dimethylaminohexadecyl methacrylate (DMAHDM)
Yield 27.52 g (99.3%); 1 H NMR (CDCl 3 ) δ 6.12 (s, 1H), 5.65 (s, 1H), 4.64 (br s, 2H), 4.13 (br s, 2H), 3.70–3.55 (m, 2H), 3.49 (s, 6H), 1.93 (s, 3H), 1.90–1.61 (m, 2H), 1.37–1.20 (m, 26H), 0.85 (t, J = 5.7 Hz, 3H); 13 C NMR (CDCl 3 ) δ 166.4, 135.3, 127.4, 65.6, 62.3, 58.3, 52.0, 32.0, 29.7, 29.6, 29.5, 29.4, 29.3, 26.4, 23.0, 22.7, 18.3, 14.2; HRMS-ESI: m / z [M−Br] + calculated for C 24 H 48 NO 2 Br: 382.3685; found: 382.3691.
2.2
Formulation of composites with antimicrobial action
For each material, Bis-GMA and TEGDMA (Esstech Inc., USA, batch TSMPOO4397) were used in a 1:1 ratio by weight, 1% camphorquinone (Esstech Inc., USA; batch TSNP004397) and 1% of amine EDMAB (Sigma–Aldrich, USA, batch MKBB3614). Furthermore, different concentrations (5% or 10%) of the antibacterial monomers listed above were also incorporated into the resin matrix. Inorganic filler particles were added in order to obtain a composite containing 50% mass load. Regarding the inorganic content, 15% of 40 nm pre-silanized spherical silica particles (Aerosil R 812, batch 3523102) and 85% of 0.7 μm pre-silanized borosilicate barium glass particles (V-258-4107, batch 699-17, Esstech Inc.) were incorporated into the composite. Thus, four resin composites were formulated, containing different antimicrobial agents in different concentrations, and one control group without antibacterial action ( Table 1 ).
