Synthesis and characterization of dimethacrylates containing quaternary ammonium functionalities for dental applications

Abstract

Objectives

The widespread incidence of recurrent caries highlights the need for improved dental restorative materials. The objective of this study was to synthesize low viscosity ionic dimethacrylate monomers (IDMAs) that contain quaternary ammoniums groups (antimicrobial functionalities) and are compatible with existing dental dimethacrylate-based monomers. Such monomers have the potential to copolymerize with other methacrylate monomers and produce antibacterial polymers.

Methods

Two monomers (IDMA-1 and IDMA-2) were synthesized using the Menschutkin reaction and incorporated at 0–30% (by mass) into a 1:1 (by mass) bisphenol A glycerolate dimethacrylate (BisGMA):triethylene glycol dimethacrylate (TEGDMA) resin. Resin viscosity was quantified using rheology, and polymer degree of conversion (DC) and surface charge density were measured using Fourier transform infrared spectroscopy (FTIR) and fluorescein binding, respectively. Effects of IDMA-1 on initial attachment of Streptococcus mutans and on viability and metabolic activity (via reductase enzymes) of RAW 264.7 macrophage-like cells were quantified.

Results

IDMA-1 and IDMA-2 were prepared and characterized. IDMA-1 was miscible with BisGMA:TEGDMA and slightly increased the resin viscosity and DC. As expected, polymeric surface charge density increased with increasing IDMA-1. Incorporation of 10% IDMA-1 into BisGMA:TEGDMA reduced bacterial colonization without affecting viability or metabolic activity of mammalian cells. Increasing IDMA-1 up to 30% had no additional effect on bacterial coverage, but ≥20% IDMA-1 significantly reduced macrophage density, viability, and metabolic activity. Leachables from polymers containing IDMA-1 were not cytotoxic.

Significance

The Menschutkin reaction provides a facile, convenient means to synthesize new monomers with quaternary ammonium groups for dental and medical applications.

Introduction

Since their introduction in the 1960s, crosslinking (meth)acrylic resins, such as bisphenol A glycerolate dimethacrylate (BisGMA), have been increasingly used in many dental applications, including restorative composites, protective sealants, and adhesive bonding agents . Over the past several decades, considerable efforts, including the utilization of photopolymerization methods to yield crosslinked polymers, have been expended to improve the quality and durability of polymeric restorative materials . These efforts, coupled with improvements in dental adhesive systems for bonding composites to tooth structure, have increased the clinical utility of polymeric restorative materials to the point that they are currently the material of choice for direct, esthetic restorations. For all applications, one major concern remains: susceptibility to bacteria colonization. Biofilm growth is especially problematic when restorative composites interfacially debond from tooth structure to create a gap prone to the development of secondary (recurrent) caries. When a conventional bonding agent is used, as in the case of orthodontic brackets, biofilm growth at the tooth-adhesive junction can lead to rapid demineralization of the tooth .

One approach to prevent recurrent caries is to use dental materials, such as polymeric adhesives or composites, with antibacterial properties. Charged moieties, such as quaternary ammonium compounds, have been shown to be effective in reducing bacterial growth in a wide range of applications including medical devices, water purification systems, and textiles . This tactic of utilizing quaternary ammonium compounds has carried over to dental materials, where cationic, monomethacrylate monomers have been developed to impart antibacterial activity to polymeric dental materials . For instance, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) are two monomers that have demonstrated bactericidal activity in dental adhesives. MDPB was also bacteriostatic in dental resins . Unlike the dimethacrylates typically used in dental resins and adhesives, these cationic monomers generally have only one methacrylate group. Incorporating high concentrations of monomethacrylate could significantly affect the overall polymer network structures and properties. In addition, some monomethacrylates with pendant quaternary ammonium moieties may present miscibility problems with hydrophobic dimethacrylates commonly used in dental composites.

Another approach to incorporate high concentrations of quaternary ammonium groups into dental composites is through the use of filler particles. Polymers derived from cationic monomers have been pulverized into a fine powder and used as filler particles with bacteriostatic properties . In addition, quaternary ammonium poly(ethylenimine) nanoparticles have also been incorporated into resin composites to impart antibacterial properties . While this approach using filler particles has shown some promise in reducing biofilm growth, the effectiveness may be difficult to reproduce, as the activity depends on particle size and uniformity of dispersion. There is also the potential for particle loss due to leaching or wear processes experienced by the composites.

The classical Menschutkin reaction (the addition reaction of tertiary amines with organo-halides) provides a facile approach to produce a wide variety of potentially antibacterial monomers, oligomers, and polymers that have potential applications in a range of dental and biomedical materials. For instance, step-growth polymerization based on the Menschutkin reaction resulted in multiple quaternary ammonium functional groups in the backbone of these ionic polymers (termed ionenes) and imparted antimicrobial properties to the linear polymers .

The objective of this study was to adapt the Menschutkin reaction for the synthesis of free radical, photo-crosslinking, dimethacrylate monomers containing quaternary ammonium functionalities. These novel, reactive monomers can be designed to have solubility parameters similar to common dental resins and therefore are expected to be miscible with such resins. The resultant copolymers are expected to have lower monomer leachability and degradability due to multiple vinyl groups that lead to tighter polymer networks with increased crosslink density. Further, the class of monomers can be designed to contain one or more quaternary ammonium functionalities for reduced bacterial growth. In this study, we demonstrate the synthesis of two dimethacrylates with quaternary ammonium functionalities. The monomers were characterized by proton nuclear magnetic resonance ( 1 H NMR) and Fourier transform infrared (FTIR) spectroscopy. The viscosity of these monomers when incorporated into common dental monomers was characterized to assess how they affect resin processability. The degree of conversion (DC) of the photo-crosslinked materials was determined by near infrared (NIR) spectroscopy, and the surface charge density was determined using a fluorescein binding assay. A model dimethacrylate system containing various amounts of one quaternary ammonium monomer was used to conduct a preliminary evaluation of the polymers’ biological response in terms of both bacterial colonization and mammalian cell viability.

Materials and methods 1

1 Certain commercial materials and equipment are identified in this article to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by NIST or that the material or equipment identified is necessarily the best available for the purpose.

Materials

2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA), 2-bromoethyl methacrylate (BEMA), 2,2′-bis(bromomethyl)-1,1′-biphenyl (BbmBP), camphorquinone, ethyl 4-N,N-dimethylaminobenzoate, and anhydrous ethanol (EtOH) were purchased from Sigma–Aldrich. BisGMA and triethylene glycol dimethacrylate (TEGDMA) were obtained from Esstech Inc. Culture reagents were purchased from Invitrogen Corp. All reagents were used as received.

Synthesis and characterization of ionic dimethacrylates (IDMAs)

The synthesis of bis(2-methacryloyloxyethyl)dimethylammonium bromide [IDMA-1] is shown in Fig. 1 A. DMAEMA (1.57 g, 10 mmol), BEMA (1.93 g, 10 mmol), and 3 g ethanol were added to a tared vial equipped with a magnetic stir bar. The vial was capped and stirred for 24 h at 60 °C. After removal of the solvent and residual reagents by evaporation with the aid of a moderate (7 kPa) vacuum, a clear, colorless, viscous product (IDMA-1) was isolated with a yield >95%. 2,2′-bis(methacryloxyloxyethyl dimethylammonium bromide-1,1′-benzyl) (IDMA-2) was synthesized in virtually quantitative yield in a similar manner from DMAEMA and BbmBP ( Fig. 1 B).

Fig. 1
Reaction scheme for the synthesis of low viscosity ionic dimethacrylates containing quaternary ammonium functionalities: (A) IDMA-1 and (B) IDMA-2.

FTIR spectra of the starting materials and the low viscosity products were collected between ThBr, I plates in the 4000 cm −1 to 400 cm −1 region with a wavenumber expanded uncertainty of 0.5 cm −1 . The spectrum of solid BbmBP was obtained from the ground powder, about 10 μm in particle size, in a KBr pellet. Water bands from the KBr pellet and from the atmosphere were removed from all spectra by subtraction. All spectra for IDMA-1 ( Fig. 2 A) were normalized to the same carbonyl absorbance at 1720 cm −1 . For IDMA-2, the total area of the three bands at 776 cm −1 , 771 cm −1 , and 756 cm −1 of BbmBP was set approximately equal (± 5%) to the area of the 770 cm −1 band of reacted BbmBP in the product IDMA-2.

Fig. 2
FTIR spectra of reactants and products for low viscosity ionic dimethacrylate monomers (A) IDMA-1 and (B) IDMA-2.

High-resolution, 270 MHz 1 H NMR spectra were taken on a 6.35 T JEOL GX270 spectrometer manufactured by JEOL, Ltd. (Akishima, Japan). Deuterated chloroform was used as a solvent and the monomer concentrations varied between 2.5% and 3.0% by mass fraction. All spectra were run at room temperature, 15 Hz sample spinning, 45° tip angle for the observation pulse, and a 10 s recycle delay, for 64 scans. The standard relative uncertainty for molecular mass calculated via 1 H NMR arises from the choice of baseline and is estimated to be 8%.

Resin formulation and viscosity

IDMA-1 was mixed with BisGMA:TEGDMA (50:50, mass fraction) for final IDMA-1 concentrations of 10%, 20%, and 30% (by mass). Control resins contained no IDMA-1. Viscosity measurements were performed at room temperature on an ARES rheometer (TA Instruments) with a cone-and-plate geometry (25 mm diameter). Steady shear viscosity was determined from 0.1 s −1 to 10 s −1 . No changes in viscosity were detected as a function of shear rate. The relative standard uncertainty for the viscosity measurements is 5%.

Photopolymerization and DC

Resins containing IDMA-1 were activated for blue light (470 nm) photopolymerization with camphorquinone (0.2% by mass) and ethyl 4-N,N-dimethylaminobenzoate (0.8% by mass). Polymer disks (diameter ≈ 4 mm, height ≈ 1 mm) were prepared by irradiating (Dentsply Triad 2000, 250 W, 120 V) the activated resin blends between two clean glass slides (1 min per side) . DC was quantified using transmission NIR spectroscopy on a Nicolet Magna 550 FTIR spectrometer (Madison, WI) configured with a white light source, CaF 2 beam splitter, and InSb detector. Spectra of the activated resin and the photopolymerized polymer disks were acquired over 7000 cm −1 to 4000 cm −1 from 64 co-added scans at 6 cm −1 resolution. The methacrylate peak height (4743 cm −1 ) was normalized against the aromatic peak height (4623 cm −1 ), and the reduction in the normalized value due to polymerization was calculated as the DC .

Surface charge density of polymers

The density of quaternary ammonium groups present on the polymer surfaces was quantified using fluorescein dye . Sample diameters (∼7 mm to 8 mm) were measured using calipers, and samples were placed in a 48-well plate. Sample heights were 1 mm. Fluorescein sodium salt (200 μL of 10 mg/mL in deionized (DI) water) was added, and samples were left for 10 min at room temperature in the dark. After removing the fluorescein solution and rinsing extensively with DI water, each sample was placed in a new well, and 200 μL of 0.1% (by mass) cetyltrimethylammonium chloride (CTMAC) in DI water was added. Samples were shaken for 20 min at room temperature in the dark to desorb the bound dye. The CTMAC solution was supplemented with 10% (by volume) 100 mM phosphate, pH 8.0 (0.94 mg/mL monosodium phosphate – monohydrate and 13.2 mg/mL disodium phosphate – anhydrous in DI water). Sample absorbance was read at 501 nm using a standard plate reader. The fluorescein concentration was calculated using Beers Law and an extinction coefficient of 77 mM −1 cm −1 . Using a ratio of 1:1 for fluorescein molecules to accessible quaternary ammonium groups, the surface charge density was calculated as the total molecules of charge per exposed surface area (sum of top, bottom and side edge area, measured independently for each polymer disk due to slight variations in disk diameters).

Bacteria inoculation and imaging

Streptococcus mutans Clarke UA159 from the American Type Culture Collection (ATCC) were cultured in brain heart infusion (BHI) broth with 0.5 μg/mL bacitracin. Polymers were sterilized with 70% (volume fraction) ethanol for 20 min, soaked in phosphate buffered saline (PBS) overnight, and inoculated with S. mutans prepared at an optical density (OD 600 ) of 0.06 in PBS containing 100 mg/L MgCl 2 ·6H 2 O and 100 mg/L CaCl 2 . After incubating at 37 °C, 5% CO 2 (by volume) for 4 h, samples were washed 3× to remove nonadherent bacteria, fixed with 37 mg/mL formaldehyde, and stained for 1 h with 1 μmol/L SYTOX green . Samples were not passed through the air–liquid interface during the rinsing and fixing steps. Samples were imaged using a Zeiss LSM 510 laser scanning confocal microscope with a 40× water immersion objective. Three samples were evaluated for each IDMA-1 concentration. Five image stacks were collected at random locations on each sample, and projection images were prepared using the manufacturer’s software. Custom macros in Image-Pro Plus software (Media Cybernetics, Inc.) were used to quantify each image in terms of the total surface area covered by bacteria, the area of each object, and the object density. Object density and surface coverage were plotted as a percentage of the control (no IDMA-1).

Macrophage viability and enzymatic activity

Cytotoxicity experiments were performed using the murine RAW 264.7 macrophage-like cell line (passages 8–12, ATCC TIB-71). Polymers containing 0–30% IDMA-1 were prepared and sterilized with 70% ethanol (by volume) and seeded with RAW 264.7 macrophages (18 000 cells/cm 2 ). Negative controls consisted of cells seeded on tissue culture polystyrene (TCPS). After 24 h, samples were evaluated for viability by staining for 10 min with 2 μmol/L calcein acetoxymethyl ester (calcein AM, live cells), 2 μmol/L ethidium homodimer-1 (EthD-1, dead cells), and 10 μmol/L Hoechst 33342 (H33342, all nuclei) . Cells were visualized on a Leica DMA upright epifluorescent microscope (Leica Microsystems AG). Images were captured using a digital camera (Hamamatsu Photonics K.K.) and analyzed with Image-Pro Plus. Four separate fields of view were imaged on each of three samples per composition, and data were combined from two separate experiments (total images per composition = 24). Cell density was calculated as the number of cells per image. Cell viability was determined as the number of live cells divided by the total number of cells per image.

Cellular metabolic activity was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, a colormetric assay measuring the enzymatic reduction of MTT to formazan . Prior to sample sterilization, the average diameter of each polymer disk was determined from three separate diameter measurements using digital calipers. Disks were then sterilized, placed in 24-well TCPS plates, and seeded with 90 000 cells/cm 2 . Controls included wells with polymers only (no cells), wells with cells only (no polymers) and wells without cells or polymers (blanks). After 24 h, the growth medium was removed. The polymer disks were transferred to a new 24-well plate, and 300 μL MTT solution (0.5 mg/mL MTT in PBS) was added to each polymer disk and each TCPS well from which the disks were removed. The plates were incubated for 1 h at 37 °C, 5% CO 2 . The MTT solution was then removed and replaced with 300 μL DMSO. The plates were incubated at room temperature with gentle mixing for 20 min. After brief mixing via pipetting, 200 μL from each well was transferred to a 96-well plate, and the optical density at 540 nm was measured. Results were normalized to the surface area exposed to cells (for disks: polymer disk area; for TCPS: well area minus the polymer disk area). Controls without cells and polymers were used as blanks and subtracted from all sample readings. Two separate experiments, each with three samples, were completed (total n = 6).

Statistical analysis

Rheology, DC, charge density, and bacterial object area data were analyzed using One-Way Analysis of Variance (ANOVA) and a post hoc Bonferroni multiple comparison test (significance level of 0.05), with charge density and bacterial object area data log-transformed prior to analysis to homogenize the variance and to approximate normality, respectively. The surface area covered by bacteria and the bacterial object density were analyzed using the non-parametric Kruskal–Wallis test at a 95% confidence interval to compare populations, as the data were not found to conform to an established statistical distribution. For RAW 264.7 cells, viability, density, and MTT assay data were analyzed using One-Way ANOVA and the Tukey/Kramer multiple comparison test at 95% confidence, with cell density data log-transformed to approximate normality prior to analysis.

Materials and methods 1

1 Certain commercial materials and equipment are identified in this article to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by NIST or that the material or equipment identified is necessarily the best available for the purpose.

Materials

2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA), 2-bromoethyl methacrylate (BEMA), 2,2′-bis(bromomethyl)-1,1′-biphenyl (BbmBP), camphorquinone, ethyl 4-N,N-dimethylaminobenzoate, and anhydrous ethanol (EtOH) were purchased from Sigma–Aldrich. BisGMA and triethylene glycol dimethacrylate (TEGDMA) were obtained from Esstech Inc. Culture reagents were purchased from Invitrogen Corp. All reagents were used as received.

Synthesis and characterization of ionic dimethacrylates (IDMAs)

The synthesis of bis(2-methacryloyloxyethyl)dimethylammonium bromide [IDMA-1] is shown in Fig. 1 A. DMAEMA (1.57 g, 10 mmol), BEMA (1.93 g, 10 mmol), and 3 g ethanol were added to a tared vial equipped with a magnetic stir bar. The vial was capped and stirred for 24 h at 60 °C. After removal of the solvent and residual reagents by evaporation with the aid of a moderate (7 kPa) vacuum, a clear, colorless, viscous product (IDMA-1) was isolated with a yield >95%. 2,2′-bis(methacryloxyloxyethyl dimethylammonium bromide-1,1′-benzyl) (IDMA-2) was synthesized in virtually quantitative yield in a similar manner from DMAEMA and BbmBP ( Fig. 1 B).

Fig. 1
Reaction scheme for the synthesis of low viscosity ionic dimethacrylates containing quaternary ammonium functionalities: (A) IDMA-1 and (B) IDMA-2.

FTIR spectra of the starting materials and the low viscosity products were collected between ThBr, I plates in the 4000 cm −1 to 400 cm −1 region with a wavenumber expanded uncertainty of 0.5 cm −1 . The spectrum of solid BbmBP was obtained from the ground powder, about 10 μm in particle size, in a KBr pellet. Water bands from the KBr pellet and from the atmosphere were removed from all spectra by subtraction. All spectra for IDMA-1 ( Fig. 2 A) were normalized to the same carbonyl absorbance at 1720 cm −1 . For IDMA-2, the total area of the three bands at 776 cm −1 , 771 cm −1 , and 756 cm −1 of BbmBP was set approximately equal (± 5%) to the area of the 770 cm −1 band of reacted BbmBP in the product IDMA-2.

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Synthesis and characterization of dimethacrylates containing quaternary ammonium functionalities for dental applications
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