Acid-modified BisGMA derivatives were synthesized and characterized.
Photopolymerization of experimental adhesives was evaluated by FTIR spectroscopy.
Resin samples with Ag nanoparticles, Zn methacrylate and triclosan were tested against Streptococcus mutans and Candida albicans .
The inhibition of microorganism growth at the contact of composite films with bacteria was evidenced.
To achieve bisphenol A glycerolate dimethacrylate (BisGMA) analogs with reduced viscosity to be used in the formulation of dental adhesives containing biocidal components.
A series of low-viscosity BisGMA derivatives ( η : 39–12 Pa s) modified with 30, 60 and, respectively 80 mol% carboxylic acid units were synthesized and characterized. Hydrogen bonding interactions in our monomers, the photopolymerization behavior and implicitly the conversion degree (DC) for some experimental adhesive formulations containing acid-modified BisGMA, commercial BisGMA (only in F1–F3), triethyleneglycol dimethacrylate and 2-hydroxyethyl methacrylate were examined by FTIR spectroscopy. The water effects on the photocrosslinked networks together with the flexural strength/modulus were also investigated. The adhesive penetration into the dentin surface was surveyed by SEM analysis, and the antimicrobial activity triggered by the incorporation of 0.5 wt% AgNO 3 , 10 wt% zinc methacrylate or 1 wt% triclosan methacrylate in selected adhesive formulations on the growth of Streptococcus mutans and Candida albicans strains was evidenced.
The contribution of the hydrogen bonding interactions was found to be lower in BisGMA derivatives than in non-modified BisGMA, and the DC varied between 56.5 (F6) and 83.7% (F1) compared with a control formulation based on BisGMA:TEGDMA (DC = 58.2%). The flexural strength and flexural modulus varied in the range 33.7 MPa (F6)–54.4 MPa (F8) MPa and 0.64 (F6)–1.43 (F8) GPa, respectively. SEM observation of adhesive-dentin interface revealed the formation of resin tags for the carboxyl-containing adhesive, while for the control adhesive they are hardly formed. Also, the microorganism development was inhibited, the proposed materials displaying antimicrobial activity.
The experimental formulations based on carboxyl-functionalized BisGMA exhibit a similar or even improved behavior over control sample, suggesting their potential applicability as antimicrobial dental adhesives.
Depending on the clinical approach, the contemporary dental adhesives were classified as either etch-and-rinse or self-etch systems, although the main goal is the achieving of a strong and durable bonding between the restorative composite and the hard dental tissue (dentin or enamel) through the infiltration of the fluid adhesive into the tooth surfaces . However, besides the application procedure, the attaining of an efficient and stable adhesive bonding is strongly influenced by several factors such as the chemical structure of the functional monomers/composition, interactions between monomers, hydrophilic/hydrophobic character, evolution of the photopolymerization process, and so on . The components included into the adhesive systems are generally based on monomers bearing both photopolymerizable and acidic groups (carboxylic, phosphoric or phosphonic acids) mixed with crosslinking dimethacrylates, solvents (ethanol, water, acetone), polymerization initiators, and other monofunctional additives . The majority of acidic monomers reported in literature are methacrylate or acrylamide type, functionalized with phosphate, phosphonate or carboxyl moieties in order to impart the desired features to the adhesive materials. In addition, the dental adhesives contain cross-linking dimethacrylates [ e.g. , 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)-phenyl] propane (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis-[2-methacryloyloxyethoxycarbonylamino]-2,4,4-trimethylhexane (UDMA), or glycerol dimethacrylate (GDMA)], that are essential in the formation of adhesive polymer networks. They favorably increased the polymerization rate through a “gel-effect” leading to improved mechanical strength of the adhesive layer and stability of the adhesive interface over time, in conjunction with a decreased swelling degree . Among these, the most frequently used not only in adhesive systems but also in resin composites is the well-known crosslinking monomer BisGMA (Bowen’s resin), due to its low polymerization shrinkage, high esthetic quality, and superior mechanical strength. Still, the main drawback of BisGMA monomer is related to its high viscosity (in the range of 500–1200 Pa s), attributed to the intermolecular hydrogen bonding between the hydroxyl groups present in the monomer structure . To overcome this disadvantage, various strategies have been proposed. In most cases the combination with lower-molecular-weight and low-viscosity monomers ( e.g. , TEGDMA) was preferred, because it reduces the viscosity of formulations but increases the curing shrinkage, water uptake and depreciates the mechanical characteristics of the final materials . Another attractive approach refers to the achievement of BisGMA derivatives with lower viscosities, through the chemical modification of hydroxyl groups with alkyl-urethane, alkoxy, urethane-methacrylate or silyl sequences that decrease the hydrogen bonding extent and consequently, diminish the amount of diluent monomer required of a workable dental mix. Moreover, up to now, for adhesive applications only a phosphoric ester of BisGMA obtained by the treatment of BisGMA with phosphorus oxychloride was reported in the literature .
On the other hand, the self-etching adhesives incorporating acidic monomers with lower pH in formulation may present a limited antibacterial effect to 24 or 48 h , and the addition of selected biocidal agents ( e.g. , chlorohexidine, fluoride, silver particles) in the matrix provide antibacterial-agent releasing materials which impede the caries occurrence. Since in such systems the physical properties were negatively affected by the release of the biocide from the polymeric network without neglecting its toxic effect and uncontrolled release kinetics, the development of non-antibacterial agent releasing materials ( e.g. , quaternary ammonium monomers) capable to generate long-term antibacterial activity remains a field of active research. As reported by Wang et al. the presence of antibacterial monomers in adhesive compositions could be advantageously because their qualities to reduce biofilm accumulation preventing the evolution of dental caries and improving the longevity of materials.
On this line, the present study describes the synthesis, characterization, and photopolymerization behavior of new acid BisGMA derivatives prepared through the functionalization of hydroxyl units from the pristine BisGMA with carboxyl sequences introduced in variable proportions (from 30 to 80 mol%) by means of succinic anhydride. The proposed monomers are intended to be used in adhesive formulations along with other photopolymerizable commercial monomers commonly encountered in such systems, in order to evaluate their influence on the specific properties for this kind of dental materials including the formation of photopolymerized resin tags. Due to the widespread susceptibility to bacteria and fungi colonization into the oral environment that may cause recurrent caries, some antibacterial agents (silver nanoparticles, zinc methacrylate or triclosan methacrylate) were included in dental adhesive resins to impart antimicrobial activity to the resulting adhesives . Furthermore, it is expected that the covalent immobilization of the monomer units with potential antimicrobial functions in the polymer backbone to inhibit bacterial growth by contact mechanism allowing an improved control of the antibacterial activity and a long-lasting effect owing to the non-leaching of them from the photocrosslinked matrix .
Materials and methods
Bisphenol A glycerolate dimethacrylate (BisGMA), lithium hydride, succinic anhydride, camphorquinone (CQ), 4-(dimethylamino) phenylacetic acid (DMPheAA), silver nitrate (AgNO 3 ) and zinc methacrylate were purchased from Sigma Aldrich Chemical Co. and used without further purification. The commercial monomers used in this study are 2-hydroxyethyl methacrylate (HEMA) and triethyleneglycol dimethacrylate (TEGDMA) (Sigma–Aldrich Chemical Co.).
Synthesis of the BisGMA derivatives modified with carboxyl groups
The synthesis conditions for the obtaining of BisGMA derivatives modified with carboxyl groups (BisGMA-COOH1, BisGMA-COOH2, BisGMA-COOH3) were similar ( Scheme 1 ), the differences arising in the molar ratio of the reactants, and therefore the detailed experimental pathway will be described only for BisGMA-COOH1 monomer. Thus, 10 g (19.5 mmol) BisGMA were dissolved in 100 mL anhydrous tetrahydrofuran (THF) and the solution was cooled to 0 °C. Next, 0.114 g (13.6 mmol) LiH was added and the stirring was continued for 30 min, at the same temperature to give a lithium alkoxide. Further, 1.41 g (13.6 mmol) succinic anhydride was poured into the reaction vessel and the temperature was kept at 0 °C for another 90 min. After that, the reaction mixture was brought to room temperature and stirred for another 24 h, and moreover it was heated at 35–37 °C for another 24 h. The reaction mixture was neutralized with HCl 37% solution (1.2 mL, 13.6 mmol) and the solution was filtered. After the removal of the solvent on a rotary evaporator, the BisGMA-COOH1 product was dissolved in CH 2 Cl 2 and the organic phase was washed 3 times with distilled water. The resulting extract was dried over anhydrous Na 2 SO 4 and the solvent was removed under reduced pressure with a rotary evaporator at 30–35 °C, the carboxyl-functionalized BisGMA being collected.
BisGMA-COOH1 . Yield: 9.1 g (86%). FTIR (KBr, cm −1 ): 3458 (OH); 2966–2874 (C H); 1719 (C O); 1637 and 815 (CH 2 = C); 1171 (C O C). pH = 3.23; η = 39 Pa s.
BisGMA-COOH2 . Yield: 9.8 g (88%). FTIR (KBr, cm −1 ): 3477 (OH); 2967–2876 (C H); 1720 (C O); 1637 and 815 (CH 2 = C); 1166 (C O C). pH = 3.02; η = 25 Pa s.
BisGMA-COOH3 . Yield: 9.4 g (81%). FTIR (KBr, cm −1 ): 3471 (OH); 2966–2876 (C H); 1719 (C O); 1636 and 815 (CH 2 = C); 1164 (C O C). pH = 2.34; η = 12 Pa s.
The synthesis of photopolymerizable triclosan derivative (TCS-UMA) was previously reported , and its structure is represented in Scheme 2 .
The structures of the monomers were confirmed by the 1 H NMR spectra (registered on a Bruker Avance DRX 400 spectrometer) recorded in CDCl 3 at room temperature. Fourier transform infrared (FTIR) spectra were made on a Bruker Vertex 70 FT-IR spectrometer by applying the samples in thin films on KBr pellets. All spectra were collected in the range 4000–400 cm −1 . The stretching vibrations of C O and OH absorptions in the FTIR spectra were deconvoluted in order to evaluate the contributions on hydrogen bonding. The carbonyl absorption was deconvoluted from 1850 cm −1 to 1650 cm −1 , while the hydroxyl absorption was deconvoluted from 3700 cm −1 to 3100 cm −1 . The baseline correction, normalization and peak areas calculations were performed by OPUS 6.5 software, while the peak positions were determined using the second derivative of the spectra. Viscosity measurements for the BisGMA-COOH1 ÷ 3 monomers were performed with a Dial Reading Brookfield viscometer at 25.0 ± 0.2 °C, in triplicate. The test was run with a RV/HA/HB-7 spindle, at spindle speeds of 6 and 12 rpm, and the viscosity readings obtained were recorded and expressed as Pascal second (Pa s).
For the FTIR photopolymerization experiments, small amounts of the experimental adhesive formulations detailed in Table 1 , containing 0.5 wt% CQ and 1 wt% DMPheAA as photoinitiator system were placed between two KBr pellets and the FTIR spectra of the uncured samples were recorded. The samples were then light cured for 60 s with a Demetron Optilux curing light (Kerr company, USA; light intensity > 500 mW/cm 2 ) and the FTIR absorption spectra were registered after the irradiation. Triplicate specimens of each monomer mixtures were polymerized and analyzed.
The degree of conversion (DC%) was determined from the ratio of the peak areas from the aliphatic C C bond (1637 cm −1 ) and the reference aromatic C C bond (1608 cm −1 ), measured by using the baseline technique according to the formula :
DC = 100 − [ [ peak area of cured aliphatic C = C peak area of cured aromatic C = C ] [ peak area of uncured aliphatic C = C peak area of uncured aromatic C = C ] × 100 ]
The pH of BisGMA derivatives modified with carboxyl groups and of the corresponding formulations was measured using an ExStik™ Waterproof pH meter (model PH100, Extech Instruments). Calibration of the pH meter was performed using commercial buffer standards at pH 4.00, 7.00, and 10.00. A drop of each sample was placed on a glass slide and positioned into a full contact with the flat contact electrode of the pH meter.
The formulations for contact angle, water sorption and water solubility determinations were prepared according to the mass percents listed in Table 1 . Disc-shaped specimens (15 mm diameter, 1 mm thickness) were photopolymerized upon their exposure to irradiation using a dental light source at room temperature for 1 min on each side. The static water contact angle measurements were made on these samples using KSV Cam 200 goniometer. 2 μL droplets of double-distilled water were placed on the disc specimen surface, the average contact angle being calculated starting from at least ten separate measurements. The water sorption and water solubility values were determined by preparing five disk specimens of reduced dimensions (15 mm diameter, 1 mm thickness) for each group of mixtures, using a Teflon split ring mold between two glass plates covered with polyethylene film. The specimens were pre-conditioned over a desiccant containing calcium sulfate at 37 °C until their weight remain constant (initial weight m 1 ). Further, specimens were placed in distilled water at 37 °C for seven days and then removed from the water, lightly blotted with a paper to eliminate the surface-adherent water, and weighed again (m 2 ). After that, the specimens were placed into a desiccator containing calcium sulfate and dried at 37 °C until their weight was constant (m 3 ). The values (%) for water sorption, water solubility, and water uptake were calculated using the equations:
Water sorption (%) = m 2 − m 1 m 1 × 100
Water solubility (%) = m 1 − m 3 m 1 × 100