Neutralization of the acidic micro-environment at the tooth/material interface is expected to provide enhanced durability for dental composite restorations. The objective of this study is to explore the effect of amine-containing monomer formulations and the crosslinking density of the resultant polymers on the neutralization capacity.
Materials and methods
The co-monomer system was varied systematically to obtain different proportions of Bisphenol A glycerolate dimethacrylate (BisGMA) and 2-hydroxyethyl methacrylate (HEMA), while maintaining a constant amount of amine-containing methacrylate monomer. A series of amine-containing monomers covering a range of p K a values were examined. Crosslinking density of formed copolymers was controlled by adjusting the weight content of the dimethacrylate monomer BisGMA. Lactic acid (LA) was used as a probe to analyze the effectiveness of the basic polymers to neutralize acid. The neutralization capacity of each amine-containing crosslinked copolymer was characterized by measuring pH as a function of time when the specimens were soaked in 1-mM LA solution, and the results were compared to the control formulations composed solely of BisGMA and HEMA. Polymer surfaces were examined using the methyl orange (MO) assay to quantify the amount of accessible amine groups.
For each amine-containing crosslinked co-polymer, the neutralization capacity is enhanced by decreasing crosslinking density (e.g., by reducing BisGMA concentration in the formulation). In addition, more amine groups were accessible when crosslinking density was decreased. For different amine-containing polymers with the same BisGMA concentration, the neutralization capacity is higher when the amino monomers with higher p K a values were used in the formulations.
This is the first time that the neutralization capacity based on crosslinked dental polymers has been studied. The information is important for future development of durable dentin adhesives.
Polymer-based composites have become the most common restorative material and are currently used more than twice as often as dental amalgam . These resin composites fulfill many of the requirements for clinical restorative applications, including excellent esthetics. The durability of composite restorations does not, however, match that of dental amalgam . The average clinical lifetime of composite resin restorations is just 5.7 years due to recurrent decay or fracture . Recurrent decay has been linked to the failure of the bond between the tooth and composite and increased levels of the cariogenic bacteria Streptococcus mutans at the perimeter of these materials .
The composite is too viscous to bond directly to the tooth; a low-viscosity adhesive is used to connect the tooth to the composite. The adhesive bonds effectively to the acid-etched enamel, but bonding to dentin has been fraught with problems. In vitro and in vivo studies suggest that several factors inhibit the formation of a durable adhesive/dentin bond. One of the important factors is water sorption and hydrolysis of the adhesive polymers . Water and saliva are always present in the mouth of healthy patients, and they are expected to penetrate into the free volume spaces between polymer chains. With water penetration, hydrolysis of ester groups in the polymer chains may occur, which shortens the clinical lifetime of polymer-based dental restoratives.
Another important factor is the polymer network structure, which is induced by photopolymerization of methacrylate monomers. Due to the rapid polymerization rate, after polymerization, highly crosslinked networks are formed, which are usually very heterogeneous due to the formation of highly crosslinked regions and loosely crosslinked regions . The more heterogeneous a material, the more likely it is to have a significantly weaker structure in the regions of lower crosslinking, increasing the risk of premature failure.
The monomers used in dental restorative materials are particularly critical because polymerization of monomers produces the crosslinked matrix in the resultant polymers. Thus, monomer selection exerts considerable influence on the properties, durability and behavior of dentin adhesives in the wet, oral environment. Much attention and effort has been devoted to developing new adhesive monomers in order to enhance the lifetime of dental composite restorations .
Introducing neutralization capacity by using amine-containing monomers offers a promising approach to enhance hydrolysis resistance. The presence of water promotes the chemical hydrolysis of ester bonds in methacrylate materials . This reaction might be relatively slow at the neutral pH typical of saliva, but excursions in pH, caused by food or cariogenic bacteria that produce lactic acid, may lead to transient acid catalysis . Degradation of methacrylate ester groups produces carboxylic acids, which contain the same functional group that is the culprit in lactic acid-induced decay. Degradation products from ester hydrolysis are more hydrophilic than the parent ester, which further enhances the local ingress of water and hydrolysis. With time, local domains of the methacrylate network may become sufficiently degraded and/or hydrophilic to permit access by esterases that greatly accelerate ester bond hydrolysis.
The chemical and enzymatic degradation of the methacrylate-based matrix could create a low pH environment at the composite/tooth interface because of the low p K a values of methacrylic acid and lactic acid, which are 4.66 and 3.86, respectively . Furthermore, acidification of the oral microenvironment promotes demineralization of tooth structure at the margin of composite restorations. The increased surface roughness of the demineralized tooth surface creates additional opportunity for adhesion by biofilm, mainly salivary proteins and pioneer pathogenic bacteria, thereby accelerating the degradation process. We proposed that the pathogenic impact of biofilm at the margin of the composite restoration could be reduced by engineering novel dentin adhesives that neutralize the acidic microenvironment and resist biofilm attachment . Integrating basic moieties with an appropriate p K a into methacrylate derivatives provides the opportunity to act as an acid-neutralizing proton sponge and/or buffer to protect against acid-induced degradation.
In our previous work, the neutralization capacity of amine-containing monomers was quantified in a water/ethanol co-solvent system and the effects of solvent environment on p K a were examined . However, to our knowledge, there are no reports of neutralization capacity having been determined for amine-containing crosslinked polymers. As such, it is necessary to determine the correlation between the interrelated properties of neutralization capacity and crosslinking density/chemical structure of amine-containing monomers within crosslinked polymer systems.
The twofold objectives of this work are: (1) to study the crosslinking effect on the neutralization capacity of the polymer, and (2) to investigate the influence of the chemical structure of amine-containing monomers on the neutralization capacity. To our knowledge, this investigation marks the first study on the neutralization capacity of crosslinked dentin adhesives. The results provide important information to enable future development of durable dental restorative materials.
Materials and methods
Camphorquinone (CQ), ethyl-4-(dimethylamino) benzoate (EDMAB) and diphenyliodonium hexafluorophosphate (DPIHP) were used as a three-component-photoinitiator system. Bisphenol A glycerolate dimethacrylate (BisGMA) and 2-hydroxyethyl methacrylate (HEMA) were used as co-monomers. 2-(dimethylamino) ethyl methacrylate (DMAEMA), 2-(diisopropylamino) ethyl methacrylate (DIPAEMA), 2-( tert -butylamino) ethyl methacrylate (TBAEMA) and 2- N -morpholinoethyl methacrylate (MEMA) were used as amine monomers. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The chemical structures of monomers are shown in Table 1 .
Preparation of adhesive formulations
The preparation of the adhesive formulations has been reported . There are three control adhesive formulations, consisting of HEMA and BisGMA with a mass ratio of 45/55 (C0-55), 70/30 (C0-30), 85/15 (C0-15), which were used for comparison to the amine-containing experimental adhesive resins. For all experimental formulations, the weight content of amine monomer is 25 wt%. The methacrylate formulations are comprised of HEMA/amine monomer/BisGMA, and differ in that the content of BisGMA was decreased from 55, 30 to 15 wt% in order to examine the crosslinking effect on neutralization capacity at a constant amine concentration. Table 2 shows the adhesive formulations with weight percentage and molar ratio. A three-component photoinitiator system was used that contains CQ (0.5 wt%), EDMAB (0.5 wt%) and DPIHP (1.0 wt%). The resin mixtures were prepared in brown glass vials. The mixtures were shaken on an orbital shaker for two days to dissolve the initiators and form a homogeneous solution.
|Samples||HEMA (wt%)||Amine monomer (wt%)||BisGMA (wt%)||Mole of amine/gram (10 −3 )||Molar ratio a|
Real-time in situ monitoring of the photopolymerization of the adhesive formulations was performed using an infrared spectrometer (Spectrum 400 Fourier transform infrared spectrophotometer, Perkin-Elmer, Waltham, MA) at a resolution of 4 cm −1 . One drop of adhesive solution was placed on the diamond crystal top-plate of an attenuated total reflectance (ATR) accessory (Pike, GladiATR, Pike Technology, Madison, WI) and covered with a mylar film. A 40-s-exposure to the commercial visible-light-polymerization unit (Spectrum 800 ® , Dentsply, Milford, DE, ∼480–490 nm ), at an intensity of 550 mW cm −2 , was initiated after 50 spectra had been recorded. Real-time IR spectra were recorded continuously for 600-s after light curing began. A time-resolved spectrum collector (Spectrum TimeBase, Perkin-Elmer) was used for continuous and automatic collection of spectra during polymerization. Three replicates were obtained for each adhesive formulation.
Preparation of polymer samples
Disc samples with a thickness of 1 mm and a diameter of 4 mm were prepared by injecting the adhesive formulations into hermetic lids (TA instruments, T 120110, USA) and each covered with a round glass coverslip (Ted pella, Inc., Prod no. 26023). Ten specimens were prepared for each formulation. The samples were light polymerized with a 40-s exposure to the commercial visible-light polymerization unit (Spectrum ® , Dentsply, Milford, DE) at an intensity of 550 mW cm −2 . The polymerized samples were stored in the dark at room temperature for two days to provide adequate time for post-cure polymerization. The resultant disc samples were used for prewash, neutralization and water sorption experiments.
Prewash of disc samples
Five disc samples for each formulation were placed in a flask containing 100 mL deionized water and shaken at 37 °C for 5 days. The water was changed daily. At day 6, the solution was removed. The specimens were rinsed with water and then dried in a vacuum oven at 37 °C for 15 days.
Neutralization experiments and pH measurements
A 2 mL volume of 1-mM lactic acid (pH 3.5) was added into the vial and mixed with 0.2 g of disc samples. The vials containing the control and experimental solutions were placed on the shaker at room temperature and the pH was measured at fixed time intervals. The pH measurements were performed with a Fisher Scientific (Waltham, MA, USA) Accumet Research AR25 pH meter equipped with a micro-probe. Calibration was done using commercial buffer standards (Fisher Scientific, pH 4.01, 7.00, and 10.01)
The water sorption protocol has been reported previously . After prewash, the dry disc samples ( m 1 dry ) were then immersed in deionized water and stored at room temperature. At fixed time intervals (6, 24, 48, 72, 120, 168 and 240 h), the polymer samples were retrieved, blotted dry to remove excess liquid, weighed ( m 2 wet ) and re-immersed in the water. The value (%) for mass change due to water sorption was calculated as follows: <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='Masschange%=100m2wet−m1drym1dry’>Masschange(%)=100m2wet−m1drym1dryMasschange%=100m2wet−m1drym1dry
Mass change % = 100 m 2 wet − m 1 dry m 1 dry
Methyl orange (MO) assay to detect the surface amine groups
The methyl orange assay was performed as reported previously . A 0.2 wt% methyl orange (Fisher Scientific) stock solution was prepared using deionized water. This solution was diluted to 0.05 wt% with 0.1 M NaH 2 PO 4 solution (Fisher Scientific) to prepare an acidic methyl orange solution, such that the final buffer concentration was 0.075 M NaH 2 PO 4 and solution pH was 4.4.
Samples were prepared in 96-well plates. To make the polymer coating, 60 μL of resin formulation was added to each well. Each resin formulation was analyzed in triplicate. Polymerization and curing in the 96-well plates was performed using oxygen-depleted conditions in which the plate was placed in a sealed enclosure and purged with nitrogen for 10 min. Monomer resins were cured in a curing chamber (Triad ® 2000™, visible light cure system, Dentsply, York, PA) at an intensity of 50 mW cm −2 with a 16-min total exposure time in which the plate was rotated by 45 degrees every 2 min to ensure complete polymerization in all wells. The plate was left in the dark for post polymerization for 48 h. At 48 h 0.3 mL of water was added to each well. The water was changed twice during each week to remove leachable components. After 5 weeks prewash, water was removed and polymers were rinsed under a stream of deionized water for approximately 10–30 s, and excess water was removed by blotting samples with a Kimwipe. 50 μL of acidic MO solution was added in each well and incubated for 5 h. The MO solution was decanted and the wells were washed with deionized water 10 times; excess water was removed by blotting samples with a Kimwipe. 200 mL 0.1 M Na 2 CO 3 (Fisher Scientific) solution, pH 11.0 was added in each well and incubated for 72 h. The final solution in each well was extracted; the solution was diluted 40 fold, and transferred to a new plate for analysis.
The 96-well plate was read using the plate reader (CYTATION 3 imaging reader, BioTek Instruments, Inc., Minooski, Vermont, USA) in bottom-read absorbance mode. Methyl orange concentration was determined from measuring absorbance at 465 nm and comparing the data to a standard calibration curve. The standard curve was linear over the concentration range investigated. Finally, the accessible amine density on the polymer surface was calculated based on the surface area of a well in a 96-well plate, which was calculated to be 0.33 cm 2 .
The results were analyzed statistically using analysis of variance (ANOVA), together with Tukey’s test at α = 0.05 (Microcal Origin Version 8.0, Microcal Software Inc., Northampton, MA).