An alternative methodology is described to study passive diffusion of residual monomers through the coronal dentinal tubules stimulated via eluent liquids surrounding the root structures only.
Measurements have been conducted at 37 °C over a 7 d period and key variables including residual dentin thickness and eluent media have been considered.
GC/MS methods have been used to detect and quantify a range of dimethacrylate monomers in the eluent media and the kinetics of elution.
Critical periods post-restoration for exposure of different monomers to pulp cells have been identified.
Ethoxylated bisphenol A dimethacrylate (bisEMA) is a base monomer in several dental resin composites. It was the main aim of the present study to determine if bisEMA can reach the dental pulp by generally passive diffusion through the coronal dentinal tubules stimulated via eluent liquids surrounding the root structures only.
In 20 human third molar teeth, standard Class-I occlusal cavities were prepared and provided either with an adhesive system alone or additionally with a composite restoration, according to the instructions of the manufacturer. The teeth were placed in an elution chamber such that the elution media only came into contact with the tooth root/tooth base where they were incubated at 37 °C for up to 7 d. Samples were taken after 1, 2, 4 and 7 d. Gas chromatography/mass spectrometry was used to identify bisEMA and other monomers in ethanol/water (3:1) and aqueous eluates.
bisEMA was only found in ethanol/water eluates, where the teeth had received a composite restoration. Traces of bisEMA with up to three ethylene oxide units could be detected in these eluates. Depending on the dentin thickness, different elution kinetics of bisEMA were determined. Regardless of the treatment of teeth, triethylene glycol dimethacrylate (TEGDMA) and tetraethylene glycol dimethacrylate (TEEGDMA) were found in ethanolic/aqueous eluates in equal amounts. Most TEGDMA and TEEGDMA diffused through the dentin within the first 24 h.
Depending on the dentin layer thickness, bisEMA was released for varied time periods, resulting in varied concentrations and exposure times for the different cells of the dental pulp. The concentrations of TEGDMA and TEEGDMA were greatest for cells of the dental pulp within the first 24 h.
Over recent decades, the number and placement of methacrylate-based dental restorative materials has risen sharply. Methacrylic acid and its derivatives are a widely used substance class for dental monomers [ ]. In addition to composite restorations they are also used in glass ionomer cements, resin-based luting composites, root fillings and adhesive systems as well as in pit and fissure sealants. The physical properties of methacrylic acid derivatives range from fluid to highly viscous liquids, from hydrophilic to lipophilic, from low to high molecular mass. Particularly widespread derivatives are low-viscosity and low-molecular mass monomers, mainly 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and/or tetraethylene glycol dimethacrylate (TEEGDMA), as well as the high-viscous and high-molecular mass substances such as bisphenol A glycidyldimethacrylate (bisGMA) and ethoxylated bisphenol A dimethacrylate (bisEMA; bisphenol A ethoxylate dimethacrylate). In addition to these substances, which are largely responsible for the mechanical and physical properties of the restorative/bonding system, there are other components such as camphorquinone (CQ), which are important for photopolymerization. The compositions of methacrylate-based materials differ according to their function. Some of the monomers used have been detected in eluates ( e.g. aqueous, methanolic, artificial saliva) after polymerization of the restorative material [ , ].
This is possible because the degree of conversion (DC) of the carbon–carbon (C C) double bond of the methacrylate-based monomers varies between 40 to 80% and therefore unreacted substances can be released [ , ]. It is well known that released monomers, co-monomers, initiators, stabilizers, decomposition-products or contaminants can lead in vitro to cytotoxic, teratogenic, estrogenic, mutagenic and/or genotoxic effects as well as to allergic reactions in vivo, such as allergic contact dermatitis and asthma in humans [ ]. The DC decreases with increasing layer thickness towards depth [ ]. This is explained by the fact that the light cannot penetrate so deeply and therefore less radicals are formed in the depth, which start the polymerization, which in turn leads to reduced conversion of the monomers to the polymer. Applied to a standard dental restoration, this means that light-induced polymerization is initiated via occlusal light delivery and the light penetration and resultant DC may decrease towards the pulp.
The released substances can be eluted by saliva and enter the gastrointestinal tract when swallowed, but can also reach the dental pulp by diffusion through the coronal dentinal tubuli. In vitro it was shown that uncured monomers leaching from composites can diffuse through dentin and reach the pulp within minutes after placement of a restorative material [ ]. Within the dental pulp some of the substances can lead to a cytotoxic response. Thereby consequences may arise such as post-operative pain, pulpitis and in worst cases pulp necrosis [ , ]. The diffusion of substances through the dentin tubuli can be simulated by different models such as teeth in a chamber [ ] or the use of dentin slices with variables such as dentin thickness, test setup, bovine or human teeth slices and further pretreatment of the teeth [ , ]. Dentin discs with a thickness of 300–500 μm are often sawn from human teeth [ , ]. These dentin discs are usually subjected, on one side, to the usual restorative preparation, as also performed in humans, and then placed in a pulp chamber model, where different simulations are performed. These include pure diffusion, after different exposure times to light, or diffusion using different elution media and/or additives to elution media. Usually the dentin discs have to be pretreated — often by removing the smear layer with 50% citric acid. The cutting process as well as the further processing afterwards could change the diffusion behavior, since the procedure could affect the dentin tubuli, by mechanical and/or thermal influences during sawing or via demineralisation by the strong chelator citric acid. In addition, the thickness of the dentin discs determines the diffusion distance, which can be significantly shorter than the physiological thickness of the dentin layer. Moreover, tubule diameters are greater closer to the pulp, so the outcome can depend upon the axial height at which the dentin disks are cut. The diffusion path could influence the amount and type of substances that can be eluted ( e.g. low or high molecular mass substances, and hydrophilic or hydrophobic molecules).
To avoid the above-mentioned limitations, we have developed a model in which human teeth without further pretreatment are treated with a standardized cavity restoration as used in routine dental treatment of patients. The closeness to real patient care is a very important point for us. For this reason, we have chosen a standard Class I restoration of a tooth with a carious lesion. As in reality, a standard restoration can leave different residual dentin thicknesses.
The cavities can be restored with an adhesive system alone or with an adhesive system and a light-cured, commercially available composite. These teeth can be placed in a special test chamber, where the diffusion of substances/monomers through the dentinal tubuli into different elution media can be determined precisely since other surfaces are not in contact with the different elution media ( Fig. 1 ). The focus of this investigation was on: (i) the detection in the eluate of high-molecular mass methacrylate-based monomers particularly bisEMA and its homologous ( Fig. 2 ) indicating a diffusion through the residual dentin barrier and (ii) the determination of temporal diffusion kinetics as a function of dentin thickness and elution medium. Furthermore, the experimental setup can be used to estimate the proportion of monomers originating from the adhesive system and the proportion of monomers originating from the composite. Two different gas chromatography/mass spectrometry (GC/MS) methods were used: (a) for the determination of the higher boiling substances such as bisEMA and (b) for the lower boiling substances such as TEGDMA.
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and were of the highest purity available. The dental restorative materials used and their compositions are listed in Table 1 .
|Materials||Manufacturer colour, batch||Resin matrix||Filler||Filler wt/vol|
|Tetric EvoCeram®||Ivoclar Vivadent/A3 Y09076||Dimethylacrylate||Barium glass, Ytterbium trifluride, mixed oxide and copolymer||17/n.s.|
|Syntac® Primer||Ivoclar Vivadent Y05344||TEGDMA|
|Syntac® Adhesive||Ivoclar Vivadent X46682||PEGDMA|
|Heliobond®||Ivoclar Vivadent X48463||bisGMA TEGDMA||60/40|
20 extracted human third molar teeth were used and prepared for our examination. Ethics approval for the extracted molars was obtained by the Commission for Medical Ethics of the Ludwig-Maximilians-University, Munich (July 2018, No 18-551DE). The teeth selected fulfilled the following criteria: no dental caries or decay, no lesions and matured tooth growth. The teeth were stored after extraction and prior to treatment in Ringer’s solution mixed with 0.1% sodium azide, without further preparation steps such as sterilization.
The preparation process was initiated after the teeth were rinsed under water for 10 min. Standardized cylindrical Class I inlay cavities (3 mm long, 3 mm wide and 3 mm deep) were drilled in each tooth using a 151 μm grain size diamond drill (Komet Dental Brasseler, Lemgo, Germany) for the initial preparation and a 46 μm grain size drill (Komet Dental Brasseler) for the finishing surface at 40,000 rpm and under constant water cooling. The treated surfaces were completely dried, corresponding to clinical drying using a rubber dam.
Each cavity was etched using a 37% phosphoric acid gel (Ivoclar Vivadent, Schaan, Liechtenstein). This was applied for 30 s on the enamel and for 15 s on the dentin surfaces. The phosphoric acid was thoroughly rinsed off with water and the surface dried using air flow.
10 teeth were treated using only the three-step adhesive system: Syntac® Classic (Ivoclar Vivadent). The adhesive system was applied according to the Manufacturer’s instructions. Briefly, a primer liquid was applied using a disposable brush for 15 s and carefully blown-off and dried. Subsequently an adhesive liquid was applied for 10 s and thoroughly dried in an air flow. Heliobond® (Ivoclar Vivadent), as a bonding layer, was applied and thinly dispersed.
The adhesive system was cured for 10 s using a light-curing unit (Satelec® Acteon®, Mini L.E.D. SN 304863181, Merignac Cedex, France) with an output irradiance of 1000 mW/cm².
10 further teeth were treated with the adhesive system (as above) and the cavities were filled with the composite: Tetric EvoCeram® (Ivoclar Vivadent). The cavities were filled via a layering technique according to the manufacturer’s instructions (max. 2 mm layer), whereby each layer was cured for 20 s. using a light-curing unit (Satelec® Acteon®) with an irradiance of 1000 mW/cm².
After preparation, the teeth were assigned into different groups according to the elution medium (ethanol/water (3:1) or distilled water) and their dental treatment. Four groups with five teeth each were formed: EW-A : Teeth only treated with the adhesive system (A) were incubated in ethanol/water (3:1; EW); EW-AC : Teeth treated with the adhesive system and composite (AC) were incubated in ethanol/water (3:1; EW); W-A : Teeth only treated with the adhesive system (A) were incubated in distilled water (W); W-AC : Teeth treated with the adhesive system and composite (AC) were incubated in distilled water (W). Subsequently, the group abbreviations were followed by the tooth number.
The teeth were mounted in 5 ml glass vials (Wagner & Munz, Munich, Germany) using a wire fixture that ensured that the root only would be completely immersed in the elution medium, while the crown remained above the surface of the elution medium. The fixture was made of a dental wire wound around the tooth neck at the enamel border ( Fig. 1 ).
Then the elution medium was added. Depending on the size of the tooth, between 3.5 and 4.5 ml of the elution liquids were added to reach the required level up to the crown of the tooth (see paragraph above). Then the glass vials were sealed and incubated at 37 °C. After 1, 2, 4 and 7 d, 100 μl from each specimen vial were taken and stored in GC-vials (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The removed liquids were replaced with the same volume of either ethanol/water (3:1) or distilled water.
The diffusion of the leached monomers depended on the thickness of the residual dentin. Therefore, it was determined. The shortest distance between the bottom of the cavity and the pulp chamber was measured by X-ray images (10 cm distance, 60 kV at 0.08 s, irona Dental Systems, Wals, Austria; ModellTyp: No 3343741) A 20 mm endodontic file (Fa VDW GmbH Munich, Germany; Typ: K-Feile-ISO 30) was used as the reference scale.
The analysis of the eluates was performed on a Thermo Scientific™ TRACE™ 1310 gas chromatograph connected to a Thermo Scientific™ ISQ™ 7000 mass spectrometer (Thermo Fisher Scientific). Analysis was conducted using two different GC/MS methods. For reasons of comparability, a standard method was applied. A second method, working at higher temperatures and with, necessarily, a suitable GC column, was used to increase the recovery of higher boiling components. With the first method “normal” substances like TEGDMA and TEEGDMA were determined. A Thermo Scientific™ TraceGOLD™ TG5Silms capillary column (length 30 m, inner diameter 0.25 mm, coating 0.25 μm; Thermo Fisher Scientific) was used as the capillary column for GC separations under standard operating conditions. The GC oven was heated from 50 °C (2 min isotherm) to 300 °C (5 min isotherm) with a rate of 10 °C/min and 1.0 μl of the solution was injected via a programmed temperature vaporization (PTV) injector. Helium was used as carrier gas at a constant flow rate of 1.0 ml/min. The temperature of the PTV injector was 50 °C during the injection phase and then increased to 300 °C with a rate of 14 °C/s. The temperature of the direct coupling (transfer line) to the MS was 300 °C. The MS was operated in electron ionisation mode (EI, 70 eV), with the ion source operated at 300 °C; only positive ions were scanned. Scans were made over the range m/z 40–600 at a scan rate of 5 scan/s for scans operated in full scan mode for identification. Caffeine was used as internal standard.
An additional analysis (second method for the determination of bisGMA as well as bisEMA and its homologous) at higher temperatures was done using a TraceGOLD TG-XLBms column, 20 m × 0.18 mm ID × 0.18 μm FT (Thermo Fisher Scientific). The GC oven was programmed from 50 °C for 1 min to 330 °C for 5 min at a rate of 15 °C/min. PTV injection (1.0 μl) was done at 50 °C increasing to 350 °C with a rate of 14 °C/s. The MS transfer line and MS ion source were heated to 320 °C. GC/MS system, carrier gas settings and scan range were the same as for the first method. In the single ion mode (SIM) targeted analysis of the bisEMA homologues was performed for the following m/z : 452, 496, 525, and 569 for quantitation of the analytes. In the case of the analytics of the dissolved composite in methanol the scans were made over a range m/z 100–800 at a scan rate of 5 scan/s.
The integration of the chromatograms was carried out over the base peak or other characteristic mass peaks of the compounds, typically M + and M + -15. Identification of the various substances was achieved by chemical analysis of their fragmentation patterns [ ].
Calculations and statistics
The analytical results are presented as means. The standard deviation (SD) was calculated from the variation of the caffeine signal. The evaluation regarding the amount of an analyte was based on the peak area under the curve for each analyte. An alternative parameter was the peak height. Due to the different thickness of the remaining dentin, the quantities of an analyte found (area under the curve) were also related to the residual thickness. For appropriate cases, the measured quantity of the analyte was divided by the measured thickness of the residual dentin. For analytes where there was a significant progressive time-dependence of the released molecules, a non-linear regression analysis was used to model the elution kinetics. For this purpose, a 3-parameter single exponential function with an increase to a maximum, a standard function in physics for the description of such kinetic processes, was used.