INTRODUCTION

Temporary restorations play an integral part in most prosthetic treatment courses as they are used from tooth preparation until placement of the final restoration [1]. During this time, temporary crowns and bridges serve to maintain the esthetics and functionality of the masticatory system and to protect teeth from thermal, mechanical, and microbial noxae [2, 3]. In prosthetic dentistry, temporary restorations are fabricated with resin-based crown and bridge (C&B) materials, which can be subdivided into methacrylate resins (liquid/powder, hand-mixed) and composite resin-based materials [4, 5]. As a result of their mechanical and esthetic properties, composites are currently being used as state-of-the-art materials [5, 6]. The organic polymer matrix of C&B composite materials is very similar to that of composites used for dental fillings [7]. The monomers predominantly used are bisphenol A-glycidyl methacrylate (Bis-GMA), urethane dimethacrylate (UDMA), and an additional co-monomer, which is usually triethylene glycol dimethacrylate (TEGDMA) [8-10].

In recent years, the release of residual monomers, and especially bisphenol A (BPA), from dental composites has been a cause for public concern [11]. However, pure BPA is not being used as a monomer in dentistry, and thus only small amounts are leachable as a result of possible contamination from the use of BPA derivatives [12, 13]. Composites for restoring dental cavities are similar to C&B composites in terms of composition but differ with regard to the initiator system used, as the latter is self-curing or dual-curing and not light-curing [14].

In contrast to light-cured materials, C&B materials show the disadvantages of self-curing composites, such as a lower degree of conversion and air porosities [15-17]. Prior studies on core build-up materials have shown that insufficiently converted composites lead to sustained monomer elution [18-20]. As a result, some manufacturers have substituted BPA derivatives with UDMA and have introduced BPA-free composites in order to avoid the release of BPA and its derivatives [21-23]. As monomer elution depends on the extraction ratio (the ratio of surface area to solvent volume) [24-26], the larger surface area of temporary crowns and bridges compared with that of fillings and the use of self- or dual-curing materials could increase biocompatibility concerns. Therefore, this study aimed to examine the monomers released from C&B materials and to compare the concentrations of monomers released from conventional and BPA-free materials. The in vitro set up applied was intended to simulate clinical practice as closely as possible. In accordance with the clinical workflow, all samples were immersed in water immediately after preparation and incubated at 37 ̊C, with mild agitation, to simulate the oral environment. Almost identical concentrations of monomers were released from samples incubated in water compared with those incubated in artificial saliva [27, 28], whereas monomer release was increased from samples incubated in strongermedia, such as ethanol/water mixtures [26, 27, 29], which is attributed to the softening of Bis-GMA-based resins and the formation of soluble units [30-32]. Instead, stronger media are better suited for assessing potential long-term health risks [33]. Although the release of BPA and its derivatives is the focus of current research and these substances are considered a potential health risk [34-36], it is known that small quantities, at most, of Bis-GMA and BPA are released in aqueous media, such as water, artificial saliva, or collected saliva [26-28]. As the investigated materials are present in the oral cavity only for a short period of time and temporary restorations are luted directly after fabrication [37], water was used as the extraction medium in the present study and all samples were immersed in water immediately after preparation. The tested null hypotheses were: (a) monomer/BPA elution is material dependent; and (b) BPA and Bis-GMA are either not released or released only in very small amounts.

MATERIAL AND METHODS Study design

To test the stated hypotheses, samples of four different C&B materials were prepared according to the manufacturers’ specifications (discussed in the next section), then immersed immediately in high-performance liquid chromatography (HPLC)-grade water (Sigma Aldrich). The incubation time-periods were 1 h, 12 h, 24 h, and 7 days. Prior to calibration with the respective reference substances (Table 1), all eluates were analyzed for Bis-GMA, TEGDMA, and UDMA by high-performance liquid chromatography coupled with ultraviolet-visible spectroscopy and mass spectrometry (HPLC-UV/Vis-MS) and for BPA by high-performance liquid chromatography coupled with ultraviolet-visible spectroscopy (HPLC-UV/Vis) with confirmation by HPLC-tandem mass spectrometry (HPLC-MS/MS). Because of the high costs of HPLC-UV/Vis-MS measurements, the period of maximum elution corresponding to 24h of incubation according to the current literature was analyzed first. The absence of a monomer after 24 h of incubation was considered as sufficient evidence that the substance was not released from the material being examined. However, if a monomer was detected, all incubation periods were investigated to evaluate the respective elution patterns. Subsequently, the results were statistically evaluated in order to reveal potential material-dependent differences.

TABLE 1. Chemicals used for derivatization and high-performance liquid chromatography (HPLC) analysis Name Abbreviation Manufacturer Molecular mass (g mol–1) CAS-Nr. Purity Urethane dimethacrylate UDMA Sigma Aldrich 470.56 72869-86-4 > 97% Triethylene glycol dimethacrylate TEGDMA Sigma Aldrich 286.32 109-16-0 99% Bisphenol A BPA Sigma Aldrich 228.29 80-05-7 ≥ 99% Bisphenol A-glycidyl methacrylate Bis-GMA Sigma Aldrich 512.59 1565-94-2 Not specified Diethyl phthalate DEP Sigma Aldrich 222.24 84-66-2 99.5% Pyridine-3-sulfonyl chloride PSC Sigma Aldrich 177.61 16133-25-8 ≥ 98.0% Bisphenol A-d16 d16BPA Sigma Aldrich 244.38 96210-87-6 98 atom % Sample preparation

According to the manufacturer's specifications, standardized cylindrical samples were prepared of two conventional temporary C&B materials, Protemp 4 (3 M ESPE) and Luxatemp Automix Plus (DMG), and of two BPA-free materials, ExperTemp (Ultradent), and Visalys Temp (Kettenbach). Information about the composition of the C&B materials is given in Table 2. Chemical polymerization was initiated by placing all samples in polytetrafluorethylene (PTFE) molds (10 mm diameter; 10 mm height) using an automix dual cartridge. Excess material was removed using a glass slide and the samples were left undisturbed for the recommended setting duration (4 min for ExperTemp,7 min for Luxatemp Automix Plus, 5 min for Protemp 4, and 4 min for Visalys Temp). This resulted in samples (n = 5 for each combination of material and incubation period) with a surface area of 4.712 cm2. Following the manufacturer's instructions, all samples were washed with ethanol to remove the oxygen inhibited layer. For standardization, the samples were fully immersed in ethanol and agitated for 20 s. To remove excess ethanol, the samples were cleaned with HPLC-grade water.

TABLE 2. Composition of the crown and bridge materials tested Material Main monomers* Contains BPA* Manufacturer ExperTemp Aliphatic dimethacrylate, poly(alkylene glycol) diacrylate, hydroquinone monomethyl ether BPA free Ultradent Luxatemp Automix Plus Dimethacrylate – DMG Protemp 4 Dimethacrylate, reaction products of 1,6-diisocyantohexane with 2-[(2-methacryloyl)ethyl]6-hydroxyhexanoate and 2-hydroxyethyl methacrylate – 3 M ESPE Visalys Temp Aliphatic dimethacrylate, poly(alkyleneglycol)diacrylate, hydroquinone monomethyl ether BPA free Kettenbach * According to manufacturers’ information. Incubation

Ultraviolet light-protected borosilicate sample containers with PTFE-coated closures were used for sample incubation. Each vial was cleaned using HPLC-grade methanol (Sigma Aldrich) and HPLC-grade water. Corresponding to the clinical workflow, all samples were immediately immersed in 1.74 mL of HPLC-grade water after preparation. The extraction ratio (surface area/solvent volume) was chosen following ISO 10993-12 and all samples were fully covered with HPLC-grade water. Incubation was performed in an incubator shaker (Excella E24; New Brunswick Scientific) for 1 h, 12 h, 24 h, and 7 days at 37°C and 112 rpm. Following each period of incubation, the samples were removed, and to prevent secondary chemical reactions the eluate was frozen at −18°C and kept in the dark.

HPLC-UV/Vis-MS analysis

After thawing at room temperature, aliquots of 0.5 mL of each eluate and 0.5 mL of the internal standard, diethyl phthalate (Sigma Aldrich) (10.0 μg mL–1), were transferred into HPLC amber glass vials. For detection of BPA, an external calibration without diethyl phthalate was performed. The HPLC-UV/Vis-MS analysis was carried out on an Agilent 1200 SL (Agilent Technologies) with a Surveyor PDA Plus Detector (Thermo Fisher Scientific) coupled to an LTQ Orbitrap XL with high-resolution MS capability (Thermo Fisher Scientific). Separation was performed in a Kinetex 100A column (Phenomenex) with 150 × 2.1 mm dimensions and a 5 μm particle size. The column was kept at 25°C, and the injection volume was 10 μL. Solvent A was water with 0.05% (v/v) formic acid, and solvent B was methanol with 0.05% (v/v) formic acid. Gradient elution was applied as follows: 0 min, 60% A; 0–15 min, 60%–0% A; 15–22 min, 0% A. The scan range of the photodiode array (PDA) was set to 200–600 nm with a scan rate of 1 nm for Bis-GMA, TEGDMA, and UDMA analysis. For BPA analysis, the scan range was adjusted to 270–280 nm. Mass spectrometric analysis for detection of Bis-GMA, TEGDMA, and UDMA was performed using electrospray ionization (ESI) (see Table 3 for a summary of all technical settings). All measurements were performed in duplicate.

TABLE 3. Mass spectrometry settings (high-performance liquid chromatography coupled with ultraviolet–visible spectroscopy and mass spectrometry [HPLC-UV/Vis-MS]) Parameter Setting Ionization source Electrospray ionization Mass range 100–1000 m/z Source voltage 4 kV Capillary temperature 275°C Capillary voltage 42 kV Tube lens 125 V Sheath gas flow 50 arb Auxiliary gas flow 0 arb Resolution 60 000 Abbreviations: arb, arbitrary unit; m/z, mass-to-charge ratio.

As proposed by the American Chemical Society, the limit of detection (LOD) of all substances investigated was determined experimentally by measuring blanks and a dilution series (0.01 μg mL–1, 0.05 μg mL–1, and 0.5 μg mL) [38]. For all substances detected, the limit of quantification (LOQ) was determined by the lowest calibration standard on the calibration curve, as proposed by the European Medicines Agency [39]. The calibration curve of UDMA and TEGDMA consisted of five concentrations from 0.5 to 20.0 μg mL–1, while the calibration curve for BPA included four concentrations from 0.5 to 10.0 μg mL–1. The dilution solvent was methanol/water (20:80; v/v). The LOD and LOQ values of all substances are listed in Table 4. Calibration was validated by the distribution of data points on the residual plot and the coefficient of determination (r2). A uniform residual plot with r2 ≥ 0.95 was taken as evidence for the linearity of the calibration. The calibration for all analytes was linear within the calibration range and, in all calibrations, r2 > 0.99. Detection and quantification of Bis-GMA, UDMA, and TEGDMA were performed using HPLC-UV/Vis-MS. Due to low ionizability, BPA analysis was performed using HPLC coupled with ultraviolet–visible spectroscopy (HPLC-UV/Vis). To avoid false-positive results, all samples in which BPA was detected were prepared again and analyzed by HPLC-coupled tandem mass spectrometry (HPLC-MS/MS), using isotope-labeled BPA as the internal standard.

TABLE 4. Limits of detection and analytical methods used for quantification of the monomers Substance Analytical method Limit of detection Limit of quantification UDMA HPLC-UV/Vis-MS 0.05 μg mL–1 0.5 μg mL–1 TEGDMA HPLC- UV/Vis-MS 0.05 μg mL–1 0.5 μg mL–1 Bis-GMA HPLC -UV/Vis-MS 0.5 μg mL–1 – BPA HPLC-UV/Vis 0.5 μg mL–1 0.5 μg mL–1 dBPA HPLC-MS/MS 0.009 ng mL–1 0.03 ng mL–1 Abbreviations: Bis-GMA, bisphenol A-glycidyl methacrylate; BPA, bisphenol A; dBPA, derivatized BPA; HPLC-MS/MS, high-performance liquid chromatography coupled with tandem mass spectrometry; HPLC-UV/Vis, high-performance liquid chromatography coupled with ultraviolet—visible spectroscopy; HPLC-UV/Vis-MS, high-performance liquid chromatography coupled with ultraviolet–visible spectroscopy and mass spectrometry; TEGDMA, triethylene glycol dimethacrylate; UDMA, urethane dimethacrylate. HPLC-MS/MS

Immediately before measurements, the eluates were thawed at room temperature. An aliquot of 1.5 mL was transferred from each eluate into HPLC amber glass vials and the eluates were dried using a speed vacuum concentrator (RVC 2–25 CD plus; Christ) at 37°C. The dry samples were derivatized and analyzed immediately. Bisphenol A was derivatized using pyridine-3-sulfonyl chloride, as described by Regueiro et al. [40]. The derivatization scheme is shown in Figure 1. Isotopically labeled BPA-d16 (d16BPA) was used as an internal standard. The chemicals used for derivatization and HPLC analysis are listed in Table 1.

Derivatization of bisphenol A (BPA) with pyridine-3-sulfonyl chloride (PSC). BPA-diPS, bisphenol A derivatized with pyridine-3-sulfonyl chloride

The HPLC-MS/MS analysis was performed using an Agilent 1290 Infinity II HPLC system (Agilent Technologies) coupled to an Agilent 6460 triple quadrupole detector (Agilent Technologies). Separation was performed using a Polaris 3 C18-Ether column (100 × 2 mm with a 3 μm particle size; Agilent Technologies). The column was kept at 40°C, and the injection volume was 10 μL. Solvent A was water with 0.1% (v/v) formic acid, and solvent B was methanol with 0.1% (v/v) formic acid. The gradient was as follows: 0–0.2 min, 30% B; 0.2–6 min, 30%–98% B; 6–10 min, 98% B; 10–10.5 min, 98%–30% B; 10.5–14 min, 30% B. The eluent was ionized using an ESI source. Nebulizer pressure was 60 psi, and the capillary voltage was 4000 V. The inert gas was nitrogen at 350°C and the flow rate was 13 L min–1. Bisphenol A and its derivatives were quantified in a multiple reactions monitoring (MRM) mode. The acquisition parameters are described in Table 5. All measurements were performed in duplicate.

TABLE 5. Acquisition parameters for bisphenol A (BPA) and its derivatives Compound Polarity Parent ion Product ions Collision energy (V) Fragmentor (V) Cell accelerator voltage (V) BPA Negative 227 212 28 110 4 133 28 d16BPA Negative 241 223 15 115 141 30 BPA-diPS Positive 511 354 35 163 290 35 276 30 d16BPA-diPS Positive 525 365 35 170 301 40 286 30 Abbreviations: BPA-diPS, bisphenol A derivatized with pyridine-3-sulfonyl chloride; d16BPA, bisphenol A-d16; d16BPA-diPS, bisphenol A-d16 derivatized with pyridine-3-sulfonyl chloride.

The calibration curve of BPA consisted of 10 concentrations from 39 to 20,000 ng mL–1, while the calibration curve for derivatized BPA (dBPA) included 16 concentrations from 0.005 to 156 ng mL–1. The dilution solvent was acetonitrile/water (40:60; v/v). The LOD and LOQ values were calculated based on the standard deviation of the blank, as described by Wenzl et al. [41]. Calibration was validated as stated before. The calibration was linear within the calibration range and r2 > 0.99. The LOD and LOQ values are shown in Table 4.

Statistics

Statistical analysis, including graphical processing, was performed using Microsoft Excel (Microsoft) and R version 3.6.1 (R Development Core Team). Statistical tests were used to determine significant differences between the materials after the period of maximal elution. Following a Shapiro–Wilk test to ensure normal distribution and a Levene's test to check for variance homogeneity, a one-way ANOVA followed by a Tukey post-hoc test was performed. The significance level was set to 0.05.

RESULTS

In the present investigation, the average concentrations of monomers eluted appeared to be material-dependent; in addition, a high degree of variability within the materials was observed, especially between BPA-free and conventional composites (see Table 6). Whereas UDMA and/or TEGDMA were detectable in the eluates of all materials after the period of maximum elution, Bis-GMA was not detected in any eluate after the period of maximum elution. In the eluates of two composites, BPA was detected and quantified using PDA. These results were not reproducible using HPLC-MS/MS and were therefore considered as false positive. A representative chromatogram of a sample in which UDMA and TEGDMA were detected is shown in Figure 2. The results for each C&B material are discussed in the remainder of this section.

TABLE 6. Concentrations of monomer released by different crown and bridge materials according to duration of incubation Material Incubation period Bis-GMA (μg mL–1) BPA (μg mL–1) TEGDMA (μg mL–1) UDMA (μg mL–1) ExperTemp 1 h – – <LOD 6.23 ± 0.8 12 h – – <LOD 6.74 ± 1.4 24 h <LOD <LOQMS/MS <LOQ 8.31 ± 1.6 7 days – – <LOD 5.46 ± 0.7 Luxatemp Automix Plus 1 h – – <LOQ <LOD 12 h – – 1.34 ± 0.4 1.24 ± 0.4 24 h <LOD <LODUV/Vis 1.03 ± 0.1 0.95 ± 0.1 7 days – – 0.94 ± 0.2 1.08 ± 0.3 Protemp 4 1 h – – <LOD <LOD 12 h – – <LOD <LOD 24 h <LOD <LODUV/Vis <LOQ <LOD 7 days – – <LOD <LOD Visalys Temp 1 h – – <LOD 1.18 ± 0.4 12 h – – <LOD 3.35 ± 1.2 24 h <LOD <LOQMS/MS <LOD 3.45 ± 1.1 7 days – – <LOD 4.11 ± 0.5 Values are given as mean ± SD. Abbreviations: Bis-GMA, bisphenol A-glycidyl methacrylate; BPA, bisphenol A; LOD, limit of detection; LOQ, limit of quantification; MS/MS, tandem mass spectrometry; TEGDMA, triethylene glycol dimethacrylate; UDMA, urethane dimethacrylate; UV/Vis, ultraviolet–visible spectroscopy. LOD: UDMA and TEGDMA, 0.05 μg mL–1; Bis-GMA, 0.5 μg mL–1. LOQ: UDMA and TEGDMA, 0.5 μg mL–1. LODUV/Vis BPA, 0.5 μg mL–1. LOQMS/MS BPA, 0.00003 μg mL–1.

Chromatogram of an ExperTemp sample obtained after an incubation period of 24 h. (a) Chromatogram: peaks identified by retention time are underlined. (b) Relative abundance corresponding to triethylene glycol dimethacrylate (TEGDMA). (c) Relative abundance corresponding to urethane dimethacrylate (UDMA). (d) Relative abundance corresponding to diethyl phthalate (internal standard); RT: retention time, MA: manual area under peak

Conventional C&B composites

Neither Bis-GMA nor BPA was detected in the eluates of Protemp 4 and Luxatemp Automix Plus. Moreover, UDMA was not detectable in the eluates of Protemp 4, and TEGDMA levels were below the LOQ after the period of maximum elution (24 h of incubation). Quantifiable amounts of UDMA and TEGDMA were eluted from Luxatemp Automix Plus, especially in the first 12–24 h of incubation.

BPA-free C&B composites

No Bis-GMA was detectable in the eluates of Visalys Temp and ExperTemp. Quantifiable amounts of BPA were detected in the eluates of both Visalys Temp and ExperTemp by HPLC-UV/Vis. After 24 h of incubation, 3.1 ± 0.18 μg mL–1 of BPA was detected in eluates of ExperTemp and 1.6±0.22 μg mL–1 in eluates of Visalys Temp. Due to the well-known false-positive results of this detection technique [42-45], all samples were reanalyzed. Using tandem mass spectrometry, the levels of BPA previously measured by HPLC-UV/Vis were not reproducible, and these results were therefore considered as false positive. After 24 hours of Incubation no TEGDMA was detectable in Visalys Temp eluates and the concentrations were below the LOQ in ExperTemp eluates. Quantifiable amounts of UDMA were detectable in the eluates of both Visalys Temp and ExperTemp, with most of the release occurring within the first 24 h of incubation.

Statistical analysis was performed solely for UDMA because only this monomer was quantifiable in the eluates of more than one material (see Figure 3). As UDMA was not detectable in the eluates of Protemp 4, it was therefore assumed that UDMA was not released from this material, which was therefore given 0-values in the following statistical evaluation. The Shapiro–Wilk test showed a normal distribution of data across all groups (ExperTemp P = 0.313; Luxatemp P = 0.06321; Visalys Temp P = 0.163). Variance homogeneity could not be refuted by a Levene test (F = 1.7295, P = 0.22). An ANOVA revealed highly significant differences between the C&B materials regarding UDMA release (P < 0.001). Tukey's post-hoc test showed significant differences between the materials studied. The amounts of UDMA released were significantly higher in ExperTemp eluates than in all other materials (Visalys Temp < 0.001; Luxatemp Automix Plus P < 0.001; Protemp 4 P < 0.001). Visalys Temp eluted significantly more UDMA than Protemp 4 (P < 0.001) and Luxatemp Automix Plus (P = 0.005), although the difference between the latter was not significant (P = 0.44). In summary, a larger amount of UDMA was released from BPA-free materials than from conventional C&B composites.

Urethane dimethacrylate (UDMA) release, after 24 h of incubation, from each crown and bridge (C&B) material investigated. Exp, ExperTemp; Lux, Luxatemp Automix Plus; Pro, Protemp 4; Vis, Visalys Temp; *Bisphenol A (BPA)-free

DISCUSSION

In the present study, the elution of BPA and the three most relevant monomers from four different C&B composite materials was investigated over a 7-day period. The monomers eluted, and their quantity, varied considerably according to the C&B material analyzed. It was shown that significantly more UDMA was eluted from BPA-free materials in aqueous media than from conventional composites containing BPA derivatives. In general, our data show that, after 24 h, almost all monomers were completely released. This is already known from other studies on different composite materials [29, 46-48]. For these reasons, the 24-h incubation period was defined as the period of maximum elution in this study. Based on other studies, we hypothesized that Bis-GMA and BPA are either not released or released only in small quantities in aqueous media, such as water, artificial saliva, or collected saliva [26-28]. For detection and exclusion of Bis-GMA and BPA release, we analyzed the eluates after the period of maximum elution because this incubation period is considered the reference time for meta-analysis [25]. After this period of incubation, Bis-GMA was not detectable, regardless of the C&B material analyzed. By contrast, BPA was detected and quantified in the eluate