1

Introduction

The degree of conversion of resin-based composites (RBCs) varies between 40% and 75% suggesting an incomplete monomer to polymer conversion . The majority of unreacted C C bonds in the cured material exist in the form of pendant, side groups chemically attached to the network . A smaller amount, up to 10% of the monomer content, may be trapped within the polymer as unreacted monomers and oligomers . These may elute from RBCs as a result of chemical biodegradation in the presence of water , salivary and bacterial enzymes .

Based on material weight loss, it was previously suggested that elution from RBCs occurs acutely within the first 24 h post-immersion . More sensitive high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) techniques have shown that elution is a continuous process over a period of several days to several months . Michelsen et al. identified as many as 32 leachable substances from resin-based materials using GC/MS.

Scarce literature data have been found on monomer elution from nanohybrid and ormocer RBCs in contrast to a substantial amount of work on microhybrid RBCs . After 24 h of immersion, Tabatabaee et al. found more Bis-GMA but less TEGDMA eluted from a nanohybrid than a flowable RBC and Seiss et al. found less TEGDMA released from a nanohybrid and an ormocer compared to a flowable and two microhybrid RBCs. Polydorou et al. investigated the effect of curing times on monomer elution from a nanohybrid and an ormocer RBC and reported a different effect of curing times on the two materials as well as more eluted monomers from the nanohybrid RBC.

Elution of monomers from RBCs may affect the biocompatibility of these materials in clinical conditions. Cytotoxic and genotoxic effects of BisGMA, TEGDMA, UDMA and HEMA have been found in various cell types in vitro . Adverse effects of methacrylate monomers on cellular homeostasis have been shown even for sub-toxic concentrations .

The aim of this study was to determine elution of four monomers (UDMA, BisGMA, TEGDMA and HEMA) from nanohybrid, ormocer and control microhybrid RBCs cured with different LCUs.

2

Materials and methods

Twenty-eight intact, human premolars extracted for orthodontic reasons were used in this study. Ethical approval was granted by the Ethics Committee Belgrade University School of Dentistry and patients’ informed consent was obtained for the use of such teeth in research purposes. The teeth were cleaned from debris and stored in 0.02% thymol at 4 °C prior to the study. The teeth were randomly allocated to one of four groups ( n = 7) according to the RBCs used in the study. All teeth from one group were embedded up to the amelo-cemental junction in super hard gypsum in a dental impression tray so that their long axes were parallel with the occlusal plane of the tray. While the gypsum was setting, the teeth were wrapped in wet cotton to prevent dehydration.

The convexity on the buccal and oral surfaces of each tooth crown was flattened with a diamond disc under water. Each dental tray was mounted on a parallelometer (Cendres & Metaux SA Dental Division, Biel-Bienne, Switzerland) and two standardized cavities were prepared in each tooth with a round diamond bur using the rotary unit of the parallelometer which allowed only vertical movements. One cavity was on the buccal and the other on the oral surface of each tooth crown. A new bur was used after 7 cavity preparations. Each cavity was 2.5 mm in diameter and 2 mm deep. The dimensions of the cavity were determined by the size of the bur and the parallelometer settings and were verified using a digital caliper ( d = 0.1 mm; PROWIN Products Inc., Jiangsu, China). A sharp probe was used to check whether or not the pulp chamber was exposed.

Materials used in the present study are listed in Table 1 . Each cavity was filled with one increment of the designated RBC without its adhesive system, covered with a Mylar strip and pressed with a glass slab to extrude excess material. The RBC restorations on the buccal surfaces were cured for 40 s with a halogen LCU (Megalux soft-start, Mega-Physik GmbH&Co. KG, Rastatt, Germany) at an intensity of 400 mW/cm ² whereas those on the oral surfaces were cured for 20 s with an LED LCU (bluephase, Ivoclar Vivadent, Schaan, Liechtenstein) at an intensity of 700 mW/cm ² . These curing times were recommended by the manufacturers with regard to the light intensity of each LCU. Both LCUs operated at standard mode and light intensity was monitored before and after curing with a radiometer (SDS, Kerr, Danbury, CT, USA). Tip-to-surface distance was standardized to 1 mm and was maintained with a custom-made light guide. Immediately after light curing, excess material was removed with a scalpel and each RBC was polished using a series of Sof-Lex discs (medium, soft, super-soft, 3 M ESPE, St. Paul, MN, USA) for 30 s each under water and standardized pressure in the experimental set-up. After polishing, each crown was sectioned in the mesial-distal direction, parallel to the tooth axis and cut off with a diamond disc. This resulted in two halves from each tooth, restored with the same RBC but cured with different LCUs.

Each sample was immersed in 1 mL of 75% ethanol/water solution (HPLC Gradient Grade solvents) in a glass vial and stored in a water bath at 37 °C. HPLC measurements were done 1 h, 6 h, 24 h, 3 days, 7 days and 28 days after immersion. After each interval, the whole solution was taken up for analysis, following which the samples were air-dried with the very mild stream of air and immersed in 1 mL of fresh ethanol/water solution.

Qualitative and quantitative analysis was performed on an HPLC instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an XDB-C18 column, 75 mm × 4.6 mm ID and 3.5 μm particle size (Zorbax Eclipse ^® , Agilent Technologies, Santa Clara, CA, USA). In front of the separation column, a pre-column was installed, 12.5 mm × 4.6 mm ID and 5 μm particle size (Zorbax Eclipse ^® , Agilent Technologies, Santa Clara, CA, USA). The mobile phase was a mixture of water and acetonitrile (HPLC Grade, Sigma–Aldrich, Dorset, UK) and a gradient was applied according to the following method (A: H ₂ O, B: CH ₃ CN): 10% B (0–2 min); 20% B (2–12 min); 70% B (12–20 min); 100% B (20–22 min); 10% B (22–29 min). The flow rate was 1 mL/min and the injection volume was 10 μL. UV detection was performed at 205 nm (for monitoring the elution of HEMA, TEGDMA, BisGMA and UDMA) and 275 nm (for monitoring the elution of BisGMA). The compounds were identified by comparison of their retention times with those of the reference compounds under the same HPLC conditions. Reference standards of BisGMA, TEGDMA and UDMA (>98% purity; Ivoclar Vivadent, Schaan, Liechtenstein) and HEMA (≥99% purity; Sigma–Aldrich, Dorset, UK) were used as obtained to produce stock solutions of 100 μg/mL each. These stock solutions were diluted with 75% ethanol to produce the final calibration solutions: 0.5, 1, 2.5, 5, 10 and 20 μg/mL. The peak area for each monomer was determined and plotted versus concentration using linear regression analysis and used to quantify monomer concentration in the sample solutions. The limit of quantification (LoQ) and detection (LoD) were calculated for a signal to noise ratio of 10 (S/N = 10) and 3 (S/N = 3), respectively. The obtained values for LoQ were 0.25 μg/mL for HEMA, 0.075 μg/mL for TEGDMA, 0.4 μg/mL for UDMA and 0.23 μg/mL for BisGMA. The obtained values for LoD were 0.075 μg/mL for HEMA, 0.022 μg/mL for TEGDMA, 0.12 μg/mL for UDMA and 0.07 μg/mL for BisGMA.

Data were analyzed using Minitab 15 (Minitab Inc., State College, PA, USA). Multivariate analysis of variance (ANOVA) was used to test the differences in eluted monomer concentrations among RBCs. Repeated measures ANOVA was used to test the differences among time intervals for each monomer and each RBC. In cases where repeated measures ANOVA showed significant differences among time intervals ( p < 0.05), paired t -tests with the Bonferroni correction were performed to identify at what time points these differences became apparent. The level of significance was set at α = 0.05.

Based on the experimental data, a mathematical model of monomer elution kinetics was calculated and expressed as a sum of two exponential regression functions representing the first order kinetics law (Eq. (1) ):

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