MICROSCOPY RESEARCH AND TECHNIQUE 78:255–259 (2015)

Do the Monomers Release From the Composite Resins After Artificial Aging? UGUR TOKAY,1* ALP ERDIN KOYUTURK,2 ABDURRAHMAN AKSOY,3 AND BILAL OZMEN2 1 2 3

Department of Pediatric Dentistry, Faculty of Dentistry, Ishik University, Erbil, Iraq Department of Pediatric Dentistry, Faculty of Dentistry, Ondokuz Mayis University, Samsun, Turkey Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ondokuz Mayis University, Samsun, Turkey

KEY WORDS

aging; composite resins; monomer; HPLC; thermal cycling

ABSTRACT Objectives: The aim of this study is to measure the effect of thermal cycling on the amount of monomer released from three different composite materials by HPLC analysis method. Experimental Design: Three different composite materials, inlay composite, posterior composite and micro-hybrid composite were used. Sixty cylinder specimens each with a dimension of approximately 1 cm width and 3 mm depth, were prepared before experiments were carried out. Inlay composite material was polymerized according to manufacturers’ instructions. Thermal cycling device was used to simulate thermal differences which occur in the mouth media. Monomers were analyzed using HPLC technic after thermal cycling process. The amount of ethoxylated Bis-GMA and urethane dimethacrylate (UDMA) in inlay composite material, the amount of ethoxylated Bis-GMA in posterior composite material, the amount of ethoxylated BisGMA and triethyleneglycol dimethacrylate (TEGDMA) in micro-hybrid composite material were investigated. Results: Monomer release of thermal cycles levels showed a linear increase in UDMA and TEGDMA (P < 0.05). In terms of thermal cycles levels, Bis-EMA released from posterior composite showed a cubic change (P < 0.001). Conclusions: It was observed that use of additional polymerization processes might have positive effect on the decrease of residual monomer. In the light of the results, we suggest that indirect composite resins have more outstanding features than direct composite resins in terms of biocompatibility. Microsc. Res. Tech. 78:255–259, 2015. V 2015 Wiley Periodicals, Inc. C

INTRODUCTION The composite resin materials have been used in dentistry, and over the years these materials have been testified to be convincing alternative for amalgam, glass-ionomer etc.(Koyuturk et al., 2013; Reichl et al., 2008). Composite resin materials usually consist of polymer matrix, inorganic filler and siloxane coupling agents (Sakaguchi, 2012). Despite their reputation, there are occasions in which the monomers released from resin-based composites may be toxic (Reichl et al., 2008). There are different systematic intakes of residual monomers (ingestion of gastro intestinal tract, diffusion to pulp through dentinal tubules, uptake in the lungs etc.). These intakes may affect different people such as dental practitioners, dental assistants and patients (Gerzina and Hume, 1996; Marquardt et al., 2009; Reichl et al., 2008; Rogalewicz et al., 2006; Van Landuyt et al., 2011). Monomers were often used in resin composite materials (Peutzfeldt, 1997). There are lots of monomers such as Bis-EMA, Bis-GMA, UMDA and TEGDMA (Hervas-Garcia et al., 2006; Sakaguchi, 2012). But composites are never fully polymerized (Van Landuyt et al., 2011). In composite resin materials, the degree of conversion can change between 50–70% (Halvorson et al., 2002; Neves et al., 2005). As a result, uncured monomers and substances could release into the medium. To decrease the uncured monomer that occur, with using C V

2015 WILEY PERIODICALS, INC.

different polymerization methods, additional curing process may be a good alternative. Polymerization shrinkage can be taken under control via indirect polymerization methods (such as inalys/onlays) in composite resins. The rate of formation of residual monomer is reduced in the composite resin polymerized by indirect method (Altintas and Usumez, 2012). In dentistry, thermal cycling has two effects on the samples; first, hot bath accelerates hydrolysis of components and provides dissolution of unpolymerized monomers and substances. Second, thermal cycling stimulates side effects of expansion and contraction on the materials (De Munck et al., 2005; Hashimoto et al., 2000). Brown et al. (1972) suggested that 10 thermal cycles equals to a day in intraoral medium. Generally, high performance liquid chromatography (HPLC), which is frequently used in medical *Correspondence to: Dr. Ugur Tokay, Head of Department, Department of Pediatric Dentistry, Faculty of Dentistry, Ishik University, Erbil, Iraq. E-mail: [email protected] Received 8 August 2014; accepted in revised form 8 January 2015 REVIEW EDITOR: Chaunbin Mao Contract grant sponsor: The Scientific and Technological Research Council of Turkey (TUBITAK); Contract grant number: 111S282. This article was published online on 28 January 2015. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 13 February 2015. DOI 10.1002/jemt.22468 Published online 28 January 2015 in Wiley Online Library (wileyonlinelibrary.com).

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Brand name

TABLE 1. Ingredients of composite resin materials used in this study Manufacturer Ingredients

Tescera DENTINE ÆLITE LS Posterior ÆLITE All Purpose Body

(Bisco, USA) (Bisco, USA) (Bisco, USA)

Ethoxylated Bis-GMA, Urethane Dimethacrylate, Glass Filler, Amorphous Silica Ethoxylated Bis-GMA, Glass Filler, Amorphous Silica Ethoxylated Bis-GMA, Triethyleneglycol Dimethacrylate, Glass Filler, Amorphous Silica

chemistry, pharmacology and clinical chemistry, has been used in order to carry out bio-analysis of natural and artificial products (Seger et al., 2013). According to monomer analysis studies, researchers have advocated that HPLC is the most convenient method in the residual monomer analysis (Ortengren et al., 2001; Van Landuyt et al., 2011). The objective of this study was to examine the effects of thermal cycling on the residual monomer released from three types of composites. Hypothesis of this study is that the thermal cycling will increase the residual monomer released from composites, but it will not increase the release of residual monomers in the indirect posterior composites. MATERIALS AND METHODS In this study, three types of composites (posterior, micro-hybrid, and indirect posterior composite) were used (Table 1). Sample Preparation According to One-way ANOVA power analyzing, power of this study which were consist of 20 samples was determined as 0.99941 (Altintas and Usumez, 2012). In this study, there were three composite groups and twelve subgroups (Table 2). Sixty composite disks were obtained from composite resin materials. Composite disk sizes were 1 cm diameter 3 3mm depth. All samples were polymerized by halogen light source (Lunar Curing Light, Benlioglu Dental Inc, Turkey). Light was applied to both the top (20 s) and bottom surfaces (20 s) for 40 s. Further, the indirect posterior composite samples were post-cured with a combination of light, heat, and pressure in an oven (Tescera ATL, Bisco). After polymerization process, polishing was carried out using low-speed micromotor and polishing burs. Each sample was placed into the opaque glass tubes. Totally, 3 mL of 75% ethanol and 25% ultrapure water was put in a tube to obtain a homogeneous mixture. In order to prevent water leakage in the thermal cycling, glass tubes’ caps were coated with paraffin. Thermal Cycling In control group, all samples were stored in 37 C for 24 h. Thermal cycling device was set up to 5 C for hot bath, and 55 C for cold bath. The period of time for the tubes to stay in hot and cold baths were calibrated to 30 s for each baths on the device, and 10 s in between (Gale and Darvell, 1999). Thousand times thermal cycle was applied on groups A2, B2, and C2; 5,000 times thermal cycle was applied on groups A3, B3, C3; 10,000 times thermal cycle was applied on groups A4, B4, C4 (Table 2). After the thermal cycling process, composite samples were removed from the solution. The solutions, from which samples were extracted, were stored at 4 C until analysis procedure.

HPLC Analysis Altintas ve Usumez (Altintas and Usumez, 2009) analysis method was modified, HPLC device (LC-20AT Prominence, Shimadzu, Kyoto-JAPAN) was used to measure the levels of residual monomer released from composite resins. HPLC device conditions during the analysis process are specified in Table 3. Pure monomers used in the study are shown in Table 4. A 0.45-lm disk was used to distill ethanol/ water (75/25, v/v) solution from which the samples were extracted. After filtration, solution was injected into HPLC with DAD. Calibration curve was obtained, and Retention Time (RT), Limit of Detection (LOD) and Limit of Quantification (LOQ) of pure monomers (Fig. 1, Table 5) were determined. Statistical Analysis Before starting the statistical evaluation of the data, the normality assumption was checked by the Shapiro-Wilk test, and the data has shown normal distribution. Statistical analysis was performed with SPSS for 20.0 V (IBM, NY). The data on residual monomers were analyzed by One-way ANOVA using Tukey’s HSD post hoc analysis at a significance level of P < 0.05. RESULTS Calibration curves were obtained for Bis-EMA, TEGDMA, UDMA monomer standards ranging between 0.5, 1, 3, 5, 7 ve 10 lg/mL (Bis-EMA calibration formula: Y 5 5386.963X-1248.32, R2 5 0.999; TEGDMA calibration formula: Y 5 62957.95X2889.196, R2 5 0.999; UDMA calibration formula: Y 5 40790.34X-3572.54, R2 5 0.999). Retention times were 5.17 min, 6.41 min, 10.96, 11.55, 12.48, 13.54 min for TEGMDA, UDMA, and Bis-EMA, respectively (Table 5). In indirect posterior groups, there were no significant differences among the groups in terms of residual monomer amount of UDMA (P > 0.05) (Table 6). However, linear development was observed as thermal cycling increased. Furthermore, there was no significant difference among the groups in terms of residual monomer amount of Bis-EMA (P > 0.05) (Table 6). When residual monomer amount of UDMA and BisEMA were taken into account, there was a significant difference only between control group and 1000 cycle group (P < 0.05) (Table 6). In terms of residual monomer amount of TEGDMA, there was a significant difference among all microhybrid composite groups (P < 0.05) (Table 6). Further, there was no significant difference among all groups in terms of residual monomer amounts of Bis-EMA (P > 0.05). When released amounts of TEGDMA and Bis-EMA were compared, there was significant difference among all groups (P < 0.05) (Table 6). Microscopy Research and Technique

EFFECT OF THERMAL CYCLING ON MOMOMER RELEASING

When the posterior composite groups were examined, there was significant difference among all groups in terms of residual monomer amount of Bis-EMA (P < 0.05) (Table 6). When the whole composite groups were appraised, there were significant differences between control groups and group 1,000 cycles in terms of residual monomer amount of Bis-EMA (P < 0.05) (Table 7).

TABLE 2. Distribution of composite resin groups

Control 1,000 cycle 5,000 cycle 10,000 cycle Total

Indirect posterior composite (n)

Posterior composite (n)

Micro-hybrid composite (n)

A1 (5) A2 (5) A3 (5) A4 (5) 20

B1 (5) B2 (5) B3 (5) B4 (5) 20

C1 (5) C2 (5) C3 (5) C4 (5) 20

Group A1, B1, C1; Control Groups, No thermal cycling. Group A2, B2, C2; 1,000 times thermal cycling was applied. Group A3, B3, C3; 5,000 times thermal cycling was applied. Group A4, B4, C4; 10,000 times thermal cycling was applied.

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DISCUSSION Different methods were used for the analysis of eluted residual monomers by researchers. Well-known methods are LC-MS, GC/MS, and HPLC (Altintas and Usumez, 2009; Ortengren et al., 2001; Seger et al., 2013; Van Landuyt et al., 2011). Due to the fact that they have the most powerful analyzing abilities, high sensitivity detection, and that they are less expensive, HPLC methods were commonly used for evaluating elution of monomers in dentistry (Van Landuyt et al., 2011). Therefore, because of its advantages, HPLC device was used for residual monomer analysis in this study. In oral cavity, many different conditions (low pH values, enzymatic activities, and erosive factors) occur, and these changes cause degradation of resin materials (Botsali et al., 2014; Van Landuyt et al., 2011). Different artificial aging tests, such as mechanical loading, thermal cycling, organic solvents, strength tests, were used to simulate oral cavity (Altintas and Usumez, 2009; De Munck et al., 2005; Gale and Darvell, 1999; Van Landuyt et al., 2011), but these tests alone are insufficient to simulate oral conditions. In

TABLE 3. HPLC device conditions Analysis column C18, 5 lm x 250 x 4.6 mm

Mobil phase

Flow speed

Detector and Wave length

Column oven temperature

Injection volume

water (75: 25, v/v)

1 mL/min. (isocratic)

DAD 205 nm

25 C

20 lL

Bis-EMA Brand name Manufacturer Product No. Formula Molecule weight CAS No.

TABLE 4. Pure monomers properties used UDMA

Bisphenol A ethoxtylate dimetachrylate Sigma Aldrich 455059 C21H28O6 376.4 g/mol 41637-38-1

Diurethane dimethacrylate Sigma Aldrich 436909 C23H38N2O8 470.56 g/mol 72869-86-4

TEGDMA Trieyhylene glycol dimethacrylate Sigma Aldrich 261548 C14H22O6 286.32 g/mol 109-16-0

Fig. 1. HPLC-DAD chromatogram for 10 lg/mL mix standard. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Microscopy Research and Technique

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this study, we focused on drawbacks of early studies. Two different artificial aging tests such as thermal cycling and organic solvents were combined. These methods were not used at the same time before. The solvents that were used to incubate in earlier studies could be divided into two groups: (1) waterbased solvents such as human or artificial saliva, cell culture etc.; and (2) organic solvents such as ethanol/ methanol-based mixtures (Van Landuyt et al., 2011). Organic solvents are most reliable methods for the analysis of residual monomer, for such materials penetrate to polymer matrix and they accelerate hydrolysis of unreacted monomers (Altintas and Usumez, 2012; Van Landuyt et al., 2011). Van Landuyt et al. (2011) suggest that the release of residual monomers was higher in organic solvents than in water or a waterbased solution. Because of these reasons, the 75% ethanol/water solution was used as storage medium. As a result, use of ethanol/water solvent resulted in the release of greater amount of residual monomers. Botsali et al. (2014) reported that HEMA releasing was higher than the TEGDMA releasing. Ak et al. (2010) reported that the amount of TEGDMA released from composites was significantly different from BisGMA at the end of the 7 days. Gioka et al. (2005) suggested that TEGDMA monomers were detected in incubation solution, but Bis-GMA monomers levels were below detection threshold. They reported that the amount of TEGDMA was significantly higher than BisGMA. Polydorou et al. (2009) reported that maximum amount of Bis-GMA release was observed 24 h later. In this study, we observed that the release of TEGDMA was much more higher than the others. These results were similar with the previous studies. High release of TEGDMA was clarified with low molecular weight and low viscosity of TEGDMA monomer. TABLE 5. Retention Time (RT), Limit of Detection (LOD) and Limit of Quantification (LOQ) of pure monomers Monomers RT (min) LOD (ng/mL) LOQ (ng/mL) TEGDMA UDMA Bis-EMA

5.17 6.41 10.964 11.553 12.481 13.548

5.2 8.0 7.5

15.6 24.1 22.6

Botsali et al. (2014) observed that release of TEGDMA from different compomers ranged between 4 min and 24 h. Danesh et al. (2012) reported that the amount of the release of monomers UDMA increased on the 1st, 3rd, and 7th days, but there were no significant differences among the groups. In this study, release of Bis-EMA from microhybrid composite increased in the control group and 1,000 cycles group, but it decreased in the 5,000 cycles group and increased again in the 10,000 cycles group. There was quadratic development in terms of the release of BisEMA. The reason for the differences among groups may be due to lack of enough activation among initiator molecules of C@C connections. Although TEGDMA was used to improve filler connections (Kramer et al., 2008), it had the highest amount of residual monomer. In this study, differences among Bis-EMA groups (quadratic development-not continuing increasing) can be attributed to crumbling of “Bis-EMA into bisphenol a dyglycidy” (Schmalz et al., 1999), as a result, BisEMA cannot be detected by HPLC. No detection of BisEMA by HPLC caused observation of low amount of release of Bis-EMA. Also, hydrolysis of dimethacrylate could be explained with increasing Bis-EMA or BisGMA’s tendency of formation of metacrylic acid (Altintas and Usumez, 2008; Schwengberg et al., 2005). Release rates of commonly used monomers are as follows; HEMA>TEGDMA>Bis-GMA>UDMA (Van Landuyt et al., 2011). In this study, the highest concentrations are 195,27 lg/mL for TEGDMA, 104,21 lg/mL for BisEMA, 3,32 lg/mL for UDMA, respectively. TABLE 7. Mean Bis-EMA values of different composites Composites Indirect posterior

Posterior

Microhybrid

Mean 6 SD (lg/mL

Mean 6 SD (lg/mL)

Mean 6 SD (lg/mL)

Groups Control 1000 5000 10000

C

B

1.7761.00 2.5361.38C 2.6261.15B 2.8261.28B

24.476 3.12 24.8267.43B 104.21630.36A 67.13627.93A

A

37.1667.28 41.14610.16A 29.80612.62B 40.6767.53A

P

Do the monomers release from the composite resins after artificial aging?

The aim of this study is to measure the effect of thermal cycling on the amount of monomer released from three different composite materials by HPLC a...
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