Analysis of trace elements released from glass ionomer restorative materials by inductively coupled plasma-mass spectrometry

Background The aim of this study was to determine the amount of trace elements released from a traditional glass ionomer, a bulk-ll glass ionomer, a resin-modied glass ionomer, a glass carbomer ll and a compomer material stored in ultra-distilled water. Ten cylindrical (10×2mm) specimens were prepared from each Each sample was in 50mL ultra-distilled at for fourteen and rinsed twice daily. The amount of elements in the solutions was determined using inductively coupled plasma-mass spectrometry. Aluminum, boron, nickel, copper, zinc, barium and lead were detected in all solutions. The traditional glass ionomer and resin-modied glass ionomer released arsenic, the bulk-ll glass ionomer and compomer released selenium, and the resin-modied glass ionomer and compomer released antimony. Only the resin-modied glass ionomer released iron. impact on the release of elements. There is a need for further in vitro studies, using different materials, different periods of experimentation and storage media, or clinical trials.

Caps, VOCO GmbH, Cuxhaven, Germany) were tested in this study (Table 1). Ten cylindrical specimens, 10mm in width and 2mm in depth, were prepared from each material according to the manufacturer's instruction at 23±2°C [8]. A syg-200 dental amalgamator (Smaco Company, Hangzhou Zhejiang, China) was used as a mixing device. The light-curing devices used for polymerization are shown in Table 2. The samples were kept at 95% humidity and 37°C for 24 hours [3,8]. Then, each sample was placed in a sterile polypropylene tube with a cover, and 50ml of ultra-distilled water (18MΩ.cm) was added. All samples were stored at 37°C for fourteen days. Each tube was rinsed twice daily. After 14 days, the samples were removed from the solutions, and the latter were analyzed for trace elements using ICP-MS.

Elemental analysis
An inductively coupled plasma-mass spectrometer (Agilent 7700, Agilent, Santa Clara, USA) was used to measure the levels of trace elements released from the ve different dental materials. First, device validation was performed with standard mixtures (High-Purity Standards, North Charleston, USA). The standard mixtures and ultra-distilled water were injected into the device, and calibration lines were obtained. Second, the solutions were sent to the mass spectrometer after ionization in inductively coupled plasma and measured as mass-to-charge ratio [20]. The basic principle was that solutions were ionized by argon plasma which was brought to a temperature of 10,000K by electromagnetic induction. The amounts of the elements were measured with an electron augmentation detector. The reactive materials were ultra-distilled water (18MΩ.cm), 60% ultrapure nitric acid at a density of 1.37kg/L, 30% hydrochloric acid at a density of 1.15kg/L, 5% nitric acid washing solution and a blank solution (0.5% nitric acid). The amount of each element was determined as parts per billion (ppb or µg/L). The laboratory temperature was 23±2ºC degrees.

pH measurements
The pH of each solution was measured with a pH meter (WTW InoLab-IDS multi 720, Probe 325). For pH measurements, device validation was performed rst using solutions with standard pH (pH 4, 7 and 10; Certipur buffer solutions, Merck KGaA, Darmatadt, Germany). Then, the device probe was held in the test solution until the pH was stabilized. The pH value was then recorded. Before each subsequent measurement, the probe was washed with distilled water and dried with a napkin.

Electrical conductivity measurements
The electrical conductivity of each solution was measured with a meter (WTW InoLab-IDS multi 9310, Probe 925). First, device validation was performed with solutions with standard conductivity (electrical conductivity of 12.85±4%mS/cm and 1412±5% µS/cm; Certipur potassium chloride solution, Merck KGaA, Darmatadt, Germany). Second, the device probe was placed in the test solution until conductivity was stabilized, and the value was then recorded. Before each subsequent measurement, the probe was washed with distilled water and dried with a napkin.

Statistical analysis
The data were statistically analyzed using one-way analysis of variance, Duncan's multiple range test and independent samples t-test for two-group comparisons (SPSS 17 for Windows, SPSS Inc., Chicago, IL, USA) with the signi cance level set at p<0.05.

Results
The mean and standard deviation of the amount of each element released are shown in Table 3. Boron, aluminum, nickel, copper, zinc, barium and lead were released from each material (p<0.05). BFGI and the compomer released selenium (p>0.05). RMGI and the compomer released antimony (p>0.05). TGI and RMGI released arsenic (p<0.001). Only RMGI released iron.
The lowest pH was observed in the compomer group (p<0.001); the pH values of the other groups were similar ( Table 4). The electrical conductivities of solutions were signi cantly different between groups (p<0.001; Table 4). The highest electrical conductivity value was observed in the RMGI group.

Discussion
GICs are used for different purposes, such as restorative lling, pit-and-ssure sealing, luting, lining, and as base cements [4]. However, they have been modi ed, mainly by the addition of resin components, to improve their mechanical properties, without an adequate evaluation of uoride release [3,8]. In GICs, there is an acid-base reaction resulting in the leaching of uoride ions to form a polysalt matrix [4]. In addition, unreacted glass particles may cause rapid release after mixing [21] which may be responsible for the fast elution process. This phenomenon is known as the 'burst effect' [15]. There is also a much smaller but steady release that can last for a long period [8]. These release processes may also be true for other elements. Previous studies have shown that dental materials can release some heavy metals [17][18][19]22]. The inclusion of heavy metals in dental materials raises concerns because most dental materials are in contact with both soft and hard tissue [17]. Therefore, dental materials should not contain heavy metals or heavy metals should not be released into the mouth. This study showed that boron, aluminum, iron, nickel, copper, zinc, arsenic, selenium, antimony, barium and lead were released from glass ionomers. When all the results of the study were considered together, there were statistically signi cant differences between glass ionomer types. Therefore, the null hypothesis was rejected.
In release studies, different storage media, such as arti cial saliva, human saliva, saline, acidic solutions, and deionized water, have often been used [15,23]. Some studies have suggested that saliva or pH-cycling models may better simulate the oral environment and, consequently, be more appropriate for studying ion release from dental materials. However, the use of ultra-distilled water is considered to give an accurate estimate of the ions released since there are no existing ions in this medium [24]. Ultra-distilled water is acceptable as a storage medium for in vitro studies, and, in this study, it was preferred for release of all elements. The number of samples in previous studies [15,25] was limited to three or six. This study attempted to obtain more precise results by increasing the number of samples.
ICP-MS is an appropriate method for identifying less frequently observed elements, and provides highly accurate results even if the sample size is small. In this method, plasma reaches a high temperature (nearly 10,000K) at which metals and metalloids are ionized, making them detectable on a mass detector [26]. Some researchers have used inductively coupled plasma-atomic emission spectrometry [22] while others prefer inductively coupled plasma-optical emission spectrometry [19] for detecting trace elements inside different types of mineral trioxide aggregate. All methods are suitable for determining trace elements in dental materials. Following previous studies [17,27] ICP-MS was used in this study.
A number of studies have reported [15,28,29] that the highest ion release from GICs was within the rst week and ion release was reduced to constant levels at about 14 days. Therefore, this study determined the cumulative number of elements released over 14 days. Although the same conditions (temperature, specimen geometry, storage medium and polishing procedure) were maintained for all samples, differences were observed in the amounts of elements released. These ndings may be related to multiple factors, such as the chemical and physical characteristics of the GICs, powder: liquid ratio, solubility of glass particles, mixing time and type of polymerization .
Kuhn and Wilson suggested three mechanisms for uoride release from GICs: diffusion through pores and microfractures, mass diffusion and super cial rinse [30]. Super cial rinse can be related to the particle size of GICs. Glass particles are milled under dry conditions to a size less than 20µm. As a result, the surface may be coated with a ne dust that can be washed off and dissolved when in contact with the ambient solution. Other release mechanisms may be related to long-term release processes. GICs absorb water over time, and ion release may occur from the cracks and body of GICs and require more time than conjectured. Similar phenomena may be true for release of elements in this study. However, depending on the differences in the content of the materials, not all element can be released from each type of glass ionomer. In this study, little iron, nickel, copper, arsenic, selenium, antimony and lead release was found; in contrast, aluminum, boron, barium and zinc were released at a higher rate.
Antibacterial activity of uoride and alumino-uoro complexes is important against cariogenic microorganisms [3,17]. However, aluminum is a heavy metal, and high exposure to it may cause neurotoxicity, and may play a role, in addition to genetic factors, in the development of Alzheimer's disease [31]. Moreover, inclusion of aluminum is not indicated in dental materials and other biomaterials owing to the risk of Alzheimer's disease from excessive exposure to the element in close contact with human tissue [16]. Gjorgievska et al. detected aluminum (10.6-26.3ppm) using atomic absorption spectrophotometry in four different dental materials (TGI, RMGI, compomer and composite) [16]. They reported that TGI generally released more aluminum than RMGI [16]. In this study, all groups released aluminum, as also found in previous studies [3,16], and the element with the highest level of release was aluminum (0.08-4.42mg/L). RMGI released more aluminum than the other glass ionomers. This nding agrees with that of Okte et al. [3]. The larger pore size and greater porosity of RMGI in comparison to TGI may explain the difference in the level of release of aluminum from the glass particles [16]. The average intake from food is 10mg/day [32]. The amounts obtained in this study are far below the daily consumption of the element. Hence, the amount of aluminum released from glass ionomers is acceptable and poses no threat to human health.
Boron is a ubiquitous non-essential element in the human body and is part of many biochemical and metabolic functions bene cial to human health and wellbeing [33]. People consume many products (vegetables, fruits, nuts, etc.) containing boron in daily life. The acceptable safe range of boron in food is 1-7mg/day. In healthy people, boron levels change between 15 and 80μg/kg [34]. In this study, boron showed the second highest release from glass ionomers.
The highest amount released (1221ppb) was in GCF and the lowest (7.86ppb) was in RMGI. The amount of boron released from GICs is not at a level that can be harmful to humans.
Barium is a heavy metal and is found in many types of food and beverages. González-Weller et al. demonstrated that barium intake in individuals from different regions was nearly 0.6-0.7mg/day [35]. Barium sulfate is often used as a radiopaque agent in the composition of various dental materials [36]. The effects of barium sulfate are debatable. Sabokbar et al. reported that it was not adequate to prevent bone resorption [37]. However, Khandaker et al. suggested that barium sulphate may be biologically useful [36]. In this study, barium was released at the highest amount in the compomer group (193.7ppb) and at the lowest (0.8ppb) in the TGI group. More barium may be added to compomers for opacity or compomers can release more barium independently of opacity.
Zinc is not stored in the body, but must be obtained from dietary sources. It has catalytic, structural, and regulatory functions in the body, participates in many enzymatic activities and has an anticancer role. Zinc de ciency is a major health problem, affecting over two billion people worldwide [38]. The daily recommendation for men is 11mg and for women 8mg [39]. In this study, all materials released zinc and the amounts released (0.57-6.82ppb) were much less than the recommended daily dose. The lowest zinc release was detected in BFGI and the highest in RMGI.
In this study, iron was detected only in the RMGI group (0.43ppb). Antimony was determined in two groups, at 4.73ppb in the compomer and at 0.09ppb in RMGI. Selenium was released in BFGI (0.16ppb) and the compomer (0.24ppb). Very small amounts of nickel (0.12-0.41ppb) and copper (0.21-1.18ppb) were observed in all groups. The amounts detected were far from the rates that threaten general health. It is believed that these metals may play a role in the hardening reactions of glass ionomers.
Previous studies have shown that some dental materials may contain toxic trace elements such as arsenic and lead [17][18][19][20]22]. Arsenic is a metalloid element and a poison. Arsenic toxicity results in multisystem disease. The European Food Safety Authority has estimated that a 70-kg adult consumes an average of 9.1-39.2μg of arsenic daily [40]. Lead, just like arsenic, is a known neurotoxic metal and lead toxicity is a major public health problem in all countries. Acute or chronic lead exposure has the potential to cause many deleterious effects in the cardiovascular, renal, nervous, immune, and gastrointestinal systems. A lead level of≥5 μg/dL in the blood is considered high for adults and children [41]. In this study, lead release was observed in all glass ionomers: RMGI (0.44ppb)>TGI (0.41ppb)>BFGI (0.35ppb)>compomer (0.16ppb)>GCF (0.14ppb). Detectable levels of arsenic were observed in TGI and RMGI at 0.38ppb and 0.10ppb, respectively. The lead and arsenic release were negligible, and was considerably lower than in previous studies [17,18,20].
Camilleri et al. identi ed lead and arsenic in ve different dental cements kept in two different solutions (acid and Hank's balanced salt solution) [17]. They stated that the quantity of acid-extractable trace elements was high for most of the materials tested, but little was released in the balanced salt solution. They reported that arsenic concentrations were 0.08-52.5mg/kg and lead concentrations were 0.03-14.5mg/kg [17]. Simsek et al. investigated the accumulation of trace elements, such as lead and arsenic, inside the organs of rats using three different dental materials (Mineral trioxide aggregate, BioAggregate and Biodentine) [20]. They found both arsenic (0.9-21.9µg/kg) and lead (0.4-2.9µg/kg) release from all materials, and detected these substances in the brain, kidney and liver of rats [20]. Similarly, Jang et al. evaluated the release of nine heavy metals from three dental materials, and reported lead (1.1-1.9ppb) and arsenic (0.1-9.3ppb) release from all materials. The researchers concluded that cements were reliable materials for dental treatment [18].
Electrical conductivity is closely correlated to the number of ions in the solution. In this study, RMGI had the highest electrical conductivity. The lowest pH was observed in the compomer, whereas the other groups had similar pH values. Hardening occurs in TGIs chemically, in RMGIs with light, and in GCF with both light and heat. These differences may have affected the amounts of elements released from the glass ionomers and their electrical conductivity and pH.

Conclusion
This study demonstrated that all dental materials tested -glass ionomers and compomer -released some trace elements, but the rates were quite low. Therefore, these materials should be considered safe to use. Elements such as lead and arsenic, which are known to be toxic and harmful to humans, should be eliminated from dental materials or their release to the body should be prevented. The composition and chemical and physical characteristics of glass ionomers are important for release of elements, pH and electrical conductivity. The type of polymerization (chemical, light or light and heat) of glass ionomers can also have an impact on the release of elements. There is a need for further in vitro studies, using different materials, different periods of experimentation and storage media, or clinical trials.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

Funding
No nancial support was received for this research.
Authors' contributions BO was supervisors and principal investigators of the study and drafted the manuscript. EA and BO revised the manuscript. All authors read and approved the nal version of the manuscript.

Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Tables