Determination of leached-elements from dental restorations has been investigated as a result of exposure to oral conditions ¹ . Several methods have been performed both in vitro and in vivo. Most of the in vitro techniques relied on storing specimens in surrounding media, such as distilled water or artificial saliva ^{1– 3} . Various analytical means had been used to analyze the leached-elements, including inductively-coupled-plasma, atomic-absorption, Fourier-transform-Infrared spectroscopies and chromatographic methods ^{1, 2, 4} .

On the other hand, in vivo methods have been used to assess the actual situation intra-orally ⁵ . In vivo techniques have been performed either in animals or humans. Cavities are prepared in the teeth of animals, such as monkeys ⁶ , pigs ^{7, 8} or rats ⁹ , then filled by dental-restorations. The released-elements are either detected after sacrificing the animals ⁷ or by analyzing secretory products, such as urine ⁹ or fecal samples ⁶ .

Yet, measuring the released elements in humans may be best. Several studies quantified the excretion of mercury in urine, hair and nails of patients with dental amalgam-restorations and found a positive correlation ^{10, 11} . In contrast, others did not find a correlation between mercury content in hair and amalgam-restoration, but mercury in hair has been correlated with fish consumption ¹² . Thus, assessing the leached-elements in the oral-cavity may be more specific to eliminate other possible systemic sources. Thus, several studies examined released ions in patients’ saliva ^{13, 14} . However, these ions may be affected by salivary secretions or washed out by swallowing. In addition, saliva collection and storage may be technique sensitive ¹⁵ . Thus, a new simple method was proposed in this study through using night-guards (polymeric occlusal-splints used to decrease bruxism and clenching teeth) in gathering leached-elements from dental-restorations.

Ten upper dental alginate impressions (Tropicalgin, Zhermack, Italy) were taken as part of routine treatment at the Dental Family Clinic (Cairo, Egypt), between February and December 2016, for ten patients suffering from bruxism, and who also had amalgam-restorations in their upper molars. This was performed after all the patients signed written informed consent forms agreeing to participate in the study. The participants were six females and four males with the following inclusion criteria: adult (>18 years), non-periodontal-affected patients suffering from bruxism, and had amalgam restoration. Medically-compromised patients were excluded. Custom-made night-guards (example shown in Figure 1) were fabricated for each patient from 2mm soft polymeric sheets (Easy-Vac-Gasket, 3A-Medes, Korea), using a vacuum forming machine (Pro-Form, Keystone, Germany). The night-guards were delivered to the patients in the clinic after one week from first visit and they were instructed to wear the night-guards when they were asleep (≈7 hours±2). Recall visits were given to the patients in the regular check-up schedule after six months. In this visit, the night-guards were taken from the patients to be analyzed. A new unused night-guard was fabricated from the same material to be used as a control.

Scanning-Electron-Microscopy (SEM) and Energy-Dispersive-X-ray-Analysis (EDXA) were used to examine structural and elemental changes in the used night-guards, and compared to the unused one. In the used night-guards, two areas were studied: the fitting surfaces contacting amalgam-restorations and the fitting surfaces not contacting amalgam-restorations. The patients’ records were used to identify teeth that had restorations. Parts from these areas of interest were cut using a scissor (Singer, Germany), mounted on coded brass stubs and sputter coated with 10Å gold platinum and observed at 20000× magnification. The SEM and EDXA (Supra40, Carl-Zeiss-NTS-GmbH, Germany) were used with an accelerating voltage of 20.0–30.0kV.

The SEM of the unused night-guard revealed a homogenous structure ( Figure 2), with its composition consisting of carbon and oxygen (C=88.9wt%±0.3 and O=11.1wt%±0.4; Figure 3). By contrast, the night guard surfaces contacting amalgam-restorations showed numerous lustrous particles ( Figure 4). Elemental analysis of these areas showed the presence of mercury and sulfur, in addition to carbon and oxygen (Hg=21.2wt%±0.6, S=2.5wt%±0.5, C=67.1wt%±0.3 and O=9.2wt%±0.5; Figure 5). The surfaces not contacting amalgam-restorations under SEM showed slight cracking, but were still homogenous without lustrous particles ( Figure 6). Their composition was carbon and oxygen (C=88.3wt%±0.6 and O=11.7wt%±0.6; Figure 7).

The oral-cavity is an aggressive environment, which may affect the integrity of dental restorations ^{16– 18} . It is essential to examine the resultant leached-elements to assess the degradation products of the filling materials in the oral-cavity and investigate if these materials could provoke an adverse systemic effect ¹⁹ .

Several studies have examined released elements by in vitro testing through fabricating specimens and soaking them in medium ^{1, 2, 20} . However, simulating the oral-cavity is difficult due to its complex nature with multifactorial variables ¹⁷ . Thus, some studies have analyzed patients’ saliva ^{21– 23} . However, saliva is composed of various components and its analysis has shown variation in methodology. In addition, contamination of saliva may occur ¹⁵ .

Accordingly, in this study, a new technique was introduced via examining night guards contacting dental restorations. Night guards are polymeric materials designed to fit the occlusal-surfaces of patients to decrease signs and symptoms of bruxism and teeth clenching. Consequently, the study was performed by providing patients who suffered from bruxism with custom-made night guards. An additional inclusion criterion was the presence of amalgam fillings in their upper molars. Amalgam restorations were selected rather than any other dental restoration, as it has been well proven in the literature that mercury is released from such material ¹⁹ . Therefore, utilizing night guards in gathering leached-elements from amalgam-restorations intra-orally was evaluated in this study.

Both the unused night guard and the used surfaces not contacting amalgam-restorations were homogenous, yet the latter showed cracking, which may be due to bruxism. This consistent structure may be attributed to the following: these areas were not contacting dental restorations and no leached elements were deposited. This was confirmed by the EDXA results, which were only carbon and oxygen in both. The night-guards are polymeric materials, with their basic structure consisting of carbon, oxygen and hydrogen ²⁴ . However, hydrogen was not detected as it did not have core electron, only one valence electron that enters in chemical bonding ²⁵ . Analysis relied on excitation of electrons in lower shells, not the valence electron, which was shared in covalent bonding in the case of hydrogen ²⁵ .

The night guard surfaces contacting amalgam restorations showed numerous lustrous particles, which were identified by EDXA as mercury. This is in agreement with numerous studies that have detected mercury released from amalgam restorations ^{19, 26} . Sulfur was also detected, which may be due to tarnish and corrosion of the amalgam ²⁷ .

Since leached elements detected by night guards matched with that reported in the literature, night-guards could be used as a simple method to detect released elements from dental restorations such as amalgam intra-orally.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2017 Abdelraouf RM

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1. SEM images for night guard surfaces contacting and not contacting amalgam restorations. 10.5256/f1000research.12311.d177383 ²⁸

Dataset 2. Raw EDXA data for night guard surfaces contacting and not contacting amalgam restorations. 10.5256/f1000research.12311.d177384 ²⁹