Could night-guards be used as a simple method to detect leached-elements from dental restorations intra-orally? A study

Detection of leached-elements from dental restorations Background: intra-orally has been a subject of prime importance in dental research. However, this is challenging as most of the present techniques have some limitations. In this study, a new simple method was proposed via using night-guards. Thus, the aim of the study was to verify if night-guards could detect leached-elements from restorations as dental amalgam. Ten upper custom-made night-guards were fabricated for patients Methods: suffering from bruxism, who had amalgam-restorations in their upper molars. The night-guards were delivered to the patients and they were instructed to wear the night-guards during when they were asleep. After six months, 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. In the used night-guards, two areas were studied: the fitting surfaces contacting the amalgam restorations and the fitting surfaces not contacting amalgam restorations. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Analysis (EDXA) were used to examine the structural and elemental changes in the night-guards. SEM of the unused night-guard revealed a homogenous structure, Results: and the composition was carbon and oxygen, as shown using EDXA (C=88.9wt% and O=11.1wt%). By contrast, the fitting surfaces of the night-guards contacting amalgam restorations showed numerous lustrous particles. Elemental analysis of these areas showed the presence of mercury and sulfur, in addition to carbon and oxygen (Hg=21.2wt%, S=2.5wt%, C=67.1wt% and O=9.2wt%). The night-guards’ fitting surfaces not contacting amalgam restorations showed slight cracking, and the composition was carbon and oxygen (C=88.3wt% and O=11.7 wt%). : Analyzing fitting surfaces of night-guards contacting dental Conclusions restorations, such as amalgam, could aid in understanding the nature of leached-elements from these restorations intra-orally. However, further studies about its application upon dental-restorations other than amalgam are recommended. 1 2

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][2][3] . Various analytical means had been used to analyze the leached-elements, including inductively-coupled-plasma, atomic-absorption, Fouriertransform-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.

Methods
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 nightguards 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.

Discussion
The oral-cavity is an aggressive environment, which may affect the integrity of dental restorations [16][17][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 occlusalsurfaces 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 custommade 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.

Author contributions
It should be noted that all the work, including the clinical steps, was performed by the author.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.
At the very least, elemental mapping should have been used, identifying the restoration borders carefully and looking for migration beyond them. Particulates should have been studied in more detail, but the experiment fails to achieve what it sets out to do, but it seems that there is no comprehension of this or why. For example, was the sulphur already on the amalgam surface as sulphide (which is likely) and simply transferred? I can think of two ways of testing this -both very simple. But, without this information, we have no idea what is going on. Indeed, why is there no silver?
The English is pretty poor, with failures of logic and sense in many places. At the very least it should have been copy-edited by a native English speaker versed in technical writing before submission. It is in the best interests of all authors that this be done where English is not the first language. It would save referees an awful lot of trouble, and authors a lot of time.
Scientifically, this paper is below the standard required in several respects. Division of the content into the proper sections is required. However, the 'review' section is of no value, and the discussion completely empty except for repetition of introductory material and other material of no value.
With some sensible direction, something useful might have been achieved with the basic idea, but this has not occurred. The remainder of this paragraph is padding. para 5: results, delete. para 6: is the totality of the discussion? Actually, a conclusion, but very weak. There is no point in Figures 3, 5, and 7 -this is raw data. Fig. 4 : elemental maps for Hg and S (as well as Ag, Sn, Cu, if ever detected locally) would make more sense. "Lustrous" is quite inappropriate for a description from an SEM image -this character is not detectable.

Is the study design appropriate and is the work technically sound? No
Are sufficient details of methods and analysis provided to allow replication by others? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? No source data required

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.

Sabry A. El-Korashy
Faculty of Science, Department of Chemistry, Suez Canal University, Ismailia, Egypt Different methods are used in determination of the leached-elements from dental restorations intra-orally are reported. Some of these methods have been performed both and , using different in vitro vivo analytical techniques. They were carried out via surrounding media, such as distilled water or artificial saliva. However, these methods still having some reservations and almost take a great effort. In the present article, the author used very simple and new technique for measuring the leached elements from amalgam restorations via fabricated polymeric night guards for the tested patients suffering from bruxism. The concentrations of the leached elements after removing the night guards were measured using SEM with EDAX spectroscopy.
In my opinion, the article is accepted for publication after answering the following questions: The results of EDAX (Fig. 5) have detected the Hg, O, S and Ca elements. The author did not