Organic Eluates Derived from Intermediate Restorative Dental Materials

A great number of different types of materials have been used in dentistry as intermediate restoratives. Among them, new resin-based bases have been released in the dental market. The present study focuses on the identification of the organic eluates released from such materials and the study of their surface microstructure in combination with their corresponding elemental composition. For this purpose, the following materials were used:ACTIVA™BioACTIVE-BASE/LINER™, Ketac™Bond Glass Ionomer, SDR™ and Vitrebond™Light Cure Glass Ionomer Liner/Base. Methanolic leachates derived from polymerized materials were analyzed by means of gas chromatography-mass spectrometry (GC-MS). Scanning electron microscopy(SEM) was used for the surface monitoring of suitably prepared specimens. The GC-MS analysis revealed the elution of twenty different substances from the three resin-based materials, while none was eluted from the glass ionomer base. The SEM analysis for Vitrebond™ presented small pits, the one for Ketac™Bond presented elongated cracks, while no voids were present for ACTIVA™BioACTIVE-BASE/LINER™ and SDR™. Moreover, the resin matrix of some dental materials may inhibit elements’ accumulation on the surface layers. Particularly, the detected organic eluents may be related to potential toxic effects.


Introduction
In restorative dentistry a wide range of materials, called intermediate restoratives, are placed upon the dentine prior to the placement of the final restoration. In this context, two main treatments may be applied in clinical practice: (a) direct pulp capping, where the material's placement is in direct contact with the pulp tissue, and (b) indirect pulp capping, where there is a remaining dentin layer between the pulp and the material.Both procedures aim to protect the dental pulp and preserve vitality [1,2]. These intermediate restoratives are often referred to as cavity liners or bases. Calcium hydroxide is the "gold standard" material, since it is traditionally used and has exhibited clinical success [3]. However, in the last decades new materials consisting mainly of calcium silicates, such as mineral trioxide aggregate (MTA), have been introduced in the clinical practice and have exhibited high success rates [4][5][6]. MTA and calcium hydroxide induce the formation of hydroxyapatite, release calcium ions and promote dentin bridge formation with less inflammatory response [1]. Additionally, both of these materials are proved to have a minimum cytotoxic action to the dental pulp tissue [7]. However, both MTA and calcium hydroxide have some disadvantages, like long-setting times or alack of setting, a high solubility, a gradual resorption, weak physical properties and poor handling [2].
(ROS) formed by these monomers [32]. As a consequence, inflammation, inhibition of cell proliferation and apoptosis may result from the release of these monomers [33]. HEMA and TEGDMA are proven to promote inflammatory responses in gingival fibroblasts and the release of prostaglandin E 2 [34,35], could cause allergic reactions [36] and may inhibit the cell proliferation or differentiation of human dental pulp cells into dentin [37]. Low molecular weight monomers such as HEMA and TEGDMA may diffuse through dentinal tubules, reach the pulp tissue and cause the above-mentioned reactions [38]. Since all these materials are placed in close proximity to the pulp and may exhibit some grade of toxicity due to the release of chemical species, the aim of this study was to identify possible organic eluates released from four intermediate bases by the use of gas chromatography-mass spectrometry (GC-MS), as well as to study the surface microstructure in combination with the elemental composition of these materials with scanning electron microscopy (SEM). To the best of our knowledge, there is no literature data on the investigation of the potential chemical activity of such materials, and the findings of the present work intend to clarify the hypothesis that the studied materials may be associated with possible toxic components. Table 1 accumulates the GC-MS identification data for the total compounds eluted from the examined dental materials. The analytes detected in the methanolic eluent for the specific intermediate base material are summarized in Table 2. Representative GC-MS chromatograms of methanol extracts from each material are depicted in Figure 1, while the mean values and standard deviations of the eluted substances are listed in Table 3. It is obvious that no substances were released from Ketac™ Bond, since this material constitutes a conventional glass ionomer cement and was considered as the control group. However, HEMA and camphorquinone (CQ) were released from both Vitrebond™ and ACTIVA™ BioACTIVE-BASE/LINER™, with Vitrebond™ presenting a significantly higher release of both HEMA and CQ than ACTIVA™ BioACTIVE-BASE/LINER™ (p < 0.05). Furthermore, butylated hydroxytoluene (BHT), ethyl 4-(dimethylamino) benzoate (DMABEE) and TEGDMA were released from both ACTIVA™ BioACTIVE-BASE/LINER™ and SDR™. Although the DMABEE release was similar among the two materials, the BHT and TEGDMA elution was significantly higher in SDR™ (p < 0.05). Moreover, methoxyphenyl acetic acid (MOPA) was detected only for ACTIVA™ BioACTIVE-BASE/LINER™, while the benzene chloride (BC) and benzene iodide (BI) compounds were unique for the Vitrebond™ organic eluent.       The surface morphological characteristics of the four studied materials captured by SEM is illustrated in Figure 2. It can be seen that Vitrebond TM exhibited small pits approximating a size of up to 10 µm, while Ketac™ Bond presented elongated cracks along with some filler aggregates. On the contrary, no voids were visible for ACTIVA™ BioACTIVE-BASE/LINER™ and SDR™, yielding structural characteristics close to a typical homogeneous surface. The SEM-EDX elemental analysis data for all materials are plotted in Figure 3. Considerable amounts of fluoride (13.2 wt-%) and calcium (14.1 wt-%) were found for Ketac™ Bond (Figure 3d

Discussion
According to manufacturer's MSDS declaration, ACTIVA TM BioACTIVE-BASE/LINER TM is a bioactive ionic resin with reactive glass filler. In particular, it is a blend of diurethane and other methacrylates with modified polyacrylic acid containing amorphous silica and sodium fluoride ( Table 4). It is also claimed that ACTIVA TM contains no Bis-GMA, BPA and relative derivatives. In the present study, no BPA could be identified in the chromatographic profile of the aforementioned material (Figure 3a). MOPA was found to be the dominant compound among the other eluted components of the ACTIVA TM extract. Previous studies have proven the fungicidal activities of MOPA [39] and its corresponding derivatives [40] against specific fungal strains. Acetophenone (ACP), and mostly its derivatives like 2,2-dimethoxy-2-phenyl acetophenone (DMPA), are widely spread as type I photoinitiators yielding free-radicals due to a unimolecular bond cleavage [41][42][43][44][45]. DMPA is found to induce cell viability [46]. α-Methylstyrene (MS) and cumene (CM) traces could be possibly associated with α-methylstyrene dimer which in turn has served as a reversible addition-fragmentation chain-transfer (RAFT) agent in the synthesis of branched nanogels, containing UDMA crosslinkers, potentially used as shrinkage and stress-limiting resin additives [47]. However, the high temperature conditions during the gas chromatography analysis process can result in the formation of ACP, MS and methyl cumyl ether (MCE) as potential thermal degradation products of cumyl hydroperoxide or dicumyl peroxide [48,49]. The latter residual constituents in the polymer matrix may be used in redox initiator systems promoting the free radical polymerizations of methacrylate monomers at low temperatures [50]. Furthermore, the DMABEE found in ACTIVA's organic eluent mixture is a well-known tertiary amine contributing to such a redox initiation system [51]. DMABEE demonstrates a moderate cytotoxic effect, and due to its lipophilic nature it may accumulate in cell membranes and disrupt their integrity [52]. After 24 hours storage in methanol, the lower eluted fraction of the CQ initiator, compared to the DMABEE and 2-(dimethylamino)ethyl methacrylate (DMAEMA) co-initiators, may be attributed to the steric bulk of the chiral structure of CQ, which could be responsible for lower diffusion rates to methanol despite the relatively low molecular weight (196.22 g/mol). HEMA was found to be the secondabundant eluate, even if it is not declared in the corresponding SDS. Provided that ACTIVA TM is considered a resin-modified glass ionomer, HEMA may finally react with the polyacid and thus contribute to the formation of the resin-modified polyacid chain, as well as act as a potential co-monomer in the crosslinking process [53]. Moreover, HEMA has been correlated with UDMA thermal fragmentation in the GC injector unit [29] and could be an indirect evidence of the unreacted UDMA monomer [1]. HEMA, due to its low molecular weight and high hydrophilicity, may diffuse through dentin, reach the pulp and cause adverse pulpal reactions [54]. In addition, the detected (trimethylolpropane trimethacrylate) TMPTMA is a tri-functional methacrylate monomer usually used in dental composite resins' formulations to prompt improved flexural strength, hardness, absorption, and crosslink density [55] and wear properties [56] by acting as a crosslinking agent in the polymer matrix. The chromatogram pattern in the elution range of 23.37-23.86 min has been also shown by other researchers for different capping materials, corresponding to high molecular weight methacrylates (tetra-EGDMA) [1].
Regarding the Vitrebond TM , GC-MS analysis revealed that HEMA was the most leachable organic component after 24 h aging in methanol. Indeed, this finding fits the MSDS information (Table 4) where HEMA is mentioned in the glass-ionomer liquid composition. BC and BI seem, rather, to be degradation products of diphenyliodonium chloride (DPICl), which has been previously reported as an accelerator of 3-component photoinitiator systems [57,58]. Iodonium salts are capable of photosensitizer's regeneration by inserting active phenyl initiating radicals, and by substituting inactive terminating radicals and concurrently adding new active phenyl radicals [59,60]. Furthermore, the presence of DPICl in the organic leachate is in line with the SDS statement. SDR™ is a bulk-fill restorative material consisting of the base monomers UDMA and ethoxylated bisphenol-A dimethacrylate (Bis-EMA) and the co-monomer TEGDMA, according to the respective MSDS (Table 4). No BPA or other relative derivatives were detected in the methanol leachate after 24 h conditioning. The organic eluent mixture was found to be enriched with oxybenzone (HMBP), which is a UV stabilizer frequently incorporated in cosmetics, personal care products, coating products, fillers, putties, plasters, modelling clay and finger paints [52]. 2-Hydroxypropyl methacrylate (HPMA) and TEGDMA were also eluted, revealing the existence of two methacrylate co-monomers in the SDR™ formulation, while HPMA played a dualrole similar to HEMA, as described above. Additives like BHT, identified in the SDR™ chromatogram, ensure the stability during storage by polymerization inhibition through consuming free radicals that are formed spontaneously [61]. The presence of the DMABEE accelerator as an eluent, accompanied by a CQ weak detection, denotes either a complete degradation of the main initiator to generate free radicals or possible obstacles dealing with diffusion phenomena. 1,6-hexamethylene diisocyanate (HMDI) is a representative starting material used in the synthesis of urethane dimethacrylate monomers [62], which are contained in the SDR™ formulation.
In contrast to the aforementioned materials, Ketac TM Bond did not provide an organic-enriched mixture in methanol, as was expected, because it is a typical glass ionomer cement (Table 4), and hence the setting reaction does not involve any organic monomers and other initiating additives. Furthermore, no traces of tartaric acid were found, indicating a full consumption during the setting process.
Regarding the SEM-EDX analysis, the occurrence of oxygen, aluminum, silicon, phosphorous and calcium in all materials could be generally attributed to the oxides of aluminosilicate glass fillers used in glass ionomer cements [63]. In particular, the obtained results for Ketac TM Bond denoted that a relatively brittle surface due to the absence of resin, as shown in Figure 2d, may favor the formation of partial higher surface areas enriched with high fractions of elements like fluoride and calcium available to be analyzed. On the other hand, the rest of the investigated intermediatebases contain resin, which probably prevents the accumulation of elements on the surface layers, while the penetration of some elements in the ionic form from the bulk to the surface might be activated through the chemical interactions in aqueous media. Indeed, Akbulut et al. conducted an SEM-EDX for SDR™ and Vitrebond TM after incubation with periodontal ligament fibroblasts (PDL) cell media, and the results showed that a higher number of elements remained on specimens' surfaces, including a high ratio of fluoride in the case of Vitrebond TM [64]. Nevertheless, the small amount of detected phosphorous for ACTIVA TM BioACTIVE-BASE/LINER TM confirms the manufacturer's claim about a water-friendly ionic resin containing phosphate acid functionality with antibacterial activity, whereas resin-glass fillers and intermediate base-tooth structure interactions are also sustained in this way. As result, a robust resin-hydroxyapatite complex can be formed through a phosphate group hydrogen ions replacement by calcium in the tooth structure. Barium found on ACTIVA TM and SDR™ surfaces is usually associated with the existence of the radiopacifiying filler agent BaO [65]. In terms of Vitrebond TM , the identified zinc has also been reported by other researchers [64], and its bacterial properties have also been studied comprehensively [66]. The large content of lanthanum for Ketac TM Bond can be attributed to its frequent incorporation in the powder constituents of glass-ionomer cements for an opaqueness increase against rays [67] and was also measured by other researchers [68].  Table 4.

Specimens' Preparation
Five specimens of each material were prepared according to the following procedure:100 mg of uncured material was used to fill Teflon molds in order to produce disks (6 mm diameter, 1 mm thickness). The polymerization of the disks was performed using a curing LED light (Bluephase style, Ivoclar/Vivadent, Amherst, New York, USA, power ranged 1100-1400 mW/cm 2 ), which was applied for 20 s directly on the surface of the samples. Three identical repetitions ofthe experiment were conducted.

Elution Evaluation
A solution of 1ml methanol (Methanol, HPLC gradient grade 99.9+%, CHEM-LAB, Zedelgem, Belgium) containing 0.1 mg/ml caffeine (Caffeine 99%, Alfa Aesar, Kandel, Germany) as internal standard was placed in separate glass tubes, and each sample was immersed in the solution.The glass tubes were secured with a ground glass stopper to prevent evaporation. After 24 hours, the solutions were transferred to separate GC vials and injected into the gas chromatograph.

Separation by Gas Chromatography and Mass Spectrometric Detection
The analyses were performed by using a gas chromatography-mass spectrometry instrument (GC-MS Clarus 500, Perkin Elmer, Shelton, Connecticut, USA) supported by a suitable software (Perkin Elmer, TurboMass version 5.4.2). The GC unit was equipped with an autosampler and a DB-5-MS capillary column (30 m, 0.25 mm id., 0.25 µm film, Agilent, Santa Clara, California, USA). The injector (split 1:20) was held at 250 • C. After a constant temperature of 50 • C for 2min, the oven's temperature increased until 300 • C and remained constant for 5 min. As a carrier, gas Helium 5.0 was used with a constant flow rate of 1 mL/min. The transfer line from GC to MS was set to 310 • C. The mass spectrometer was operated in electron ionization mode (E.I.), while the ion source was operated at 220 • C. Only positive ions were scanned. The syringe was rinsed two times before and after injection.
The identification and quantification of the analytes were performed by using a mass spectrometer in full scan mode scanning from 50 to 450 m/z at a rate of 0.2 scans per second.NIST library (National Institute of Science and Technology, Gaithersburg, MD, USA), retention time and literature data were used for the identification of different compounds. The internal standard, caffeine, was analyzed at the m/z ratio of 194 and was used for quantification, and each unknown peak was normalized to the caffeine peak. Reagent blank samples, only containing caffeine dissolved in methanol, were also analyzed. In order to prevent carry-over effects, methanol was injected between all samples.

Scanning Electron Microscopy Characterization (SEM)
Two specimens of each material (5 × 5 × 5 mm) were prepared and stored at 37 • C. Then, the samples were mounted in resin and were ground using discs and pastes with an automatic polishing machine. Prior to mounting on aluminum stubs, they were carbon-coated to avoid charging under the electron beam. They were viewed under the scanning electron microscope (JEOL, JSM-6390LV, JEOL USA, Inc., Peabody, Massachusetts, US) equipped with an energy dispersive X-ray (EDX) microanalytical system (INCA PentaFETx3, Oxford Instruments, Abingdon, England). Scanning electron micrographs of the different material microstructural components at different magnifications in back-scatter electron mode were captured. The elemental analysis of the specimens' surfaces was carried out by an Energy Dispersive X-ray microanalysis.

Statistical Analysis
The results are presented as mean values with associated standard deviations. A statistical analysis was performed for eluted substances from at least two materials using IBM SPSS software, version 25 with an assumed level of significance p < 0.05.

Conclusions
Different organic substances were eluted from each material, and the substances' elution was dependent on the material's composition. Moreover, the resin matrix of some intermediate dental materials may inhibit the elements' accumulation on the surface layers. The findings, based on the leachate's chemical analysis, confirmed the initial hypothesis that the examineddental materials could be associated with chemical species that may potentially exhibittoxicity.