Abstract
Objectives
Unpolymerized (co)monomers and additives can be released from resin based composites (RBCs) and can enter the human organism. In this study, the binding of ingredients from composites to salivary proteins and plasma proteins was investigated.
Methods
The composites investigated were Admira ® flow, Venus ® Diamond flow, Filtek™ Supreme XTE flow, Tetric EvoCeram ® , Tetric EvoFlow ® . The samples ( n = 4) were polymerized according to the instructions of the manufacturer of RBCs. The samples were immersed into native saliva, protein-free saliva (artificial saliva), water and ethyl acetate, and incubated at 37 °C for 24 h or 72 h. The eluates were analyzed by gas chromatography/mass spectrometry. To determine the binding to salivary proteins, the concentration of (co)monomers and additives detected in native saliva was compared to the concentration of (co)monomers and additives detected in protein-free saliva, water and ethyl acetate respectively.
To assess the affinity of TEGDMA, EGDMA, DEGDMA, PMGDMA, BPA, and DCHP to human serum albumin (HSA) and human α 1 -acid glycoprotein (AGP), a plasma protein binding assay (ABNOVA, Transil XL PPB Prediction Kit TMP-0212-2096) was performed.
The statistical significance ( p < 0.05) of the difference between the experimental groups was tested using the one-way-analysis of variance (ANOVA), followed by Tukey‘s analysis.
Results
The concentration of TEGDMA, GMA and CyHEMA released in native saliva was significantly lower than the concentration released in protein-free saliva or water (Admira ® flow: concentration of TEGDMA after 72 h: 0.08 mmol/L (native saliva), 0.34 mmol/L (protein-free saliva), 0.39 mmol/L (water)). The concentrations of HEMA, EGDMA, DDDMA and CQ released in native saliva remained even below the detection limit, compared to the other extraction media.
Protein binding of the tested methacrylates to HSA + AGP was 82–85%, the binding of DCHP was 96.6%, and the binding of BPA was 95.2%.
Significance
Artificial saliva or water as extraction medium does not reflect the real physiological situation in the body. Salivary and plasma proteins may bind (co)monomers and additives and may thereby contribute to a lower bioavailability of leachables from RBCs in vivo than previously thought.
1
Introduction
In the last decade the use of resin based composites (RBCs) has increased tremendously. RBCs, consisting of a number of (co)monomers and additives, belong to the most commonly used filling materials. Due to the incomplete (co)monomer-polymer conversion, a release of the unpolymerized (co)monomers from the dental composite is possible . There are many in vitro studies on the toxicity and biocompatibility, which have shown that some of the eluted (co)monomers and additives even have estrogenic, mutagenic, teratogenic and genotoxic effects . Previous in vivo studies have demonstrated that HEMA, TEGDMA and BisGMA can be metabolized to the expoxy compound 2,3-epoxymethacrylic acid in hepatic microsomes . Epoxides are regarded as mutagenic and carcinogenic agents .
A previous study has shown that the amount of eluted (co)monomers and their recovery rate is dependent on the medium used for extraction. Thus, the recovery rate of TEGDMA by HPLC analysis is lower if a cell culture medium with fetal calf serum is used, which contains a high amount of proteins .
To assess the toxicity and biocompatibility of dental composites, it is not only important to know which (co)monomers and additives are released and in what quantity, but whether these substances may also bind to proteins. This protein binding could lead to a reduced bioavailability in vivo . Only the free, non-protein-bound fraction of an active agent may exert pharmacological or toxicological effects, penetrate cell membranes and can be eliminated .
Proteins are present in a concentration of 2.5 g/L in saliva and they have a decisive influence on the properties of saliva . Overall, saliva contains more than 1000 proteins with a molecular weight from 40 to 1000 kDa and are almost exclusively glycoproteins . In quantity, the mucins dominate, which occur in two basic forms in saliva. Mucins are generally classified in high molecular weight proteins (MUC5B, former MG1) and low molecular weight proteins (MUC7, former MG2) . Biologically active and antibacterial salivary proteins are: agglutinin, lactoferrin, lysozyme, peroxidases, histidine-rich proteins (histatins), defensins and cystatins .
The aim of this study was to examine the binding of releasable ingredients from the investigated RBCs to the above-mentioned proteins. For this purpose native saliva (with proteins) was used as an extraction medium for the elution experiments in addition to protein-free saliva, water or ethyl acetate.
The concentration of (co)monomers and additives released in native saliva was compared to the concentration of (co)monomers and additives released in protein-free saliva, water and ethyl acetate, respectively. (Co)monomers and additives can also be swallowed and can enter the blood and finally the whole organism by absorption via the intestinal tract . In addition to the knowledge of the binding of (co)monomers to salivary proteins, the binding to plasma proteins is also important to assess the toxicity of dental materials. Therefore, the binding of some methacrylates and additives to specific plasma proteins was also examined. The null hypotheses tested was that the recovery rate of (co)monomers and additives via GC–MS would not be influenced by salivary or plasma proteins.
2
Materials and methods
2.1
Chemicals
All chemicals and reagent products were obtained from Merck, Darmstadt, Germany and were of highest purity available. RBCs tested in the elution study are shown in Table 1 . Four flowable composites and Tetric Evo Ceram ® (a nano-hybrid composite) were investigated. The flowable composites were chosen because preliminary tests have shown, that the tested flowables can elute more (co)monomers which were considered more relevant for our study.
| Composite; Manufacturer; [LOT in parenthesis] | Composition of material based on manufacturer’s data | Curing time recommended by manufacturer |
|---|---|---|
| Admira Flow; VOCO GmbH, Cuxhaven, Germany; [1232535] | N/A | 40 s for shade A3 in layers of max. 2 mm |
| Venus ® Diamond Flow; Heraeus Kulzer GmbH; Hanau; Germany; [010100] | UDMA, Bis-EMA, Ba-Al-F silicate glass, YbF3; SiO 2 | 20 s for shade A3 in layers of max. 2 mm |
| Filtek™ Supreme XTE Flow; 3 M ESPE, Seefeld, Germany; [N336909] | Bis-GMA, TEGDMA and Bis-EMA, silanized ceramic; silanized silicic acid; dimethacrylate-polymer (functionalized); zirconium oxide powder glass (silanized), additives | 20 s for shade A3 in layers of max. 2 mm |
| Tetric Ceram ® ; Ivoclar Vivadent, Ellwangen, Germany; [R09964] | Bis-GMA, UDMA, and Bis-EMA, barium glass, ytterbium trifluoride, mixed oxide, prepolymer, additives, catalysts, stabilizers, pigments | 20 s for shade A3 in layers of max. 2 mm |
| Tetric Flow ® ; Ivoclar Vivadent, Ellwangen, Germany; [R54703] | Bis-GMA, UDMA and DDDMA, barium glass, ytterbium trifluoride, mixed oxide,highly dispersed silicon dioxide, copolymer, additives, catalysts, stabilizers, pigments | 20 s for shade A3 in layers of max. 2 mm |
To assess the plasma protein binding to human serum albumin (HSA) + α 1 -glycoprotein (AGP), a protein binding assay (Transil XL PPB Prediction Kit TMP-0212-2096) from ABNOVA (Taipei, Taiwan) was performed.
All substances detected in the elution study and tested in the protein binding assay with abbreviations are shown in Table 2 .
| Compound Abbreviation | Compound | Limit of detection [mmol/L] |
|---|---|---|
| CyHEMA (comonomer) | Cyclohexylmethacrylate | 0.001 |
| DDDMA (comonomer) | 1,10-decandiol dimethacrylate | 0.0005 |
| DEGDMA (comonomer) | Diethylene glycol dimethacrylate | 0.004 |
| EGDMA (comonomer) | Ethylene glycol dimethacrylate | 0.004 |
| GMA (comonomer) | Glycidyl methacrylate | 0.003 |
| HEMA (comonomer) | Hydroxyethyl methacrylate | 0.001 |
| PMGMA (comonomer) | Pentamethylene glycol methacrylate | 0.0005 |
| TEGDMA (comonomer) | Triethylene glycol dimethacrylate | 0.001 |
| BPA (decomposition product of monomers) | Bisphenol A | 0.00008 |
| BHT (inhibitor, antioxidant) | 2,6-Di-t-butyl-4-methyl phenol | 0.00005 |
| CQ (photoinitiator) | Camphorquinone | 0.0006 |
| DMABEE (coinitiatior) | 4-N,N-dimethylaminobenzoic acid butyl ethoxy ester | 0.0003 |
| DCHP (softener) | Dicyclo hexyl phthalate | 0.0004 |
| DEDHTP (softener) | Diethyl-2,5-dihydroxyterephthalate | 0.0002 |
2.2
Preparation of samples
From each of the five light-curable RBCs ( Table 1 ), 32 samples ( n = 4) of approximately 100 mg (thickness of 2 mm; diameter of 5 mm; surface of the cylinder of 70.65 mm 2 ) were prepared according to standard procedure as follows. For the preparation of the samples, polytetrafluoroethylene (PTFE) rings were used. The PTFE rings were filled with uncured dental material, covered with plastic strips (Frasaco, Tettnang, Germany) to prevent the formation of an oxygen inhibition layer and were finally polymerized with a LED-lamp (Elipar S™10 ® high intensity halogen light, 1200 mW/cm 2 , 3 M ESPE, Seefeld, Germany) in accordance with the manufacturer‘s instructions ( Table 1 ). The curing unit was directly applied on the sample’s surface. The light intensity was controlled with Demetron ® Radiometer (Kerr, USA) and was always between 1100 and 1200 mW/cm 2 .
Directly after curing, the samples were immersed into 500 μL of native saliva, protein-free saliva, water (LC–MS-Grade, ROTISOLV ® , Roth, Karlsruhe, Germany) and ethyl acetate (LC–MS-Grade, ROTISOLV ® ≥ 99.9%, Roth, Karlsruhe, Germany), respectively. The samples were stored in the absence of light in brown GC-vials (Macherey-Nagel, Düren, Germany) at 37 °C for 24 h and 72 h, respectively.
Native saliva was collected from three volunteers as described in previous studies : the volunteers were asked not to swallow and spat into a 50 mL Falcon-Tube. The saliva was immediately used as extraction medium without prior freezing. To produce protein-free saliva as extraction medium, native saliva was centrifuged for 45 min by using Amicon filter (Cut-off 3 kDa) (Amicon Ultra-4 Centrifugal Filter Devices, Merck, Darmstadt, Germany). Ethyl acetate was used as a slightly polar and aprotic solvent since it dissolves even higher lipophilic ingredients from dental composites.
2.2.1
Preparation of samples in protein-free saliva and water
After 24 h and 72 h, 10 μL of an aqueous caffeine solution (0.01 mg/mL) was added as internal standard to determine the relative quantities of substances released from RCBs (see Section 2.3 ) and the sample was mixed for 30 s. The resulting eluate was transferred to 15 mL tubes and extracted with 200 μL of ethyl acetate. To optimize layer separation, the samples were centrifuged at 2800 rpm for 10 min. A total of 100 μL of the organic layer was analyzed by gas chromatography/mass spectrometry (GC–MS).
2.2.2
Preparation of samples in native saliva
The internal standard was added according to Section 2.2.1 . The eluate was subsequently transferred into the Amicon filter and was centrifuged for 45 min at 4000 rpm. The filtrate was then extracted with 200 μL of ethyl acetate and centrifuged at 2800 rpm for 10 min. A total of 100 μL of the organic layer was analyzed by GC–MS.
2.2.3
Preparation of samples in ethyl acetate
After 24 h and 72 h, a caffeine solution (0.01 mg/mL) in ethyl acetate was added as internal standard and the sample was mixed. Afterwards, 100 μL of the eluate was analyzed by GC–MS.
2.3
Analytical procedure
The analysis of the eluates was performed on a Finnigan Trace GC ultra gas chromatograph connected to a Dual-Stage Quadrupole (DSQ) mass spectrometer (Thermo Electron, Dreieich, Germany). A Factor Four ® capillary column (length 25 m, inner diameter 0.25 mm; coating 0.25 μm; Varian, Darmstadt, Germany) was used as the capillary column for GC. The GC oven was heated from 50 °C (2 min isotherm) to 280 °C (5 min isotherm) with a rate of 25 °C/min, and 1 μL of the solution was injected with a split ratio of 1:30. Helium 5.0 was used as carrier gas at a constant flow rate of 1 mL/min. The temperature of the split–splitless injector, as well as of the direct coupling to the mass spectrometer, was 250 °C. MS was operated in the electron ionization mode (EI, 70 eV), ion source was operated at 240 °C; only positive ions were scanned. The performed scan ran over the range m/z 50–600 at a scan rate of 4 scan/s for scans operated in full scan mode in order to qualify the analytes.
The results were referred to an internal caffeine standard (0.01 mg/mL caffeine = 100%), which allows to determine the relative quantities of substances released from various resin-based materials as described in previous studies . All eluates were analyzed five times ( n = 5). Values of integrated areas of the relevant signals of the compounds were compared to the corresponding reference standards and normalized by means of the caffeine standard. Identification of the various substances was achieved by comparing their mass spectra with those of reference standards, the NIST/EPA library and literature data . Detection limit for each compound is listed in Table 2 .
2.4
Plasma protein binding assay
To determine the plasma protein binding of TEGDMA, EGDMA, DEGDMA, PMGDM, BPA and DCHP to HSA + AGP (mixed in each well at a physiological ratio of 24:1), a protein binding assay (ABNOVA, Transil ® XL PPB Prediction Kit TMP 0212-2096) was performed. As a positive control propranolol hydrochloride was used due to its high protein binding and as a negative control caffeine was chosen due to its low protein binding properties .
The assay was performed exactly according to the manufacturer’s instructions. Plasma protein binding was determined by incubating a fixed concentration of the above mentioned substances with varying concentrations of the plasma proteins HSA and AGP. The compounds tested were dissolved in dimethyl sulfoxide (DMSO) and a stock solution was prepared (80 μM). The final DMSO concentration was 2%. The frozen plate was thawed at 25 °C for 3 h and then centrifuged for 5 s at 750 g. Fifteen μL from a stock solution with 80 μM of the test compound were added, thus retaining the final assay concentration at 80 μM. The substances were incubated for 12 min on a plate shaker at 1000 rpm. After incubation, the plate was centrifuged for 10 min at 750 g. The supernatant (150 μL) was removed and extracted with 100 μL of ethyl acetate and then analyzed by GC–MS.
2.4.1
Derivatization of propranolol hydrochloride with pentafluorobenzoyl chloride
In order to analyze the positive control propranolol of the protein binding assay by GC–MS, the process of derivatization was necessary. For derivatization of propranolol, pentafluorobenzoyl chloride was used. First, the propranolol dissolved in wells was transferred into the corresponding base by 10% NaOH aq. Afterwards the free base was extracted with 100 μL of dichloromethane. The propranolol base was derivatized in the presence of trimethylamine with pentafluorobenzoyl chloride. Ten μL of the propranolol base were mixed with 150 μL of the derivatization reagent and incubated at 25 °C for 2 h. After 2 h the sample was reduced under a gentle stream of N 2 to dryness. A total of 100 μL of dichloromethane was added and analyzed by GC–MS .
2.5
Calculations and statistics
The results are presented as the mean ± standard deviation (SD). The statistical significance ( α = 0.05) of the differences between the experimental groups was tested using the one-way-ANOVA, followed by Tukey‘s analysis .
2
Materials and methods
2.1
Chemicals
All chemicals and reagent products were obtained from Merck, Darmstadt, Germany and were of highest purity available. RBCs tested in the elution study are shown in Table 1 . Four flowable composites and Tetric Evo Ceram ® (a nano-hybrid composite) were investigated. The flowable composites were chosen because preliminary tests have shown, that the tested flowables can elute more (co)monomers which were considered more relevant for our study.
| Composite; Manufacturer; [LOT in parenthesis] | Composition of material based on manufacturer’s data | Curing time recommended by manufacturer |
|---|---|---|
| Admira Flow; VOCO GmbH, Cuxhaven, Germany; [1232535] | N/A | 40 s for shade A3 in layers of max. 2 mm |
| Venus ® Diamond Flow; Heraeus Kulzer GmbH; Hanau; Germany; [010100] | UDMA, Bis-EMA, Ba-Al-F silicate glass, YbF3; SiO 2 | 20 s for shade A3 in layers of max. 2 mm |
| Filtek™ Supreme XTE Flow; 3 M ESPE, Seefeld, Germany; [N336909] | Bis-GMA, TEGDMA and Bis-EMA, silanized ceramic; silanized silicic acid; dimethacrylate-polymer (functionalized); zirconium oxide powder glass (silanized), additives | 20 s for shade A3 in layers of max. 2 mm |
| Tetric Ceram ® ; Ivoclar Vivadent, Ellwangen, Germany; [R09964] | Bis-GMA, UDMA, and Bis-EMA, barium glass, ytterbium trifluoride, mixed oxide, prepolymer, additives, catalysts, stabilizers, pigments | 20 s for shade A3 in layers of max. 2 mm |
| Tetric Flow ® ; Ivoclar Vivadent, Ellwangen, Germany; [R54703] | Bis-GMA, UDMA and DDDMA, barium glass, ytterbium trifluoride, mixed oxide,highly dispersed silicon dioxide, copolymer, additives, catalysts, stabilizers, pigments | 20 s for shade A3 in layers of max. 2 mm |
To assess the plasma protein binding to human serum albumin (HSA) + α 1 -glycoprotein (AGP), a protein binding assay (Transil XL PPB Prediction Kit TMP-0212-2096) from ABNOVA (Taipei, Taiwan) was performed.
All substances detected in the elution study and tested in the protein binding assay with abbreviations are shown in Table 2 .
