Abstract
Objectives
The study was aimed at investigating the cytotoxicity of different composites as a function of composite shade and the light curing unit (LCU) employed.
Methods
Non-polymerized and polymerized samples of the composites Grandio ® (VOCO, Cuxhaven), Solitaire ® (Heraeus Kulzer, Hanau) and Filtek Z 250 ® (3M/Espe, Seefeld) in two markedly differing shades (A2, C2) were prepared. Polymerization was performed with two LCUs: Heliolux II (Ivoclar/Vivadent, Ellwangen) and Swiss Master Light (EMS, Nyon, Switzerland). To obtain composite extracts, the samples were immersed in cell culture medium (DMEM – Dulbecco‘s Modified Eagle Medium), which was replaced daily up to the 7th day of the experiment, and then on the 14th, 21st and 28th day. After incubation of human gingival fibroblasts (HGF) with the extracts obtained, cytotoxicity was determined using the MTT test.
Results
With the non-polymerized samples, essentially no influence of the composite shades investigated on HGF viability was detected, with the exception of the Solitaire material, where a higher cytotoxicity of the shade C2 in the non-polymerized state was found at the end of the observation period. After polymerization of the different composites, the cytotoxic reaction observed for the extracts of shade C2 was stronger than that observed for A2. After polymerization with the Heliolux II (HLX) LCU, the extracts of composites Grandio and Solitaire C2 were significantly more toxic than those of the A2 shade ( p < 0.01). Polymerization with the Swiss Master Light (SML) reduces this cytotoxic effect. The extracts of the Grandio composite showed the least cytotoxic effect throughout the observation period, irrespective of the LCU used. For the extracts of the Z250 specimens, the cytotoxicity observed was generally higher.
Significance
The results show that the shade of the composite has an influence on its cytotoxicity and that this cytotoxicity is also influenced by the light curing unit used. It was observed that composites of the darker shade (C2) had a higher cytotoxicity, which varied with the LCU employed.
1
Introduction
In routine dental practice, biomaterials are applied in filling therapies, which interact with the surrounding tissues, but may also have an influence on the human organism as a whole .
Composites are used in a great number of color shades. While this is especially due to esthetic reasons, the biocompatibility of these shades also plays an important role . Composites have been proved to have a cytotoxic effect on a variety of tissues and cells . Recently, our team established that, besides the composite and the dental adhesives used, also the light curing unit (LCU) employed can influence the cytotoxicity of the material .
It is known that the successful use of the composites is subject also to various technical parameters involved in their application . Several studies have proved the influence of the polymerization conditions, such as light wavelength, exposure duration, distance of the light exit and light intensity, on the degree of polymerization . These factors mentioned also determine the degree of hardness of the composite solid, the polymerization depth and, indirectly, the release of cytotoxic residual monomers . However it is not only the extent of monomer conversion but also the density of polymeric cross-linking that can influence the release of unconverted components .
In this way, also the chemical composition of the composites, irrespective of the polymerization conditions, has an influence on the degree of polymerization . Given the wide range of composite shades and the strong inherent yellowish hue of the standard photoinitiator camphorquinone, especially the production of brighter or darker shades often requires the admixture of further photoinitiators . These are, for example, Lucirin TPO or phenyl-propanedion (DPD), which have their absorption maximum at lower light wavelengths. The amount of these co-photoinitiators in the photoinitiator system of the composites, together with the spectral light intensity of the light curing unit (LCU), can have a significant influence on the polymerization degree and, in the end, on the residual monomer content . Moreover, the use of several pigments, e.g. for darker composite shades, modifies not only the chemical composition but also the optical properties of the materials . This might influence the light scattering and reflection properties of the composite, possibly resulting in an attenuation of the light intensity and, thus, a loss in the efficiency of light curing. Especially for thick increments this means a change in polymerization in deeper increment layers, whereas this influence is lacking on the surface . No influence of the LCU used has been documented so far.
The present study was aimed at investigating in an in vitro model whether, under identical polymerization conditions, different composite shades can have an influence on the cytotoxicity of the material and whether this varies with the LCU employed.
2
Materials and methods
2.1
Materials and sample preparation
Two different light curing units ( Table 1 ) were used to polymerize three different composites in two different shades (A2, C2) currently used in dental practice ( Table 2 ). The cytotoxicity of the extracts and its variation with power density were investigated in vitro.
| Heliolux II | Swiss master light | |
|---|---|---|
| Manufacturer | Vivadent, Germany | EMS (Electro Medical Systems), Switzerland |
| Light source | 1 halogen lamp, 35 W | 1 halogen lamp, 260 W |
| Optical fiber [mm] | 8.0 | 11.0 |
| Radiation intensity (Radiant energy density) [mW/cm 2 ] | 676 | 3000 |
| Exposure time [s] | 40 | 4 |
| Light energy [J] | 13.6 | 11.4 |
| Emission spectrum [nm] | 390–490 | 390–520 |
| Name | Z250 | Solitaire 2 | Grandio | |
|---|---|---|---|---|
| Type | Fine hybrid composite | Fine-grain hybrid composite | Nanohybrid composite | |
| Manufacturer | 3M, St. Paul, MN, USA | Heraeus Kulzer Dormagen, Germany | Voco, 27457 Cuxhaven Germany | |
| Shade | A2 C2 |
A2 C2 |
A2 C2 |
|
| Organic matrix | BIS-GMA UDMA BIS-EMA |
BIS-GMA TEGDMA |
BIS-GMA TEGDMA |
|
| Inorganic fillers | Zirconium–silicon particles 0.01–3.5 (average: 0.6) |
Barium–aluminum– silicate glasses, Porous silicate glass |
Spherical nanoparticles of silicon dioxides 0.02–0.05 Ceramics |
|
| Filler content [vol.%] | 60 | 58 | 72 | |
| Filler content | 82.1 | 75.1 | 87.0 | |
| [wt.%] | ||||
| Photoinitiator content with absorption maximum within | 450–500 nm | Yes b | Yes a | |
| <450 nm | No | Yes c,d | ||
For each composite–LCU combination we prepared five samples, 2 mm thick and 4 mm in diameter, in a Teflon (PTFE) mold. The samples were polymerized for 40 s with the Heliolux II LCU (HLX) or 4 s with the Swiss Master Light (SML), according to the instructions of the manufacturers. Polymerization was carried out without a polyester film covering the samples, so as to create worst-case conditions. In the same way, 5 test specimens per composite, of the same dimensions, were prepared of non-polymerized material. The samples, each with 200 μl cell culture medium, were placed into 96-well microplates. The ratio of the sample surface area to the volume of the solution was adjusted to approximately 2.5 cm 2 /ml, which is within the 0.5–6.0 cm 2 /ml range recommended by ISO . The test batches were stored in the dark at 37 °C. The extracts of the 1st to 7th days and the 14th, 21st and 28th days were individually collected in test-tubes and stored at −20 °C until further use. After every removal of extract, the sample bodies were rinsed with 200 μl PBS (phosphate-buffered balanced salt solution) and again immersed in fresh 200 μl medium.
2.2
Cell culture
After approval (1881-10/06) by the Ethics Committee of the Friedrich-Schiller-University Jena, human gingival fibroblasts (HGFs) were obtained from a healthy gingival biopsy of a female patient aged 42 by the explant method . The HGFs were cultivated by several passages in DMEM (Dulbecco’s Modified Eagle Medium) with 10% FCS (fetal calf serum) and 0.1% AAS (antibiotic antimycotic solution) at 37 °C and 5% CO 2 . Only cells of the 6–8th passages were used to minimize the differences in cell cycle.
2.3
Cytotoxicity
The cells of the HGF culture were pipetted into 96-well microplates with a cell density of approx. 10,000 cells per well. To increase the sensitivity of our test setting, we immediately topped up with 100 μl composite extract per well. In this way, 10 parallel test batches were obtained from each composite/LCU combination and from the unpolymerized composites, plus 10 control batches with DMEM. Incubation of the cell cultures with the test substances was done for 48 h at 37 °C and 5% CO 2 . Subsequently, we used the Roche MTT Test to determine the cell viability of the HGFs which depends on the amount and the activity of mitochondrial dehydrogenases in the living cells. The mitochondrial dehydrogenases reduce the yellow MTT dye (tetrazolium salt) into the insoluble blue formazan. After adding the solubilization solution, we measured the optical densities (OD) with a microplate reader at 570 nm, and calculated the cell viability according to the following equation:
Cell viability [ % ] = 100 × OD mean of control groups OD mean of control groups
2.4
Statistics
The data were analyzed with the SPSS 17.0 statistics program for windows. We calculated the means and standard deviations. The numbers of samples were 10 for each composite/LCU combination, i.e. 30 for each material. Each experiment was independently repeated five times. Significance was tested by analysis of variance with repeated measurements, and correction according to Bonferroni was applied in multiple comparisons. The significance level was determined as p < 0.01.
2
Materials and methods
2.1
Materials and sample preparation
Two different light curing units ( Table 1 ) were used to polymerize three different composites in two different shades (A2, C2) currently used in dental practice ( Table 2 ). The cytotoxicity of the extracts and its variation with power density were investigated in vitro.
| Heliolux II | Swiss master light | |
|---|---|---|
| Manufacturer | Vivadent, Germany | EMS (Electro Medical Systems), Switzerland |
| Light source | 1 halogen lamp, 35 W | 1 halogen lamp, 260 W |
| Optical fiber [mm] | 8.0 | 11.0 |
| Radiation intensity (Radiant energy density) [mW/cm 2 ] | 676 | 3000 |
| Exposure time [s] | 40 | 4 |
| Light energy [J] | 13.6 | 11.4 |
| Emission spectrum [nm] | 390–490 | 390–520 |
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