Cytotoxic Effects of Bulk-Fill Composites on L 929 Fibroblast Cells

Objective: Unlike traditional composite resins, bulk-fill composite resins could be polymerized as thicker layers. This study aims to contribute to the field by investigating the cytotoxic effects of various bulk-fill composite resins on L929 mouse fibroblast cells in vitro. Material and Methods: In our study, six bulk fill and one conventional composite resin were used. Composite resin samples (8×4 mm) were prepared in a sterile cabinet by using a glass mod and polymerizing with a led light device (DTE LUX E, Germany). Composite samples (n:3) of which surface area was calculated according to ISO 10993-12: 2012 standards (3 cm2/ml), were kept in media for 24 h and 72 h in 37 oC incubator, their extracts were filtered in 1:1 and 1:2 proportion and were added on L929 mouse fibroblast cells. Cell viability was examined by the MTT assay and cell death by the LDH test. Cell viability results were evaluated using one-way analysis of variance (ANOVA) test (p<0.05). Results: When the 1:1 extracts from 4 mm thick bulk-fill composite samples were applied on L929 mouse fibroblast cells, cell viability rates showed significant differences compared to the control group at the end of 24 h and 72 h (except for Estelite Bulk Fill Flow). Although the extracts of the tested composite samples at 1:1 and 1:2 ratio at the end of 72 hours caused a decrease in L929 mouse fibroblast cell viability, the cell viability rate of only PRG-containing bulk fill composite and conventional composite remained below the cell viability ratio (70%) specified in ISO standards. Bulk fill composites did not produce toxic effects (except Beautifil Bulk Restorative) according to the LDH test. Conclusions: Despite decreasing in general the cell viability, bulk-fill RESUMO


INTRODUCTION
A dvancements in restorative materials used in dentistry have enabled the use of composite resins in large cavities in posterior teeth [1]. The fact that composite resins are in tooth color makes these materials advantageous in an esthetic sense [2,3]. However, these materials have also disadvantages such as micro leakage and sensitivity occurring due to the polymerization shrinkage [4,5]. Also, as the polymerization depth of conventional composite resins is limited to 2 mm, they are recommended to be used in the layering technique for the restoration of the teeth [6]. The incremental placement of the materials requires longer times in restoration and entails certain risks such as air inflow and contamination between the layers [7]. Furthermore, application of the conventional resins into the deep cavities is more difficult due to the limited depth of cure [8].
In recent years, in order to provide composites that are applicable to the cavity in larger masses and as thicker layers, "bulkfill" composites have been introduced. As the new-generation bulk-fill composites allow for higher degrees of polymerization than the conventional composites due to their advanced translucent structures, they could be placed into the cavity in larger masses (4-6 mm) [9,10]. In a study evaluating the clinical performance of bulk-fill composite composite resins used in 4 mm thick layers provided cell viability rates over the acceptability level, except PRG-containing bulk fill composite (Beautifil Bulk Restorative), which was cytotoxic to L929 mouse fibroblasts.

KEYWORDS
Bulk fill composite; Cytotoxicity; L929 cells; LDH assay. resins in the restoration of cavities in the posterior teeth, it was stated that there was no difference between conventional and bulkfill composites [11].

PALAVRAS-CHAVE
Despite the increasing popularity of bulk-fill composite resins, there are concerns about the biocompatibility of these materials. These materials could release monomers in their structures depending on the physical and chemical conditions in oral environment [12]. It is stated in the literature that bisphenol-A glycidyl methacrylate (Bis-GMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate [UDMA] the essential monomers included in the organic matrix of composite resins -create cytotoxic and mutagenic effects on cells [13]. Recently, a nanohybrid ormocer that includes both nanofillers and glass-ceramic fillers has been introduced by the composite industry. Several studies have shown that ormocers release fewer monomer particles and have less cytotoxic effects than the dymethacrylatebased conventional composites. Those studies have carried out the cytotoxicity tests in mouse fibroblasts [14,15].
The aim of this study is to provide a more detailed comparative perspective on the cytotoxicity of bulk-fill composite resins of different contents through an investigation on L929 mouse fibroblast cells using the MTT test in vitro according to ISO 10993-12:2012. The zero hypothesis of the study is that extracts from 4 mm samples of bulk-fill composites will not show cytotoxic effects on L929 mouse fibroblast cells.
Cylinder-shaped samples of composite having a 3 cm2/ml surface area, which is calculated according to ISO 10993-12: 2012 standards [16], were incubated in 2 ml serum-free Dulbecco's modified eagle medium (DMEM) (HyClone Laboratories, Inc., Logan, UT, USA) (control group in serum-free medium) for 24 and 72 h, at 37 oC, in an incubator with 5% CO2. The tubes were covered with pieces of aluminum foil to prevent the composite samples immersed into the serum-free DMEM medium from being exposed to light. The extracts of composite samples were filtered after 24 h and 72 h periods, diluted with DMEM medium (1:1 and 1:2) and cytotoxicity experiments were conducted.

Cell Culture
The L929 fibroblast cell line stored at -196 °C was let thaw in a water bath at 37 °C and centrifuged. The cells were kept in DMEM, which is supplemented with 10% fetal bovine serum (PAA Laboratories, Linz, Austria), at 37 °C and 5% CO 2 in a humidified incubator. Once the cells reached the optimal density (1×105 cells/ml), the cell suspension was prepared according to the descriptions in ISO 10993-5: 2009 [17] by calculating the cell number of the desired density for a 96well cell culture plate using DMEM medium including 10% FBS and 1% antibiotic. Then the cell suspension was allocated into the 96-well cell culture plate [100 µl/ well] and incubated for 24 h in a 5% CO2 incubator. After the incubation, DMEM was removed and the media remaining of the two different dilutions, in which the composites were immersed, were similarly allocated into wells (100 µl/well) and the materials were incubated for another 24 h in a 5% CO2 incubator. Finally, the MTT assay was performed.

Cytotoxicity Test
USA] was combined with PBS, homogenized and an MTT solution with a final concentration of 5 mg/ml became ready for the cell viability test. The secreted media in 96-well cell culture plate that were incubated for 24 h was removed after incubation, then 100 µl/well DMEM medium and 13µl/well MTT solution were filled in clusters and incubated at 37 °C in a dark environment for 4 h. After that, the MTT solution was removed from the medium by aspiration. 100 µl/well Ammonia-Dimethyl sulfoxide (5:100) mixture was poured into 96-well cell culture plate; and at the optical reader, the absorbance rates were read at 550 nm (BIO-TEK µQuant, BIO-TEK Instruments, Inc, USA) then, the results were compared with the control wells.

Lactate dehydrogenase (LDH) Leakage Assay
The lactate dehydrogenase assay (LDH) for cytotoxicity was performed on the extracts (1:1 ratio) of the bulk fill composite samples after 72 h according to the instructions given in the commercial kit. In brief, L929 cells were cultured in a 96-well plate at 1×104 cells/ well density and incubated in a humidified atmosphere (5% CO2) at 37 °C for 24 h. After removing the culture media, cells were exposed to extracts of the composite samples.
After collecting the existing culture media, it was centrifuged at 600 g for 10 min. Then, 100 µl of LDH reaction mix was added onto 10-µl supernatant of sample and incubated for another 30 min at room temperature. Absorbance levels of the samples were read at 450 nm and 650 nm reference wavelength in microplate spectrophotometer (BIO-TEK µQuant, BIO-TEK Instruments, Inc, USA). All experiments were triplicated. Cytotoxicity percent was calculated as: (absorbance of sample − absorbance of control sample) / (absorbance of high control sample − absorbance of control sample) × 100.

Statistical Analysis
Statistical analysis of the data was performed using the SPSS 22.0 program (SPSS Inc., Chicago, IL, USA). Cell viability rates belonging to the 1:1 and 1:2 diluted extracts of the composite samples obtained at the end of 24 h and 72 h periods were compared by using one-way analysis of variance (ANOVA) and Tukey multiple comparison tests (p<0.05).

Bulk fill composite resins tested, only
Filtek Bulk Fill Posterior and Beautifil Bulk Restorative extracts (1:1) at the end of 24 h showed significant differences in cell viability compared to the control group (p <0.05). The cell viability values of the extracts (1:2) of these composite resins (except Beautifil Bulk Restorative) did not differ significantly compared to the control group (p>0.05), (Table II).  (Table III). When the extracts of bulk-fill composites at the end of 24 and 72 were diluted by 1:2 the cell viability increased (Table II and III). At the end of 72 h, Beautifil Bulk Restorative and conventional composite (Filtek Z250) remained below the ISO cell viability standard (70%) at 1:1 (Table III and Figure 1) and 1:2 dilution (Table III).
When the LDH test results of the extracts of composites (1:1 ratio after 72 h) are examined; The increase in the LDH activity of the Beautifil Bulk Restorative and conventional composite (Filtek Z250) groups was statistically significant compared to the DISCUSSION Bulk-fill composite resins are commonly preferred by dentists for the restoration of   teeth as they could be applied in thick layers. However, due to the failure of led light to reach a sufficient depth during the polymerization of these materials, the insufficiently polymerized monomer particles may remain free in their structure. It has been reported that the biocompatibility of composite resins is correlated with the amount and structure of the organic substances released [18] and monomers released from the resin matrix because of the insufficient polymerization may produce cytotoxic results over time [19,20]. In our in vitro study, we tried to examine the cytotoxic effects occurring on L929 mouse fibroblast cells depending on the use of bulk-fill resins of different contents in 4 mm thickness.
ISO 10993-12: 2012 proposed several cell culture testing models to evaluate the cytotoxicity of dental materials [16]. These are direct contact (direct method), indirect contact with a barrier (indirect method), and the extract method in which the extracts from biomaterials are added onto the cells. In the ISO 10993-5: 2009 standard, it was stated that the tested materials may have toxic potential if the cell vitality is below 70% after MTT test [17]. Lim et al. [21] compared those in vitro test models use to evaluate the cytotoxicity of composite resins and suggested the extract test due to its higher sensitivity if a single test model is planned to be used in the studies.
L929 mouse fibroblast cell lines are the most widely used cells to evaluate the in vitro cytotoxicity of dental materials [22,23]. Among the major advantages of mouse fibroblast cell lines it is stated that they are practical to use, contain one single type of cell and provide more accurate cytotoxic responses [23]. Therefore, in our study L929 mouse fibroblast cell line was preferred.
It is stated that the extent to which the polymerization of the restorative composite resins is accomplished, has an impact on the toxicity [24], and the oxygen inhibition layer formed on the surface of the composites after polymerization increases the monomer release [25]. In their study evaluating the cytotoxicity of the composites, Couchman et al. [26] suggested that the curing time decreased cytotoxicity by increasing the degree of polymerization. In the literature, it is also reported that there is no correlation between the oxygen inhibition layer formed during the polymerization of the samples by covering them with a glass and the amount of monomer release [27]. In our study, composite materials were covered with 1 mm glass coverslip and polymerized for 20 seconds with high intensity (DTE LUX E, Germany, 1200 mW/cm²) led light device.
In a study on the toxicity of bulk-fill composites carried out on mouse fibroblast cells, Toh et al. [28] reported that extracts obtained from 4 mm samples showed more cytotoxicity than 2 mm samples. In a similar study on the toxicity of fluid and paste bulkfill composite resins conducted on L929 mouse fibroblasts by Demirel et al. [29] it was reported that at the end of 72 h composite extracts caused a statistically significant decrease in cell viability level, which is in line with the former. In a study on human pulp cells, by examining the toxic effect of bulkfill composite samples in terms of whether it change at the layers of different polymerization depths [0-2, 2-4 and 4-6 mm] Lee et al. [30] stated that as the irradiation depth increased the more toxicity occurred, and the highest cytotoxicity was observed in the layer of 4-6 mm depth. However, Nascimento et al. [31] reported that bulk-fill resins exhibited low level and/or no cytotoxicity on L929 cells, except for Opus, which showed more moderate cytotoxicity, as pointed out in the MTT assay index.
In our study, at the end of 72 h, the cell viability rate of the Estelite Bulk Fill Flow composite used as 4 mm layers did not cause a statistically significant difference compared to the control group. However, ormocer-based (Admira Fusion x-tra), PRG-containing bulkfill composite (Beautifil Bulk Restorative) and the other bulk-fill composites (GrandioSO x-tra, Tetric N-Ceram Bulk Fill, Filtek Bulk Fill Posterior) caused a significant reduction in the cell viability. As the bulk-fill composites diminished the viability of L929 mouse fibroblast cells, the null hypothesis of the study was rejected.
Legraand et al. [32] the release of LDH culture medium as a result of damage to the cell membrane indicates cell death. Increased LDH activity is associated with an increase in dead cell numbers and a decrease in glucose consumption. In our study, Beautiful Bulk Restorative and conventional composite (Filtek Z250) showed more LDH activity than the control group with extracts in the ratio of 1:1 after 72 h. Our results were in accordance with Legrand.
Schubert et al. [33] found that Admira Fusion had significantly less cytotoxic effect on mouse L929 cells and human gingival fibroblasts than Filtek Supreme XTE and GrandioSO. The absence of certain classic resin monomers in Admira Fusion apparently allowed for lower cytotoxicity and better biocompatibility, compared to resinbased dental restoratives, which is of great importance for the clinical practice. In our study, bulk-fill composite (Admira Fusion x-tra) containing ormocer caused a significant decrease in cell viability at the end of 72 h compared to the control group, even if it did not at the end of 24 h.
It is stated that both the resin content of the composites and the degree of monomer conversion play a determining role in cytotoxicity levels [34]. Although bulk-fill composites have many advantages, there could remain some unpolymerized monomers at a depth of 4 mm. Those monomers, Bis-GMA, TEGDMA and UDMA, released from the structure of composites have been proven to be cytotoxic in many studies [35,36]. The toxicity grading of these monomers has been reported to be as Bis-GMA> UDMA> TEGDMA [36]. In our study, Estelite Bulk Fill Flow, which has similar monomers (Bis-GMA, Bis-MPEPP, TEGDMA, UDMA), showed the highest cell viability, while Beautifil Bulk Restorative showed the lowest one.
In this study, Beautifil Bulk Restorative was observed to cause a significant reduction in cell viability in both 24 and 72 h of experimentation periods. This product contains PRG filler in its resin matrix, unlike the other tested bulk-composites. The fluoro-aluminosilicate glass is pre-reacted with polyacid by forming a glass-ionomer matrix structure and blended with resin. Resin-based restorative materials containing PRG filler have been reported to provide higher fluoride release than compomers due to their glass ionomer hydrogel matrices [37]. In line with the results of this study, in a previous study, Toh et al. [28] found that Beautifil Bulk Restorative was to be more cytotoxic than all other tested bulk-fill composites. They suggested that the cytotoxic effects of Beautifil Bulk Restorative might be caused by the release of fluoride and other ions, such as PRG fillers including aluminum, boron, sodium, silicon, strontium, and zinc.
In the previous literature on the toxic effects of conventional composites on L929 mouse fibroblasts, it was stated that the decrease in cell viability after the first 24 h was not significant, while it was observed to reach more significant levels after 72 h [33,38]. The data obtained in our study shows that composites keep releasing cytotoxic materials after the first 24 h following polymerization. Furthermore, the reduction in cell viability turned out to be higher in the 72 h extracts.
The findings of this in vitro study, which aimed to examine the cytotoxic effects of bulk-fill composites, are limited to the data collected on a single type of cell line and a two types of cytotoxicity test applied. Then, our study provides only a general and elementarylevel evaluation about the cytotoxicity of bulkfill composites. The tests models applied onto different cell lines or cells that are sourced from humans' oral environment could give different responses in terms of cytotoxicity.

CONCLUSION
At the end of 72 h, the majority of bulkfill composites decreased the cell viability but they did not cause unacceptable cytotoxic effects to L929 mouse fibroblasts, except PRGcontaining bulk fill composite (Beautifil Bulk Restorative), which was cytotoxic.