Abstract
Purpose
To compare the cytotoxicity, gelatinolytic activity, and protein levels (MMP-2 and MMP-9) produced by 3T3 fibroblasts cells after stimulation with GuttaFlow 2 and AH Plus.
Methods
3T3 fibroblasts were incubated with elutes of GuttaFlow 2 and AH Plus for 24 h. The cytotoxicity of tested materials was determined using the MTT and the LDH assay. Supernatants of cell cultures incubated with sealers were collected to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Cell lysates were used to determine MMP-2 and MMP-9 protein levels by Western Blot. Data were analyzed using ANOVA and Tukey test (P<0.05).
Results
AH Plus showed significantly less cell viability (mitochondrial activity of cells) than GuttaFlow 2 (P<0.01). Moreover, GuttaFlow 2 was noncytotoxic, showing no statistically significant difference in LDH leakage levels compared to the control group (p>0.05). Specific characterization of MMPs demonstrated that GuttaFlow 2 seemed not to affect MMP-2 levels compared with the control group, while AH Plus had elevated gelatinolytic activity and protein levels of MMP-2 as confirmed by quantitative measurements. No detectable gelatinolytic activity or protein levels of MMP-9 (92 kDa) was observed in any tested group.
Conclusions
GuttaFlow 2 did not showed cytotoxic effects and did not induce MMP-2 or MMP-9 expression.
Keywords
Introduction
Root canal sealers are widely used in endodontic procedures to improve sealing ability at the dentin-root canal space interface. Although they should be contained within the root canal space, unintentional extrusion through the apical constriction may occur or eluents from the sealers may come into contact with periradicular tissues. This might cause irritation, inflammation, and possible delay in wound healing after endodontic procedures (1). In this case, degeneration of matrix proteins is thought to occur during periapical inflammation, and matrix turnover requires the activity of many different endopeptidases. Regarding this, recent studies have demonstrated the potential of endodontic sealers to activate matrix metalloproteinases (MMPs), which play an important role in the pathogenesis of root canal sealer-induced periapical inflammation (2-3-4).
Silicone is an inert and biocompatible material widely used in medicine as an implant material (5). To overcome the cytotoxic effects of traditional endodontic sealers, silicon-based sealers have been recently introduced; the polydimethylsiloxane-based root canal filling material (GuttaFlow 2; Coltène/Whaledent, Langenau, Germany) is one example of this group of materials. According to the manufacturer, GuttaFlow 2 is an improvement of the existing GuttaFlow material in capsules with similar properties. GuttaFlow 2 contains very small gutta-percha particles in powder form, with a particle size of less than 30 µm, and sealer in its bulk mass. Furthermore, the manufacturer claims better sealing and good adaptability of the material because of its increased flowability and slight expansion on setting. It is supplied ready to use in a dual-barrel syringe (6). Although silicone is well tolerated as an implant material and can seal perfectly even in moist environments, there is little evidence regarding the biocompatibility of the silicone-based root canal sealer GuttaFlow 2 (7).
The aim of the present study was to compare the cytotoxicity, gelatinolytic activity and protein levels (MMP-2 and MMP-9) produced by 3T3 fibroblasts cells after stimulation with GuttaFlow 2. AH Plus was used as a reference material for comparison. The null hypothesis tested was that there are no differences on the cytotoxicity and MMP-2 and -9 expression between the sealers in a cell-culture model.
Materials and methods
Sample preparation
The following endodontic sealers were tested: GuttaFlow 2 (Roeko, Colténe/Whaledent, Altstätten, Switzerland) and AH Plus (Dentsply, Konstanz, Germany). Product names and composition of the sealers, as provided by manufacturers, are shown in Table I. Sealer extracts were prepared according to ISO Standards 10993-5 (surface/medium rate, 0.5-6.0 cm/ml; extraction time, 1-3 days) (8). Under aseptic conditions, the sealers were mixed according to the manufacturer's instructions and placed in Teflon rings (5 mm in diameter, 2 mm high). After an initial set (4 h) the cement specimens were removed from the Teflon rings and placed in serum-free Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY, USA) using a 1.25 cm2 mL−1 ratio between the sample surfaces and the medium volume (9). Undiluted extracts were used for the test.
Composition of endodontic sealers as provided by the manufacturer
Cell culture
Fibroblast cells (lineage 3T3) were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 µg mL−1 streptomycin and 100 mg mL−1 penicillin at 37°C in a humidified incubator under ambient air pressure atmosphere containing 5% CO2. Confluent cells were detached with 0.25% trypsin and 0.05% ethylenediaminetetraaceticacid (Gibco) for 5 min, and aliquots were subcultured. For the experimental set, 3 · 105 cells were placed in each well of a 6-well plate and allowed to achieve 80% confluence. Cells were cultured for 24 h, and after that, the culture medium was replaced with fresh DMEM without serum. The cells were then exposed to the materials elute, for a period of 24 h. For the control group, 3 · 105 cells were cultured for 24 h, and after this, given fresh DMEM without serum for another 24 h period (3, 4, 9). After the experimental period, cell culture supernatants and lysates were collected for further analysis.
Cytotoxicity assay
Cell viability was determined through MTT and LDH assays. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma, St Louis, MO, USA) solution was prepared as 1 mg mL−1 in complete medium just before use. Then, 0.6 mL of this solution was added to each well, and cells incubated in a humidified atmosphere of 5% CO2 in air at 37°C for 4 h. After the incubation period, the supernatant was removed, and dark blue formazan crystals were dissolved in 0.6 mL of ethanol. The plates were then shaken for 5 min and after that, the blue solution was transferred to a 96-well plate and the optical densities were read at 570 nm in a multiwell spectrophotometer (EPOCH; Biosystems, Curitiba, PR, Brazil) (3, 4, 9).
Lactate dehydrogenase (LDH) leakage from dead cells was analyzed using an LDH Assay kit (Promega, Madison, WI, USA). A total of 50 mL of the supernatant was mixed with 50 mL of reagent and incubated for 30 min at room temperature. A stop solution of 50 mL was then added, and absorbance at 490 nm was measured using a spectrophotometer.
Zymography
The activities of MMP-2 and MMP-9 of the cell culture supernatants were measured by a gelatin zymogram protease assay, as previously described (3, 4). Aliquots of supernatants (20 µL) were mixed with sample buffer (2% SDS; 125 mmol L) 1 Tris–HClpH 6.8, 10% glycerol and 0.001% bromophenol blue) and loaded on the gel. Then, prepared samples were subjected to electrophoresis on 8% SDS–polyacrylamide gels containing 0.1% gelatin. Following electrophoresis, the gels were washed twice in 2% Triton X-100 for 30 min at room temperature to remove SDS. The gels were then incubated at 37°C for 18 h in substrate buffer containing 50 mmol L)1 Tris–HCl and 10 mmol L)1 CaCl2 at pH 8.0 and stained with 0.5% Coomassie blue R250 in 50% methanol and 10%glacial acetic acid. Protein standards were run concurrently, and approximate molecular weights were determined by plotting the relative mobilities of known proteins. Gelatinolytic activities were detected as unstained bands against the background of Coomassie blue-stained gelatin. Enzyme activity was assayed by densitometry using open-source computer software (ImageJ, NIH, Bethesda, Maryland, USA) and the intensities of digitalized bands were normalized with regard to an internal standard (FBS) to allow intergel analysis and comparison.
Western blot analysis
Cells were washed with cold PBS and lysed in standard RIPA (radioimmunoprecipitation assay) buffer (Thermo Scientific, Rockford, IL, USA). After centrifugation, soluble protein concentrations were measured using Bicinchoninic Acid (BCA) assay. Total protein (50 µg) was resolved using SDS-PAGE and transferred onto nitrocellulose membranes. Anti-MMP-2 (ab2462) and anti-MMP-9 (ab3159) were purchased from ABcam (Cambridge, MA, USA). Rabbit anti-β-actin (DB070; Delta Biolabs, Gilory, CA) was used as control. Membranes were visualized using PIERCE ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and exposed to Amersham Hyperfilm ECL film (GE Healthcare Biosciences, Piscataway, NJ, USA).
Statistical Analysis
The experiments were performed in triplicate throughout the study to ensure reproducibility. Data from the assays are presented as means ± standard deviation (SD). After testing for normality (Kolmogorov-Smirnov test), data were analyzed using 1-way Analysis of Variance (ANOVA). Statistical differences between the groups were analyzed using Tukey test at a significance level of 5%. All statistical analyses were performed using a commercially available statistical software package (SPSS, Chicago, IL, USA).
Results
As shown in Figures 1A and B, the cytotoxicity of AH Plus and GuttaFlow 2 elutes were evaluated using MTT assay and LDH. AH Plus showed significantly less cell viability (mitochondrial activity of cells) than GuttaFlow 2 (P<0.01). Moreover, GuttaFlow 2 was noncytotoxic, showing no statistically significant difference in LDH leakage levels compared to the control group (P>0.05).
Cytotoxicity effects of endodontic sealers on 3T3 cells by MTT (
Specific characterization of MMPs in the cell culture supernatants by gelatin zymography demonstrated that all groups induced different expression of MMP-2 (72 kDa) by 3T3 cells. GuttaFlow 2 did not seem to affect MMP-2 levels compared with the control group, while AH Plus had elevated gelatinolytic activity of MMP-2 as confirmed by quantitative measurements (Fig. 1C). No detectable gelatinolytic activity of MMP-9 (92 kDa) was observed in any tested group. Similar results were observed by the Western Blot analysis. No MMP-9 protein level was observed in any tested group (data not shown). MMP-2 protein levels were higher in the AH Plus group when compared to the control and GuttaFlow 2 group (Fig. 1D).
Discussion
In the present study, AH Plus was more cytotoxic and induced higher gelatinolytic activity and protein levels of MMP-2 than GuttaFlow 2. Therefore, the null hypothesis was rejected. The current results are partially in accordance with previous studies, which demonstrated the cytotoxic effects of AH Plus (2, 3, 10-11-12-13-14-15-16) and the possibility of MMP-2 activation (3). A recent study demonstrated similar results with GuttaFlow 2, demonstrating the higher biocompatibility of this material compared to AH Plus Jet. However, to the best our knowledge, this is the first attempt to evaluate gelatinolityc activity of a silicon-based endodontic sealer.
Cell viability was determined by MTT assay in this study. MTT assay is based on the ability of mitochondrial dehydrogenase enzymes in living cells to convert the yellow water-soluble tetrazolium salt MTT into dark blue formazan crystals. The MTT assay is a simple, fast, precise, sensitive and reproducible experimental model for measuring in vitro cytotoxicity and cell proliferation (17). Although a positive correlation between cell number and MTT reduction does not always exist, its isolated use to attest the toxicity of a given biomaterial is not universally accepted. Therefore, we also evaluated the cytotoxicity using the LDH assay. LDH is a soluble cytosolic enzyme present in most eukaryotic cells that is released into culture medium up on cell death due to damage of plasmatic membrane. The increase of the LDH activity in culture supernatant is proportional to the number of lysed cells. Therefore, high LDH activities represent high cytotoxicity effects. The association of different methods may increase the chance of detection of cytotoxic effects, allowing the correlation of different parameters and also providing hints for the mechanisms of toxicity (18).
It has been claimed that biocompatibility assessment through primary cell culture are appealing, because the biomaterials will interact with such kind of cells after in vivo implantation (19). However, in the present study, the biological properties of endodontic sealers were evaluated in 3T3 fibroblast cells. Studies with established cell lines are used due to the high reproducibility of the results, the high rate of cell multiplication compared to primary culture cells, and their unlimited life span, allowing higher reproducibility of results (14, 15). Moreover, fibroblasts are the major constituents of connective tissue, the predominant cell type of periodontal ligament and are the most important collagen producers in this tissue (20, 21). Finally, 3T3 cell line is endorsed by ISO experts to be suitable for cytotoxicity screenings. Fibroblasts secrete MMPs that are capable of initiating the degradation of extracellular matrix macromolecules, and this seems to be a key event for the progression of the inflammatory process. In this study, 3T3 cells were found to produce MMP-2 after exposure to AH Plus when compared to GuttaFlow 2. This MMP-2 expression may induce an extracellular matrix proteolysis, and it seems to be a key initiating event for the progression of the inflammatory process (2).
GuttaFlow 2 is a new version of the silicon-based sealer GuttaFlow, manufactured by adding gutta-percha powder to a silicone matrix. One of its main components is polydimethyl siloxane. Polydimethyl siloxane has been used widely in several prosthodontics and maxillofacial applications because of its low dimensional change (about 0.6-0.15%) and low water absorption. Also silicon-low temperature isotropic carbon (LTI) alloy implants, which are usually placed on a metal graphite substrate in the form of either a subperiosteal implant or an endosteal blade, are found to be very biocompatible (22). Several previous studies demonstrated the optimal biocompatibility of GuttaFlow (14, 16, 23-24). Miletić et al (16) found no cytotoxic effects of the silicon-based sealer Roekoseal on HeLa cells and mouse skin fibroblasts (L929). GuttaFLow demonstrated comparatively low cytotoxicity in human gingival fibroblasts and mouse fibroblast cell line compared with EndoREZ, AcroSeal and Apexit (23). In another study, GuttaFlow also exhibited a comparatively lower cytotoxicity than Resilon/Epiphany system or AH Plus (14). An in vivo study also demonstrated that GuttaFlow showed good compatibility and acceptable tissue toxicity (24).
The results of the present study confirm the optimal biocompatibility potential of the new endodontic sealer GuttaFlow 2. Future studies should evaluate other properties of this endodontic sealer.
Footnotes
Financial support: Dr. Emmanuel JNL Silva and Dr. Aline de Almeida Neves are eligible with a JCNE grant from FAPERJ, Rio de Janeiro, Brazil. Dr. Gustavo De-Deus is eligible with a CNE grant from FAPERJ.
Conflict of interest: The authors deny any conflicts of interest.