/home/opencode/cpanel/stomaeduj_hacked/uploads/article_Atay-2020.pdf DENTAL MATERIALS www.stomaeduj.com CYTOTOXICITY OF INDIRECT RESTORATIVE MATERIALS Original Articles ON FIBROBLAST CELLS: IN-VITRO STUDY Ayşe Atay1a , Vildan Bozok Çetintaş2b , Pelin Guneri3c , Beste Becerikli Kivrak4d , Ebru Cal5e 1 Department of Prosthodontics, Faculty of Dentistry, Altinbaș University, TR-34147, Bakırkoy/Istanbul, Turkey 2 Department of Medical Biology, Faculty of Medicine, Ege University, TR-35100, Bornova/Izmir, Turkey 3 Department of Oral and Maxillofacial Radiology, Faculty of Dentistry, Ege University, TR-35100, Bornova/Izmir, Turkey 4 Private Clinic, İzmir/Turkey 5 Department of Prosthodontics, Faculty of Dentistry, Ege University, TR-35100, Bornova/Izmir, Turkey 1a DDS, PhD, Assistant Professor; email: ayseatay82@hotmail.com; https://orcid.org/0000-0002-5358-0753 2b DDS, Associate Professor; email: vildanbcetintas@gmail.com; https://orcid.org/0000-0003-3915-6363 3c DDS, PhD, Professor; email: peleen_2000@yahoo.com; https://orcid.org/0000-0001-9423-919 4d DDS, PhD; email: beste_becerikli@hotmail.com; https://orcid.org/ 0000-0002-7650-9749 5e DDS, PhD, Professor; email: ebrucal68@gmail.com; https://orcid.org/0000-0002-1908-9733 ABSTRACT https://doi.org/10.25241/stomaeduj.2020.7(3).art.1 Introduction The aim of this study was to assess the cytotoxicity profiles of eight different fixed prosthetic restoration materials [Gold-based alloy (A), Chromium-Cobalt alloy (B) and Nickel-Chromium alloy (C), fiber- reinforced-resin-blocks (D), resin-nano-ceramic (E), lithium-disilicate-glass-ceramics (F), monolithic-zirconia (G) and feldspathic-ceramic (H)] by using cell culture methods on the L929 mouse fibroblast cells. Methodology 36 disc-shaped samples of each test material were prepared (5x2mm, N=288). After sterilization, discs were placed in EMEM and incubated at 37°C. Mediums were collected and filtered from each of four samples in 1st and 7th days. After 24hours incubation, cells were treated with 100 µl medium extracts of materials. Viability of cells was measured after 48 hours. Cytotoxicity was assessed with XTT and xCELLigence tests. Apoptosis was analysed using Annexin-V/PI staining. All data were statistically analysed with One-way ANOVA and Tukey’s multiple range tests (p<0.05). Results Considering the cell viability and apoptosis rate significant differences were found after the 1st and 7th days of incubation periods for each material group (p<0.05). Among the material groups significant differences were observed (p<0.05). F group showed the lowest cell viability and showed highest apoptosis rate (p<0.05). Along the entire test period, E group showed the highest cell viability and lowest apoptosis rate (p<0.05). Conclusion All fixed restoration materials investigated in the study exposed various levels of cytotoxicity, with significant differences among the test groups. KEYWORDS Dental Alloys; CAD-CAM Materials; Cytotoxicity; Apoptosis; Fibroblasts. 1. INTRODUCTION dentistry are widely used as restorative materials in dental applications [1]. Recently, the use of cast alloys In parallel with the improvements in dental has become very limited due to the improvements technology and material science, innovative mate- of full ceramic restorations and more durable resin- rials are being developed to be used in fixed prosthetic based composites. Nevertheless, for fixed prosthetic restorations, which are in direct contact with bone, restorations, dental alloys have continued to be used connective tissue or oral epithelium. Dental casting as the primary material [2]. In general, alloys usually alloys that play an important role in restorative include at least four and often six or more metals, OPEN ACCESS This is an Open Access article under the CC BY-NC 4.0 license. Peer-Reviewed Article Citation: Atay A, Bozok Cetintas V, Guneri P, Becerikli Kivrak B, Cal E. Cytotoxicity of indirect restorative materials on fibroblast cells: in-vitro study. Stoma Edu J. 2020;7(3):155-162. Received: June 23, 2020; Revised: July 27, 2020; Accepted: August 06, 2020; Published: August 10, 2020 *Corresponding author: Assistant Professor Dr. Ayșe Atay, Department of Prosthodontics, Faculty of Dentistry, Altinbaș University, İncirli Avenue No:11/A, 34147, Bakırkoy, Istanbul, Turkey Tel.: +90-212-709 45 28, Fax: +90-212-445 81 71; e-mail: ayse.atay@altinbas.edu.tr Copyright: © 2020 the Editorial Council for the Stomatology Edu Journal. Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 155 Atay A, et al. www.stomaeduj.com Original Articles Table 1. The codes, brand names, compositions and manufacturers of the materials. Code Brand Material type Chemical composition Manufacturer Au 86.5%, Pt 11.5%, Zn 1.4%, Rh, Fe, A PX Premium Bio Au alloy PX Dental SA, Marin, Switzerland Mn, Ta <1% Co 59%, Cr 25%, W 9.5%, Mo 3.5%, B Strabond CoS Cr-Co alloy Scheftner Dental Alloys, Germany Si %1, C, Fe, Mn, N <1% Ni 62%, Cr 24%, Mo 11%, Si 1.6%, C MoguCera N Ni-Cr alloy Scheftner Dental Alloys, Germany Mn <1% Epoxy resin matrix and Fiber reinforced D TriLor® multidirectional integrated Bioloren, S.r.l., Italy resin block fiberglass Matrix: Bis-GMA, UDMA, Bis-EMA, Resin nano TEGDMA E Lava Ultimate 3M-ESPE, Seefeld, Germany ceramic Filler: SiO2, ZrO2, aggregated ZrO2/ SiO2 cluster (80wt%) Lithium Li2O 10-15%, SiO2 71-80%, B2O3 F Rosetta SuperMill disilicate glass- 0-6%, P2O5 2-5%, Al2O3 2-5%, other Hass Corporation, Korea ceramic oxides and colorants 5-12% ZrO2+HfO2+Y2O3 ≥99.0%, Y2O3 5.4%, Monolithic Sirona Dental Systems GmbH, G InCoris TZI Al2O3<0.35%, F2O3 < 0.01%, other zirconia Bensheim, Germany oxides < 0.2% SiO2 56-64%, Al2O3 20-23%, Na2O Feldspathic Sirona Dental Systems GmbH, H CEREC locks 6-9%, K2O 6-8%, CaO 0.3-0.6%, TiO2 ceramic Bensheim, Germany 0.0-0.1% Abbreviations: Au: gold; Pt: platinum; Zn: zinc; Rh: rhodium, Fe: iron; Mn: manganese; Ta: tantalum; Cr: chromium; Co: cobalt; W: tungsten; Mo: molybdenum; Ni: nickel; Si: silicon; Bis-GMA: bisphenol A-glycidyl methacrylate; UDMA: urethane dimethacrylate; Bis-EMA: ethoxylated bisphenol A-glycol dimethacrylate; TEGDMA: triethylene glycol dimethacrylate; SiO2: silicon dioxide; ZrO2: zirconium dioxide; Li2O: lithium oxide; B2O3: Boron trioxide; P2O5: Phosphorus pentoxide; Al2O3: aluminium oxide; ZrO2: zirconium dioxide; HfO2: hafnium dioxide, Y2O3: yttrium Oxide; F2O3: ferric oxide; Na2O: sodium oxide; K2O: potassium oxide; CaO: calcium oxide; TiO2: titanium dioxide. but metallurgically, considering the periodic table composites have been added to the previous CAD/ various elements can be used in dental alloys and CAM materials [4,5]. Glass-fiber-composite tech- they are even more complex materials. The intricacy nology has been introduced to the dental practice and variety of these alloys complicate to understand for well over 20 years. Fiber reinforced composite their biocompatibility, since the body can be materials like TRILOR are alternative materials for affected by any element released from the alloy [1]. permanent and temporary dental restorations. According to the American Dental Association (ADA) Copings, substructures, frameworks for anterior or (1986), dental cast alloys can be: (1) high noble alloys posterior crowns, bridges, telescopic restorations, (≥60% Gold (Au), Platinum (Pt), Palladium (Pd) and bar attachments on implants and drilling guide for ≥40% Au), (2) noble alloys (≥25% Au, Pt, Pd) and implant surgery are among the indications for these (3) predominantly base metal alloys (<25% Au) [3]. materials [6]. Dental alloys are defined by their composition, but Since dental materials used in fixed prosthodontics composition can be explained in two ways, either as are contact with oral tissues, the biocompatibility of in the alloy percent of the number of atoms of each these materials is very critical and dentists, especially element (atomic percentage = at%) or percentage prosthodontics, should focus on dental biomaterials of weight (wt%) of elements. Even though the alloy [7]. The release of copper or nickel from cast alloys manufacturers and standards organizations describe has been suggested as the main (toxic) cause of an alloy’s composition by weight percentage, atomic oral tissue reactions, such as gingival inflammation percentage of these materials determine their [8]. In order to study the cytotoxicity of restorative biological properties [1]. In recent decades, esthetic materials, various in vitro systems including organ and durable restorations have been designed cultures and cells in culture have been utilized. and produced with computer-aided design and However, for in-vitro toxicity test of dental materials, computer-aided manufacturing (CAD/CAM) techno- the most commonly used biological system is logy. Feldspathic ceramics, glass ceramics containing the cell culture method. Two different types of leucite and lithium disilicate or yttrium tetragonal cells are generally preferred; permanent cell lines zirconia polycrystals are the examples of CAD/ derived from type-culture collections (L929 or 3T3 CAM high quality CAD/CAM ceramic materials. mouse fibroblasts), and primary cells derived from Recently, nano-hybrid ceramics, zirconia-reinforced mucosal or gingival explants and established in each lithium silicate ceramics, composites and glass-fiber individual laboratory. However, permanent cell lines 156 Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 Cytotoxicity of indirect restorative materials www.stomaeduj.com Original Articles Figure 1. Mean and standard deviation (SD) values of cell viability Figure 2. Mean and SD values of cell viability comparison among the 1st comparison among the 1st and 7th day medium extracts of restorative and 7th day medium extracts of restorative materials obtained from materials obtained from XTT assay. xCELLigence assay. are preferred since they are well defined and easily available [9]. Prior to introduction to dental clinical practice, physical property and biocompatibility accuracy assessment of dental materials is imperative [7]. Within this context, restorative materials entail thorough evaluation with respect to their interaction with the vital tissues, because they may release substances resulting in allergic reactions and inflammation [8]. The aim of the study was to assess the cytotoxic and apoptotic effects of eight indirect restorative materials by using XTT cell proliferation assay, xCELLigence real-time cell analysis system and Figure 3. Apoptosis analysis for 1st and 7th day medium extracts of restorative materials. Bar graphs showing the percentage of cell Annexin-V PI staining on fibroblast cells. populations (early apoptosis, late apoptosis and necrosis) in treated cells. 2. MATERIALS AND METHODS with the manufacturer’s instructions. Subsequently, the specimens were placed into an ultrasonic Three metal framework restoration materials [Gold- water bath (Whaledent Biosonic Jr, Whaledent based alloy (A), Chromium-Cobalt alloy (B) and International, New York, NY) for 10 minutes and Nickel-Chromium alloy (C)] and five metal-free CAD/ then dried. A total of 288 specimens (n = 36 per test CAM materials [fiber-reinforced-resin-blocks (D), material) were prepared. Each group was divided resin-nano-ceramic (E), lithium-disilicate-reinforced- into three (n =12 per group) randomly; two groups ceramics (F), monolithic-zirconia (G) and feldspathic- were assessed with the cytotoxicity assays, while the ceramics (H)] were studied. They are shown in Table other group was used for the apoptosis assay. 1. 36 disc-shaped specimens (h=2mm, Ø=5mm) were prepared in accordance with ISO 10993–5: 2.1. Preparation of medium extracts Tests for Cytotoxicity - In Vitro Methods [10] for The sterilization process was made with 16 kGy each material group. For the A, B and C groups; the gamma irradiation (Gamma-Pak Sterilization Ind., disc-shaped wax patterns (2x5mm) were produced Tekirdag, Turkey). Then the sterilized disc samples with conventional lost-wax technique using an were transferred into 96-well plate and each well induction-casting machine (Argonocaster-C, Shofu, was filled with 150 μL Eagle's Minimum Essential Japan). The casting method was performed under Medium (EMEM) containing 10% fetal calf serum the pressure of argon gas and vacuum suction, and (FBS) with 100 U/mL of penicillin-streptomycin. afterwards standard dental laboratory procedures In control group, there were no specimens in the were performed. well plate. All plates were incubated in a highly After casting, air particle abrasion with 100µm humidified atmosphere containing 5% CO2 at 37°C; aluminum oxide particles (80 psi=5.62 kgf/cm2) medium extracts of the test materials were collected was applied to the discs. For polishing process, at 1st and 7th days and were stored in −20°C until 400, 600, 1200 and 2000 grit silicon carbide papers cytotoxicity experiments. were utilized and the polishing was completed with diamond and aluminum oxide pastes. For the D, E, 2.2. Cytotoxicity assay F, G, and F groups, the CAD/CAM blocks were cut L929 cell line was obtained from the American Type using a low-speed diamond saw (Mecatome T180; Culture Collection (ATCC, Manassas, VA) and grown Presi, Grenoble, France). F and G discs were sintered in EMEM culture medium that was supplemented according to the manufacturer’s instructions. with 10% FBS and 1% penicillin-streptomycin in a Then, OptraFine ceramic polishing system (Ivoclar humidified atmosphere with 5% CO2 at 37°C. XTT Vivadent, Schaan, Liechtenstein) was used for Cell Proliferation Kit (Roche Applied Science, Basel, polishing the surfaces of the specimens, complying Switzerland) and xCELLigence real-time cell analyzer Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 157 Atay A, et al. www.stomaeduj.com Original Articles Figure 2. Annexin V/PI staining of indicated cell groups. Cells were treated with 1st, 3rd and 7th day medium extracts of restorative materials for 48h and then subjected to flow cytometric analysis. (LL: Vital cells, LR: Early apoptosis, UR: Late apoptosis, UL: Necrosis). (Roche Applied Science, Basel, Switzerland) were the cell viability, the classification used by Sjogren et used to assess the cytotoxicity of the samples. al [11], was utilized. If cell viability was below 30%, For the XTT assay; 3 × 104 cells/well were plated in the material was accepted as severely cytotoxic. a 96-well plate for 24 hours. The next day, 100 μL of Moderately cytotoxic materials scored 30–59% cell culture medium extract of each test material was viability, while slightly cytotoxic materials scored pipetted immediately into each well containing L929 60–90% and non-cytotoxic materials scored above cells. Formazan formation was quantified spectro- 90% [11]. photometrically at 450 nm with a microplate reader (Thermo, Vantaa, Finland) following 48 hours of 2.3. Apoptosis assay incubation. xCELLigence real-time cell analyzer L929 cells were seeded into 96-well plate at a measures electrical impedance across micro- density of 3×104 cells/well. Following 24 hours electrodes integrated on the bottom of tissue incubation period, 100 µL of the medium culture e-plates. 3x104 cells/well were seeded in 100 was aspirated and the cells were treated with μL medium and incubated for 24 hours. 100 µL medium extracts of test materials. Next day, 100 μL medium extracts of each test The cells were gathered after 48h of the treatment, materials were added to the wells. Cell impedance washed with phosphate buffered saline (PBS) and was measured every 15 minutes for a period of assessed with apoptosis detection kit (Annexin V 3 days. All experiments were applied in triplicate FITC/PI, Roche Applied Science) using BD Accuri C6 and the data was assessed with the xCELLigence Flow Cytometer (BD Biosciences, San Jose, CA). software (ACEA Biosciences). In order to determine 158 Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 Cytotoxicity of indirect restorative materials www.stomaeduj.com 2.4. Statistical analysis 3.2. Apoptotic effects of prosthetic restoration materials Original Articles Three replicated spectrophotometric measurements Within different incubation periods, apoptosis of XTT assay were completed to calculate the cell rates of the prosthetic restoration materials varied viability rates of the samples. Real-time cell analyze significantly (p<0.05). data was evaluated with the XCELLigence software. F group presented the highest apoptosis rates for The normality of the data distribution was tested by both 1st and 7th days (54.27±3.95; 63.27±2.96) using the Kolmogorov-Smirnov test. whereas the lowest apoptosis rates were observed in The data were normally distributed. One-way E (17.01±2.02; 21.73±1.55) and H group (21.80±1.71; ANOVA and Tukey’s multiple range tests were used 33.03±2.66). C and B groups followed with the with SPSS for Windows (22.0, SPSS Inc., Chicago, IL), 45.97±2.59, 40.53±2.83 and 51.03±3.66, 52.36±2.57 p<0.05 was considered significant. apoptosis rates on the 1st and 7th days, respectively (Figs. 3, 4). 3. RESULTS 4. DISCUSSION The survival, early and late apoptosis, and necrosis cell rates of 1st and 7th days medium extracts of Currently, wide ranges of restorative materials for eight different prosthetic restoration materials fixed prosthodontics are available in the market which were obtained by XTT, xCELLigence test (Figs. for dental practitioners’ use [7]. Dental casting 1, 2) and apoptosis analysis (Figs. 3, 4) are presented. alloys have played a major role in the restorative treatment of the patients, but this role has changed 3.1. Cytotoxic effects of prosthetic restoration materials considerably in recent years due to the development Regarding cell viability, among the material groups of more durable resin-based composites and the statistically significant differences were observed improvement of all-ceramic restorations. Never- between the 1st day and 7th day medium extracts theless, alloys will continue to be a commonly measurements (p<0.05). The lowest cell viability used material for fixed prosthetic restorations for was seen in F group both xCELLigence and XTT the upcoming years [12], despite the fact that experiments (p<0.05). their mutual shortcoming remainis the long-term On the 1st day, the highest cell viability values presence of all fixed prosthodontic materials in the were observed in E and H groups, whereas C and F oral cavity [7]. Considering that biocompatibility is groups displayed the lowest cell viability with both one of the critical factors affecting the treatment xCELLigence and XTT tests (p<0.05). No significant outcome, the biomaterials that are used for partial differences were observed between A and B; and D or complete substitutions of tooth and/or oral and G groups (p>0.05). tissues should be examined thoroughly before On the 7th day, the cell viability was significantly clinical applications [13-15]. Fortunately, increasing affected by the material type (p<0.05). E group development of the innovative materials in dental showed the highest viability value and had an applications has led to an improved awareness of the enhanced effect on cell survival, while F group biological risks and restrictions of these materials. displayed the lowest cell viability both with Monitoring the cell viability is crucial for biomedical xCELLigence and XTT tests (p<0.05). study both from a systematic view to comprehend There were no substantial differences among A, the biochemical and molecular pathways regulating G and H; B and C groups, respectively in all test cell viability, and from a therapeutic approach to methods (p>0.05). acquire agents that modulate cell viability [16]. The cell viability of B, F, G and H groups decreased Cell culture method is considered as a coordinated, over time both with XTT and xCELLigence tests reproducible, and cost-effective technique to (p<0.05). However, no significant differences were investigate the biocompatibility [17]. Dental discerned among A, C, D and E groups regarding materials’ biocompatibility is commonly investigated time periods (p>0.05). with cytotoxicity and apoptosis tests [18]. In the Considering the 7-day observation period, among present study, XTT and xCELLigence systems were the tested materials E group showed the highest used to assess the cytotoxicity. The xCELLigence cell survival values, whereas F group displayed the system uses impedance as readout and provides lowest cell viability, both with xCELLigence and XTT dynamic and real-time monitoring of cellular tests (Figs. 1, 2). phenotypic changes. By means of continuous Throughout the entire test period, A, D, E, G and H monitoring, this system perceives between various groups continued to exhibit cell viability between disturbances of cell viability, for instance senescence, 60–90%. Hence, these materials were considered as cell cycle arrest, and cell death. Additionally, the slightly cytotoxic. time perseverance of the xCELLigence system The C and F groups were moderately cytotoxic at all provides determination of the optimal time points to incubation periods (Figs. 1, 2). On the other hand; accomplish standard cell viability assays, alongside Group B was slightly cytotoxic on the 1st day but with other end-point assays to comprehend action moderately cytotoxic on the 7th day. mode [16]. XTT cell proliferation is a colorimetric Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 159 Atay A, et al. www.stomaeduj.com assay system that measures formazan products trials, are needed due to possible exceptions. Original Articles produced by metabolically active cells and is used as Although dental ceramics are known as chemically a common cell culture method [19]. The programmed inert materials, a specific inert property of a ceramic death of cells is apoptosis, and it is essential for the cannot be attributed as a general feature to all sustenance of homeostasis. The flow cytometric ceramics since different ceramics have distinctive apoptosis assay is another method that specifies the chemical configurations [28]. In addition to the cells that have started the apoptotic pathway [20,21]. diverse constituents and microstructures of the Al-Hiyasat et al. [22], investigated the cytotoxicity of ceramics and the corrosive properties, the period high-noble alloy and base-metal alloys by the di- and the temperature of the environment they are Methyl Thiazol diphenyl-Tetrazolium (MTT) method exposed to may negatively affect their chemical on Balb/C 3T3 fibroblasts, and revealed that the behavior [29]. Because of the structure and pH of difference in the composition of alloys markedly saliva, pH of foods, plaque amount and the presence affected their cytotoxicity potential. They stated of abdominal acids, oral environment is considered that the high concentration of chromium (Cr) and corrosive. As a result of deterioration of chemical molybdenum (Mo) have reduced the cytotoxicity stability, release of potential toxic inorganic ions [22]. On the contrary, in the present study, the cell from dental ceramics may increase [28]. Milleding et viability was lower in Group C with 11%Mo than al. [29], investigated the corrosive behaviors of crystal Group B with 3.5% Mo in the 1st day of the tests, and and oxide ceramics in liquid and acidic mediums, also, higher apoptosis rate was observed in Group and reported that crystal ceramics like Empress was C. Tai et al. [23], investigated both the corrosion prone to corrosion more than oxide ceramics like rates and Cr, nickel (Ni) and beryllium (Be) release zirconia and alumina. Nevertheless, massive loss of alloys in artificial oral environments. According of ceramics is very hard to investigate technically to their study, nickel release was higher than that of due to the oxidation of the released elements and chromium. Parallel to their findings, in the present the phenomenal precision of atomic absorption study, on the 7th day tests, the decrease in the cell methods [30]. In their investigations concerning the viability of Group C may be attributed to the Ni cytotoxicity of disilicate materials, Messer et al. [30] release. On the contrary, Schedle et al. [24], stated and Bracket et al. [31], stated that, regardless of the that Cobalt (Co) ions are more toxic than Ni ions, and materials fabrication methods and minor structural the increase in Co content in an alloy would increase differences, Empress 2 is biologically precarious. In the toxicity of the material. In the present study, a previous study, IPS e.max CAD material was not one of the highest apoptosis induced groups on the found toxic [32]. Nevertheless, in the present study, first day was the Ni-Cr-Mo alloy group (45.97%). In a lithium disilicate material Rosetta SuperMill was similar study where biocompatibility and apoptosis considered as moderately toxic. This contradiction effects of Au, Titanium (Ti) and Ni-Cr alloy on L929 may be attributed to the distinctive material compo- fibroblast cells were investigated, apoptosis was sitions in different brands. inducted via Caspase-3 and Caspase-9 mRNA Y-TZP based materials produced by CAD/CAM expressions increase in Ni-Cr material [25]. systems are introduced to be utilized both in esthetic Wataha et al. [26], tested different gold alloys for and load bearing areas. With superior esthetics and element release into cell-culture medium, and physical features, zirconia is the preferred material in reported that Au and Pd ions generally did not current procedures [33]. Shin et al. [34] investigated dissolve into the medium, but silver (Ag), copper the cytotoxicity of the zirconia posts cemented with (Cu) and zinc (Zn) ions were frequently dissolved. In different materials on L929 cells, and reported that another study, Sjörgen et al. [11], investigated the zirconia posts alone did not reveal toxicity. cytotoxicity of 15 different metals, dental alloys and Frese et al. [35] declared that composites with fiber ceramic materials, and reported that the Au alloy content exhibited minor toxicity. In the present study, with 0.6% Zn showed moderate cytotoxicity. In the fiber reinforced resin material exhibited less toxicity present study, slight cytotoxicity was revealed for compared to the other materials tested. This finding the Au alloy with 1.4% Zn content. may be attributed to the controlled polymerization Faria et al. [27], investigated the cytotoxicity of of the CAD/CAM resin materials under optimum Ti6Al4V, CpTi, Ni-Cr and Co-Cr alloys on SCC-9 cell pressure and temperature during manufacturing lines with cell viability and quantity. Surprisingly, process [32]. The major limitation of this study is they found that Co-Cr alloy was cytotoxic but Ni-Cr that it is an in-vitro study accomplished in laboratory alloy was not. In the present study, Cr-Co alloy was conditions, and the results cannot be directly slightly cytotoxic, as well. Similarly, in-vitro cyto- valid for clinical practice. However, the results may toxic effects of elements released from gold alloy provide additional information for clinicians during are also reported [7]. Therefore, clinicians should material selection. Permanent cell lines from mouse be aware that Au alloy is not completely inert fibroblasts were used in this study, but in future and biocompatible with oral tissues. The clinical studies primary cells (e.g., gingival fibroblasts) may relevance of these findings remains unclear and be preferred due to their better mimicking ability of further in-vitro studies, as well as controlled clinical the oral environment. 160 Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 Cytotoxicity of indirect restorative materials www.stomaeduj.com 5. CONCLUSIONS CONFLICT OF INTEREST Original Articles The authors declare no conflict of interest. Within the limitations of this in vitro study, the following conclusions were drawn: AUTHOR CONTRIBUTIONS 1) Rosetta SuperMill (Lithium disilicate ceramic material group (F)) revealed the highest apoptosis AA: Data gathering, analysis and interpretation of the results, rate and the lowest cell viability at all incubation manuscript writing. VBC: experimental design, analysis and inter- periods. pretation of the results. 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J Prosthet uncoated fibre-reinforced composites. Acta Odontol Scand. Dent. 2003;90(5):452-458. doi:10.1016/s0022-3913(03)00533-x. 2014;72(5):321-330. doi:10.3109/00016357.2013.826381. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS 31. Brackett MG, Lockwood PE, Messer RL, et al. In vitro cytotoxic response to lithium disilicate dental ceramics. Dent Mater. 2008;24(4):450-456. doi:10.1016/j.dental.2007.06.013. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS Ayșe ATAY DDS, PhD, Assistant Professor Department of Prosthodontics Faculty of Dentistry Altinbaș University TR-34147, Bakırkoy/Istanbul, Turkey CV Ayşe Atay graduated from Ege University, Faculty of Dentistry, Izmir, Turkey in 2004. She enrolled on her PhD degree in 2006 and she was awarded her PhD degree by Ege University in 2010. Since 2014, she has been working as an assistant professor at the Department of Prosthodontics within the Faculty of Dentistry of the Altınbaș University. Questions 1. Choose the content of the high noble alloys below: qa. <60% Au, Pd; qb. ≥60% Au, Pt, Pd and ≥40% Au; qc. ≥25% Au, Pt, Pd; qd. <25% Au. 2. Which of the following is the element released from cast alloys and shown as the main (toxic) cause of oral tissue reactions such as gingival inflammation? qa. Au; qb. Ni; qc. Cr; qd. Co. 3. Which of the following is a colorimetric assay that measures formazan products produced by metabolically active cells and is used as a common cell culture method? qa. XTT assay; qb. MTT assay; qc. xCELLigence system; qd. Apoptosis assay. 4. According to the results of this study, which materials showed favorable viability results? qa. Cr-Co alloy; qb. Lithium disilicate glass-ceramic; qc. Fiber reinforced composite material; qd. Ni-Cr alloy. 162 Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285