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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.
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Atay A, et al.
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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
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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
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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
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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
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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.
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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. PG: Study design, manuscript proof-
2) Cr-Co alloy material group (B), Ni-Cr alloy material reading. BBK: sample preparation. EC: Study and experimental
group (C) and F group had moderate cytotoxic design, analysis and interpretation of the results, manuscript
effects on the day 7. proofreading.
3) Au alloy material group (A) showed similar cell
viability result with Cr-Co alloy material group (B) ACKNOWLEDGMENTS
on the 1st day, whereas B group showed moderate
cytotoxicity at the end of the 7th day. This study was supported by the Ege University, Scientific
4) CAD/CAM restorative materials with fiber and Research Project Coordination Unit (Project Number: DİŞ-022).
resin content had favorable viability results.
5) All fixed restoration materials presented a variable
degree of cytotoxicity potential.
REFERENCES
1. Wataha JC. Biocompatibility of dental casting alloys: a 15. Scott A, Egner W, Gawkrodger DJ, et al. The national survey
review. J Prosthet Dent. 2000;83(2):223-234. doi:10.1016/s0022- of adverse reactions to dental materials in the UK: a preliminary
3913(00)80016-5. study by the UK Adverse Reactions Reporting Project. Br Dent J.
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS 2004;196(8):471-477. doi:10.1038/sj.bdj.4811176.
2. Wataha JC, Messer RL. Casting alloys. Dent Clin North Am. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
2004;48(2):vii-512. doi:10.1016/j.cden.2003.12.010. 16. Ke N, Wang X, Xu X, et al. The xCELLigence system for real-
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus time and label-free monitoring of cell viability. Methods Mol Biol.
3. Classification system for cast alloys. Council on Dental 2011;740:33-43. doi:10.1007/978-1-61779-108-6_6.
Materials, Instruments, and Equipment. J Am Dent Assoc. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
1984;109:766. doi:10.14219/jada.archive.1984.0185. 17. Hensten-Pettersen A. Comparison of the methods available
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus for assessing cytotoxicity. Int Endod J. 1988;21(2):89-99.
4. Aktas G, Yerlikaya H, Akca K. Mechanical failure of endocrowns doi:10.1111/j.1365-2591.1988.tb00961.x.
manufactured with different ceramic materials: an in vitro [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus
biomechanical study. J Prosthodont. 2018;27(4):340-346. 18. Malkoc S, Ozturk F, Corekci B, et al. Real-time cell analysis of
doi:10.1111/jopr.12499. the cytotoxicity of orthodontic mini-implants on human gingival
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS fibroblasts and mouse osteoblasts. Am J Orthod Dentofacial
5. Lauvahutanon S, Takahashi H, Shiozawa M, et al. Mechanical Orthop. 2012;141(4):419-426. doi:10.1016/j.ajodo.2011.12.009.
properties of composite resin blocks for CAD/CAM. Dent Mater J. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
2014;33(5):705-710. doi:10.4012/dmj.2014-208. 19. Shehata M, Durner J, Eldenez A, et al. Cytotoxicity and
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus induction of DNA double-strand breaks by components leached
6. Bioloren [Internet]. Website: https://bioloren.com/en/prodotti/ from dental composites in primary human gingival fibroblasts.
trilor-en/trilor-block/Accessed 20/05/2020. Dent Mater. 2013;29(9):971-979. doi:10.1016/j.dental.2013.07.007.
7. Elshahawy W, Watanabe I. Biocompatibility of dental alloys [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
used in dental fixed prosthodontics. Tanta Dental Journal 20. McCracken M. Dental implant materials: commercially pure
2014;11(2):150-159. doi:10.1016/j.tdj.2014.07.005. titanium and titanium alloys. J Prosthodont. 1999;8(1):40-43.
[Full text links] [CrossRef ] Google Scholar doi:10.1111/j.1532-849x.1999.tb00006.x.
8. Taylor TD, Morton TH Jr. Ulcerative lesions of the palate [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus
associated with removable partial denture castings. J Prosthet 21. Sedarat C, Harmand MF, Naji A, et al. In vitro kinetic
Dent. 1991;66(2):213-221. doi:10.1016/s0022-3913(05)80050-2. evaluation of titanium alloy biodegradation. J Periodontal Res.
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS 2001;36(5):269-274. doi:10.1034/j.1600-0765.2001.360501.x.
9. Schmalz G. Use of cell cultures for toxicity testing of dental [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
materials-advantages and limitations. J Dent. 1994; 22 Suppl 22. Al-Hiyasat AS, Darmani H, Bashabsheh OM. Cytotoxicity of
2:S6-S11. doi:10.1016/0300-5712(94)90032-9. dental casting alloys after conditioning in distilled water. Int J
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS Prosthodont. 2003;16(6):597-601.
10. . International Standards Organization (ISO) 10993–5:1992. [PubMed] Google Scholar Scopus WoS
Biological evaluation of medical devices - Part 5: Tests for in 23. Tai Y, De Long R, Goodkind RJ, et al. Leaching of nickel,
vitro cytotoxicity. International Standards Organization, Geneva; chromium and beryllium ions from base metal alloy in an
Switzerland, 1992. artificial oral environment. J Prosthet Dent. 1992;68(4):692-697.
11. Sjogren G, Sletten G, Dahl JE. Cytotoxicity of dental alloys, doi:10.1016/0022-3913(92)90388-q.
metals, and ceramics assessed by millipore filter, agar overlay, [Full text links] [CrossRef ] [PubMed] Google Scholar
and MTT tests. J Prosthet Dent. 2000;84(2):229-236. doi:10.1067/ 24. Schedle A, Samorapoompichit P, Rausch-Fan XH, et al.
mpr.2000.107227. Response of L-929 fibroblasts, human gingival fibroblasts and
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS human tissue mast cells to various metal cations. J Dent Res.
12. Wataha JC. Alloys for prosthodontic restorations. J Prosthet 1995;74(8):1513-1520. doi:10.1177/00220345950740081301.
Dent. 2002;87(4):351-363. doi:10.1067/mpr.2002.123817. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus 25. Meng H, Li XM, Xu YL, et al. [Effect of three dental alloys on
13. Hondrum SO. A review of the strength properties of dental cytotoxicity and apoptosis related gene expression in L929 cells].
ceramics. J Prosthet Dent. 1992;67(6):859-865. doi:10.1016/0022- Shanghai Kou Qiang Yi Xue. 2013; 22(1):30-34.
3913(92)90602-7. [PubMed] Google Scholar Scopus
[Full text links] [CrossRef ] [PubMed] Google Scholar 26. Wataha JC, Hanks CT, Craig RG. The in vitro effects of metal
14. Browne RM. The in vitro assessment of the cytotoxicity of cations on eukaryotic cell metabolism. J Biomed Mater Res.
dental materials-does it have a role? Int Endod J. 1988;21(2):50- 1991;25(9):1133-1149. doi:10.1002/jbm.820250907.
58. doi:10.1111/j.1365-2591.1988.tb00955.x. [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
[Full text links] [CrossRef ] [PubMed] Google Scholar
Stoma Edu J. 2020;7(3): 155-162 pISSN 2360-2406; eISSN 2502-0285 161
Atay A, et al.
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27. Faria ACL, Rosa AL, Rodrigues RC, et al. In vitro cytotoxicity 32. Atay A, Gürdal I, Bozok Çetıntas V, et al. Effects of new
Original Articles of dental alloys and cpTi obtained by casting. J Biomed Mater generation all-ceramic and provisional materials on fibroblast
Res Part B Appl Biomater. 2008;85(2):504-508. doi:10.1002/ cells. J Prosthodont. 2019;28(1):e383-e394. doi:10.1111/
jbm.b.30972. jopr.12915.
[Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS [Full text links] [CrossRef ] [PubMed] Google Scholar Scopus WoS
28. Jakovac M, Zivko-Babic J, Curkovic L, et al. Measurement 33. Miyazaki T, Nakamura T, Matsumura H, et al. Current status
of ion elution from dental ceramics. J Eur Ceram Soc. of zirconia restoration. J Prosthodont Res. 2013;57(4):236-
2006;26(9):1695-1700. doi:10.1016/j.jeurceramsoc.2005.03.242. 261. doi:10.1016/j.jpor.2013.09.001.[Full text links] [CrossRef ]
[CrossRef ] Google Scholar Scopus WoS [PubMed] Google Scholar Scopus WoS
29. Milleding P, Haraldsson C, Karlsson S. Ion leaching from dental 34. Shin H, Ko H, Kim M. Cytotoxicity and biocompatibility of
ceramics during static in vitro corrosion testing. J Biomed Mater Zirconia (Y-TZP) posts with various dental cements. Restor Dent
Res. 2002;61(4):541-550. doi:10.1002/jbm.10109. Endod. 2016;41(3):167-175. doi:10.5395/rde.2016.41.3.167.
[CrossRef ] [PubMed] Google Scholar Scopus WoS [Full text links] [CrossRef ] [PubMed] Google Scholar
30. Messer RL, Lockwood PE, Wataha JC, et al. In vitro cytotoxicity 35. Frese C, Wolff D, Zingler S, et al. Cytotoxicity of coated and
of traditional versus contemporary dental ceramics. 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