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Cobalt–chromium alloys fabricated with

four different techniques: Ion release,
toxicity of released elements and surface
roughness

Maria Kassapidoua,b,∗, Lars Hjalmarssona,c,d, Carina B. Johanssona, Petra

Hammarström Johanssona, Else Morisbake, Ann Wennerberga,
Victoria Franke Stenporta
a
Department of Prosthodontics/Dental Materials Science, Institute of Odontology, The Sahlgrenska Academy,
University of Gothenburg, Box 450, SE-405 30 Göteborg, Sweden
b
Department of Prosthetic Dentistry, Institute for Postgraduate Dental Education, Box 1030, SE-551 11 Jönköping,
Sweden
c
Specialist Dental Clinic, Folktandvården Sörmland AB, The Mälar Hospital, SE-631 88 Eskilstuna, Sweden
d
Centre for Clinical Research Sörmland, Uppsala University, SE-631 88 Eskilstuna, Sweden
e
NIOM, Nordic Institute of Dental Materials, Sognsveien 70 A, 0855 Oslo, Norway

A R T I C L E I N F O A B S T R A C T

Keywords: Objective. To investigate the metal ion release, surface roughness and cytoxicity for Co–Cr
Prosthodontics alloys produced by different manufacturing techniques before and after heat treatment. In
Dental alloys addition, to evaluate if the combination of materials affects the ion release.
Manufacturing technique
Methods. Five Co–Cr alloys were included, based on four manufacturing techniques. Com-
Cobalt–chromium
mercially pure titanium, CpTi grade 4 and a titanium alloy were included for comparison.
Titanium
The ion release tests involved both Inductive Coupled Plasma Optical Emission Spectrometry
Corrosion
and Inductive Coupled Plasma Mass Spectrometry analyses. The surface analysis was con-
Metal ion release
ducted with optical interferometry. Cells were indirectly exposed to the materials and cell
Surface roughness
viability was evaluated with the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-
Cytotoxicity
diphenyltetrazolium bromide) method.
Results. All alloys showed a decrease of the total ion release when CpTi grade 4 was
present. The total ion release decreased over time for all specimens and the highest ion
release was observed from the cast and milled Co–Cr alloy in acidic conditions.
The cast and laser-melted Co–Cr alloy and the titanium alloy became rougher after heat
treatment. All materials were within the limits of cell viability according to standards.

∗
Corresponding author at: Department of Prosthodontics/Dental Materials Science, Institute of Odontology, The Sahlgrenska Academy,
University of Gothenburg, Box 450, SE-405 30 Gö teborg, Sweden.
E-mail addresses: maria.kassapidou@rjl.se (M. Kassapidou), lars.hjalmarsson@gu.se (L. Hjalmarsson),
carina.johansson@odontologi.gu.se (C.B. Johansson), petra.h.johansson@gu.se (P. Hammarströ m Johansson),
niom@niom.no, else.morisbak@niom.no (E. Morisbak), ann.wennerberg@odontologi.gu.se (A. Wennerberg),
victoria.stenport@odontologi.gu.se (V. Franke Stenport).
https://doi.org/10.1016/j.dental.2020.08.012
0109-5641/© 2020 The Authors. Published by Elsevier Inc. on behalf of The Academy of Dental Materials. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363 e353

Significance. The ion release from Co–Cr alloys is influenced by the combination of materials,
pH and time. Surface roughness is influenced by heat treatment. Furthermore, both ion
release and surface roughness are influenced by the manufacturing technique and the
alloy type. The clinical implication needs to be further investigated.
© 2020 The Authors. Published by Elsevier Inc. on behalf of The Academy of
Dental Materials. This is an open access article under the CC BY-NC-ND license
(http://
creativecommons.org/licenses/by-nc-nd/4.0/).

and SLM Co–Cr alloys [23]. Xin et al. also demonstrated an

1. Introduction improved corrosion resistance after firing of SLM Co–Cr
speci- mens compared to cast ones in an acidic environment
Results from a previous survey pointed out that more than
(pH 2.5) [23].
30 different Co–Cr alloys were reported as being used in
To investigate the biocompatibility of a dental material
fixed prosthodontics (FP) in Sweden [1]. Still, there is limited
in vitro, cell culture tests are often used [8]. One such test is
knowl- edge of the longevity of Co–Cr alloys in FP [2,3] and
the MTT test that measures the cytotoxicity of a material by
the biocompatibility of Co–Cr has been questioned [4–7].
quantifying the viability of cells via metabolic activity [24].
One important factor related to the biocompatibility of a
No cytotoxic effect on mouse fibroblasts and human
prosthetic materials is the amount of substances released
epithelial and fibroblast cells were reported, related to
from the material [8]. Metal degradation may impair not
different six dif- ferent cast Co–Cr alloys [25–28]. The in vitro
only the material surface but also affect adjacent tissues and
biocompatibility of titanium on mouse fibroblasts and
trig- ger an adverse biological reaction [9,10]. The knowledge
human epithelial cells is well documented [4]. However,
about the amount of released elements from dental materials
Hjalmarsson et al. showed that both human epithelial cells
and the possible cytotoxic effects is scarce [11–14].
and mouse fibroblasts around Co–Cr were less viable
Results from a previous study demonstrated that fre-
compared to exposure to Ti [4]. Still, there have been
quently used Co–Cr alloys had variations both in their
concerns about possible toxic/allergic reac- tions to Ti
chemical composition and the way they were manufactured
[29,30].
[1]. Yet, the effect on corrosiveness because of chemical
Thus, there is a need to further explore possible metal ion
com- position and manufacturing is sparsely examined.
release and cell viability amongst the most commonly used
Various in vitro studies have shown a general low metal ion
Co–Cr alloys. Moreover, the various novel production tech-
release from Co–Cr alloys. Still, differences could be seen
niques used nowadays need to be investigated and
between Co–Cr alloys that were manufactured by various
compared to traditional manufacturing techniques.
techniques. Thus, the cast alloys were more prone to metal
The aim of the present study was to explore Co–Cr spec-
ion release compared to the SLM (Selective Laser Melting)
imens manufactured by different techniques and compare
alloys [15,16]. Another study showed increased corrosion
them to CpTi4 and Ti6Al4V with respect to
resistance for Co–Cr–Mo alloys manufactured by DMLS
(direct metal laser sintering) compared to the cast alloy [17].
1) Metal ion release in media with different pH.
Yet, another in vitro study showed a low ion release of Co
2) Metal ion release for Co–Cr with the presence of CpTi4.
and Cr from a cast Co–Cr–Mo alloy and a low ion release of Ti
3) Surface roughness.
from Ti grade 2 and Ti, Al and V from Ti-6Al-4V [18]. Both
4) The possible impact of heat treatment on metal ion
Luccheti et al. and Puskar et al. showed a higher total ion
release and surface roughness.
release from cast Co–Cr alloys when the pH changed from
5) Viability of cells indirectly exposed to released ions.
6.8 to 2.3, an increased release of Co, Cr and Fe [16] and Co, Cr
and Mo [17] from cast Co–Cr alloy after incubation in an
acidic solution. The null hypothesis was that the
It has been reported that the contact surfaces (after saliva
exposure), of both Ti implants and frameworks of both Co– - total ion release does not differ among the materials
Cr and Ti became rougher after saliva exposure, indicating a - total ion release does not differ with the presence or not of
pos- sible process of ongoing material degradation [19]. In CpTi4
another study, no statistically significant differences were - surface roughness does not differ among the materials
observed in surface roughness between cast polished pure - heat treatment does not affect the surface roughness
titanium and cast Co–Cr frameworks [20]. However, Lovgren - viability of cells (exposed to released ions) does not differ
et al. observed a rougher surface on the laser-sintered Co–Cr among the materials.
alloy compared to the cast one [21].
The effect of repeated porcelain firings were evaluated by 2. Material and methods
Tuncdemir et al. who found that the corrosion resistance of
a cast Co–Cr alloy was higher when the alloy was fired up 2.1. Specimens preparation
to seven times and decreased as the firing cycles increased
(up to 11 repeated firing cycles) [22]. Moreover, an Co–Cr alloys manufactured with four different techniques
increased oxide layer thickness was reported after firing were included in the study (Table 1).Two shapes of speci-
both for cast
e354 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363

Table 1 – Technical information of the used specimens.

Tradename Manufacturing Processing Manufacturer Composition (%)e
technique
Wirobond 280® (W280) Cast In housea Bego, Bremen, Germany Co 60.2, Cr 25, Mo 4.8, W
http://www.bego.com/ 6.2, Ga 2.8,Si <1, Mn <1
remanium® star (Rc) Cast In housea Dentaurum GmbH &
Co 60,5, Cr 28, Si 1,5, W
remanium® star MDII Milled Milled (rectangular Co., Ispringen, Germany
9, Mn <1, N <1, Nb <1
(Rm) specimen) turned https://www.dentaurum.de/eng/
(cylindrical
specimen)b
c
remanium® star CL Laser-sintered
(Rlm)
Zirkonzahn® Presintered milled Milled (rectangular Zirkonzahn, Gais, Italy Co 62–68, Cr 26–30, Mo
Sintermetall (Zz) specimen) turned http://www.zirkonzahn.com 5–7, N <0.5, C <0.5
(cylindrical
specimen)d
CpTi4 Cold drawn Milled (rectangular 1. Timet, Exton, USA, - Rectangular-shaped: C
specimen) turned http://www.timet.com 0.008–0,5, N 0.004–0.05,
(cylindrical (rectangular-shaped)1. O 0.37–0.4, Fe 0.02–0.5,
specimen)b 0.003–0.0125, Ti balance.
2. Zapp Precision Metals Gmbh, - Cylinder-shaped: C
Schwerte, Germany, 0.001, N 0.001, O 0.33, Fe
https://www.zapp.com/us.html 0.1, Ni 0.005, Ti balance.
(cylinder-shaped)
Ti6Al4V Drawn, annealed Milled (rectangular 1.Baoji Titanium Industry CO., - Rectangular-shaped:
specimen) turned LTD., Shaanxi, China Al ≤ 5.960, V ≤ 4.190, Fe
(cylindrical (rectangular-shaped). ≤ 0.146, C ≤ 0.009, N ≤
specimen)b 0.025, O ≤ 0.125, H ≤
0.001.
2.Zapp Precision Metals - Cylinder-shaped: Al
Gmbh(cylinder-shaped)- 5.96-6.05, V 4.11-4.02, Fe
Rectangular-shaped: 0.140-0.170, C
(cylinder-shaped) 0.008-0.009, N
0.004-0.005, O 0-0.1200,
H 0- 0.0018.
a
The cast specimens; W280 and Rc were cast by an experienced dental technician at the Dental Laboratory Technology in the Institute of
Odontology, University of Gothenburg, Sahlgrenska Academy, Sweden, according to the manufacturer’s recommendation.
b
Processed by Kullberg Mikroteknik, Lycke, Sweden.
c
Received prepared from the manufacturer.
d
Final sintering at Sä ffle Dental AB, Sä ffle, Sweden. Processed by Kullberg Mikroteknik, Lycke, Sweden.
e
The composition in Co–Cr alloys of Co ranges from 60.2 to 68.0%, Cr 25.0–30.0%, W 6.2–9.0%, Mo <1–7.0%, Ga 2.8%, Si <1–1.5%, Nb <1%, Mn,
<1%, N <1%, C <0.5%.

mens were used: (a) rectangular-shaped for the immersion
test I (pH 2.3) and surface roughness test and (b) cylinder-
shaped for the cell viability test and immersion test II
(pH 7.03) (Fig. 1). The specimens were ground on both flat
sides with Silicon Carbide (SiC) grinding paper 320–1200 grit
size (Struers A/S, Ballerup, Denmark), using a wet-grinding
equipment (Exakt-Apparatebau, Norderstedt, Germany). The
post-grinding cleaning process included; (a) 10 min in ultra-
sonic bath at 60 ◦C, in mixture of 1% Extran®AP 15 (Merck
KGaA, Darmstadt, Germany), and 99% deionisized ultra-pured
water, (b) rinsed in deionized ultrapure water for 30 s, (c) fol-
lowed by packing in a sterile bag.
2.2. Ion release tests
2.2.1. Immersion test I (acidic conditions, heat treatment)
The test was conducted according to ISO 22674:2016 and ISO
10271:2011. Six specimens (34 mm × 13 mm × 1.5 mm)
from each group (W280, Rc, Rm, Rlm, Zz, CpTi4 and Ti6Al4V)
were used. They were divided into 2 groups; (a) three non-
heat
treated and (b) three heat treated i.e. with four firing processes
to simulate the oxidation process and porcelain firing. The
highest firing temperature (830–960 ◦C) was set from the
rec- ommended porcelain to be used from each
manufacturer. The firing process was performed in a
vacuum furnace (Jelenko Commodore 100 VPF, New York,
USA). After the firing pro- cesses the specimens were
maintained on the ceramic plate to cool down in room
temperature. The heat treated specimens were then wet
grinded at all six flat surfaces with SiC grinding paper
(Struers A/S, Ballerup, Denmark), from 320 to 1200 grit size,
using Knuth Rotor grinding equipment (Struers, Ballerup,
Denmark). All flat surfaces of the specimens were measured
before and after grinding to ensure that at least 0.1 mm was
removed from all sides. The final measurements of the total
area of the specimens were recorded to ensure the
equivalent volume of lactic acid to a ratio of 1 ml/cm 2
specimen surface area. This was followed by cleaning in 96%
ethanol for 2 min in an ultra-sonic bath, followed by one rinse
with distilled water and finally air-dried. Lactic acid 0.1
mol/l and sodium chlo- ride 0.1 mol/l was mixed to ensure
the pH of 2.3 ± 0.1. Each
 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363 e355

min. The specimens were dried at 60◦ in heating cabinet (Bak-

Fig. 1 – Illustration of the specimens used in the present
study.
Rectangular-shaped (34 × 13 × 1.5 mm) for the ion release
tests and surface analysis and cylinder-shaped (8 × 8 mm)
for the cell viability tests.
specimen was covered by the corresponding volume of lactic
acid in a closed tube and stored for 7 days in 37 ◦C. One con-
trol tube (without a specimen) was treated in similar
manner.
After 7 days the specimens were removed, and the
immersion solutions were extracted from the tubes and
placed in indi- vidual sterile tubes. The extracts were
analyzed with ICP-OES (ARL 3580, Thermo-Optek, Ecublens,
Switzerland), by Sheffield Analytical Services Limited
(Sheffield, UK). In addition to the elements included in the
alloys as described by the manufac- turer (Table 1) the
following elements were included in the present study: Ni,
Cd, Be and Pb. The detection limit was set to <1 µg/cm2.
teriologskå p TE, B7151, Termaks, Norway) and then divided
into two test groups; A and B. A: six specimens each of
W280, Rc, Rm, Rlm, Zz, CpTi4 and Ti6Al4V were placed in
separate glass tubes (Borosilicate, 125 × 16 mm, Marienfeld
Lauda- Kö nigshofen, Germany. In each glass tube, 3 ml of
artificial saliva was added (Fig. 2). B: six specimens each of
W280, Rc, Rm, Rlm, Zz and Ti6Al4V were placed in separate
glass tubes (Borosilicate, 125 × 16 mm, Marienfeld Lauda-
Kö ningshofen, Germany), together with one specimen of
CpTi4/tube. Finally, 6 ml of artificial saliva was added in
each glass tube (Fig. 2).
The release of metal ions was measured at five differ-
ent occasions: after 1 day (d) ± 1 hour(h), 4 d ± 1 h, 7 d
± 1 h, 14 d ± 1 h and 21 d ± 1 h. A total of 900 µl of
extract was retrieved from each glass tube. The extracts were
added in Eppendorf® tubes (Polypropylene Transparent 1.5
ml, Sarstedt, Nü mbrecht, Germany) and 100 µl 65% HNO3
was added (to prevent bacterial growth) so that the total vol-
ume in each Eppendorf® tube (Polypropylene Transparent 1.5
ml) reached the volume of 1000 µl. Before the final analysis,
the tubes were stored in 37 ◦C in a heating cabinet (Bakteri-
ologskå p TE B7151, Termaks). The specimens were removed
rinsed in ethanol 99.7% (Solveco, Rosersberg, Sweden) for 15

2.2.2. Immersion test II (artificial saliva, physiologic

condition, with and without CpTi4)
The test was conducted according to modified ISO standards
10993-15:2009 and 10271:2011. Six (6) cylinder-shaped speci-
mens (8 × 8 mm) per material were used for the test. The
final measurements of the total area of the specimens were
recorded to ensure the equivalent volume of artificial saliva
to a ratio of 1 ml/cm2 per specimen. The total area of each
cylin- der was approximately 3.0 cm 2. In total, 2.5 l artificial
saliva was prepared with a composition of: (a) 0.815 g
Na2HPO4 × 2H2O, (b)1.75 g NaCl, (c) 0.825 g KSCN, (d) 0.5 g
KH2PO4, (e)
3.75 g NAHCO and (f) 3 g KCl. The pH value of the artificial
saliva was adjusted with 10 ml 37% HCl and was measured
with a pH-meter (ATI Orion Perphect Meter model 370, Scan-
dinovata, USA) to ensure a final pH of 7.03. The artificial saliva
was preserved in a closed tube during the test (21 days) at
37 ◦C in a heating cabinet (Bakteriologskå p TE (B7151, Ter-
maks, Norway). The specimens were placed in separate
tubes
(Borosilicate, 125 × 16 mm, Marienfeld Lauda-Kö nighofen,
Germany) and cleaned in an ultrasonic bath (Bandelin Sonorex
RK 100, Bandelin Electronic, Germany) at 700 ◦C using to
the following procedure: (a) Extran MA01 2.5% (Merck KGaA,
Darmstadt, Germany) in ultrapure water for 15 min, (b) rinsed
twice in ultrapure water and ultrasonic bath for 10 min and (c)
from the used tubes and cleaned with milliQ water, ethanol
70% (Solveco, Rosersberg, Sweden) and then left to self-dry
on filter paper (Munktell Swedish filter paper, Grycksbo
Pappers- bruk, Sweden). The used glass tubes were cleaned
with 10% of HNO3 in 24 h and washed with milliQ water four
times. After this procedure, new artificial saliva and
specimens were added into the cleaned tubes and the next
measurement started. Each measurement involved one
control; i.e. a tube that only contained artificial saliva. The
extracts were analyzed by ICP- MS (Inductive Coupled Plasma
Mass Spectrometry) (Thermo Fisher Scientific Inc., Bremen,
Germany) at RISE (Research Institute of Sweden, Borå s,
Sweden, https://www.sp.se). The elements of interest for
analysis were Co, Cr, Mn, Fe, Mo Ti, Al and V. The detection
limit was 1 of 1015.

2.3. Surface roughness analysis

Six rectangularly milled specimens (three before treat-

ment, and three after heat treatment) from each group
were randomly selected and examined regarding surface
topography with an optical interferometer (smartWLI-
extended, GBS, Ilmenau, Germany). Three randomly selected
regions/specimen were measured i.e. in total a mean
number of 9 measurements per material were registrered.
Accord- ing to recommendations by Wennerberg and
Albrektsson a high-pass Gaussian filter 50 µm × 50 µm
was used to sepa- rate roughness from errors of form and
waviness [31]. Three surface parameters were calculated
according to previous rec- ommendations [31]:

1. Sa (µm)–the average height deviation of each point com-

pared to the arithmetical mean of the surface.
2. Sds (1/µm2)–the density of the summits, number of
sum- mits per area.
3. Sdr (%)–developed inter facial area ratio, the percentage of
the definition area’s additional surface area contributed
to the texture as compared to the planar unit definition
area.
e356 denTAL MATERIALs
Fig. 2 – Flow chart for the specimens used for ion release analysis.
In total, 114 specimens were divided into two groups.
The surface evaluation was performed with Surfascan
soft- ware (Somicronic Instrument, Lyon, France).
2.4. Cell viability test
The cell viability was measured with MTT [24] according to ISO
10993-5:2009 using two different cell lines: (a) mouse fibrob-
lasts (L929) and (b) human bronchial epithelial cells (BEAS-
2B). As a positive control the 2-hydroxyethylmethacrylate,
(HEMA, 10 mM) (Fluka Chemie AG, Swtzerland) was used.
As a nega- tive control, medium without a specimen was
utilized. Three cylinder-shaped specimens (8 × 8 mm) from
each group were evaluated ; W280, Rc, Rm, Rlm, Zz, CpTi4,
Ti6Al4V. The experiment was repeated three times for each
material. New specimens were applied for each test.
2.4.1. Cell cultures
Both cell lines were purchased from European Collection of
Authenticated Cell Cultures (ECACC, Public Health, England):
a) Mouse fibroblasts (L929) cultivated in minimum
essential medium Eagle, with Eagle’s balanced salt
solution (EMEM) supplemented with L-Glutamine
(LonzaVerviers, Belgium), and 5% fetal bovine serum
(FBS,Sigma-Aldrich, St. Louis, USA). The confluent cells
were harvested by trypsin/EDTA (Lonza, Verviers
Belgium). Cells were plated at a density of 12,000
cells/well in a 96-well plate and incubated at 37 ◦C, 5%
CO2 and 95% humidity (Sanyo Incubator, Tokyo, Japan) 24
h before the start of the experiment.
b) The human bronchial epithelial cell line (BEAS-2B), a SV40
hybrid (Ad12-SV40)-transformed cell line were cultured in
serum-free Lechner and LaVeck (LHC9, GIBCO (Life Tech-
nologies, Foster City, CA, USA) which was replaced every
second day (maximum 20 passages). The cells were pas-
saged by trypsin/EDTA when confluence had reached
>85%. The culture flask and the 24 well-plate were
precoated with collagen (Advanced Biomatrix, INAMED
Corp., Fre- mont, USA) 30 µg/ml diluted in HEPES buffered
saline (HBS), HEPES (Lonza, Verviers, Belgium). The cells
(60,000 cells in
0.5 ml medium) were plated in a 24 well-plate and incu-
3 6 ( 2 0 2 0 ) e352–e363
bated at 37 ◦C, 5% CO2 and 95% humidity for 24 h. One
hour before the experiment started, the cell medium was
replaced with fresh medium in the 24 well-plate.
2.4.2. Specimens
2.4.2.1. Day 1. All 21 specimens were placed in separate
glass bottles in respective cell culture medium (pH 7.2–7.4)
as described above. The extract volume of the cell culture
medium was set to 3 cm2/ml (according to ISO 10993-
12:2012). As for the exact volume of the medium, it was
calculated in relation to the total surface of each specimen,
0.99 ml medium in each bottle. The bottles were sealed and
placed in agitated water bath (Julabo, Gö teborg, Sweden) in
37 ◦C for 24 h. After that the specimens were removed and
the extract was sterile filtered (Spritzen Syringen-Filter 0.22
µm, TPP Switzerland). The extract was then ready for the
MTT assay according to ISO 10993-5:2009.
2.4.2.2. Day 2. The cell medium from the well-plates was
dis- carded, leaving only the attached cells on the plates. In
each well 100 µl (L929) and 500 µl (BEAS-2B) of the specimen
extract was added. Following this procedure, the well-plates
were incubated for 24 h again at 37 ◦C, 5% CO2 and 95%
humidity.
2.4.2.3. Day 3. The extract and control medium were replaced
with 100 µl (L929) and 400 µl (BEAS-2B) of MTT (0.5 mg/ml
diluted in phosphate buffered saline, Sigma, St Louis, USA)
and placed for one hour incubation at 37 ◦C, 5% CO2 and
95% humidity. According to Edmondson et al. [24] only the
enzyme mitochondrial dehydrogenases in viable cells
converts MTT to a insoluble purple-coloured product;
Formasan. At the end of the incubation, the plates were
inspected and photographed using Olympus C7070 camera
(Olympus Europe, Hamburg, Germany) in a phase contrast
microscope, Olympus CKX41 (Olympus Europe, Hamburg,
Germany) (Fig. 3). The culture medium was removed and 0.1
ml (L929) and 0.4 ml (BEAS-2B) dimethyl sulfoxide (DMSO,
VWR, Life science, Radnor, USA) was added in each well, to
dissolve Formasan. Afterwards the plates were agitated for
20 min in room temperature and the
 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363 e357

Fig. 3 – MTT on human bronchial epithelial cells and mouse fibroblasts. No quantative examination of the cellular response
to the various materials were performed. Morphological changes on both cells were observed for all materials. Note the
impact of HEMA. As the cell viability decreased, the cells were changed to a more round and compact appearance,
indicating the presence of less viable cells [44]. Though all materials were approved as being “non-toxic” according to the
standards, the test revealed rounded cells from all materials. However, no obvious difference regarding the cellular
appearance could be observed between the various test specimens.

absorbance was measured at 570 nm in a

spectrophotometer (Synergi H1, Biotek, Vermont, USA).
2.5. Statistical analysis
The statistical analysis included a parametric test; one-
way ANOVA and independent samples t-test, non-parametric
tests; Mann–Whitney U, Friedman’s and Kruskal Wallis. The
Bonferroni correction method was used for post-hoc testing.
Data processing was performed using SPSS version 25 (IBM
Corp., Armonk, New York, USA). The significance value was
set to p < 0.01.
est amount of Co release was observed from the Rc. Cr ions
were only detected in Rc and Si ions were only observed in
Rc and Rm. The results indicated a statistically significant
differ- ence (p < 0.01) in the release of Si ion for the Rc
compared to the other materials (W280 and Rm). The ion
release from the tested materials were below the limit of the
total ion sum of 200 µg/cm2 per 7 days according to ISO
22674:2016.
3.2. Immersion test II (physiological condition, with
and without CpTi4) with ICP-MS

3. Results The results from the quantitative corrosion analysis with

ICP- MS showed that the amount of ions released decreased
3.1. Immersion test I (acidic conditions heat treatment over time and a lower ion release was observed when CpTi4
with ICP-EOS) was present (Tables 3 and 4).

The results from the quantitative corrosion analysis with
ICP- OES are presented in Table 2. Overall, the results
showed that the cast remanium® star (Rc) released the
highest total amount of ions followed by the cast W280 Ht
(Heat treated) and milled Rm Ht. No statistically significant
differences could be observed between the materials before
and after heat treat- ment.
Small amounts of ions of Co, Cr and Si were detected in
the cast Co–Cr alloys; W280, Rc and the milled (Rm). The high-
3.2.1. Test A (without CpTi4)
3.2.1.1. Total ion release. The results from the ICP-MS showed
that the tested materials (the controls included) had a total
ion release between 0.08–0.65 µg/cm2 with a general decrease
over time, when comparing day 1 to day 21. The presintered
milled Zz had the highest ion release, 0.65 µg/cm2 and the
largest decrease in ion release after day 14, 0.5 µg/cm2 (p <
0.01). The titanium materials, i.e. CPTi4 and TI6Al4V (together
with the control) and Rm presented the lowest ion release,
0.08–0.1 µg/cm2 (p < 0.01).
e358 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363

Table 2 – Ion release after 7 days, µg/cm2 in pH 7.03.

µg/cm2 (mean) Co Cr W Mo Si Ni Cd Be Pb Ce Nb Mn Ti V Al Sum
Cast W280 2.6 2.6
Cast W280 Ht* 1.0 1.0

Cast Rc 2.2 1.1 8.3 11.6

Cast Rc Ht* –

Milled Rm 1.1 1.1

Milled Rm Ht* 3.1 3.1

Laser-melted Rlm –
Laser-melted Rlm Ht* –

Presintered milled Zz –
Presintered milled Zz Ht* –

CpTi4 –
CpTi4 Ht* –

Ti6Al4V –
Ti6Al4V Ht* –

Blank cells represents non detectable ions (detection limit was set to 1 µg/cm2). The sum represents minimum detectable values.
–The sum of ion release could not be calculated due to non detectable ions.
Ht* = Heat treated.

Table 3 – Total ion release difference between test A and test B (µg/cm2) in pH 2.3.
Day 1 Day 4 Day 7 Day 14 Day 21
W280 −0.1
Rc −0.3 −0.1 −0.1 −0.06
Ti6Al4V −0.03
The results are presented as median values test B-test A, p < 0.01. No statistical significant differences in total ion release for Rm, Rlm and Zz.

Table 4 – Ion release difference between test A and test B (µg/cm2) in pH 2.3.
Materials Day 1 Day 4 Day 7 Day 14 Day 21
W280 −0.1
Co Rc −0.2 −0.1 −0.1 −0.1 −0.001 No differences in Co
Rm −0.03 −0.005 release for Rlm, Zz,
Rc −0.002 −0.001 Ti6Al4V (p < 0.01)
Cr Zz −0.003 No differences in Cr
release for W280,
Rm, Rlm (p < 0.01)
Ti6Al4V −0.0007
W280 −0.002
Rc 0.003 0.005
Ti
Rm −0.005
Ti6Al4V −0.008
Al Ti6Al4V −0.02 No differences in Al
release for W280, Rc,
Rm, Rlm, Zz and
Ti6Al4V (p < 0.01)
The results are presented as median values test B-test A, p < 0.01.

3.2.1.2. Ions. The highest amount of detected ion release
was observed for the Co ion, which was detected in all Co–Cr
mate- rials, 0.002−0.5 µg/cm2. Al and V ions were detected in
very low concentrations in all materials. In general, the ion
release test showed an overall decrease of ions over time.
3.2.2. Test B (CpTi4 present)
3.2.2.1. Total ion release. The results from the ICP-MS
showed that all tested materials (the controls included) had
a lower total ion release compared to test A, 0.08−0.4
µg/cm2. All
materials showed a gradually reduced ion release over time
with the highest decrease of 0.3 µg/cm2 for Zz (not statisti-
cally significant, p > 0.01). Similarly, as in test A the presintered
milled Zz showed the highest total metal ion release, i.e. 0.4
µg/cm2. The lowest total ion release, 0.08 µg/cm2 was
observed from Ti6Al4V.
3.2.2.2. Ions. In accordance to test A a general decrease of
ions was shown over time. Co was detected at the highest
amount of all ions in all Co–Cr materials. Ti, Al and V ions
 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363 e359

Table 5 – Results of surface analysis; Sa, Sdr and Sds. The results are presented as mean values, p < 0.01.
Sa (µm) Sds 1/µm2) Sdr (%)

Before After Difference Before After Difference Before After Difference

heat heat heat heat heat heat
treat- treat- treat- treat- treat- treat-
ment ment ment ment ment ment
Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD)
p < 0.01 p < 0.01 p < 0.01
W280 0.07 (0.01) 0.10 (0.01) 0.04 (0.01) 0.2 (0.03) 0.4 (0.02) 0.2 (0.04) 1.4 (0.2) 5.9 (1.2) 4.5 (1.3)
Rc 0.1 (0.01) 0.1 (0.02) 0.2 (0.03) 0.2 (0.04) 3.1 (0.3) 4.1 (2.1)
Rm 0.1 (0.01) 0.1 (0.01) 0.2 (0.01) 0.2 (0.01) 0.03 (0.01) 4.6 (0.4) 4.8 (0.7)
Rlm 0.06 (0.003) 0.09 (0.01) 0.02 (0.01) 0.2 (0.02) 0.2 (0.03) 0.06 (0.03) 1.6 (0.2) 2.8 (1.1) 1.2 (1.0)
Zz 0.06 (0.01) 0.07 (0.02) 0.2 (0.01) 0.2 (0.05) 1.6 (0.5) 1.8 (0.8)
CpTi4 0.2 (0.05) 0.2 (0.02) 0.2 (0.07) 0.2 (0.03) 5.7 (3.2) 4.7 (1.0)
Ti6Al4V 0.1 (0.02) 0.2 (0.07) 0.1 (0.07) 0.1 (0.02) 0.3 (0.07) 0.2 (0.07) 2.3 (0.7) 18.0 (12.4) 15.7 (12.3)
Standard deviations within parenthesis.

were detected in extremely low concentrations in all materi-

als 0.01–0.09 µg/cm2. The test also detected a small increase
of Al (from Rm) and V (from Rc, Rlm and Zz) of <0.003–0.05
µg/cm2 over time (p < 0.01).
3.2.3. Comparisons between test A–B
When the results from tests A and B were compared, test B
revealed a lower ion release (for all ions) for W280, Rm and
Ti6Al4V (except for Rc). Further, less Ti ions were released
in test B as compared to test A for the same three materials.
In addition, Ti6Al4V had a lower Al ion release in test B than
in test A (Tables 3 and 4).
3.3. Surface roughness
3.4. MTT (Cytotoxicity) assay
Irrespective of cell-line (L929 and BEAS-2B) none of the
materials tested could be rated cytotoxic according to ISO
10993-5:2009. The “cut-off” value (according to ISO 10993-
5:2009) for reduction of cell viability is 30% (Fig. 4). However,the
results showed that Ti6Al4V was rated slightly more cyto-
toxic compared to both cast CoCr alloys (Rc and W280)
when exposed on epithelial cells (BEAS-2B) (p < 0.05). A
cellular effect with respect to morphology could be observed
microscopically with more rounded cell shapes for all
materials tested (Fig. 3). These reactions were in accordance
with the cell morphology on the HEMA exposed cells, which
served as a positive control and was rated cytotoxicly as
expected.

All materials tested showed a smooth surface, Sa < 0.5 µm [32].
The results demonstrated differences between the materials
in all surface parameters, p < 0.01. The titanium materi-
als, CpTi4 (before heat treatment) and Ti6Al4V(Ht) showed
a rougher surface (Sa, Sdr) compared to all materials, p <
0.01 (except in comparison to CpTi4 and Rm, p > 0.01). For
the Sds parameter did W280(Ht) show the highest value of
0.4/µm2 compared to the other materials, p < 0.01. The test
also showed rougher surface (Sa) of the milled Rm compared
to cast W280, laser melted Rlm and presintered milled Zz
and rougher sur- face of cast Rc compared to presintered
milled Zz, p < 0.01 (Tables 5 and Table 6).
3.3.1. Heat treatment
When comparing the difference in surface roughness
between the materials before and after heat treatment the test
revealed that W280, Rlm and Ti6Al4V showed a rougher
surface after heat treatment in all parameters (Sa, Sds, Sdr),
p < 0.01. More specifically, the increase for the Sdr
parameter was seven times higher for Ti6Al4V and four
times higher for W280. The test revealed a minor increase for
Rlm after heat treatment, p <
0.01. A minor increase in Sds and Sdr values was also
demon- strated for Rm after heat treatment, p < 0.01 (Tables
5 and 6).
4. Discussion
The results from the present in vitro study showed a reduced
ion release from the various tested alloys when CpTi4 was
present. The ions release decreased over time and a higher
ion release was observed for all materials in the acidic envi-
ronment. Furthermore, the highest ion released was
observed from the cast material (Rc). All materials had a
total amount of metal ions released below the limit of 200
µg/cm2 (ISO 22674:2016).
In the clinical praxis, a combination of various materials
is routinely used. The present study indicated that it may,
the- oretically, be favorable to use the two materials (Co–Cr
and pure titanium) together in implant prosthetics, since the
total ion release decreased when these materials were
combined. This finding is in accordance with the results
from Hjalmars- son et al. who observed that the release
of Co, Cr and Ti decreased when the supra constructions
were coupled to tita- nium implants [19]. The results may
indicate that titanium passivate the Co–Cr alloys resulting in
a decreased ion leakage. Moreover, Pettersson et al.
demonstrated with ICP-AES, Inductive Coupled Plasma
Atomic Emission Spectroscopy that Ti in the presence of Co
neutralized the proinflammatory cytokine IL(interleukin)-
1β from human macrophages [33].
However, the mechanisms underlying these findings are not
yet fully understood.
e360 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363

Table 6 – Differences in Sa, Sdr and Sds values before and after heat treatment.

All materials tested showed a smooth surface, Sa < 0.5 µm [

Fig. 4 – Cell viability in relation to the control. Measured as succinate dehydrogenase activity in BEAS-2B and L929 cells. The
cells were exposed to extracts of material for 24 h. The results are shown as mean ± SD (n ≥ 3). Minor mean differences
could be observed between epithelial cells for Ti6Al4V ELI-W280 (p < 0.01**) and Ti6Al4V ELI-Rc (p < 0.05*).

In the present study, heat treatment with four firing-

According to the results from the present study, heat
cycles was used as a simulation for porcelain firing. No
treat- ment may affect the surface of the materials. In
statis- tically significant differences in ion release was
particular W280, Rlm and Ti6Al4V demonstrated a rougher
observed between the materials before or after heat
surface after heat treatment (Tables 5 and 6).
treatment. These results must be taken with caution because
Tuncdemir et al. demonstrated an increased oxide layer
of few data registrations (n = 3). Other studies that
after firing for the cast and SLM Co–Cr alloys [22]. This
investigated changes in ion release after heat treatment,
change could eventually affect the porcelain binding to the
performed up to eleven firing cycles and the results were
Co–Cr alloy. This was however not examined in the present
contradictory [22]. This result indicates that further
study.
studies are needed to evaluate the possible effect of heat
The results obtained in this study showed that the sur-
treatment on ion release.
face of all Co–Cr alloys tested were smoother compared to
the control materials, CpTi4 and Ti6Al4V (Sa).
 denTAL MATERIALs 3 6 ( 2 0 2 0 ) e352–e363 e361

Other studies using different evaluation techniques, e.g.

rials released Pb, Cd, Ni or Be, which are considered harmful
AFM (atomic force microscopy) did not find any statistically
elements [42]. The ICP-MS also detected Al, Ti and in some
significant differences in roughness (Sa) between titanium
cases V ions released from all Co–Cr alloys. Moreover, the ICP-
materials and cobalt–chromium alloys [20,34]. The results
MS detected Cr, Mo and Mn in both CpTi4 and Ti6Al4V. This
from the present study also showed that the surface of
phenomena was described by Harloff et al. who found that all
milled Co–Cr alloy was rougher than one of the cast Co–Cr
implant materials (both Ti alloys and CpTi grade 1) contained
alloys tested (W280) compared to laser-melted and
traceable amounts of “impurities” i.e. Al, Be, Cd, Co, Cr, Cu,
presintered milled Co–Cr alloy.
Hf, Mn, Ni, Pd and V (0.01–0.034% weight) irrespective of pro-
Other in vitro studies that investigated the internal
ducer [43]. Maybe “impurities” can explain the presence of ions
surface roughness of Co–Cr crowns fabricated by different
that were not expected to be found in the metal ion release
manufac- turing techniques found that the laser-sintered
test. Harloff et al. suggest a better refinement of the produc-
Co–Cr alloys had a rougher surface than milled and cast
tion process of these materials to attain materials with fewer
crowns [21,35]. Because of different evaluation techniques
impurities [43]. In patients with nickel, palladium or chrome
(one study used profilometry) [35] and different surface
allergy these “impurities” may trigger allergic reactions [29,43].
alterations (i.e. inter- nal sandblasting procedures in both
Corrosion is one of the factors that relates to bio-
studies), comparisons between the results are made
compatibility. Still the connection between corrosion and
difficult.
biocompatibility is complex and difficult to evaluate. Further
The results showed that the ion containing elutions from
research is needed to explore the long-term effects of corro-
the materials tested had no measurable cytotoxic effect
sion.
according to the standards. With respect to cell viability,
according to ISO standard levels, the results from the present
study are in agreement with the other studies evaluating cast
5. Conclusions
Co–Cr alloys [25–27] and laser-sintered Co–Cr alloys [28]. How-
ever, the results showed a slightly higher cytotoxic effect on
Within the limitations of the present in vitro study, it can be
the epithelial cells which were exposed to Ti6Al4V ELI com-
concluded that metal ion release from the tested materials is
pared to the cast Co–Cr specimens (p < 0.05). However, in vitro
influenced by
tests which evaluate biocompatibility are used as screening
tests and are not able to imitate the clinical environment [36].
- the combination of materials; a lower ion release when
The present study showed that the cast Co–Cr alloys (Rc,
tita- nium was present
W280) and the milled Rm released the highest amounts of
- the pH; an acidic environment increase ion release from
ions compared to the other materials in acidic conditions. This
dental Co–Cr-alloys. The highest total ion release was
result is in accordance with other studies showing that laser-
obtained from the cast and milled specimens in acidic con-
sintered Co–Cr can withstand acidogenic conditions better
ditions.
than cast Co–Cr [15–17]. Other studies, using other detecting
methods, have also reported that the laser-sintered Co–Cr has
The surface roughness is influenced by
enhanced corrosion properties compared to cast Co–Cr [37,38].
In contrast, another study showed that laser-sintered Co–Cr
- heat treatment; rougher surface after heat treatment for
had similar corrosion resistance as cast Co–Cr [23]. In the
cast and laser-melted Co–Cr alloys and Ti6Al4V.
present study, the ion release test results differed among the
environments (physiological and acidic). In acidic conditions,
The ion release and the surface roughness of the
the cast and milled Co–Cr alloy demonstrated the highest total
materials tested are influenced by
ion release and in the test were the conditions were physio-
logical (pH 7.03), did the presintered milled Co–Cr alloy have
- the manufacturing technique of the material;
the highest total ion release.
a) highest total ion release for cast and presintered/milled
Based on the results from the present study and other stud-
Co–Cr alloy
ies, one may consider using Co–Cr alloys manufactured with
b) rougher surface for cast and milled Co–Cr alloys-the alloy
recently implemented techniques, i.e. presintered milled and
type.
laser sintered frameworks in patients with a known history
of acidic attacks or erosion problems to avoid potential
The null hypotheses were rejected since there was a sta-
negative effects because of corrosion.
tistically significant difference in (a) total ion release among
The present study used two different methods to investi-
the materials and especially in the presence of CpTi grade 4
gate metal ion release; ICP-EOS, under acidic conditions (pH
and (b) in surface roughness before and after heat treatment
2.3) (ISO 22674:2016) and ICP-MS, under physiological condi-
(p < 0.01). Also, the null hypothesis concerning cell viability for
tions (pH 7.03). Both methods use wavelength
epithelial cells (exposed to released ions) between cast Co–
characteristics for the specific elements, by measuring
Cr alloys and Ti6Al4V was rejected (p < 0.05).
excited atoms and ions (ICP-OES) or by measuring an atom’s
mass (ICP-MS) [39]. Fe was discarded from the analysis
because of high degree of uncer- tainty since it interferes with Funding
argon (from the ICP-MS) and may, therefore, be difficult to
detect [40]. This work was supported by Futurum-the Academy for Health
The ICP-MS offers high sensitivity, detection capability and Care, Jö nkö ping County Council, Jö nkö ping, Sweden,
and is more accurate compared to ICP-OES [41]. None of the
mate-
e362 denTAL MATERIALs
Swedish Dental Society, Stockholm, Sweden, Sylvan Foun-
dation, Gö teborg, Sweden and Folktandvå rden Sö rmland AB,
Nykö ping, Sweden.
Acknowledgments
The authors wish to thank: Dentaurum GmbH&Co. KG,
Germany for their contribution of material and specimens.
Engineer Fredrik Kullberg at Kullbergs Mikroteknik,
Lycke, Sweden and dental technician Jetmir Kazaat, Sä ffle
Dental, Sä ffle, Sweden for their help in specimen
preparation. Senior engineer Morten Syverud, NIOM, Nordic
Institute of Dental Materials, Norway for his help in metal
ion release test. Dental technician Pia Karlsson, Gö teborg,
Sweden for her help with specimen preparation.
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