d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.intl.elsevierhealth.com/journals/dema

Cobalt–chromium alloys fabricated with four

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

Maria Kassapidou a,b,∗ , Lars Hjalmarsson a,c,d , Carina B. Johansson a ,

Petra Hammarström Johansson a , Else Morisbak e , Ann Wennerberg a ,
Victoria Franke Stenport a
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-diphenyltetrazolium
Cytotoxicity 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 Göteborg, 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. Hammarström 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/).
 d e n t a l m a t e r i a l s 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 (pH 2.5)
Results from a previous survey pointed out that more than
[23].
30 different Co–Cr alloys were reported as being used in fixed
To investigate the biocompatibility of a dental material
prosthodontics (FP) in Sweden [1]. Still, there is limited knowl-
in vitro, cell culture tests are often used [8]. One such test is
edge of the longevity of Co–Cr alloys in FP [2,3] and the
the MTT test that measures the cytotoxicity of a material by
biocompatibility of Co–Cr has been questioned [4–7].
quantifying the viability of cells via metabolic activity [24]. No
One important factor related to the biocompatibility of a
cytotoxic effect on mouse fibroblasts and human epithelial
prosthetic materials is the amount of substances released
and fibroblast cells were reported, related to different six dif-
from the material [8]. Metal degradation may impair not only
ferent cast Co–Cr alloys [25–28]. The in vitro biocompatibility
the material surface but also affect adjacent tissues and trig-
of titanium on mouse fibroblasts and human epithelial cells
ger an adverse biological reaction [9,10]. The knowledge about
is well documented [4]. However, Hjalmarsson et al. showed
the amount of released elements from dental materials and
that both human epithelial cells and mouse fibroblasts around
the possible cytotoxic effects is scarce [11–14].
Co–Cr were less viable compared to exposure to Ti [4]. Still,
Results from a previous study demonstrated that fre-
there have been concerns about possible toxic/allergic reac-
quently used Co–Cr alloys had variations both in their
tions to Ti [29,30].
chemical composition and the way they were manufactured
Thus, there is a need to further explore possible metal ion
[1]. Yet, the effect on corrosiveness because of chemical com-
release and cell viability amongst the most commonly used
position and manufacturing is sparsely examined. Various
Co–Cr alloys. Moreover, the various novel production tech-
in vitro studies have shown a general low metal ion release
niques used nowadays need to be investigated and compared
from Co–Cr alloys. Still, differences could be seen between
to traditional manufacturing techniques.
Co–Cr alloys that were manufactured by various techniques.
The aim of the present study was to explore Co–Cr spec-
Thus, the cast alloys were more prone to metal ion release
imens manufactured by different techniques and compare
compared to the SLM (Selective Laser Melting) alloys [15,16].
them to CpTi4 and Ti6Al4V with respect to
Another study showed increased corrosion resistance for
Co–Cr–Mo alloys manufactured by DMLS (direct metal laser
1) Metal ion release in media with different pH.
sintering) compared to the cast alloy [17]. Yet, another in vitro
2) Metal ion release for Co–Cr with the presence of CpTi4.
study showed a low ion release of Co and Cr from a cast
3) Surface roughness.
Co–Cr–Mo alloy and a low ion release of Ti from Ti grade 2 and
4) The possible impact of heat treatment on metal ion release
Ti, Al and V from Ti-6Al-4V [18]. Both Luccheti et al. and Puskar
and surface roughness.
et al. showed a higher total ion release from cast Co–Cr alloys
5) Viability of cells indirectly exposed to released ions.
when the pH changed from 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
The null hypothesis was that the
after incubation in an acidic solution.
It has been reported that the contact surfaces (after saliva
exposure), of both Ti implants and frameworks of both Co–Cr - total ion release does not differ among the materials
and Ti became rougher after saliva exposure, indicating a pos- - total ion release does not differ with the presence or not of
sible process of ongoing material degradation [19]. In another CpTi4
study, no statistically significant differences were observed in - surface roughness does not differ among the materials
surface roughness between cast polished pure titanium and - heat treatment does not affect the surface roughness
cast Co–Cr frameworks [20]. However, Lovgren et al. observed - viability of cells (exposed to released ions) does not differ
a rougher surface on the laser-sintered Co–Cr alloy compared among the materials.
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 increased Co–Cr alloys manufactured with four different techniques
oxide layer thickness was reported after firing both for cast were included in the study (Table 1).Two shapes of speci-
e354 d e n t a l m a t e r i a l s 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 Säffle Dental AB, Säffle, 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/cm2 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
 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363
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 ␮g/cm2 .
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 cm2 . In total, 2.5 l artificial saliva
was prepared with a composition of: (a) 0.815 g Na2 HPO4 ×
2H2 O, (b)1.75 g NaCl, (c) 0.825 g KSCN, (d) 0.5 g KH2 PO4 , (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 (Bakteriologskåp TE (B7151, Ter-
maks, Norway). The specimens were placed in separate tubes
(Borosilicate, 125 × 16 mm, Marienfeld Lauda-Könighofen,
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)
rinsed in ethanol 99.7% (Solveco, Rosersberg, Sweden) for 15
e355
min. The specimens were dried at 60◦ in heating cabinet (Bak-
teriologskåp 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-
Königshofen, 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-Köningshofen,
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 ␮l of
extract was retrieved from each glass tube. The extracts were
added in Eppendorf® tubes (Polypropylene Transparent 1.5
ml, Sarstedt, Nümbrecht, Germany) and 100 ␮l 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 ␮l. Before the final analysis,
the tubes were stored in 37 ◦ C in a heating cabinet (Bakteri-
ologskåp TE B7151, Termaks). The specimens were removed
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, Borås, 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 ␮m × 50 ␮m 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 (␮m)–the average height deviation of each point com-
pared to the arithmetical mean of the surface.
2. Sds (1/␮m2 )–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 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363
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 ␮g/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-
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, Göteborg, Sweden) in 37 ◦ C for 24 h. After
that the specimens were removed and the extract was sterile
filtered (Spritzen Syringen-Filter 0.22 ␮m, 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 ␮l (L929) and 500 ␮l (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 ␮l (L929) and 400 ␮l (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
 d e n t a l m a t e r i a l s 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 est amount of Co release was observed from the Rc. Cr ions
(Synergi H1, Biotek, Vermont, USA). were only detected in Rc and Si ions were only observed in Rc
and Rm. The results indicated a statistically significant differ-
2.5. Statistical analysis 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
The statistical analysis included a parametric test; one-
tested materials were below the limit of the total ion sum of
way ANOVA and independent samples t-test, non-parametric
200 ␮g/cm2 per 7 days according to ISO 22674:2016.
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 3.2. Immersion test II (physiological condition, with
Corp., Armonk, New York, USA). The significance value was and without CpTi4) with ICP-MS
set to p < 0.01.
The results from the quantitative corrosion analysis with ICP-
MS showed that the amount of ions released decreased over
3. Results time and a lower ion release was observed when CpTi4 was
present (Tables 3 and 4).
3.1. Immersion test I (acidic conditions heat treatment
with ICP-EOS)
3.2.1. Test A (without CpTi4)
The results from the quantitative corrosion analysis with ICP-
3.2.1.1. Total ion release. The results from the ICP-MS showed
that the tested materials (the controls included) had a total
OES are presented in Table 2. Overall, the results showed
ion release between 0.08–0.65 ␮g/cm2 with a general decrease
that the cast remanium® star (Rc) released the highest total
over time, when comparing day 1 to day 21. The presintered
amount of ions followed by the cast W280 Ht (Heat treated)
milled Zz had the highest ion release, 0.65 ␮g/cm2 and the
and milled Rm Ht. No statistically significant differences could
largest decrease in ion release after day 14, 0.5 ␮g/cm2 (p <
be observed between the materials before and after heat treat-
0.01). The titanium materials, i.e. CPTi4 and TI6Al4V (together
ment.
with the control) and Rm presented the lowest ion release,
Small amounts of ions of Co, Cr and Si were detected in
0.08–0.1 ␮g/cm2 (p < 0.01).
the cast Co–Cr alloys; W280, Rc and the milled (Rm). The high-
e358 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363

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

␮g/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 ␮g/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 (␮g/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 (␮g/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 ␮g/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 ␮g/cm2 . All
materials showed a gradually reduced ion release over time
with the highest decrease of 0.3 ␮g/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
␮g/cm2 . The lowest total ion release, 0.08 ␮g/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
 d e n t a l m a t e r i a l s 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 (␮m) Sds 1/␮m2 ) 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 ␮g/cm2 . The test also detected a small increase
of Al (from Rm) and V (from Rc, Rlm and Zz) of <0.003–0.05
␮g/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).
3.4. 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.

3.3. Surface roughness

All materials tested showed a smooth surface, Sa < 0.5 ␮m [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/␮m2
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 ␮g/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)-1␤ from human macrophages [33].
However, the mechanisms underlying these findings are not
yet fully understood.
e360 d e n t a l m a t e r i a l s 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 ␮m [32].

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-cycles
was used as a simulation for porcelain firing. No statis-
tically significant differences in ion release was observed
between the materials before or after heat treatment.
These results must be taken with caution because of few
data registrations (n = 3). Other studies that investigated
changes in ion release after heat treatment, performed up
to eleven firing cycles and the results were contradictory
[22]. This result indicates that further studies are needed
to evaluate the possible effect of heat treatment on ion
release.
According to the results from the present study, heat treat-
ment may affect the surface of the materials. In particular
W280, Rlm and Ti6Al4V demonstrated a rougher surface after
heat treatment (Tables 5 and 6).
Tuncdemir et al. demonstrated an increased oxide layer
after firing for the cast and SLM Co–Cr alloys [22]. This change
could eventually affect the porcelain binding to the Co–Cr
alloy. This was however not examined in the present study.
The results obtained in this study showed that the sur-
face of all Co–Cr alloys tested were smoother compared to the
control materials, CpTi4 and Ti6Al4V (Sa).
 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363
Other studies using different evaluation techniques, e.g.
AFM (atomic force microscopy) did not find any statistically
significant differences in roughness (Sa) between titanium
materials and cobalt–chromium alloys [20,34]. The results
from the present study also showed that the surface of
milled Co–Cr alloy was rougher than one of the cast Co–Cr
alloys tested (W280) compared to laser-melted and presintered
milled Co–Cr alloy.
Other in vitro studies that investigated the internal surface
roughness of Co–Cr crowns fabricated by different manufac-
turing techniques found that the laser-sintered Co–Cr alloys
had a rougher surface than milled and cast crowns [21,35].
Because of different evaluation techniques (one study used
profilometry) [35] and different surface alterations (i.e. inter-
nal sandblasting procedures in both studies), comparisons
between the results are made difficult.
The results showed that the ion containing elutions from
the materials tested had no measurable cytotoxic effect
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
Co–Cr alloys [25–27] and laser-sintered Co–Cr alloys [28]. How-
ever, the results showed a slightly higher cytotoxic effect on
the epithelial cells which were exposed to Ti6Al4V ELI com-
pared to the cast Co–Cr specimens (p < 0.05). However, in vitro
tests which evaluate biocompatibility are used as screening
tests and are not able to imitate the clinical environment [36].
The present study showed that the cast Co–Cr alloys (Rc,
W280) and the milled Rm released the highest amounts of
ions compared to the other materials in acidic conditions. This
result is in accordance with other studies showing that laser-
sintered Co–Cr can withstand acidogenic conditions better
than cast Co–Cr [15–17]. Other studies, using other detecting
methods, have also reported that the laser-sintered Co–Cr has
enhanced corrosion properties compared to cast Co–Cr [37,38].
In contrast, another study showed that laser-sintered Co–Cr
had similar corrosion resistance as cast Co–Cr [23]. In the
present study, the ion release test results differed among the
environments (physiological and acidic). In acidic conditions,
the cast and milled Co–Cr alloy demonstrated the highest total
ion release and in the test were the conditions were physio-
logical (pH 7.03), did the presintered milled Co–Cr alloy have
the highest total ion release.
Based on the results from the present study and other stud-
ies, one may consider using Co–Cr alloys manufactured with
recently implemented techniques, i.e. presintered milled and
laser sintered frameworks in patients with a known history of
acidic attacks or erosion problems to avoid potential negative
effects because of corrosion.
The present study used two different methods to investi-
gate metal ion release; ICP-EOS, under acidic conditions (pH
2.3) (ISO 22674:2016) and ICP-MS, under physiological condi-
tions (pH 7.03). Both methods use wavelength characteristics
for the specific elements, by measuring 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 argon (from the ICP-MS) and may,
therefore, be difficult to detect [40].
The ICP-MS offers high sensitivity, detection capability and
is more accurate compared to ICP-OES [41]. None of the mate-
e361
rials released Pb, Cd, Ni or Be, which are considered harmful
elements [42]. The ICP-MS also detected Al, Ti and in some
cases V ions released from all Co–Cr alloys. Moreover, the ICP-
MS detected Cr, Mo and Mn in both CpTi4 and Ti6Al4V. This
phenomena was described by Harloff et al. who found that all
implant materials (both Ti alloys and CpTi grade 1) contained
traceable amounts of “impurities” i.e. Al, Be, Cd, Co, Cr, Cu,
Hf, Mn, Ni, Pd and V (0.01–0.034% weight) irrespective of pro-
ducer [43]. Maybe “impurities” can explain the presence of ions
that were not expected to be found in the metal ion release
test. Harloff et al. suggest a better refinement of the produc-
tion process of these materials to attain materials with fewer
impurities [43]. In patients with nickel, palladium or chrome
allergy these “impurities” may trigger allergic reactions [29,43].
Corrosion is one of the factors that relates to bio-
compatibility. Still the connection between corrosion and
biocompatibility is complex and difficult to evaluate. Further
research is needed to explore the long-term effects of corro-
sion.
5. Conclusions
Within the limitations of the present in vitro study, it can be
concluded that metal ion release from the tested materials is
influenced by
- the combination of materials; a lower ion release when tita-
nium was present
- the pH; an acidic environment increase ion release from
dental Co–Cr-alloys. The highest total ion release was
obtained from the cast and milled specimens in acidic con-
ditions.
The surface roughness is influenced by
- heat treatment; rougher surface after heat treatment for
cast and laser-melted Co–Cr alloys and Ti6Al4V.
The ion release and the surface roughness of the materials
tested are influenced by
- the manufacturing technique of the material;
a) highest total ion release for cast and presintered/milled
Co–Cr alloy
b) rougher surface for cast and milled Co–Cr alloys-the alloy
type.
The null hypotheses were rejected since there was a sta-
tistically significant difference in (a) total ion release among
the materials and especially in the presence of CpTi grade 4
and (b) in surface roughness before and after heat treatment
(p < 0.01). Also, the null hypothesis concerning cell viability for
epithelial cells (exposed to released ions) between cast Co–Cr
alloys and Ti6Al4V was rejected (p < 0.05).
Funding
This work was supported by Futurum-the Academy for Health
and Care, Jönköping County Council, Jönköping, Sweden,
e362 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363
Swedish Dental Society, Stockholm, Sweden, Sylvan Foun-
dation, Göteborg, Sweden and Folktandvården Sörmland AB,
Nyköping, 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, Säffle Dental,
Säffle, 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, Göteborg, Sweden for her help with
specimen preparation.
references
[1] Kassapidou M, Franke Stenport V, Hjalmarsson L, Johansson
CB. Cobalt-chromium alloys in fixed prosthodontics in
Sweden. Acta Biomater Odontol Scand 2017;3:53–62.
[2] Svanborg P, Langstrom L, Lundh RM, Bjerkstig G, Ortorp A. A
5-year retrospective study of cobalt-chromium-based fixed
dental prostheses. Int J Prosthodont 2013;26:343–9.
[3] Eliasson A, Arnelund CF, Johansson A. A clinical evaluation
of cobalt-chromium metal-ceramic fixed partial dentures
and crowns: a three- to seven-year retrospective study. J
Prosthet Dent 2007;98:6–16.
[4] Hjalmarsson L, Smedberg JI, Aronsson G, Wennerberg A.
Cellular responses to cobalt-chrome and CP titanium—an
in vitro comparison of frameworks for implant-retained oral
prostheses. Swed Dent J 2011;35:177–86.
[5] Arvidson K, Cottler-Fox M, Hammarlund E, Friberg U.
Cytotoxic effects of cobalt-chromium alloys on fibroblasts
derived from human gingiva. Scand J Dent Res
1987;95:356–63.
[6] Song H, Yin W, Ma Q. Allergic palmoplantar pustulosis
caused by cobalt in cast dental crowns: a case report. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:e8–10.
[7] Kim EC, Kim MK, Leeungbok R, Lee SW, Ahn SJ. Co-Cr dental
alloys induces cytotoxicity and inflammatory responses via
activation of Nrf2/antioxidant signaling pathways in human
gingival fibroblasts and osteoblasts. Dent Mater
2016;32:1394–405.
[8] Anusavice KJ, Shen C, Rawls HR. Phillips’ science of dental
materials. St. Louis, MO: Elsevier/Saunders; 2013.
[9] Wataha JC. Alloys for prosthodontic restorations. J Prosthet
Dent 2002;87:351–63.
[10] Jacobs JJ, Gilbert JL, Urban RM. Corrosion of metal
orthopaedic implants. J Bone Joint Surg Am 1998;80:268–82.
[11] Lucas LC, Lemons JE. Biodegradation of restorative metalloc
systems. Adv Dent Res 1992;6:32–7.
[12] Brune D. Metal release from dental biomaterials.
Biomaterials 1986;7:163–75.
[13] Schmalz G, Garhammer P. Biological interactions of dental
cast alloys with oral tissues. Dent Mater 2002;18:396–406.
[14] Elshahawy W, Watanabe I. Biocompatibility of dental alloys
used in dental fixed prosthodontics. Tanta Dent J
2014;11:150–9.
[15] Hedberg YS, Qian B, Shen Z, Virtanen S, Odnevall Wallinder
I. In vitro biocompatibility of CoCrMo dental alloys
fabricated by selective laser melting. Dent Mater 2014.
[16] Lucchetti MC, Fratto G, Valeriani F, De Vittori E, Giampaoli S,
Papetti P, et al. Cobalt-chromium alloys in dentistry: an
evaluation of metal ion release. J Prosthet Dent
2015;114:602–8.
[17] Puskar T, Jevremovic D, Williams RJ, Eggbeer D, Vukelic D,
Budak I. A comparative analysis of the corrosive effect of
artificial saliva of variable pH on DMLS and cast Co-Cr-Mo
dental alloy. Materials (Basel) 2014;7:6486–501.
[18] Okazaki Y, Gotoh E. Comparison of metal release from
various metallic biomaterials in vitro. Biomaterials
2005;26:11–21.
[19] Hjalmarsson L, Smedberg JI, Wennerberg A. Material
degradation in implant-retained cobalt-chrome and
titanium frameworks. J Oral Rehabil 2011;38:61–71.
[20] Jang KS, Youn SJ, Kim YS. Comparison of castability and
surface roughness of commercially pure titanium and
cobalt-chromium denture frameworks. J Prosthet Dent
2001;86:93–8.
[21] Lovgren N, Roxner R, Klemendz S, Larsson C. Effect of
production method on surface roughness, marginal and
internal fit, and retention of cobalt-chromium single
crowns. J Prosthet Dent 2017;118:95–101.
[22] Tuncdemir AR, Karahan I, Polat S, Malkoc MA, Dalkiz M. The
effect of repeated porcelain firings on corrosion resistance of
different dental alloys. J Adv Prosthodont 2013;5:44–50.
[23] Xin XZ, Chen J, Xiang N, Gong Y, Wei B. Surface
characteristics and corrosion properties of selective laser
melted Co-Cr dental alloy after porcelain firing. Dent Mater
2014;30:263–70.
[24] Edmondson JM, Armstrong LS, Martinez AO. A rapid and
simple MTT-based spectrophotometric assay for
determining drug sensitivity in monolayer cultures. J Tissue
Cult Methods 1988;11:15–7.
[25] Al-Hiyasat AS, Darmani H, Bashabsheh OM. Cytotoxicity of
dental casting alloys after conditioning in distilled water. Int
J Prosthodont 2003;16:597–601.
[26] Holm C, Morisbak E, Kalfoss T, Dahl JE. In vitro element
release and biological aspects of base–metal alloys for
metal-ceramic applications. Acta Biomater Odontol Scand
2015;1:70–5.
[27] Forster A, Ungvari K, Gyorgyey A, Kukovecz A, Turzo K, Nagy
K. Human epithelial tissue culture study on restorative
materials. J Dent 2014;42:7–14.
[28] Puskar T, Lapcevic A, Jevremovic D, Arandjelovic S,
Radulovic S, Budak I, et al. Comparative study of cytotoxicity
of direct metal laser sintered and cast Co-Cr-Mo dental alloy.
Metalurgija 2015;54:481–4.
[29] Fage SW, Muris J, Jakobsen SS, Thyssen JP. Titanium: a review
on exposure, release, penetration, allergy, epidemiology, and
clinical reactivity. Contact Dermatitis 2016;74:323–45.
[30] Kim KT, Eo MY, Nguyen TTH, Kim SM. General review of
titanium toxicity. Int J Implant Dent 2019;5:10.
[31] Wennerberg A, Albrektsson T. Suggested guidelines for the
topographic evaluation of implant surfaces. Int J Oral
Maxillofac Implants 2000;15:331–44.
[32] Albrektsson T, Wennerberg A. Oral implant surfaces: part
1—review focusing on topographic and chemical properties
of different surfaces and in vivo responses to them. Int J
Prosthodont 2004;17:536–43.
[33] Pettersson M, Pettersson J, Molin Thorén M, Johansson A.
Effect of cobalt ions on the interaction between
macrophages and titanium. J Biomed Mater Res A
2018;106(9):2518–30.
[34] Bezzon OL, Pedrazzi H, Zaniquelli O, da Silva TBC. Effect of
casting technique on surface roughness and consequent
mass loss after polishing of NiCr and CoCr base metal alloys:
a comparative study with titanium. J Prosthet Dent
2004;92:274–7.
[35] Kilicarslan MA, Ozkan P. Evaluation of retention of
cemented laser-sintered crowns on unmodified straight
 d e n t a l m a t e r i a l s 3 6 ( 2 0 2 0 ) e352–e363
narrow implant abutments. Int J Oral Maxillofac Implants
2013;28:381–7.
[36] Wataha JC. Predicting clinical biological responses to dental
materials. Dent Mater 2012;28:23–40.
[37] Tuna SH, Ozcicek Pekmez N, Kurkcuoglu I. Corrosion
resistance assessment of Co-Cr alloy frameworks fabricated
by CAD/CAM milling, laser sintering, and casting methods. J
Prosthet Dent 2015;114:725–34.
[38] Takaichi A, Suyalatu, Nakamoto T, Joko N, Nomura N,
Tsutsumi Y, et al. Microstructures and mechanical
properties of Co-29Cr-6Mo alloy fabricated by selective laser
melting process for dental applications. J Mech Behav
Biomed Mater 2013;21:67–76.
[39] https://www.thermofisher.com/se/en/home/industrial/
environmental/environmental-learning-center/contaminant
-analysis-information/metal-analysis/comparison-icp-oes-
icp-ms-trace-element-analysis.html. [updated 190102].
e363
[40] Segura M, Madrid Y, Cámara C. Elimination of calcium and
argon interferences in iron determination by ICP-MS using
desferrioxamine chelating agent immobilized in sol–gel and
cold plasma conditions. J Anal At Spectrom 2003;18:1103–8.
[41] Wolf RE, Available from: What is ICP-MS?. . .and more
importantly, what can it do?; 2005
https://docplayer.net/85421-What-is-icp-ms-and-more-
importantly-what-can-it-do.html.
[42] Dentistry — metallic materials for fixed and removable
restorations and appliances; 2016.
[43] Harloff T, Hönle W, Holzwarth U, Bader R, Thomas P, Schuh
A. Titanium allergy or not? “Impurity” of titanium implant
materials; 2010. p. 306–10.
[44] Al-Nazhan S, Spangberg L. Morphological cell changes due
to chemical toxicity of a dental material: an electron
microscopic study on human periodontal ligament
fibroblasts and L929 cells. J Endod 1990;16:129–34.