2.1

Specimens preparation

Co–Cr alloys manufactured with four different techniques were included in the study ( Table 1 ).Two shapes of specimens 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 ultrasonic 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) followed by packing in a sterile bag.

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.

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 recommended 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 processes 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 chloride 0.1 mol/l was mixed to ensure the pH of 2.3 ± 0.1. Each specimen was covered by the corresponding volume of lactic acid in a closed tube and stored for 7 days in 37 °C. One control 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 individual 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 manufacturer ( Table 1 ) the following elements were included in the present study: Ni, Cd, Be and Pb. The detection limit was set to <1 μg/cm ² .

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 specimens (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/cm ² per specimen. The total area of each cylinder was approximately 3.0 cm ² . In total, 2.5 l artificial saliva was prepared with a composition of: (a) 0.815 g Na ₂ HPO ₄ × 2H ₂ O, (b)1.75 g NaCl, (c) 0.825 g KSCN, (d) 0.5 g KH ₂ PO ₄ , (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, Scandinovata, 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, Termaks, 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 min. The specimens were dried at 60° in heating cabinet (Bakteriologskåp 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 ).

Flow chart for the specimens used for ion release analysis.

In total, 114 specimens were divided into two groups.

The release of metal ions was measured at five different 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, Nümbrecht, Germany) and 100 μl 65% HNO ₃ was added (to prevent bacterial growth) so that the total volume 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 (Bakteriologskåp 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 Pappersbruk, Sweden). The used glass tubes were cleaned with 10% of HNO ₃ 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 10 ¹⁵ .

2.3

Surface roughness analysis

Six rectangularly milled specimens (three before treatment, 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. According to recommendations by Wennerberg and Albrektsson a high-pass Gaussian filter 50 μm × 50 μm was used to separate roughness from errors of form and waviness [ ]. Three surface parameters were calculated according to previous recommendations [ ]:

• 1.

  Sa (μm)–the average height deviation of each point compared to the arithmetical mean of the surface.

• 2.

  Sds (1/μm ² )–the density of the summits, number of summits 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.

The surface evaluation was performed with Surfascan software (Somicronic Instrument, Lyon, France).

2.4

Cell viability test

The cell viability was measured with MTT [ ] according to ISO 10993-5:2009 using two different cell lines: (a) mouse fibroblasts (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 negative 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% CO ₂ 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 Technologies, Foster City, CA, USA) which was replaced every second day (maximum 20 passages). The cells were passaged by trypsin/EDTA when confluence had reached >85%. The culture flask and the 24 well-plate were precoated with collagen (Advanced Biomatrix, INAMED Corp., Fremont, 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 incubated at 37 °C, 5% CO ₂ 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 cm ² /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 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 discarded, 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% CO ₂ 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% CO ₂ and 95% humidity. According to Edmondson et al. [ ] 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 absorbance was measured at 570 nm in a spectrophotometer (Synergi H1, Biotek, Vermont, USA).

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 [ ]. 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.

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.

3.1

Immersion test I (acidic conditions heat treatment with ICP-EOS)

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 treatment.

Table 2

Ion release after 7 days, μg/cm ² 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*																–

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