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Electrochemical comparison and biological performance of a new CoCrNbMoZr alloy with commercial CoCrMo alloy
Materials Science and Engineering C 59 (2016) 346–355
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrochemical comparison and biological performance of a new CoCrNbMoZr alloy with commercial CoCrMo alloy M. Andrei a, B. Galateanu b,c, A. Hudita a, M. Costache b, P. Osiceanu d, J.M. Calderon Moreno d, S.I. Drob d,⁎, I. Demetrescu a a
Department of General Chemistry, University Politehnica Bucharest, Spl. Independentei 313, 060042 Bucharest, Romania Department of Biochemistry and Molecular Biology, University of Bucharest, 91–95 Spl. Independentei, Bucharest 050095, Romania Institute of Life Sciences, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romania d Institute of Physical Chemistry “Ilie Murgulescu” of Romanian Academy, Spl. Independentei 202, 060021 Bucharest, Romania b c
a r t i c l e
i n f o
Article history: Received 27 May 2015 Received in revised form 20 September 2015 Accepted 11 October 2015 Available online xxxx Keywords: New CoCrNbMoZr alloy Microstructure Surface features Corrosion resistance Biocompatibility Adipose stem cells (hASCs)
a b s t r a c t A new CoCrNbMoZr alloy, with Nb and Zr content is characterized from the point of view of surface features, corrosion resistance and biological performance in order to be proposed as dental restorative material. Its properties are discussed in comparison with commercial Heraenium CE alloy based on Co, Cr and Mo as well. The microstructure of both alloys was revealed by scanning electron microscopy (SEM). The composition and thickness of the alloy native passive films were identified by X-ray photoelectron spectroscopy (XPS). The surface characteristics were analyzed by atomic force microscopy (AFM) and contact angle techniques. The quantity of ions released from alloys in artificial saliva was evaluated with inductively coupled plasma-mass spectroscopy (ICPMS) measurements. The electrochemical stability was studied in artificial Carter–Brugirard saliva, performing open circuit potentials, polarization resistances and corrosion currents and rates. The biological performance of the new alloy was tested in vitro in terms of human adipose stem cells (hASCs) morphology, viability and proliferation status. The new alloy is very resistant to the attack of the aggressive ions from the artificial saliva. The surface properties, the roughness and wettabiliy sustain the cell behavior. The comparison of the new alloy behavior with that of existing commercial CoCrMo alloy showed the superior properties of the new metallic biomaterial. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Analyzing the state of the art in the field, we can observe a multitude of dental alloys for restorative prosthodontics available on the market and their biocompatibility problems [1,2]. Most of the metallic alloys are placed in the oral cavity for long term use, where they have to resist to oral corrosion, sometimes to temperature fluctuations (from about 10 °C to 45 °C), pH fluctuations, and mechanical stress [3,4]. In such conditions, the restorations with alloys need a selection in their elaboration, in order to offer resistance to corrosion and degradation with a minimum denaturation in long term exploitation [5], preserving the oral health. Having better tribological properties in comparison with those of titanium alloys and a much better price, CoCrMo alloys are one of the preferred choice for articular applications with metal-on-metal contact [6–10]. The electrochemical stability due to the passive oxide film grown on their surface [11] is a recommendation for biocompatibility as well, but it is to notice the problems with ion release in their working ⁎ Corresponding author. E-mail address: sidrob@chimfiz.icf.ro (S.I. Drob).
http://dx.doi.org/10.1016/j.msec.2015.10.031 0928-4931/© 2015 Elsevier B.V. All rights reserved.
time [12,13]. Comparing with Ti and Ti alloys which have a large passive range [14,15], CoCrMo alloys reveal a transition to transpassive behavior at lower values of electrode potentials which could be the result of fast dissolution through the passive film [16] or due to film breakdown [17]. The dependence of corrosion rate by the heat processing [18] was investigated indicating a significant decrease of corrosion rate as well. Such demerits as ion release and corrosion increase for oral cavity alloys which are subject to mechanical stress involving surface microstructure changes lead to the need of alloy modifications taking into account two approaches as surface and composition. At surface level in the last decades for CoCrMo alloys various techniques were proposed and studied from simple cheap coatings elaborated via immersion [19] or electrochemically [20], until sophisticated and expensive procedures as plasma spraying and ion bombardment [19,21]. Surface modification is recommended nowadays for enhancing properties at micro or nanolevel [22, 23], which is very important in the solving of the osseointegration problems essential at interface of the implant material with tissue [24]. In order to improve in bulk the mechanical, electrochemical stability and biological performance, the modification of alloy content is recommended and for CoCrMo alloys various compositions based on Co, Cr, and Mo elements have been the subject of patents and standards
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[25,26]. The release of Cr ions into human biofluid can influence the cell viability and can decrease the AND synthesis [27]. Co and Cr ions can cause “metal allergy”, can promote the granuloma formation and carcinoma [28]. Considering that the CoCrMo dental alloys are used at mass level favoring ion release and absorption due to their imperfections and leading to certain pigment metallic lesions or even more aggressive diseases [29] in the oral cavity, the present paper is introducing a new improved alloy with modified composition by selecting as new added component, the biocompatible Nb and Zr elements [30,31]. Niobium is a high passive metal due to its Nb2O5 protective oxide that covers its surface for the whole pH range from − 2 to 16 on a large potential range from −0.7 V (vs. SCE) to +2 V (vs. SCE) [32]; thus, this element will increase the passivation domain of the new CoCrNbMoZr alloy. Although Niobium is present in living organisms and might affect biological mechanisms, poor information regarding it's cytotoxicity is available to date. There are no information documenting its mutagenic potential, but on the other hand there are studies suggesting that some niobium compounds may have antitumor activity [33]. While niobium dust is an eye and skin irritant and a potential fire hazard, elemental niobium on a larger scale is physiologically inert and thus hypoallergenic and harmless. This is why it is frequently used in jewelry and has been tested for use in some medical implants [34]. Zirconium has a native passive film formed by ZrO2 oxide that is very resistant from pH = 3 to pH = 13 and from −1.6 V (vs. SCE) till + 1.4 V (vs. SCE) [32] and this oxide is very compatible with the blood [35]; so, this alloying element will enhance the corrosion resistance and biocompatibility of the new alloy. Despite the presence and retention in relatively high quantities in biological systems, Zirconium has not yet been associated with any specific metabolic function. Very little information is available to date about its interaction with the compounds of the genetic systems, such as nucleic acids. Some studies reveal that in vivo Zr retention is initially located in soft tissues and then slowly in the bones. The metal seems to be able to cross the blood brain-barrier, the placental barrier and also enters the milk [36]. Additionally, Wang et al., showed that Zr is a nontoxic element and that Zr containing alloys display a very good hemocompatibility [37]. The novelty and the mission of the present paper are to realize a complete and complex characterization of the new alloy in comparison with the existing CoCrMo alloy. The alloy microstructure was showed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS); the composition and thickness of the native passive films on the alloy surfaces were identified by X-ray photoelectron spectroscopy (XPS). Also, the surface roughness was revealed by atomic force microscopy (AFM) and the contact angle evinced the alloys hydrophilic properties. The amounts of ions released in artificial Carter– Brugirard saliva were evaluated by the inductively coupled plasmamass spectroscopy (ICP-MS) technique for the commercial existing CoCrMo alloy (Heraenium CE) and for the new improved CoCrNbMoZr alloy. The corrosion rate was obtained by the linear polarization method. The trial of these materials was performed by the in vitro cell culture as well, in order to establish the improvement of cell response as a prognosis of its better behavior in vivo in the future.
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2.1. Physical chemical characterization The physical chemical characterization of the both new CoCrNbMoZr alloy and commercial CoCrMo, Heraenium CE alloy involves SEM, EDS, XPS, AFM, contact angle techniques. The microstructure of the alloys was studied in a scanning electron microscope FEI Quanta 3D FEG equipped with an EDS detector. SEM micrographs were obtained with a back-scattered electron detector, using an accelerating voltage of 10 kV. EDS elemental analysis was performed using an accelerating voltage of 20 kV. The composition and the thickness of the native passive films on both studied alloys were analyzed by X-ray photoelectron spectroscope Quantera SXM that used AlKα radiation and an overall energy resolution of 0.65 eV by the full width at half-maximum (FWHM) of the Au4f7/2. The elemental composition was identified with accuracy for the binding energies (BEs) assignments of ± 0.2 eV, after a gentle Ar + ion etching (1 keV, 3 × 3 mm rastered area, 0.5 min. sputter time) on the surface, to remove the contaminant monolayer without destroying the chemical species. The thickness of the surface layer was determined by the XPS depth profiling, layer by layer experiment under 2 keV, on a 2 × 2 mm scanning area, at 45 0 incidence angle and 200 μm spot size of X-ray beam. These parameters were used to minimize the induction effect of the ion beam on the oxides [38,39]. The surface roughness was examined with atomic force microscope (Electrochemical AFM from A.P.E. Research Company) and the three-dimensional surface roughness expressed as the root mean square roughness (RMS) and average roughness (Rav) of the samples were estimated. The measurements have been performed in contact mode. The contact angle determinations were carried out with an 100 Optical Contact Angle Meter — CAM 100; the resulted value is the average of minimum three measurements for each determination and all the tests were realized with an accuracy of ±1° at 25 °C. 2.2. Ion release rate The quantity of ions released in time from alloys in tested electrolyte, Carter–Brugirard saliva was measured with an inductively plasma mass spectrometer (ICP-MS), equipment Perkins Elmer ELAN DRC-e that works with the liquid sample introduction by a micro-nebulizer and the calibration was against the aqueous multi-element solutions. The tests were performed by the periodical analyses till 168 h of immersion in artificial Carter–Brugirard saliva. 2.3. Electrochemical characterization The electrodes preparation before each determination included the grinding with abrasive paper having different granulations, polishing to mirror surface, washing with water and 30 min ultrasonication in acetone. As electrolyte for electrochemical stability tests Carter–Brugirard saliva with the following composition has been selected (g/L): KCl − 1.2; NaCl — 0.7; KH2PO4 — 0.26; KSCN — 0.33; Na2HPO4 — 0.19; urea — 0.13; NaHCO3 — 1.5. The procedures has been:
2. Materials and methods The studied alloys have the compositions presented in Table 1. Table 1 Composition of studied alloys. Material
Heraenium CE CoCrNbMoZr
wt.% Co
Cr
Mo
Mn
Si
C
N
Nb
Zr
63.5 60
27.8 26.5
6.6 4.5
0.6 0.8
1.0 1.0
0.3 0.4
0.2 0.2
– 6.0
– 0.8
‐ the monitoring (by periodical verifications till 200 immersion hours) of the open circuit potential using a performing Hewlett-Packard multimeter; ‐ the potentiodynamic linear polarization applied from a more electronegative potential with 50 mV than the open circuit potential till to a more electropositive potential with 50 mV than the open circuit potential with a scan rate of 0.1 mV/s; the samples were kept about 30 min. before the polarization tests. From Tafel plots, the main corrosion parameters Ecorr (corrosion potential), jcorr (corrosion current density), P (penetration index, corrosion rate)
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Fig. 1. SEM micrograph (a) and EDS elemental analysis (b) of CoCrNbMoZr alloy.
and Rp (polarization resistance) were determined; equipment was potentiostat Radiometer PGZ 80.
The electrochemical cell was the one with three electrodes as following: the working electrode (the commercial and the new alloy), the Pt electrode as contra electrode and a saturated calomel electrode (SCE) as reference electrode. 2.4. Cell culture model Human adipose derived stem cells (hASCs) were previously isolated from human subcutaneous adipose tissue as described by Galateanu et al. [40]. Briefly, the lipoaspirates (LAs) were processed by collagenase digestion and the cells obtained were resuspended in Dulbecco's modified Eagle's medium (DMEM), supplemented with fetal bovine serum (FBS). The mesenchymal stem cells surface markers panel was confirmed by flow cytometry starting with the 3rd passage. The human adipose tissue was obtained from female patients undergoing elective liposuction and all the medical procedures were performed in compliance with the Helsinki Declaration, with the approval of Proestetica Medical Center Committee (reference number 112/23.10.2013).
2.5. Biocompatibility assessment 2.5.1. Cellular morphology The protein expression of actin was studied at 6 h and 5 days postseeding of the hASCs on the samples by fluorescence microscopy using an Olympus IX71 inverted microscope. In this view, the hASCs were fixed with 4% paraformaldehyde for 1 h and cell membranes were permeabilized with 2% bovine serum albumin/0.1% Triton X-100 solution at 4 °C. Next, the samples were incubated 30 min at 37 °C with Alexa Fluor 546 Phalloidin for actin labeling. After cell nuclei were stained with DAPI for 15 min, the resulting labeled samples were inspected in fluorescence microscopy. Images were captured with Cell F Imaging Software (Olympus). 2.5.2. Cellular viability and proliferation potential Cell proliferation and viability within the cell-material culture systems was evaluated by fluorescence microscopy using Live/Dead Kit (Invitrogen, Life Technologies, Foster City, CA). Briefly, at 24 h and 5 days post seeding, the samples (pre-seeded with hASCs) were exposed to a staining solution, containing calcein AM and ethidium bromide at room temperature and darkness. After 10 min, the samples were analyzed by fluorescence microscopy using an Olympus IX71
Fig. 2. SEM micrograph (a) and EDS elemental analysis (b) of CoCrMo, Heraenium CE alloy.
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elemental analysis evinced that Cr and Mo are enriched in the inclusions. 3.2. Composition and thickness of the native passive film
Fig. 3. XPS survey spectrum for the native passive film existing on new CoCrNbMoZr alloy surface.
inverted microscope and images were captured with Cell F Imaging Software (Olympus). hASCs capacity to proliferate on the surface of the samples was quantitatively assessed by MTT test at 24 h and 5 days post seeding. Cell-material culture systems were incubated in 1 mg/ml MTT solution for 4 h and the formazan crystals obtained in the metabolically active cells were solubilized in isopropanol. The concentration of the resulted solution was spectrophotometrically quantified at 550 nm (Appliskan Thermo Scientific). 2.5.3. Material's cytotoxic potential The cytotoxic potential of the samples on hASCs was evaluated using in vitro toxicology LDH-based assay (Sigma Aldrich, Steinheim, Germany). The culture media were harvested at 24 h and 5 days postseeding and mixed with test solution according to the manufacturer's recommendation. After 20 min of incubation, the reaction was stopped with 1 N hydrochloric acid (HCl) and the spectrophotometric detection of LDH concentration was determined by measuring the optic density of the resulting solution at 490 nm (AppliskanThermo Scientific). 2.5.4. Statistical analysis The spectrophotometric data were statistically analyzed using GraphPad Prism 3.03 Software, one-way ANOVA, Bonferroni test. The experiments were performed with n = 3 biological replicates and each data set is presented as the average of three replicates (mean ± standard deviation). 3. Results and discussions 3.1. Alloy microstructure Microstructural analysis of the cast new CoCrNbMoZr alloy is characterized by a multicomponent, fine dendritic structure (Fig. 1a) with typical interdendritic distances of a few microns. EDS spectra (Fig. 1b) detected the alloying elements, Co, Cr, Nb, Mo, Mn, and Si (Zr main feature at ~2 keV is masked by the secondary Nb and Mo peaks). EDS elemental analysis revealed that Mo, Nb, Si and C are enriched in the interdendritic regions. Microstructural analysis of the cast CoCrMo Heraenium CE alloy (Fig. 2a) shows a main matrix phase (~90 vol.%) containing inclusions, most of them being isolated and with globular shape, sized ~ 5 μm, and well dispersed in the matrix, with typical distance between inclusions of about 20 μm, except for the formation of some linear aggregates, with lengths up to several tens of microns. EDS spectra (Fig. 2b) detected the main alloying elements, Co, Cr, Mo, and Si. EDS
XPS survey spectrum (Fig. 3) for the new CoCrNbMoZr alloy showed a complex feature that included the peaks for Co 2p, Cr 2p, Nb 3d, Mo 3d, Zr 3d, and O 1s [38]. High resolution spectra (Fig. 4) revealed the presence of Co (Fig. 4a) as metallic Co, of Cr (Fig. 4b) as Cr 2 O3 oxide (68.6%) and metallic Cr (31.4%), of Nb (Fig. 4c) as Nb 2 O 5 oxide (49.0%) and NbO (51.0%), of Mo (Fig. 4d) as MoO 3 oxide (14.0%), MoO2 oxide (25.9%) and metallic Mo (60.1%), of Zr (Fig. 4e) as ZrO 2 oxide (54.9%) and metallic Zr (45.1%), of Mn (Fig. 4f) as MnO 2 oxide (26.4%) and metallic Mn (73.6%), of Si (Fig. 4g) as SiO2 oxide and elemental Si (61.2%) and of O (Fig. 4h) as O2 − ion (87.6%) and OH− ion (12.4%); the high concentration of O 2 − ion appears from oxides. It is a thick oxide layer (about 6.5 nm–8 nm), that can confer very good protection to the substrate [41,42]; this fact will be proved by the lower values of the penetration indexes (Subchapters 3.5 and 3.6.2). It has be observed that the native passive film on the new CoCrNbMoZr alloy surface is composed from chromium, niobium, molybdenum, zirconium, manganese, and silicon oxides, thus being thicker, more compact and enhanced with oxides. Milosev et al. [11] appreciated that the increase of the oxide layer thickness on the CoCrMo alloy surface is due to the entering of Mo and Co oxides into its passive layer; thus, for the new CoCrNbMoZr alloy, the oxides Nb2 O 5 and ZrO2 which are the constituents of the passive film, also increase its thickness. For the commercial Heraenium CE alloy, the survey spectrum (Fig. 5) comprises the peaks for Co 2p, Cr 2p, Mo 3d and O 1s. This film has a thickness of 5 nm; it is a very thin layer and its protective capacity will be determined further. Also, high resolution spectra (Fig. 6) for Co (Fig. 6a) evinced only metallic Co, for Cr (Fig. 6b), Cr2O3 oxide (72.8%) and metallic Cr (27.2%), for Mo (Fig. 6c), MoO3 oxide (21.5%), MoO2 oxide (21.8%) and metallic Mo (56.7%), for O (Fig. 6d), O2 − ion (78.9%) and OH− ion (21.1%). It results that the native passive film on the Heraenium alloy surface contains chromium and molybdenum oxides which confer its resistance against to corrosion. 3.3. Surface topography and roughness In Fig. 7, the AFM images of investigated alloys have permitted evaluation of their topography and average roughness values. The both alloys revealed a similar topography; the root mean square, RMS parameter has the value of 20.2 nm for the new alloy and 20.8 nm for the commercial alloy; the average roughness, Rav is 15.5 nm for the new alloy and 16.3 nm for the commercial alloy; these values are close to each-other. This roughness is very proper for the cell adhesion [43–45]. 3.4. Contact angle An increase in the contact angle value for the new CoCrNbMoZr alloy has been observed compared to the commercial Heraenium CE alloy. The increase from 75.92 to 86.79 values indicates that both alloys have hydrophilic character but the stronger one is for the commercial one. 3.5. Ion release The quantity of ions (determined by ICP-MS method) released in artificial saliva for both alloys is relatively small, but for the new alloy, the amount is significant lower. The quantified amounts in ppb (partsper-billion, 10−9) are presented in Table 2 and sustain increased stability for the new alloy.
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Fig. 4. XPS high resolution spectra for the native passive film existing on new CoCrNbMoZr alloy surface: a) Co 2p; b) Cr 2p; c) Nb 3d; d) Mo 3d; e) Zr 3d; f) Mn 2p; g) Si 2s; h) O 1s.
3.6. Electrochemical behavior 3.6.1. Monitoring of open circuit potentials The evolution of open circuit potential in time for both alloys is presented in Fig. 8. As can be seen the curves aspect denotes a trend to steady state sooner after immersion in both cases; for the Heraenium CE, after about 20 h and for the new alloy after 60 h. It is a specific aspect for passivity due to the protective oxides formed on the surface of the both alloys [41,42]. The new alloy reached its stable passive state after a larger time period than Heraenium CE alloy; its passivation process has a lower passivation rate. This fact was observed by Robin et al.
[46] for Ti–5Nb–13Zr and Ti–20Nb–13Zr alloys which revealed “a slower growth rate of the passivating films” in comparison with “Ti, Ti–6Al–4 V and Ti–13Nb–13Zr alloys”. But, after about 200 immersion hours, the both alloys realized about the same values of the open circuit potentials, around −40 mV (vs. SCE).The open circuit potentials tend to more electropositive values, indicating an increase of passive film thickness [41,42]. 3.6.2. Linear polarization results The Tafel results are reported for the both alloys in Fig. 9 and Table 3. In Table 3 are listed corrosion potential, (Ecorr), corrosion current
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Fig. 5. XPS survey spectrum for the native passive film existing on commercial Heraenium alloy surface.
density (jcorr), penetration index (P), and polarization resistance (Rp). From these data it resulted that the new alloy at initial time is included in conventional stability scale “Perfect Stable” while the commercial alloy in “Very Stable” in the corrosion resistance group (corrosion classification) [47,48]. With the increase of immersion time, the penetration index, corrosion rate decreased. The minimum of the penetration index is after 200 h for the commercial alloy, the value being a bit less than a half of the initial value. It is necessary to mention that for the new alloy, the ratio between the value at initial time and at 200 h is around 36. Also, the polarization resistance, Rp values for the new alloy increased in time as result of the passive film thickening [41,42]. For the commercial alloy, polarization resistance decreased in time, denoting some dissolution and repassivation processes [41,42] at the interface between alloy and artificial saliva. Rp have higher values from about 8 times to 175 times than those of Heraenium CE alloy; correlating with the lower values of the corrosion current densities, it results a better corrosion resistance, a more resistant passive film. Milosev et al. [49] showed that for Ti–6Al–7Nb alloy, its passive layer is stabilized by the incorporation of Al and Nb oxides, thus increasing its resistance.
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So, it is reasonable to presume that the better corrosion resistance of the new alloy in comparison with the commercial Heraenium CE alloy is due to its passive film that contains beside chromium oxide, niobium (Nb2O5, 49.8%) and zirconium oxides (ZrO2, 54.9%), as was showed by XPS analysis. The corrosion potentials, Ecorr obtained from linear polarization tests (Fig. 9 and Table 3) have lower values than those of the open circuit potentials (Fig. 8), due to the fact that, the polarization tests began at a more electronegative potential with 50 mV than the open circuit potential; thus, the passive film was partially dissolved. This behavior was observed by other authors [12,50,51] who gave the same explanation of the passive film dissolution; they added that the cathodic partial reaction of the hydrogen evolution shifts the potential to more electronegative values. Tafel plots from Fig. 9 confirm the in time ennobling of Ecorr values both for the new alloy and for Heraenium CE alloy and the fact that after about 200 immersion hours, both alloys reached similar values (around −130 mV) for their corrosion potentials, Ecorr. 3.7. Cell morphology and distribution hASCs morphology on the alloys surfaces and their ability to interact with the substrate material in terms of adhesion, cytoskeleton development were investigated after 6 h days of culture. Cellular distribution was investigated at 5 days post-seeding. As shown in Fig. 10, at 6 h post-seeding, hASCs displayed long and distinctive actin filaments surrounding the nuclei on conventional plastic cell culture surface and already adopted their characteristic spindle-like shape. Unlikely, hASCs on both Heraenium CE and CoCrNbMoZr alloys did not display such morphology at 6 h postseeding, but significant differences were noticed between the two samples. Consequently, on Heraenium CE, short actin filaments were observed surrounding hASCs nuclei and the overall cellular shape was still round, while hASCs morphology on the surface of the CoCrNbMoZr alloy was round with elongations in different directions. Thus, over time, only hASCs on plastic and CoCrNbMoZr alloy surfaces exhibited a well-defined spindle-shaped morphology, as
Fig. 6. XPS high resolution spectra for the native passive film existing on commercial Heraenium alloy surface: a) Co 2p; b) Cr 2p; c) Mo 3d; d) O 1s.
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Fig. 7. AFM images of new CoCrNbMoZr alloy (a) and of the commercial Heraenium CE alloy (b).
Table 2 Ion release rate obtained after 168 h of immersion of the studied alloys in artificial Carter– Brugirard saliva at 37 °C. Analyzed element
Co Cr Mo Mn Nb Zr
hASCs on the surface of CoCrNbMoZr display a regular pattern both at 24 h and 5 days post-seeding as compared to hASCs random distribution on the Heraenium CE specimen.
Mean concentration (ppb) CoCrNbMoZr
Heraenium CE
0.185 3.643 4.843 0.339 1.350 1.0
8.006 12.197 23.234 2.500 – –
shown at 5 days post-seeding. Additionally, a random distribution of the cells on the plastic and Heraenium CE alloy surfaces was observed as compared to a very clear array of hASCs on the CoCrNbMoZr specimens at 5 days post-seeding; this fact is due to the more stable passive film that releases (Table 3) a lower quantity of the cytotoxicelements on the new alloy surface comparing with the commercial Heraenium CE alloy.
3.9. Quantification of the cellular viability and proliferation status hASCs viability and proliferation potential on Heraenium CE and CoCrNbMoZr alloys was quantitatively determined by MTT spectrophotometric assay. The obtained spectrophotometric data were graphically represented in Fig. 12. Our data revealed that significant differences were detected between the hASCs viability on Heraenium CE surface versus CoCrNbMoZr surface, both at 24 h (###p b 0.0001) and 5 days (##p b 0.001) post-seeding; the hASCs viability is more higher on the
3.8. Cellular viability, live/dead fluorescent microscopy assay In order to examine hASCs survival on the tested biomaterials, cellular the viability was evaluated at 24 h and 5 days post-seeding by fluorescence microscopy (Fig. 11), based on the simultaneous staining of live (green labeled) and dead (red labeled) cells. After 24 h and 5 days post-seeding only bright green-labeled cells were observed on the surface of both Heraenium CE and CoCrNbMoZr alloys as a proof of their survival. Furthermore, no red nuclei were observed. Live/Dead staining also confirmed the above observations regarding the cell distribution. As shown in Fig. 11, bright green living
Fig. 8. Evolution of open circuit potentials of studied alloys in artificial Carter–Brugirard saliva at 37 °C.
Fig. 9. Tafel plots obtained in artificial Carter–Brugirard saliva at 37 °C for: a) CoCrNbMoZr alloy; b) Heraenium alloy.
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Table 3 Main corrosion parameters obtained after different immersion periods of the studied alloys in artificial Carter–Brugirard saliva at 37 °C. Time (h)
0 24 48 72 96 120 144 168 200
jcorr (nA/cm2)
Ecorr (mV)
Rp (kΩ·cm2)
P (μm/Year)
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
−287.62 −158.83 −144.59 −147.62 −149.52 −153.17 −179.18 −166.74 −125.61
−315.89 −237.23 −221.99 −229.57 −238.78 −237.54 −227.83 −195.20 −128.71
251.26 168.05 156.37 162.98 175.38 114.72 151.62 176.37 128.82
46.52 8.29 3.78 2.00 4.45 3.81 1.52 1.72 1.355
5.772 3.860 3.592 3.744 4.028 2.635 3.483 4.051 2.729
1.171 0.209 0.095 0.050 0.112 0.096 0.038 0.043 0.032
76.23 71.54 66.92 62.02 58.51 58.16 46.65 48.48 45.49
593.77 2602.7 4191.6 5291.7 5427.3 4834.7 7075.4 7609.1 7964.7
new alloy surface as result of the more lower level of Co, Cr Mo and Mn ions released into environment (Table 2). Regarding the proliferative status, hASCs displayed a significant increase of proliferation during 5 days of culture both on Heraenium CE alloy (***p b 0.0001) and CoCrNbMoZr alloy surfaces (***p b 0.0001). 3.10. Cytotoxic potential of Heraenium CE and CoCrNbMoZr alloys on hASCs The cytotoxic potential of Heraenium CE and CoCrNbMoZr alloys was evaluated by spectrophotometric quantification of the LDH enzyme release in the culture media by hASCs seeded in direct contact with the samples. The quantitative data obtained are represented in Fig. 11b and reveal low and constant values of LDH activity in both tested cell cultures at 24 h post-seeding suggesting that none of the samples induced cell toxicity. Unlikely, after 5 days of culture a significant difference (#p b 0.05) was detected in terms of LDH activity in the culture media harvested from Heraenium CE versus CoCrNbMoZr, indicating a slightly increased rate of cell death on Heraenium CE as compared with CoCrNbMoZr; this fact can be explained by the quantity of Co, Cr, Mo,Mn ions released by the studied alloys: for the Heraenium CE alloy surface were released Co ions of about 43 times higher, Cr ions of 3.3 times higher, Mo ions of 4.8 times higher and Mn ions of 7.4 times higher, as results from Table 2. 4. Conclusions SEM observations evidenced a finer microstructure of the new CoCrNbMoZr alloy in comparison with the commercial Heraenium CE alloy.
The native passive film existing on the new CoCrNbMoZr alloy surface is thicker, more compact than that on the commercial Heraenium CE alloy surface because it contains both chromium and molybdenum oxides, and, in addition, the niobium and zirconium oxides (XPS data). The topography and roughness parameters (AFM data) are similar for the two studied alloys and very proper for the cell adhesion. The contact angle values for both studied alloys are expressions of their balanced hydrophilic–hydrophobic nature which indicates clearly affinity for cells. The quantity of ions released in solution is significantly lower for the new alloy, showing its better corrosion resistance. The open circuit potentials tended to more electropositive values, denoting the increase of the passive film thickness. The new CoCrNbMoZr alloy exhibits better stability in tested Carter– Brugirard saliva having a much lower value for corrosion current density and rate and higher value of the polarization resistance than that for the commercial CoCrMo alloy, type Heraenium CE. The biocompatibility assessment of Heraenium CE and CoCrNbMoZr alloys revealed that hASCs need more time to adhere and to acquire their characteristic spindle-like shape when seeded on Heraenium CE as compared with CoCrNbMoZr surface. Furthermore, the cells were distributed randomly on the surface of Heraenium CE alloy while on CoCrNbMoZr they followed a linear pattern. HASCs viability on CoCrNbMoZr was significant higher both at 24 h and 5 days of culture in comparison with hASCs on Heraenium CE. Furthermore, CoCrNbMoZr better sustained cellular proliferation than Heraenium CE as shown by the fluorescence microscopy micrographs
Fig. 10. Fluorescence microscopy micrographs of hASCs actin filaments network (red fluorescence) on plastic surface and Heraenium CE and CoCrNbMoZr specimens at 6 h and 5 days post-seeding; DAPI-stained nuclei are blue. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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132397. This work was supported by Romanian UEFISCDI, Project PCE no. 130/2014 as well. The authors thank Dr. Andrei Bogdan Stoian for AFM analysis. The authors thank Assoc. Prof. MD PhD Dana Jianu from the ProEstetica Medical Clinic who kindly supplied this study with human subcutaneous adipose tissue.
References
Fig. 11. Fluorescence microscopy micrographs revealing live and dead cells on Heraenium CE and CoCrNbMoZr alloys surfaces at 24 h and 5 days post-seeding. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
and by the spectrophotometric data. Lastly, Heraenium CE exerted a significant higher cytotoxic effect on hASCs at 5 days of culture as comparing with CoCrNbMoZr. In conclusion, CoCrNbMoZr alloy displayed an increased corrosion resistance and overall biocompatibility as compared with Heraenium CE and could be further employed for in vitro cell differentiation studies. Acknowledgments The work has been funded by the Sectorial Operational Programme Human Resources Development 2007–2013 of the Ministry of European Funds through the Financial Agreement POSDRU/159/1.5/S/
[1] W. Elshahawy, I. Watanabe, Biocompatibility of dental alloys used in dental fixed prosthodontics, Tanta Dent. J. 11 (2014) 150–159. [2] P. Imirzalioglu, E. Alaaddinoglu, Z. Yilmaz, B. Oduncuoglu, B. Yilmaz, S. Rosenstiel, Influence of recasting different types of dental alloys on gingival fibroblast cytotoxicity, J. Prosthet. Dent. 107 (2012) 24–33. [3] S.C. Wang, M. Browne, H.S. Ubhi, M.J. Starink, Determination of the fatigue fracture planes of Co–Cr–Mo biomedical alloys using electron backscatter diffraction, J. Microsc. 217 (2005) 118–121. [4] T. Hanava, A. Igua, S. Muñoz, S. Mischler, Effect of the environment on wear ranking and corrosion of biomedical CoCrMo alloys, J. Mater. Sci. Mater. Med. 22 (2011) 437–450. [5] J.C.M. Souza, S.L. Barbosa, E. Ariza, J.-P. Celis, L.A. Rocha, Simultaneous degradation by corrosion and wear of titanium in artificial saliva containing fluorides, Wear 292–293 (2012) 82–88. [6] J.C. Walker, R.B. Cook, J.W. Murray, A.T. Clare, Pulsed electron beam surface melting of CoCrMo alloy for biomedical applications, Wear 301 (2013) 250–256. [7] Y. Bedolla-Gil, M.A.L. Hernandez-Rodriguez, Tribological behavior of a heat-treated cobalt-based alloy, J. Mater. Eng. Perform. 22 (2013) 541–547. [8] G. Barucca, E. Santecchia, G. Majni, E. Girardin, E. Bassoli, L. Denti, A. Gato, L. Iuliano, T. Moskalewicz, P. Mengucci, Structural characterization of biomedical Co–Cr–Mo components produced by direct metal laser sintering, Mater. Sci. Eng. C 48 (2015) 263–269. [9] Y. Lu, S. Wu, Y. Gan, J. Li, C. Zhao, D. Zhou, J. Lin, Investigation on the microstructure, mechanical property and corrosion behavior of the selective laser melting CoCrW alloy for dental application, Mater. Sci. Eng. C 49 (2015) 517–525. [10] S. Dittrick, V.K. Balla, N.M. Davies, S. Bose, A. Bandyopadhyay, In vitro wear rate and Co ion release of compositionally and structurally graded CoCrMo–Ti6Al4V structures, Mater. Sci. Eng. C 31 (2011) 809–814. [11] I. Milošev, H.H. Strehblow, The composition of the surface passive film formed on CoCrMo alloy in simulated physiological solution, Electrochim. Acta 48 (2003) 2767–2774. [12] F. Contu, B. Elsener, H. Böhni, Corrosion behaviour of CoCrMo implant alloy during fretting in bovine serum, Corros. Sci. 47 (2005) 1863–1875. [13] N. Rincic, A. Celebic, I. Baucic, J. Stipetic, E. Prohic, S. Miko, The release of ions from the base Co–Cr–Mo casting alloy in vitro into the phosphate buffer at pH 6.0, Acta Stomatol. Croat. 37 (2003) 13–16.
Fig. 12. (a) Quantification of: hASCs viability on Heraenium CE and CoCrNbMoZr alloys surfaces, as revealed by MTT test at 24 h and 5 days post-seeding. [***p b 0.0001 (hASCs on Heraenium CE surface at 24 h vs. 5 days of culture) and (hASCs on CoCrNbMoZr surface at 24 h vs. 5 days of culture)]; [###p b 0.0001 (hASCs on Heraenium CE alloy surface at 24 h of culture vs. hASCs on CoCrNbMoZr surface at 24 h of culture)] and [##p b 0.001 (hASCs on Heraenium CE alloy surface at 5 days of culture vs. hASCs on CoCrNbMoZr surface at 5 days of culture)] and (b) Heraenium CE and CoCrNbMoZr alloys' cytotoxic potential effect on hASCs after 24 h and 5 days of culture as revealed by LDH assay [#p b 0.05 ((hASCs on Heraenium CE alloy surface at 5 days of culture vs. hASCs on CoCrNbMoZr surface at 5 days of culture)].
M. Andrei et al. / Materials Science and Engineering C 59 (2016) 346–355 [14] A. Cimpean, E. Vasilescu, P. Drob, I. Cinca, C. Vasilescu, M. Anastasescu, V. Mitran, S.I. Drob, Enhancement of the electrochemical behaviour and biological performance of Ti–25Ta–5Zr alloy by thermo-mechanical processing, Mater. Sci. Eng. C 38 (2014) 127–142. [15] D. Ionita, M. Grecu, C. Ungureanu, I. Demetrescu, Modifying the TiAlZr biomaterial surface with coating, for a better anticorrosive and antibacterial performance, Appl. Surf. Sci. 257 (2011) 9164–9168. [16] A.W.E. Hodgson, S. Kurz, S. Virtanen, V. Fervel, C.-O.A. Olsson, S. Mischler, Passive and transpassive behaviour of CoCrMo in simulated biological solutions, Electrochim. Acta 49 (2004) 2167–2178. [17] M. Metikos -Hukovic, R. Babic, Some aspects in designing passive alloys with an enhanced corrosion resistance, Corros. Sci. 51 (2009) 70–75. [18] F. Golgovici, M. Prodana, A. Popescu, Effect of reprocessing treatment on the corrosion of Co–Cr alloy in acid media, Rev. Chim. 66 (2015) 660–667. [19] D.A. Cortés, A. Medina, J.C. Escobedo, S. Escobedo, M.A. López, Effect of wollastonite ceramics and bioactive glass on the formation of a bonelike apatite layer on a cobalt base alloy, J. Biomed. Mater. Res. A 70 (2004) 341–346. [20] D. Ionita, I. Man, I. Demetrescu, The behaviour of electrochemical deposition of phosphate coating on CoCr bioalloys, Key Eng. Mater. 330–332 (2007) 545–548. [21] L. Reclaru, P.Y. Eschler, R. Lerf, A. Blatter, Electrochemical corrosion and metal ion release from Co–Cr–Mo prosthesis with titanium plasma spray coating, Biomaterials 26 (2005) 4747–4756. [22] V.-H. Pham, Improving osseointegration of Co–Cr by nanostructured titanium coatings, Pham. Springer Plus 3 (2014) 197–202. [23] R.J. Narayan, Big possibilities for small scale implants, Mater. Today 16 (2013) 204–205. [24] C.E. Bates, V. Marino, N.L. Fazzalari, P.M. Bartold, Soft tissue attachment to titanium implants coated with growth factors, Clin. Implant. Dent. Relat. Res. 15 (2013) 53–63. [25] ASTM F75-12 Standard Specification for Cobalt-28 Chromium-6 Molybdenum Alloy Castings and Casting Alloy for Surgical Implants (UNS R30075), ASTM International, West Conshohocken, PA, 2012 www.astm.org. [26] Biocompatible CoCrMo alloy US20140271317 A1, 2014. [27] J.W. Nocholson, The chemistry of medical and dental materials, R. Soc. Chem. (2002) 107–115. [28] H. Matusiewicz, Potential release of in vivo trace metals from metallic medical implants in the human body. From ions to nanoparticles — a systematic analytical review, Acta Biomater. 10 (2014) 2379–2403. [29] J. Laine, K. Kalimo, R.P. Happonen, Contact allergy to dental restorative materials in patients with oral lichenoid lesions, Contact Dermatitis 36 (1997) 141–146. [30] E. Eisenbarth, D. Velten, M. Muller, R. Thull, J. Breme, Biocompatibility of β stabilizing elements of titanium alloys, Biomaterials 25 (2004) 5705–5713. [31] P. Thomsen, C. Larsson, L.E. Ericson, L. Senneby, J. Lausama, B. Kasemo, Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium, J. Mater. Sci. 8 (1997) 653–665. [32] M. Pourbaix, Atlas of electrochemical equilibria in aqueous solutions, NACE, Houston, 1974. [33] K. Rydzynski, D. Pakulska, Vanadium, niobium, and tantalum, Patty's Toxicol. 37 (2012) 511–564.
355
[34] J. Vilaplana, C. Romaguera, New developments in jewellery and dental materials, Contact Dermatitis 39 (1998) 55–57. [35] M. Niinomi, Recent research and development in titanium alloys for biomedical applications and healthcare goods, Sci. Technol. Adv. Mater. 4 (2003) 445–454. [36] S. Ghosh, A. Sharma, G. Talukder, Zirconium. An abnormal trace element in biology, Biol. Trace Elem. Res. 35 (1992) 247–271. [37] B.L. Wang, L. Liand, Y.F. Zheng, In vitro cytotoxicity and hemocompatibility studies of Ti–Nb, Ti–Nb–Zr and Ti–Nb–Hf biomedical shape memory alloys, Biomed. Mater. 5 (2010) 04410. [38] J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-ray Photoelectron Spectroscopy, Physical Electronics USA, Inc., Chamhassen, 1995. [39] D.R. Baer, M.H. Engelhard, A.S. Lea, P. Nachimuthu, T.C. Drobay, J. Kim, B. Lee, C. Mathews, R.L. Opila, L.V. Saraf, W.F. Stickle, R.M. Wallace, B.S. Wright, Comparison of the sputter rates of the oxide films relative to the sputter rate of SiO2, J. Vac. Sci. Technol. A 28 (2010) 1060–1072. [40] B. Galateanu, S. Dinescu, A. Cimpean, A. Dinischiotu, M. Costache, Modulation of adipogenic conditions for prospective use of hADSCs in adipose tissue engineering, Int. J. Mol. Sci. 13 (2012) 15881–15900. [41] J. Black, Biological Performance of Materials: Fundamentals of Biocompatibility, M. Decker Inc., NY, 1992. [42] D.J. Blackwood, A.W.C. Chua, K.H.W. Seah, R. Thampuran, Corrosion behaviour of porous titanium–graphite composite designed for surgical implants, Corros. Sci. 42 (2000) 481–503. [43] J. Jakubowicz, G. Adamek, M.U. Jurczyk, M. Jurczyk, 3D surface topography study of the biofunctionalised nanocrystalline Ti–6Zr–4Nb/Ca–P, Mater. Charact. 70 (2012) 55–62. [44] E. Palaez-Abellan, L. Rocha-Sousa, W.D. Muller, A.C. Guastaldi, Electrochemical stability of anodic titanium oxide film grown at potentials higher than 3 V in a simulated physiological solution, Corros. Sci. 49 (2007) 1645–1655. [45] J. Park, S. Bauer, K. Van der Mark, P. Schmuki, Nanosize and vitality: nanotube diameter directs cell fate, Nano Lett. 7 (2007) 1686–1691. [46] A. Robin, O.A.S. Carvalho, S.G. Schneider, S. Schneider, Corrosion behavior of Ti– xNb–13Zr alloys in Ringer's solution, Mater. Corros. 59 (2008) 929–933. [47] E. Vasilescu, P. Drob, D. Raducanu, E. Cinca, D. Mareci, J.M. Calderon Moreno, M. Popa, C. Vasilescu, J.C. Mirza Rosca, Effect of thermo-mechanical processing on the corrosion resistance of Ti6Al4V alloys in biofluids, Corros. Sci. 51 (2009) 2885–2896. [48] ISO 8044/2000. Corrosion of metals and alloys. Basic terms and definitions Status: Revised, Withdrawn Published: April 2000 Replaced By: BS EN ISO 8044, 2015. [49] I. Milosev, T. Kosec, H.-H. Strenhblow, XPS and EIS study of the passive film formed on orthopaedic Ti–6Al–7Nb alloy in Hank's physiological solution, Electrochim. Acta 53 (2008) 3547–3558. [50] S.L. de Assis, S. Wolynec, I. Costa, Corrosion characterization of titanium alloys by electrochemical techniques, Electrochim. Acta 51 (2006) 1815–1819. [51] I. Cvijovic-Alagic, Z. Cvijovic, S. Mitrovic, V. Panic, M. Rakin, Wear and corrosion behaviour of Ti–13Nb–13Zr and Ti–6Al–4 V alloys in simulated physiological solution, Corros. Sci. 53 (2011) 796–808.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrochemical comparison and biological performance of a new CoCrNbMoZr alloy with commercial CoCrMo alloy M. Andrei a, B. Galateanu b,c, A. Hudita a, M. Costache b, P. Osiceanu d, J.M. Calderon Moreno d, S.I. Drob d,⁎, I. Demetrescu a a
Department of General Chemistry, University Politehnica Bucharest, Spl. Independentei 313, 060042 Bucharest, Romania Department of Biochemistry and Molecular Biology, University of Bucharest, 91–95 Spl. Independentei, Bucharest 050095, Romania Institute of Life Sciences, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romania d Institute of Physical Chemistry “Ilie Murgulescu” of Romanian Academy, Spl. Independentei 202, 060021 Bucharest, Romania b c
a r t i c l e
i n f o
Article history: Received 27 May 2015 Received in revised form 20 September 2015 Accepted 11 October 2015 Available online xxxx Keywords: New CoCrNbMoZr alloy Microstructure Surface features Corrosion resistance Biocompatibility Adipose stem cells (hASCs)
a b s t r a c t A new CoCrNbMoZr alloy, with Nb and Zr content is characterized from the point of view of surface features, corrosion resistance and biological performance in order to be proposed as dental restorative material. Its properties are discussed in comparison with commercial Heraenium CE alloy based on Co, Cr and Mo as well. The microstructure of both alloys was revealed by scanning electron microscopy (SEM). The composition and thickness of the alloy native passive films were identified by X-ray photoelectron spectroscopy (XPS). The surface characteristics were analyzed by atomic force microscopy (AFM) and contact angle techniques. The quantity of ions released from alloys in artificial saliva was evaluated with inductively coupled plasma-mass spectroscopy (ICPMS) measurements. The electrochemical stability was studied in artificial Carter–Brugirard saliva, performing open circuit potentials, polarization resistances and corrosion currents and rates. The biological performance of the new alloy was tested in vitro in terms of human adipose stem cells (hASCs) morphology, viability and proliferation status. The new alloy is very resistant to the attack of the aggressive ions from the artificial saliva. The surface properties, the roughness and wettabiliy sustain the cell behavior. The comparison of the new alloy behavior with that of existing commercial CoCrMo alloy showed the superior properties of the new metallic biomaterial. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Analyzing the state of the art in the field, we can observe a multitude of dental alloys for restorative prosthodontics available on the market and their biocompatibility problems [1,2]. Most of the metallic alloys are placed in the oral cavity for long term use, where they have to resist to oral corrosion, sometimes to temperature fluctuations (from about 10 °C to 45 °C), pH fluctuations, and mechanical stress [3,4]. In such conditions, the restorations with alloys need a selection in their elaboration, in order to offer resistance to corrosion and degradation with a minimum denaturation in long term exploitation [5], preserving the oral health. Having better tribological properties in comparison with those of titanium alloys and a much better price, CoCrMo alloys are one of the preferred choice for articular applications with metal-on-metal contact [6–10]. The electrochemical stability due to the passive oxide film grown on their surface [11] is a recommendation for biocompatibility as well, but it is to notice the problems with ion release in their working ⁎ Corresponding author. E-mail address: sidrob@chimfiz.icf.ro (S.I. Drob).
http://dx.doi.org/10.1016/j.msec.2015.10.031 0928-4931/© 2015 Elsevier B.V. All rights reserved.
time [12,13]. Comparing with Ti and Ti alloys which have a large passive range [14,15], CoCrMo alloys reveal a transition to transpassive behavior at lower values of electrode potentials which could be the result of fast dissolution through the passive film [16] or due to film breakdown [17]. The dependence of corrosion rate by the heat processing [18] was investigated indicating a significant decrease of corrosion rate as well. Such demerits as ion release and corrosion increase for oral cavity alloys which are subject to mechanical stress involving surface microstructure changes lead to the need of alloy modifications taking into account two approaches as surface and composition. At surface level in the last decades for CoCrMo alloys various techniques were proposed and studied from simple cheap coatings elaborated via immersion [19] or electrochemically [20], until sophisticated and expensive procedures as plasma spraying and ion bombardment [19,21]. Surface modification is recommended nowadays for enhancing properties at micro or nanolevel [22, 23], which is very important in the solving of the osseointegration problems essential at interface of the implant material with tissue [24]. In order to improve in bulk the mechanical, electrochemical stability and biological performance, the modification of alloy content is recommended and for CoCrMo alloys various compositions based on Co, Cr, and Mo elements have been the subject of patents and standards
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[25,26]. The release of Cr ions into human biofluid can influence the cell viability and can decrease the AND synthesis [27]. Co and Cr ions can cause “metal allergy”, can promote the granuloma formation and carcinoma [28]. Considering that the CoCrMo dental alloys are used at mass level favoring ion release and absorption due to their imperfections and leading to certain pigment metallic lesions or even more aggressive diseases [29] in the oral cavity, the present paper is introducing a new improved alloy with modified composition by selecting as new added component, the biocompatible Nb and Zr elements [30,31]. Niobium is a high passive metal due to its Nb2O5 protective oxide that covers its surface for the whole pH range from − 2 to 16 on a large potential range from −0.7 V (vs. SCE) to +2 V (vs. SCE) [32]; thus, this element will increase the passivation domain of the new CoCrNbMoZr alloy. Although Niobium is present in living organisms and might affect biological mechanisms, poor information regarding it's cytotoxicity is available to date. There are no information documenting its mutagenic potential, but on the other hand there are studies suggesting that some niobium compounds may have antitumor activity [33]. While niobium dust is an eye and skin irritant and a potential fire hazard, elemental niobium on a larger scale is physiologically inert and thus hypoallergenic and harmless. This is why it is frequently used in jewelry and has been tested for use in some medical implants [34]. Zirconium has a native passive film formed by ZrO2 oxide that is very resistant from pH = 3 to pH = 13 and from −1.6 V (vs. SCE) till + 1.4 V (vs. SCE) [32] and this oxide is very compatible with the blood [35]; so, this alloying element will enhance the corrosion resistance and biocompatibility of the new alloy. Despite the presence and retention in relatively high quantities in biological systems, Zirconium has not yet been associated with any specific metabolic function. Very little information is available to date about its interaction with the compounds of the genetic systems, such as nucleic acids. Some studies reveal that in vivo Zr retention is initially located in soft tissues and then slowly in the bones. The metal seems to be able to cross the blood brain-barrier, the placental barrier and also enters the milk [36]. Additionally, Wang et al., showed that Zr is a nontoxic element and that Zr containing alloys display a very good hemocompatibility [37]. The novelty and the mission of the present paper are to realize a complete and complex characterization of the new alloy in comparison with the existing CoCrMo alloy. The alloy microstructure was showed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS); the composition and thickness of the native passive films on the alloy surfaces were identified by X-ray photoelectron spectroscopy (XPS). Also, the surface roughness was revealed by atomic force microscopy (AFM) and the contact angle evinced the alloys hydrophilic properties. The amounts of ions released in artificial Carter– Brugirard saliva were evaluated by the inductively coupled plasmamass spectroscopy (ICP-MS) technique for the commercial existing CoCrMo alloy (Heraenium CE) and for the new improved CoCrNbMoZr alloy. The corrosion rate was obtained by the linear polarization method. The trial of these materials was performed by the in vitro cell culture as well, in order to establish the improvement of cell response as a prognosis of its better behavior in vivo in the future.
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2.1. Physical chemical characterization The physical chemical characterization of the both new CoCrNbMoZr alloy and commercial CoCrMo, Heraenium CE alloy involves SEM, EDS, XPS, AFM, contact angle techniques. The microstructure of the alloys was studied in a scanning electron microscope FEI Quanta 3D FEG equipped with an EDS detector. SEM micrographs were obtained with a back-scattered electron detector, using an accelerating voltage of 10 kV. EDS elemental analysis was performed using an accelerating voltage of 20 kV. The composition and the thickness of the native passive films on both studied alloys were analyzed by X-ray photoelectron spectroscope Quantera SXM that used AlKα radiation and an overall energy resolution of 0.65 eV by the full width at half-maximum (FWHM) of the Au4f7/2. The elemental composition was identified with accuracy for the binding energies (BEs) assignments of ± 0.2 eV, after a gentle Ar + ion etching (1 keV, 3 × 3 mm rastered area, 0.5 min. sputter time) on the surface, to remove the contaminant monolayer without destroying the chemical species. The thickness of the surface layer was determined by the XPS depth profiling, layer by layer experiment under 2 keV, on a 2 × 2 mm scanning area, at 45 0 incidence angle and 200 μm spot size of X-ray beam. These parameters were used to minimize the induction effect of the ion beam on the oxides [38,39]. The surface roughness was examined with atomic force microscope (Electrochemical AFM from A.P.E. Research Company) and the three-dimensional surface roughness expressed as the root mean square roughness (RMS) and average roughness (Rav) of the samples were estimated. The measurements have been performed in contact mode. The contact angle determinations were carried out with an 100 Optical Contact Angle Meter — CAM 100; the resulted value is the average of minimum three measurements for each determination and all the tests were realized with an accuracy of ±1° at 25 °C. 2.2. Ion release rate The quantity of ions released in time from alloys in tested electrolyte, Carter–Brugirard saliva was measured with an inductively plasma mass spectrometer (ICP-MS), equipment Perkins Elmer ELAN DRC-e that works with the liquid sample introduction by a micro-nebulizer and the calibration was against the aqueous multi-element solutions. The tests were performed by the periodical analyses till 168 h of immersion in artificial Carter–Brugirard saliva. 2.3. Electrochemical characterization The electrodes preparation before each determination included the grinding with abrasive paper having different granulations, polishing to mirror surface, washing with water and 30 min ultrasonication in acetone. As electrolyte for electrochemical stability tests Carter–Brugirard saliva with the following composition has been selected (g/L): KCl − 1.2; NaCl — 0.7; KH2PO4 — 0.26; KSCN — 0.33; Na2HPO4 — 0.19; urea — 0.13; NaHCO3 — 1.5. The procedures has been:
2. Materials and methods The studied alloys have the compositions presented in Table 1. Table 1 Composition of studied alloys. Material
Heraenium CE CoCrNbMoZr
wt.% Co
Cr
Mo
Mn
Si
C
N
Nb
Zr
63.5 60
27.8 26.5
6.6 4.5
0.6 0.8
1.0 1.0
0.3 0.4
0.2 0.2
– 6.0
– 0.8
‐ the monitoring (by periodical verifications till 200 immersion hours) of the open circuit potential using a performing Hewlett-Packard multimeter; ‐ the potentiodynamic linear polarization applied from a more electronegative potential with 50 mV than the open circuit potential till to a more electropositive potential with 50 mV than the open circuit potential with a scan rate of 0.1 mV/s; the samples were kept about 30 min. before the polarization tests. From Tafel plots, the main corrosion parameters Ecorr (corrosion potential), jcorr (corrosion current density), P (penetration index, corrosion rate)
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Fig. 1. SEM micrograph (a) and EDS elemental analysis (b) of CoCrNbMoZr alloy.
and Rp (polarization resistance) were determined; equipment was potentiostat Radiometer PGZ 80.
The electrochemical cell was the one with three electrodes as following: the working electrode (the commercial and the new alloy), the Pt electrode as contra electrode and a saturated calomel electrode (SCE) as reference electrode. 2.4. Cell culture model Human adipose derived stem cells (hASCs) were previously isolated from human subcutaneous adipose tissue as described by Galateanu et al. [40]. Briefly, the lipoaspirates (LAs) were processed by collagenase digestion and the cells obtained were resuspended in Dulbecco's modified Eagle's medium (DMEM), supplemented with fetal bovine serum (FBS). The mesenchymal stem cells surface markers panel was confirmed by flow cytometry starting with the 3rd passage. The human adipose tissue was obtained from female patients undergoing elective liposuction and all the medical procedures were performed in compliance with the Helsinki Declaration, with the approval of Proestetica Medical Center Committee (reference number 112/23.10.2013).
2.5. Biocompatibility assessment 2.5.1. Cellular morphology The protein expression of actin was studied at 6 h and 5 days postseeding of the hASCs on the samples by fluorescence microscopy using an Olympus IX71 inverted microscope. In this view, the hASCs were fixed with 4% paraformaldehyde for 1 h and cell membranes were permeabilized with 2% bovine serum albumin/0.1% Triton X-100 solution at 4 °C. Next, the samples were incubated 30 min at 37 °C with Alexa Fluor 546 Phalloidin for actin labeling. After cell nuclei were stained with DAPI for 15 min, the resulting labeled samples were inspected in fluorescence microscopy. Images were captured with Cell F Imaging Software (Olympus). 2.5.2. Cellular viability and proliferation potential Cell proliferation and viability within the cell-material culture systems was evaluated by fluorescence microscopy using Live/Dead Kit (Invitrogen, Life Technologies, Foster City, CA). Briefly, at 24 h and 5 days post seeding, the samples (pre-seeded with hASCs) were exposed to a staining solution, containing calcein AM and ethidium bromide at room temperature and darkness. After 10 min, the samples were analyzed by fluorescence microscopy using an Olympus IX71
Fig. 2. SEM micrograph (a) and EDS elemental analysis (b) of CoCrMo, Heraenium CE alloy.
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elemental analysis evinced that Cr and Mo are enriched in the inclusions. 3.2. Composition and thickness of the native passive film
Fig. 3. XPS survey spectrum for the native passive film existing on new CoCrNbMoZr alloy surface.
inverted microscope and images were captured with Cell F Imaging Software (Olympus). hASCs capacity to proliferate on the surface of the samples was quantitatively assessed by MTT test at 24 h and 5 days post seeding. Cell-material culture systems were incubated in 1 mg/ml MTT solution for 4 h and the formazan crystals obtained in the metabolically active cells were solubilized in isopropanol. The concentration of the resulted solution was spectrophotometrically quantified at 550 nm (Appliskan Thermo Scientific). 2.5.3. Material's cytotoxic potential The cytotoxic potential of the samples on hASCs was evaluated using in vitro toxicology LDH-based assay (Sigma Aldrich, Steinheim, Germany). The culture media were harvested at 24 h and 5 days postseeding and mixed with test solution according to the manufacturer's recommendation. After 20 min of incubation, the reaction was stopped with 1 N hydrochloric acid (HCl) and the spectrophotometric detection of LDH concentration was determined by measuring the optic density of the resulting solution at 490 nm (AppliskanThermo Scientific). 2.5.4. Statistical analysis The spectrophotometric data were statistically analyzed using GraphPad Prism 3.03 Software, one-way ANOVA, Bonferroni test. The experiments were performed with n = 3 biological replicates and each data set is presented as the average of three replicates (mean ± standard deviation). 3. Results and discussions 3.1. Alloy microstructure Microstructural analysis of the cast new CoCrNbMoZr alloy is characterized by a multicomponent, fine dendritic structure (Fig. 1a) with typical interdendritic distances of a few microns. EDS spectra (Fig. 1b) detected the alloying elements, Co, Cr, Nb, Mo, Mn, and Si (Zr main feature at ~2 keV is masked by the secondary Nb and Mo peaks). EDS elemental analysis revealed that Mo, Nb, Si and C are enriched in the interdendritic regions. Microstructural analysis of the cast CoCrMo Heraenium CE alloy (Fig. 2a) shows a main matrix phase (~90 vol.%) containing inclusions, most of them being isolated and with globular shape, sized ~ 5 μm, and well dispersed in the matrix, with typical distance between inclusions of about 20 μm, except for the formation of some linear aggregates, with lengths up to several tens of microns. EDS spectra (Fig. 2b) detected the main alloying elements, Co, Cr, Mo, and Si. EDS
XPS survey spectrum (Fig. 3) for the new CoCrNbMoZr alloy showed a complex feature that included the peaks for Co 2p, Cr 2p, Nb 3d, Mo 3d, Zr 3d, and O 1s [38]. High resolution spectra (Fig. 4) revealed the presence of Co (Fig. 4a) as metallic Co, of Cr (Fig. 4b) as Cr 2 O3 oxide (68.6%) and metallic Cr (31.4%), of Nb (Fig. 4c) as Nb 2 O 5 oxide (49.0%) and NbO (51.0%), of Mo (Fig. 4d) as MoO 3 oxide (14.0%), MoO2 oxide (25.9%) and metallic Mo (60.1%), of Zr (Fig. 4e) as ZrO 2 oxide (54.9%) and metallic Zr (45.1%), of Mn (Fig. 4f) as MnO 2 oxide (26.4%) and metallic Mn (73.6%), of Si (Fig. 4g) as SiO2 oxide and elemental Si (61.2%) and of O (Fig. 4h) as O2 − ion (87.6%) and OH− ion (12.4%); the high concentration of O 2 − ion appears from oxides. It is a thick oxide layer (about 6.5 nm–8 nm), that can confer very good protection to the substrate [41,42]; this fact will be proved by the lower values of the penetration indexes (Subchapters 3.5 and 3.6.2). It has be observed that the native passive film on the new CoCrNbMoZr alloy surface is composed from chromium, niobium, molybdenum, zirconium, manganese, and silicon oxides, thus being thicker, more compact and enhanced with oxides. Milosev et al. [11] appreciated that the increase of the oxide layer thickness on the CoCrMo alloy surface is due to the entering of Mo and Co oxides into its passive layer; thus, for the new CoCrNbMoZr alloy, the oxides Nb2 O 5 and ZrO2 which are the constituents of the passive film, also increase its thickness. For the commercial Heraenium CE alloy, the survey spectrum (Fig. 5) comprises the peaks for Co 2p, Cr 2p, Mo 3d and O 1s. This film has a thickness of 5 nm; it is a very thin layer and its protective capacity will be determined further. Also, high resolution spectra (Fig. 6) for Co (Fig. 6a) evinced only metallic Co, for Cr (Fig. 6b), Cr2O3 oxide (72.8%) and metallic Cr (27.2%), for Mo (Fig. 6c), MoO3 oxide (21.5%), MoO2 oxide (21.8%) and metallic Mo (56.7%), for O (Fig. 6d), O2 − ion (78.9%) and OH− ion (21.1%). It results that the native passive film on the Heraenium alloy surface contains chromium and molybdenum oxides which confer its resistance against to corrosion. 3.3. Surface topography and roughness In Fig. 7, the AFM images of investigated alloys have permitted evaluation of their topography and average roughness values. The both alloys revealed a similar topography; the root mean square, RMS parameter has the value of 20.2 nm for the new alloy and 20.8 nm for the commercial alloy; the average roughness, Rav is 15.5 nm for the new alloy and 16.3 nm for the commercial alloy; these values are close to each-other. This roughness is very proper for the cell adhesion [43–45]. 3.4. Contact angle An increase in the contact angle value for the new CoCrNbMoZr alloy has been observed compared to the commercial Heraenium CE alloy. The increase from 75.92 to 86.79 values indicates that both alloys have hydrophilic character but the stronger one is for the commercial one. 3.5. Ion release The quantity of ions (determined by ICP-MS method) released in artificial saliva for both alloys is relatively small, but for the new alloy, the amount is significant lower. The quantified amounts in ppb (partsper-billion, 10−9) are presented in Table 2 and sustain increased stability for the new alloy.
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Fig. 4. XPS high resolution spectra for the native passive film existing on new CoCrNbMoZr alloy surface: a) Co 2p; b) Cr 2p; c) Nb 3d; d) Mo 3d; e) Zr 3d; f) Mn 2p; g) Si 2s; h) O 1s.
3.6. Electrochemical behavior 3.6.1. Monitoring of open circuit potentials The evolution of open circuit potential in time for both alloys is presented in Fig. 8. As can be seen the curves aspect denotes a trend to steady state sooner after immersion in both cases; for the Heraenium CE, after about 20 h and for the new alloy after 60 h. It is a specific aspect for passivity due to the protective oxides formed on the surface of the both alloys [41,42]. The new alloy reached its stable passive state after a larger time period than Heraenium CE alloy; its passivation process has a lower passivation rate. This fact was observed by Robin et al.
[46] for Ti–5Nb–13Zr and Ti–20Nb–13Zr alloys which revealed “a slower growth rate of the passivating films” in comparison with “Ti, Ti–6Al–4 V and Ti–13Nb–13Zr alloys”. But, after about 200 immersion hours, the both alloys realized about the same values of the open circuit potentials, around −40 mV (vs. SCE).The open circuit potentials tend to more electropositive values, indicating an increase of passive film thickness [41,42]. 3.6.2. Linear polarization results The Tafel results are reported for the both alloys in Fig. 9 and Table 3. In Table 3 are listed corrosion potential, (Ecorr), corrosion current
M. Andrei et al. / Materials Science and Engineering C 59 (2016) 346–355
Fig. 5. XPS survey spectrum for the native passive film existing on commercial Heraenium alloy surface.
density (jcorr), penetration index (P), and polarization resistance (Rp). From these data it resulted that the new alloy at initial time is included in conventional stability scale “Perfect Stable” while the commercial alloy in “Very Stable” in the corrosion resistance group (corrosion classification) [47,48]. With the increase of immersion time, the penetration index, corrosion rate decreased. The minimum of the penetration index is after 200 h for the commercial alloy, the value being a bit less than a half of the initial value. It is necessary to mention that for the new alloy, the ratio between the value at initial time and at 200 h is around 36. Also, the polarization resistance, Rp values for the new alloy increased in time as result of the passive film thickening [41,42]. For the commercial alloy, polarization resistance decreased in time, denoting some dissolution and repassivation processes [41,42] at the interface between alloy and artificial saliva. Rp have higher values from about 8 times to 175 times than those of Heraenium CE alloy; correlating with the lower values of the corrosion current densities, it results a better corrosion resistance, a more resistant passive film. Milosev et al. [49] showed that for Ti–6Al–7Nb alloy, its passive layer is stabilized by the incorporation of Al and Nb oxides, thus increasing its resistance.
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So, it is reasonable to presume that the better corrosion resistance of the new alloy in comparison with the commercial Heraenium CE alloy is due to its passive film that contains beside chromium oxide, niobium (Nb2O5, 49.8%) and zirconium oxides (ZrO2, 54.9%), as was showed by XPS analysis. The corrosion potentials, Ecorr obtained from linear polarization tests (Fig. 9 and Table 3) have lower values than those of the open circuit potentials (Fig. 8), due to the fact that, the polarization tests began at a more electronegative potential with 50 mV than the open circuit potential; thus, the passive film was partially dissolved. This behavior was observed by other authors [12,50,51] who gave the same explanation of the passive film dissolution; they added that the cathodic partial reaction of the hydrogen evolution shifts the potential to more electronegative values. Tafel plots from Fig. 9 confirm the in time ennobling of Ecorr values both for the new alloy and for Heraenium CE alloy and the fact that after about 200 immersion hours, both alloys reached similar values (around −130 mV) for their corrosion potentials, Ecorr. 3.7. Cell morphology and distribution hASCs morphology on the alloys surfaces and their ability to interact with the substrate material in terms of adhesion, cytoskeleton development were investigated after 6 h days of culture. Cellular distribution was investigated at 5 days post-seeding. As shown in Fig. 10, at 6 h post-seeding, hASCs displayed long and distinctive actin filaments surrounding the nuclei on conventional plastic cell culture surface and already adopted their characteristic spindle-like shape. Unlikely, hASCs on both Heraenium CE and CoCrNbMoZr alloys did not display such morphology at 6 h postseeding, but significant differences were noticed between the two samples. Consequently, on Heraenium CE, short actin filaments were observed surrounding hASCs nuclei and the overall cellular shape was still round, while hASCs morphology on the surface of the CoCrNbMoZr alloy was round with elongations in different directions. Thus, over time, only hASCs on plastic and CoCrNbMoZr alloy surfaces exhibited a well-defined spindle-shaped morphology, as
Fig. 6. XPS high resolution spectra for the native passive film existing on commercial Heraenium alloy surface: a) Co 2p; b) Cr 2p; c) Mo 3d; d) O 1s.
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Fig. 7. AFM images of new CoCrNbMoZr alloy (a) and of the commercial Heraenium CE alloy (b).
Table 2 Ion release rate obtained after 168 h of immersion of the studied alloys in artificial Carter– Brugirard saliva at 37 °C. Analyzed element
Co Cr Mo Mn Nb Zr
hASCs on the surface of CoCrNbMoZr display a regular pattern both at 24 h and 5 days post-seeding as compared to hASCs random distribution on the Heraenium CE specimen.
Mean concentration (ppb) CoCrNbMoZr
Heraenium CE
0.185 3.643 4.843 0.339 1.350 1.0
8.006 12.197 23.234 2.500 – –
shown at 5 days post-seeding. Additionally, a random distribution of the cells on the plastic and Heraenium CE alloy surfaces was observed as compared to a very clear array of hASCs on the CoCrNbMoZr specimens at 5 days post-seeding; this fact is due to the more stable passive film that releases (Table 3) a lower quantity of the cytotoxicelements on the new alloy surface comparing with the commercial Heraenium CE alloy.
3.9. Quantification of the cellular viability and proliferation status hASCs viability and proliferation potential on Heraenium CE and CoCrNbMoZr alloys was quantitatively determined by MTT spectrophotometric assay. The obtained spectrophotometric data were graphically represented in Fig. 12. Our data revealed that significant differences were detected between the hASCs viability on Heraenium CE surface versus CoCrNbMoZr surface, both at 24 h (###p b 0.0001) and 5 days (##p b 0.001) post-seeding; the hASCs viability is more higher on the
3.8. Cellular viability, live/dead fluorescent microscopy assay In order to examine hASCs survival on the tested biomaterials, cellular the viability was evaluated at 24 h and 5 days post-seeding by fluorescence microscopy (Fig. 11), based on the simultaneous staining of live (green labeled) and dead (red labeled) cells. After 24 h and 5 days post-seeding only bright green-labeled cells were observed on the surface of both Heraenium CE and CoCrNbMoZr alloys as a proof of their survival. Furthermore, no red nuclei were observed. Live/Dead staining also confirmed the above observations regarding the cell distribution. As shown in Fig. 11, bright green living
Fig. 8. Evolution of open circuit potentials of studied alloys in artificial Carter–Brugirard saliva at 37 °C.
Fig. 9. Tafel plots obtained in artificial Carter–Brugirard saliva at 37 °C for: a) CoCrNbMoZr alloy; b) Heraenium alloy.
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Table 3 Main corrosion parameters obtained after different immersion periods of the studied alloys in artificial Carter–Brugirard saliva at 37 °C. Time (h)
0 24 48 72 96 120 144 168 200
jcorr (nA/cm2)
Ecorr (mV)
Rp (kΩ·cm2)
P (μm/Year)
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
Heraenium CE alloy
New alloy
−287.62 −158.83 −144.59 −147.62 −149.52 −153.17 −179.18 −166.74 −125.61
−315.89 −237.23 −221.99 −229.57 −238.78 −237.54 −227.83 −195.20 −128.71
251.26 168.05 156.37 162.98 175.38 114.72 151.62 176.37 128.82
46.52 8.29 3.78 2.00 4.45 3.81 1.52 1.72 1.355
5.772 3.860 3.592 3.744 4.028 2.635 3.483 4.051 2.729
1.171 0.209 0.095 0.050 0.112 0.096 0.038 0.043 0.032
76.23 71.54 66.92 62.02 58.51 58.16 46.65 48.48 45.49
593.77 2602.7 4191.6 5291.7 5427.3 4834.7 7075.4 7609.1 7964.7
new alloy surface as result of the more lower level of Co, Cr Mo and Mn ions released into environment (Table 2). Regarding the proliferative status, hASCs displayed a significant increase of proliferation during 5 days of culture both on Heraenium CE alloy (***p b 0.0001) and CoCrNbMoZr alloy surfaces (***p b 0.0001). 3.10. Cytotoxic potential of Heraenium CE and CoCrNbMoZr alloys on hASCs The cytotoxic potential of Heraenium CE and CoCrNbMoZr alloys was evaluated by spectrophotometric quantification of the LDH enzyme release in the culture media by hASCs seeded in direct contact with the samples. The quantitative data obtained are represented in Fig. 11b and reveal low and constant values of LDH activity in both tested cell cultures at 24 h post-seeding suggesting that none of the samples induced cell toxicity. Unlikely, after 5 days of culture a significant difference (#p b 0.05) was detected in terms of LDH activity in the culture media harvested from Heraenium CE versus CoCrNbMoZr, indicating a slightly increased rate of cell death on Heraenium CE as compared with CoCrNbMoZr; this fact can be explained by the quantity of Co, Cr, Mo,Mn ions released by the studied alloys: for the Heraenium CE alloy surface were released Co ions of about 43 times higher, Cr ions of 3.3 times higher, Mo ions of 4.8 times higher and Mn ions of 7.4 times higher, as results from Table 2. 4. Conclusions SEM observations evidenced a finer microstructure of the new CoCrNbMoZr alloy in comparison with the commercial Heraenium CE alloy.
The native passive film existing on the new CoCrNbMoZr alloy surface is thicker, more compact than that on the commercial Heraenium CE alloy surface because it contains both chromium and molybdenum oxides, and, in addition, the niobium and zirconium oxides (XPS data). The topography and roughness parameters (AFM data) are similar for the two studied alloys and very proper for the cell adhesion. The contact angle values for both studied alloys are expressions of their balanced hydrophilic–hydrophobic nature which indicates clearly affinity for cells. The quantity of ions released in solution is significantly lower for the new alloy, showing its better corrosion resistance. The open circuit potentials tended to more electropositive values, denoting the increase of the passive film thickness. The new CoCrNbMoZr alloy exhibits better stability in tested Carter– Brugirard saliva having a much lower value for corrosion current density and rate and higher value of the polarization resistance than that for the commercial CoCrMo alloy, type Heraenium CE. The biocompatibility assessment of Heraenium CE and CoCrNbMoZr alloys revealed that hASCs need more time to adhere and to acquire their characteristic spindle-like shape when seeded on Heraenium CE as compared with CoCrNbMoZr surface. Furthermore, the cells were distributed randomly on the surface of Heraenium CE alloy while on CoCrNbMoZr they followed a linear pattern. HASCs viability on CoCrNbMoZr was significant higher both at 24 h and 5 days of culture in comparison with hASCs on Heraenium CE. Furthermore, CoCrNbMoZr better sustained cellular proliferation than Heraenium CE as shown by the fluorescence microscopy micrographs
Fig. 10. Fluorescence microscopy micrographs of hASCs actin filaments network (red fluorescence) on plastic surface and Heraenium CE and CoCrNbMoZr specimens at 6 h and 5 days post-seeding; DAPI-stained nuclei are blue. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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132397. This work was supported by Romanian UEFISCDI, Project PCE no. 130/2014 as well. The authors thank Dr. Andrei Bogdan Stoian for AFM analysis. The authors thank Assoc. Prof. MD PhD Dana Jianu from the ProEstetica Medical Clinic who kindly supplied this study with human subcutaneous adipose tissue.
References
Fig. 11. Fluorescence microscopy micrographs revealing live and dead cells on Heraenium CE and CoCrNbMoZr alloys surfaces at 24 h and 5 days post-seeding. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
and by the spectrophotometric data. Lastly, Heraenium CE exerted a significant higher cytotoxic effect on hASCs at 5 days of culture as comparing with CoCrNbMoZr. In conclusion, CoCrNbMoZr alloy displayed an increased corrosion resistance and overall biocompatibility as compared with Heraenium CE and could be further employed for in vitro cell differentiation studies. Acknowledgments The work has been funded by the Sectorial Operational Programme Human Resources Development 2007–2013 of the Ministry of European Funds through the Financial Agreement POSDRU/159/1.5/S/
[1] W. Elshahawy, I. Watanabe, Biocompatibility of dental alloys used in dental fixed prosthodontics, Tanta Dent. J. 11 (2014) 150–159. [2] P. Imirzalioglu, E. Alaaddinoglu, Z. Yilmaz, B. Oduncuoglu, B. Yilmaz, S. Rosenstiel, Influence of recasting different types of dental alloys on gingival fibroblast cytotoxicity, J. Prosthet. Dent. 107 (2012) 24–33. [3] S.C. Wang, M. Browne, H.S. Ubhi, M.J. Starink, Determination of the fatigue fracture planes of Co–Cr–Mo biomedical alloys using electron backscatter diffraction, J. Microsc. 217 (2005) 118–121. [4] T. Hanava, A. Igua, S. Muñoz, S. Mischler, Effect of the environment on wear ranking and corrosion of biomedical CoCrMo alloys, J. Mater. Sci. Mater. Med. 22 (2011) 437–450. [5] J.C.M. Souza, S.L. Barbosa, E. Ariza, J.-P. Celis, L.A. Rocha, Simultaneous degradation by corrosion and wear of titanium in artificial saliva containing fluorides, Wear 292–293 (2012) 82–88. [6] J.C. Walker, R.B. Cook, J.W. Murray, A.T. Clare, Pulsed electron beam surface melting of CoCrMo alloy for biomedical applications, Wear 301 (2013) 250–256. [7] Y. Bedolla-Gil, M.A.L. Hernandez-Rodriguez, Tribological behavior of a heat-treated cobalt-based alloy, J. Mater. Eng. Perform. 22 (2013) 541–547. [8] G. Barucca, E. Santecchia, G. Majni, E. Girardin, E. Bassoli, L. Denti, A. Gato, L. Iuliano, T. Moskalewicz, P. Mengucci, Structural characterization of biomedical Co–Cr–Mo components produced by direct metal laser sintering, Mater. Sci. Eng. C 48 (2015) 263–269. [9] Y. Lu, S. Wu, Y. Gan, J. Li, C. Zhao, D. Zhou, J. Lin, Investigation on the microstructure, mechanical property and corrosion behavior of the selective laser melting CoCrW alloy for dental application, Mater. Sci. Eng. C 49 (2015) 517–525. [10] S. Dittrick, V.K. Balla, N.M. Davies, S. Bose, A. Bandyopadhyay, In vitro wear rate and Co ion release of compositionally and structurally graded CoCrMo–Ti6Al4V structures, Mater. Sci. Eng. C 31 (2011) 809–814. [11] I. Milošev, H.H. Strehblow, The composition of the surface passive film formed on CoCrMo alloy in simulated physiological solution, Electrochim. Acta 48 (2003) 2767–2774. [12] F. Contu, B. Elsener, H. Böhni, Corrosion behaviour of CoCrMo implant alloy during fretting in bovine serum, Corros. Sci. 47 (2005) 1863–1875. [13] N. Rincic, A. Celebic, I. Baucic, J. Stipetic, E. Prohic, S. Miko, The release of ions from the base Co–Cr–Mo casting alloy in vitro into the phosphate buffer at pH 6.0, Acta Stomatol. Croat. 37 (2003) 13–16.
Fig. 12. (a) Quantification of: hASCs viability on Heraenium CE and CoCrNbMoZr alloys surfaces, as revealed by MTT test at 24 h and 5 days post-seeding. [***p b 0.0001 (hASCs on Heraenium CE surface at 24 h vs. 5 days of culture) and (hASCs on CoCrNbMoZr surface at 24 h vs. 5 days of culture)]; [###p b 0.0001 (hASCs on Heraenium CE alloy surface at 24 h of culture vs. hASCs on CoCrNbMoZr surface at 24 h of culture)] and [##p b 0.001 (hASCs on Heraenium CE alloy surface at 5 days of culture vs. hASCs on CoCrNbMoZr surface at 5 days of culture)] and (b) Heraenium CE and CoCrNbMoZr alloys' cytotoxic potential effect on hASCs after 24 h and 5 days of culture as revealed by LDH assay [#p b 0.05 ((hASCs on Heraenium CE alloy surface at 5 days of culture vs. hASCs on CoCrNbMoZr surface at 5 days of culture)].
M. Andrei et al. / Materials Science and Engineering C 59 (2016) 346–355 [14] A. Cimpean, E. Vasilescu, P. Drob, I. Cinca, C. Vasilescu, M. Anastasescu, V. Mitran, S.I. Drob, Enhancement of the electrochemical behaviour and biological performance of Ti–25Ta–5Zr alloy by thermo-mechanical processing, Mater. Sci. Eng. C 38 (2014) 127–142. [15] D. Ionita, M. Grecu, C. Ungureanu, I. Demetrescu, Modifying the TiAlZr biomaterial surface with coating, for a better anticorrosive and antibacterial performance, Appl. Surf. Sci. 257 (2011) 9164–9168. [16] A.W.E. Hodgson, S. Kurz, S. Virtanen, V. Fervel, C.-O.A. Olsson, S. Mischler, Passive and transpassive behaviour of CoCrMo in simulated biological solutions, Electrochim. Acta 49 (2004) 2167–2178. [17] M. Metikos -Hukovic, R. Babic, Some aspects in designing passive alloys with an enhanced corrosion resistance, Corros. Sci. 51 (2009) 70–75. [18] F. Golgovici, M. Prodana, A. Popescu, Effect of reprocessing treatment on the corrosion of Co–Cr alloy in acid media, Rev. Chim. 66 (2015) 660–667. [19] D.A. Cortés, A. Medina, J.C. Escobedo, S. Escobedo, M.A. López, Effect of wollastonite ceramics and bioactive glass on the formation of a bonelike apatite layer on a cobalt base alloy, J. Biomed. Mater. Res. A 70 (2004) 341–346. [20] D. Ionita, I. Man, I. Demetrescu, The behaviour of electrochemical deposition of phosphate coating on CoCr bioalloys, Key Eng. Mater. 330–332 (2007) 545–548. [21] L. Reclaru, P.Y. Eschler, R. Lerf, A. Blatter, Electrochemical corrosion and metal ion release from Co–Cr–Mo prosthesis with titanium plasma spray coating, Biomaterials 26 (2005) 4747–4756. [22] V.-H. Pham, Improving osseointegration of Co–Cr by nanostructured titanium coatings, Pham. Springer Plus 3 (2014) 197–202. [23] R.J. Narayan, Big possibilities for small scale implants, Mater. Today 16 (2013) 204–205. [24] C.E. Bates, V. Marino, N.L. Fazzalari, P.M. Bartold, Soft tissue attachment to titanium implants coated with growth factors, Clin. Implant. Dent. Relat. Res. 15 (2013) 53–63. [25] ASTM F75-12 Standard Specification for Cobalt-28 Chromium-6 Molybdenum Alloy Castings and Casting Alloy for Surgical Implants (UNS R30075), ASTM International, West Conshohocken, PA, 2012 www.astm.org. [26] Biocompatible CoCrMo alloy US20140271317 A1, 2014. [27] J.W. Nocholson, The chemistry of medical and dental materials, R. Soc. Chem. (2002) 107–115. [28] H. Matusiewicz, Potential release of in vivo trace metals from metallic medical implants in the human body. From ions to nanoparticles — a systematic analytical review, Acta Biomater. 10 (2014) 2379–2403. [29] J. Laine, K. Kalimo, R.P. Happonen, Contact allergy to dental restorative materials in patients with oral lichenoid lesions, Contact Dermatitis 36 (1997) 141–146. [30] E. Eisenbarth, D. Velten, M. Muller, R. Thull, J. Breme, Biocompatibility of β stabilizing elements of titanium alloys, Biomaterials 25 (2004) 5705–5713. [31] P. Thomsen, C. Larsson, L.E. Ericson, L. Senneby, J. Lausama, B. Kasemo, Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium, J. Mater. Sci. 8 (1997) 653–665. [32] M. Pourbaix, Atlas of electrochemical equilibria in aqueous solutions, NACE, Houston, 1974. [33] K. Rydzynski, D. Pakulska, Vanadium, niobium, and tantalum, Patty's Toxicol. 37 (2012) 511–564.
355
[34] J. Vilaplana, C. Romaguera, New developments in jewellery and dental materials, Contact Dermatitis 39 (1998) 55–57. [35] M. Niinomi, Recent research and development in titanium alloys for biomedical applications and healthcare goods, Sci. Technol. Adv. Mater. 4 (2003) 445–454. [36] S. Ghosh, A. Sharma, G. Talukder, Zirconium. An abnormal trace element in biology, Biol. Trace Elem. Res. 35 (1992) 247–271. [37] B.L. Wang, L. Liand, Y.F. Zheng, In vitro cytotoxicity and hemocompatibility studies of Ti–Nb, Ti–Nb–Zr and Ti–Nb–Hf biomedical shape memory alloys, Biomed. Mater. 5 (2010) 04410. [38] J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-ray Photoelectron Spectroscopy, Physical Electronics USA, Inc., Chamhassen, 1995. [39] D.R. Baer, M.H. Engelhard, A.S. Lea, P. Nachimuthu, T.C. Drobay, J. Kim, B. Lee, C. Mathews, R.L. Opila, L.V. Saraf, W.F. Stickle, R.M. Wallace, B.S. Wright, Comparison of the sputter rates of the oxide films relative to the sputter rate of SiO2, J. Vac. Sci. Technol. A 28 (2010) 1060–1072. [40] B. Galateanu, S. Dinescu, A. Cimpean, A. Dinischiotu, M. Costache, Modulation of adipogenic conditions for prospective use of hADSCs in adipose tissue engineering, Int. J. Mol. Sci. 13 (2012) 15881–15900. [41] J. Black, Biological Performance of Materials: Fundamentals of Biocompatibility, M. Decker Inc., NY, 1992. [42] D.J. Blackwood, A.W.C. Chua, K.H.W. Seah, R. Thampuran, Corrosion behaviour of porous titanium–graphite composite designed for surgical implants, Corros. Sci. 42 (2000) 481–503. [43] J. Jakubowicz, G. Adamek, M.U. Jurczyk, M. Jurczyk, 3D surface topography study of the biofunctionalised nanocrystalline Ti–6Zr–4Nb/Ca–P, Mater. Charact. 70 (2012) 55–62. [44] E. Palaez-Abellan, L. Rocha-Sousa, W.D. Muller, A.C. Guastaldi, Electrochemical stability of anodic titanium oxide film grown at potentials higher than 3 V in a simulated physiological solution, Corros. Sci. 49 (2007) 1645–1655. [45] J. Park, S. Bauer, K. Van der Mark, P. Schmuki, Nanosize and vitality: nanotube diameter directs cell fate, Nano Lett. 7 (2007) 1686–1691. [46] A. Robin, O.A.S. Carvalho, S.G. Schneider, S. Schneider, Corrosion behavior of Ti– xNb–13Zr alloys in Ringer's solution, Mater. Corros. 59 (2008) 929–933. [47] E. Vasilescu, P. Drob, D. Raducanu, E. Cinca, D. Mareci, J.M. Calderon Moreno, M. Popa, C. Vasilescu, J.C. Mirza Rosca, Effect of thermo-mechanical processing on the corrosion resistance of Ti6Al4V alloys in biofluids, Corros. Sci. 51 (2009) 2885–2896. [48] ISO 8044/2000. Corrosion of metals and alloys. Basic terms and definitions Status: Revised, Withdrawn Published: April 2000 Replaced By: BS EN ISO 8044, 2015. [49] I. Milosev, T. Kosec, H.-H. Strenhblow, XPS and EIS study of the passive film formed on orthopaedic Ti–6Al–7Nb alloy in Hank's physiological solution, Electrochim. Acta 53 (2008) 3547–3558. [50] S.L. de Assis, S. Wolynec, I. Costa, Corrosion characterization of titanium alloys by electrochemical techniques, Electrochim. Acta 51 (2006) 1815–1819. [51] I. Cvijovic-Alagic, Z. Cvijovic, S. Mitrovic, V. Panic, M. Rakin, Wear and corrosion behaviour of Ti–13Nb–13Zr and Ti–6Al–4 V alloys in simulated physiological solution, Corros. Sci. 53 (2011) 796–808.