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Corrosion behavior and in vitro biocompatibility of Zr–Al–Co–Ag bulk metallic glasses: An experimental case study
Journal of Non-Crystalline Solids 358 (2012) 1599–1604
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Corrosion behavior and in vitro biocompatibility of Zr–Al–Co–Ag bulk metallic glasses: An experimental case study Nengbin Hua a, Lu Huang a, Jianfeng Wang a, Yu Cao b, Wei He b, c, Shujie Pang a, Tao Zhang a,⁎ a b c
Key Laboratory of Aerospace Materials and Performance (Ministry of Education), School of Materials Science and Engineering, Beihang University, Beijing 100191, China Department of Materials Science and Engineering, The University of Tennessee, Knoxville, TN 37996–2200, USA Department of Mechanical, Aerospace and Biomedical Engineering, The University of Tennessee, Knoxville, TN 37996–2200, USA
a r t i c l e
i n f o
Article history: Received 16 February 2012 Received in revised form 6 April 2012 Available online 15 May 2012 Keywords: Metallic glasses; Zirconium alloys; Corrosion; Biocompatibility
a b s t r a c t Ni- and Cu–free Zr–Al–Co–Ag bulk metallic glasses (BMGs) were synthesized by copper mold casting. The effects of Ag addition for partially replacing Co of Zr53Al16Co31 BMG on the corrosion behavior, surface chemistry and in vitro biocompatibility of BMGs were investigated. The Zr–Al–Co–Ag BMGs are spontaneously passivated with low passive current densities in phosphate buffered saline (PBS) solution. Partial substitution of Co by Ag is effective in improving the corrosion resistance of the Zr–Al–Co BMG. X-ray photoelectron spectroscopy (XPS) measurements reveal that the Ag addition increases the concentration of Zr and decreases the concentration of Al in the surface passive film of BMGs, which is responsible for the enhanced corrosion resistance of Zr–Al–Co–Ag BMGs. Mouse MC3T3-E1 pre-osteoblast cell proliferation results and morphology observations show that the Zr–Al–Co–Ag BMGs exhibit comparable cell viability and proliferation activity with those of Ti–6Al–4V alloy, demonstrating their good biocompatibility. The high corrosion resistance in PBS and low in vitro cytotoxicity of Zr–Al–Co–Ag BMGs suggest an initial biocompatibility for biomedical applications. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Zirconium-based bulk metallic glasses (BMGs) exhibit high strength and hardness, low Young's modulus, high fatigue limit, good wear and corrosion resistance due to their amorphous structure, which makes them candidates as biomaterials [1–6]. However, elements with high toxicity to cellular metabolism, such as Ni and Cu, are usually adopted in Zr-based BMGs with high glass-forming ability (GFA), which is disadvantageous to their practical applications as biomaterials [7,8]. Therefore, in the last decade, great efforts have been devoted to develop Zr-based BMGs with less hazardous elements so as to further improve their biocompatibility. Ni-free glass forming systems such as Zr–Al–Cu [9], Zr–Al–Co–(Cu) [10–13], Zr–Al–Co–Nb [14,15], Zr–Al–Co–Ag [16], Zr–Al–Cu–Fe [17], Zr–Al–Cu–Ag [18], Zr–Cu–Pd–Al–Nb [19], and Zr–Al–Cu–Fe–(Ti/Nb) [20] have been developed. Among the Ni-free Zr-based BMGs, Zr–Al–Co system BMGs are attractive due to the absence of copper, which is also a toxic element to cellular metabolism and may result in high cytotoxicity if released excessively during corrosion [7]. In addition, the Zr–Al–Co BMGs are spontaneously passivated with low passive current densities and wide passive potential regions in physiologically relevant environments, indicating their high corrosion resistance [13,15]. Although Co ions are known to be cytotoxic, the total mass of Co ions released by a unit surface area of glassy
⁎ Corresponding author. Tel.: + 86 10 8233 9705; fax: + 86 10 8231 4869. E-mail address: [email protected] (T. Zhang). 0022-3093/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2012.04.022
Zr56Al16Co28 alloy (0.3 ng/mm2) is significantly lower than the Co–Cr– Mo alloy (5.8 ng/mm 2) [13]. Moreover, the corrosion resistance of Zr–Al–Co BMGs can be further improved by the Nb addition due to the formation of highly protective passive film on the alloys' surface [14,15,21]. However, the addition of Nb to the Zr-based BMG shows a detrimental effect on their glass-forming ability, which may hinder their wide application [14,21]. It has been found in previous study that the addition of Ag to Zr-based glassy alloys can improve the GFA and mechanical properties [16,22]. By alloying appropriate amount of Ag in Zr53Al16Co31 alloy, the Zr53Al16(Co0.75Ag0.25)31 BMG with a critical diameter of 20 mm can be fabricated by copper mold casting. The Zr–Al–Co–Ag BMGs exhibit good mechanical biocompatibility with a combination of higher compressive yielding strength and lower Young's modulus compared to those of commercial metallic biomaterials such as Ti– 6Al–4V, 316 L stainless steel and Co–Cr–Mo alloys [16]. Meanwhile, from the viewpoint of biomedical applications, silver is a potent antibacterial agent with a very broad spectrum of activity, and the Ag-containing materials have proved their effectiveness in reducing infections [23]. Consequently, Ag-bearing Zr-based BMGs are considered to be more promising biomaterials. As a potential biomaterial, which interacts with in vivo environment, the corrosion resistance and the biocompatibility are two of the key properties, which require comprehensive examination. Thus, the critical issues of this work are (1) to investigate the effect of Ag addition to the Zr–Al–Co BMG on the corrosion behavior of alloys in a physiologically relevant environment; (2) to study the effect of Ag addition on the surface chemistry
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of Zr-based BMGs and propose mechanistic understanding of their corrosion behavior; (3) to study the in vitro biocompatibility of Zr-based BMGs and compare with that of Ti–6Al–4V alloy. All of the results provide the foundational information of this family of Ni and Cu-free Zr-based BMGs for further biomedical applications.
2. Experimental Master alloys with nominal compositions of Zr53Al16(Co1 − xAgx)31 (described in atomic percentage, x = 0, 0.1, 0.25) were prepared by arc melting the mixtures of pure Zr, Al, Co, and Ag metals under Tigettered high-purity argon atmosphere. From the master alloys, the rods of 3 mm in diameter and 50 mm in length and sheets with a size of 10 mm × 50 mm × 1 mm were prepared by injection copper mold casting under argon atmosphere. The glassy structure of the specimens was verified by a X-ray diffractometer (XRD; Bruker AXS D8) with Cu–Kα radiation for transverse cross-section of alloy rods and square surface of alloy sheets. The XRD is operating at reflection mode at 40 kV and 40 mA with a beam size of 1.5 mm × 10 mm. Corrosion behavior of the Zr53Al16(Co1 − xAgx)31 alloys were tested by corrosion rates tests and electrochemical measurements in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min. All samples for corrosion behavior were prepared to have a size of about ϕ 3 mm × 6 mm with two end-surfaces being polished to #2000. Corrosion rates were estimated from the weight loss after immersion in PBS at 37 °C for one week. At least three samples of each composition were tested to confirm reproductility. The surfaces of the samples before and after the weight loss measurements were observed by scanning electron microscopy (SEM, CS 3400) in backscattered (BS) mode. Electrochemical measurements were conducted in a three-electrode cell using a platinum counter electrode and a saturated calomel reference electrode (SCE). Prior to tests, the specimens were mechanically polished in cyclohexane with silicon carbide paper up to #2000, and then degreased in acetone, washed in distilled water, dried in air, and further exposed to air for 24 h for good reproducibility. Anodic polarization curves were measured at a potential sweep rate of 50 mV/min after open-circuit immersion for about 20 min when the open-circuit potential became almost steady. The anodic polarization curves were measured at least three times to confirm reproducibility. In order to characterize the composition and chemical states of elements on the passive films of BMGs, the specimens were immersed in PBS for 1 h after mechanical polishing and then taken out for X-ray photoelectron spectroscopy (XPS, ESCALab250) analysis using a photoelectron spectrometer with Al Kα radiation (hv = 1486.6 eV). The potential cytotoxicity of the BMGs was evaluated via a Mouse MC3T3-E1 pre-osteoblast (ATCC, USA) cell culture for one week followed by a WST-1 (a water-soluble tetrazolium salt) assay on triplicate samples (n = 3). All samples for WST-1 assay were prepared to have a size of about 10 mm × 10 mm × 1 mm with the square surface being well polished to #3000. This assay is based on the reduction of tetrazolium salts to formazan (a chromogenic by-product) by mitochondrial dehydrogenases. The resulting formazan can be quantified spectrophotometrically and correlated with the number of viable cells and metabolic activity. Cells were cultured on the substrates in 96-well plates in a final medium volume of 100 μl. The seeding density was 5 × 10 3 cell cm − 2. After 7-day incubation, 10 μl of cell proliferation reagent WST-1 (Roche Applied Science, USA) was added to each well and incubated at 37 °C in a 5 vol.% CO2 balanced air atmosphere for 4 h. Afterwards, the media were mixed thoroughly for 1 min on a shaker. The absorbance of the supernatants was measured against a background control blank at 440 nm, using a Wallac 1420 Victor 2 multilabel counter (Perkin Elmer, USA). Cells were dehydrated and fixed as described by Ref. [24]. Dry specimens were mounted on aluminum stubs, coated with gold in an SPI sputtering
device for 10 s at 20 Ma and examined using a scanning electron microscope (SEM, LEO 1525). 3. Results Fig. 1 shows the XRD patterns of transverse cross-section of ascast Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) rods with a diameter of 3 mm. The patterns exhibit a characteristic of board diffraction hump without any distinct crystalline peaks, indicating the glassy structure of the rod samples. The corrosion rate of all alloys in PBS at 37 °C was estimated from the weight loss after immersion in PBS for one week according to the following equation: Rcorr = 8.76 × 10 4 w/adt (mm per year). Where w refers to the weight loss in gram, a and d are the total surface area exposed in mm 2 and the density of the sample in g/cm 3, respectively, and t is the immersed time in hour. All specimens show no observable weight loss with an accuracy of 10 − 5 g of electronic balance after immersed in PBS at 37 °C for one week, indicating a high corrosion resistance of those BMGs in PBS. After immersed in PBS for one week, the surfaces of all BMGs were observed by SEM. Fig. 2(a) and (b) present the BS-SEM images of the surfaces for Zr53Al16(Co0.75Ag0.25)31 BMG exposed to air and immersed in PBS for one week, respectively. The BMG surfaces are homogeneous and no crystalline phase can be observed, which corresponds to the XRD results. No pitting corrosion can be seen on the alloy surface after the immersion. The SEM results of the immersed Zr53Al16Co31 and Zr53Al16(Co0.9Ag0.1)31 BMGs (no shown here) also show no pitting corrosion was occurred on alloy surface. The corrosion rates measurements and the SEM morphology results demonstrate that Zr–Al–Co–Ag BMGs exhibit good corrosion resistance in PBS. Fig. 3 shows anodic polarization curves of Zr53Al16(Co1 − xAgx)31 (x =0, 0.1, 0.25) BMGs in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min for 1 h prior to testing. The open-circuit potential increases from −500 mV to −390 mV with the increase in Ag content from x =0 to x =0.25. The alloys are spontaneously passivated, and their passive current densities remain lower than 10− 1 A/m2 before the occurrence of pitting corrosion, which is characterized by an abrupt rise in the current density. The pitting potential increases significantly from 30 mV to 230 mV with the increase in Ag content from x = 0 to x= 0.25. The electrochemical results indicate that partial replacement of Co by Ag is effective in improving the corrosion resistance of the glassy Zr–Al–Co alloy in PBS.
Fig. 1. XRD patterns of transverse cross-section of as-cast Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) rods with a diameter of 3 mm.
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Fig. 2. BS-SEM images of the surface for Zr53Al16(Co0.75Ag0.25)31 BMG (a) exposed to air and (b) immersed in PBS for one week, respectively.
To clarify the effects of Ag alloying on the corrosion resistance and other surface-related chemical characteristics of the BMGs, XPS analysis was performed for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens after the open-circuit immersion in PBS at 37 °C for 1 h with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min. Fig. 4 shows the XPS spectra for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens: (a) survey spectra and narrow scans for (b) Zr3d, (c)Al2p, (d) Co2p, and (e) Ag3d. The XPS survey spectra of Zr53Al16(Co0.9Ag0.1)31 and
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Zr53Al16(Co0.75Ag0.25)31 BMGs over the wide binding energy region showed peaks of zirconium, aluminum, cobalt, silver, oxygen, and carbon, while no peaks of silver was observed for Zr53Al16Co31 BMG. The C 1s spectrum showing a peak at around 285.2 eV is from contaminant carbon on the top surfaces of the specimens. The O 1s spectrum consists of peaks originating from oxygen in metal\O\metal bond, metal\OH bond and/or bound water [25]. The peaks of Zr 3d, Al 2p, Co 2p, and Ag 3d are composed of peaks corresponding to the species in the oxidized states in the surface films and the metallic states in the underlying alloy surfaces just below the surface films. Fig. 4(b–e) shows the narrow scans of Zr 3d, Al 2p, Co 2p, and Ag 3d spectra measured for Zr–Al– Co–Ag BMGs. The Zr 3d peaks consist of two doublets: the Zr 3d5/2 and Zr 3d3/2 peaks corresponding to the Zr4+ oxide state appeared at 182.3 and 184.7 eV, respectively, and those from the Zr0 metallic state located at 178.6 and 180.9 eV, respectively. The Al 2p spectrum consists of two peaks located at 71.6 and 74.1 eV correspond to the Al0 metallic state and Al 3+ oxide state, respectively. The Co 2p spectrum consists of two doublets: the peaks located at 779.8 and 795.0 eV correspond to the Co 2p3/2 and Co 2p1/2 electrons from the Co2+ oxide state, and those appeared at 777.9 and 792.3 eV correspond to the Co 2p3/2 and Co 2p1/2 electrons from Co0 metallic state. The Ag 3d spectrum consists of two peaks at 368.0 and 374.0 eV, which correspond to the Ag 3d5/2 and Ag 3d3/2 electrons originating from the Ag0 metallic state. Fig. 5 shows the cationic contents in the surface films for Zr53Al16 (Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs after immersion in PBS for 1 h. In the figure, superscripts “ox” represents the oxidized states of the species, “nominal” represents the nominal content in atomic percentage of the species. The XPS analysis for the immersed alloys shows that the surface films are enriched in Zr and Al, deficient in Co, and no Ag was detected in the surface films for all alloys. With increasing Ag content from x = 0 to x = 0.25, the concentration of Zr in the surface films on the BMGs immersed in PBS increases from 70.7% to 72.6%, while that of Al decreases from 26.9% to 25.2%. Therefore, the enhancement of corrosion resistance of Zr–Al–Co–Ag BMGs is attributed to the change in surface composition. The WST-1 assay results, corresponding to cell proliferation after 7day growth of MC3T3-E1 cell, are illustrated in Fig. 6. Ti–6Al–4V alloy, known as a widely used biomaterial based on its excellent biocompatibility and high corrosion resistance, was employed as a reference material. The absorbance of Zr53Al16Co31 and Zr53Al16(Co0.75Ag0.25)31 BMGs is comparably high with that of the Ti–6Al–4V alloy, indicating their high cell viability and proliferation activity. Fig. 7(a) and (b) presents the SEM images for the MC3T3-E1 cell morphology on the surface of Zr53Al16(Co0.75Ag0.25)31 BMG and Ti–6Al–4V alloy. The SEM images on the surface of the two alloys show good cell viability and proliferation activity of MC3T3-E1 cell. The inset images show that the MC3T3-E1 cells can closely adhere to and extend across the surface of those two alloys. This further confirms the good biocompatibility of the BMGs studied in this work. 4. Discussion
Fig. 3. Anodic polarization curves of Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) BMGs in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min for 1 h prior to testing.
It can be seen form Fig. 5 that the surface films of the immersed alloys are enriched in Zr and Al, deficient in Co, and no Ag was detected in the surface films for all alloys. It has been reported that preferentially oxidation of Zr and Al in the surface film of the Zr-based BMGs occurs when they are exposed to air [14]. The Standard Electrode Potential (SEP), which is evaluated relative to the standard oxidation of hydrogen gas, measures the ability of the metal atoms to get oxidized. The SEP is −1.66 V for the reaction Al3+ + 3e− ↔ Al(s), −1.45 V for the reaction Zr4+ + 4e− ↔ Zr(s), −0.28 V for the reaction Co2+ + 2e− ↔ Co(s), and 0.7996 V for the reaction Ag + + e − ↔ Ag(s) [26]. A lower SEP corresponds to an easier oxidization reaction. When the Zr–Al–Co–Ag alloys were exposed to air, Zr and Al are preferentially oxidation in the surfaces due to their lower SEPs compared to those of Co and Ag. The SEP of the reaction forming Ag+ is the highest, which
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Fig. 4. XPS spectra for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens: (a) survey spectra and narrow scans for (b) Zr3d, (c)Al2p, (d) Co2p, and (e) Ag3d.
can be responsible for the absence of Ag+ ions in the surface film. Similar results can be observed in the XPS studies on the Ti–Zr–Cu–Pd–Sn and Ni–Pd–P–B BMGs [27,28], which found that the noble metal Pd element stays completely in the underlying alloy surface as a result of its highest SEP (0.915 V) of the reaction forming Pd2+ among all the constituents in the alloy systems. Immersion in the corrosive solutions results in the change in the composition in the surface films of specimens. The corrosion behavior of the Zr–Al–Co–Nb BMGs showed that Zr is further concentrated while Al content decreases in the spontaneously passivated films on the alloys after immersion in chloride medium compared to those exposed to air [14]. Therefore, after immersion in
PBS for 1 h, the surface films of Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs are enriched in Zr and Al and largely deficient in Co and Ag. Meanwhile, with increasing Ag content from x = 0 to x = 0.25, the concentration of Zr in the surface films on the BMGs immersed in PBS increases from 70.7% to 72.6%, while that of Al decreases from 26.9% to 25.2%. It has been reported that the high corrosion resistance of Zr–Al–Co–Nb BMGs is attributed to the formation of Zr 4+- and Al 3+-enriched oxide surface film in the corrosive solutions. With increasing Zr content and decreasing Al content in the surface films, the corrosion resistance of those Zr-based BMGs in chloride medium enhanced [14]. Moreover, a direct comparison on the corrosion resistance
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Fig. 5. the cationic contents in the surface films for the Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs after open-circuit immersion in PBS for 1 h.
of pure Zr and pure Al in chloride medium revealed higher pitting resistance of Zr than that of Al, which indicates that Zr4+ oxide is more stable than Al3+ oxide [29]. Thus, the addition of Ag is favorable for the alloys to form a protective surface film with higher chemical stability resulting in its higher corrosion resistance in PBS. The WST-1 assay results showed that the BMGs exhibited good biocompatibility. In the Zr–Al–Co–(Ag) system, Co ions are recognized to be the most cytotoxic among the constituents [13,16]. The low cytotoxicity of Zr–Al–Co–Ag BMGs can partly be attributed to their high corrosion resistance. The corrosion behavior and biocompatibility of metallic glasses or commercial metallic biomaterials have been extensively studied, the results show that high corrosion resistance of alloys can decrease the ions release in corrosive solution, which may resulting in their good biocompatibility [30–34]. For example, the study of metal ions release during corrosion of FeCobased amorphous metallic materials show that the total metal ions release of the amorphous alloys in PBS solution decreases with increasing corrosion resistance of those alloys. The Fe3Co67Cr3Si15B12
Fig. 7. SEM images for the MC3T3-E1 cell morphology on the surface of (a) Zr53Al16 (Co0.75Ag0.25)31 BMG and (b) Ti–6Al–4V alloy.
exhibits the best corrosion behavior in PBS and the fewest mass of metal ions released [31]. It has been reported that nanocrystalline 304 stainless steel (SS) shows higher corrosion resistance than that of microcrystalline 304 SS in artificial saliva, and the released potentially toxic ions such as Ni, Cr and Fe ions into artificial saliva from nanocrystalline 304 SS samples are much fewer than that of microcrystalline 304 SS, which resulting in the good biocompatibility of nanocrystalline 304 SS [32]. In addition, surface modification such as sandblasting or laser surface treatment (LSM) can significantly improve the corrosion resistance of NiTi alloy and reduce the release of toxic Ni ions in physiologically relevant environments, and the surface modified NiTi alloys exhibit an improved cytocompatibility [33,34]. As a result, it can be presumed that with the increase in Ag content, the corrosion resistance of Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs significantly improves, and fewer Co ions will be released to the solutions. Meanwhile, partial replacement of Co by Ag reduces the nominal Co content of the BMGs. Therefore, Co substitution with Ag is expected to further reduce the potential cytotoxicity of Zr–Al– Co BMGs, although no obvious improvement was observed based on our 7 day proliferation experiments. Future efforts will be made on further evaluations regarding the long term biocompatibility of Zr– Al–Co–Ag BMGs. 5. Conclusions
Fig. 6. WST-1 assay results of Zr53Al16Co31 and Zr53Al16(Co0.75Ag0.25)31 BMGs after MC3T3-E1 cell proliferation for 7 days in comparison of the Ti–6Al–4V alloy.
The electrochemical properties, surface chemistry and cytotoxicity of Zr–Al–Co–Ag BMGs were investigated. The BMGs were spontaneously passivated with a low passive current density of the order of 10 − 1 A/m 2 and a wide passive potential region, indicating their high corrosion resistance in PBS. The addition of Ag to the alloy enhanced the pitting corrosion resistance in PBS. XPS analysis results show that a Zr- and Al-enriched passive film is formed on the BMGs
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after immersion in the solution. The increase in Ag content of the alloys results in an increase in Zr concentration and a decrease in Al concentration of the passive film, which is responsible for the high corrosion resistance of the alloys. WST-1 assay results show that Zr53Al16(Co0.75Ag0.25)31 BMG exhibits comparable cell viability and proliferation activity with those of Ti–6Al–4V alloy, demonstrating its good biocompatibility. Addition of Ag to the Zr–Al–Co BMG improved its corrosion resistance in PBS, which facilitates the MC3T3-E1 cell viability and proliferation activity. The high corrosion resistance and low in vitro cytotoxicity of Zr–Al–Co–Ag BMGs suggest an initial biocompatibility for biomedical applications. Future efforts will be made on further evaluations regarding the biocompatibility of Zr– Al–Co–Ag BMGs, in order to achieve a thorough understanding on the suitability of these materials to serve as biomaterials. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 51161130526). References [1] W.H. Wang, C. Dong, C.H. Shek, Bulk metallic glasses, Mater. Sci. Eng. R 44 (2004) 45–89. [2] A.L. Greer, E. Ma, Bulk metallic glasses: at the cutting edge of metals research, MRS Bull. 32 (2007) 611–619. [3] A. Inoue, Bulk amorphous alloys: practical characteristics and applications, Trans Tech Publications Ltd, Switzerland, 1999. [4] L. Huang, D.C. Qiao, B.A. Green, P.K. Liaw, J.F. Wang, S.J. Pang, T. Zhang, Biocorrosion study on zirconium-based bulk-metallic glasses, Intermetallics 17 (2009) 195–199. [5] M.Z. Ma, R.P. Liu, Y. Xiao, D.C. Lou, L. Liu, Q. Wang, W.K. Wang, Wear resistance of Zr-based bulk metallic glass applied in bearing rollers, Mater. Sci. Eng. A 386 (2004) 326–330. [6] W.H. Peter, R.A. Buchanan, C.T. Liu, P.K. Liaw, The fatigue behavior of a zirconiumbased bulk metallic glass in vacuum and air, J. Non-Cryst. Solids 317 (2003) 187–192. [7] S. Buzzi, K. Jin, P.J. Uggowitzer, S. Tosatti, I. Gerber, J.F. Löffler, Cytotoxicity of Zr-based bulk metallic glasses, Intermetallics 14 (2006) 729–734. [8] A. Yamamoto, R. Honma, M. Sumita, Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells, J. Biomed. Mater. Res. 39 (1998) 331–340. [9] A. Inoue, D. Kawase, A.P. Tsai, T. Zhang, T. Masumoto, Stability and transformation to crystalline phases of amorphous Zr–Al–Cu alloys with significant supercooled liquid region, Mater. Sci. Eng. A 178 (1994) 255–263. [10] T. Zhang, A. Inoue, New glassy Zr–Al–Fe and Zr–Al–Co alloys with a large supercooled liquid region, Mater. Trans. JIM 43 (2002) 267–270. [11] T. Wada, T. Zhang, A. Inoue, Formation, thermal stability and mechanical properties in Zr–Al–Co bulk glassy alloys, Mater. Trans. JIM 43 (2002) 2843–2846. [12] T. Wada, T. Zhang, A. Inoue, Formation and high mechanical strength of bulk glassy alloys in Zr–Al–Co–Cu system, Mater. Trans. JIM 44 (2003) 1839–1844. [13] T. Wada, F.X. Qin, X.M. Wang, M. Yoshimura, A. Inoue, N. Sugiyama, R. Ito, N. Matsushita, Formation and bioactivation of Zr–Al–Co bulk metallic glasses, J. Mater. Res. 24 (2009) 2941–2948.
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Corrosion behavior and in vitro biocompatibility of Zr–Al–Co–Ag bulk metallic glasses: An experimental case study Nengbin Hua a, Lu Huang a, Jianfeng Wang a, Yu Cao b, Wei He b, c, Shujie Pang a, Tao Zhang a,⁎ a b c
Key Laboratory of Aerospace Materials and Performance (Ministry of Education), School of Materials Science and Engineering, Beihang University, Beijing 100191, China Department of Materials Science and Engineering, The University of Tennessee, Knoxville, TN 37996–2200, USA Department of Mechanical, Aerospace and Biomedical Engineering, The University of Tennessee, Knoxville, TN 37996–2200, USA
a r t i c l e
i n f o
Article history: Received 16 February 2012 Received in revised form 6 April 2012 Available online 15 May 2012 Keywords: Metallic glasses; Zirconium alloys; Corrosion; Biocompatibility
a b s t r a c t Ni- and Cu–free Zr–Al–Co–Ag bulk metallic glasses (BMGs) were synthesized by copper mold casting. The effects of Ag addition for partially replacing Co of Zr53Al16Co31 BMG on the corrosion behavior, surface chemistry and in vitro biocompatibility of BMGs were investigated. The Zr–Al–Co–Ag BMGs are spontaneously passivated with low passive current densities in phosphate buffered saline (PBS) solution. Partial substitution of Co by Ag is effective in improving the corrosion resistance of the Zr–Al–Co BMG. X-ray photoelectron spectroscopy (XPS) measurements reveal that the Ag addition increases the concentration of Zr and decreases the concentration of Al in the surface passive film of BMGs, which is responsible for the enhanced corrosion resistance of Zr–Al–Co–Ag BMGs. Mouse MC3T3-E1 pre-osteoblast cell proliferation results and morphology observations show that the Zr–Al–Co–Ag BMGs exhibit comparable cell viability and proliferation activity with those of Ti–6Al–4V alloy, demonstrating their good biocompatibility. The high corrosion resistance in PBS and low in vitro cytotoxicity of Zr–Al–Co–Ag BMGs suggest an initial biocompatibility for biomedical applications. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Zirconium-based bulk metallic glasses (BMGs) exhibit high strength and hardness, low Young's modulus, high fatigue limit, good wear and corrosion resistance due to their amorphous structure, which makes them candidates as biomaterials [1–6]. However, elements with high toxicity to cellular metabolism, such as Ni and Cu, are usually adopted in Zr-based BMGs with high glass-forming ability (GFA), which is disadvantageous to their practical applications as biomaterials [7,8]. Therefore, in the last decade, great efforts have been devoted to develop Zr-based BMGs with less hazardous elements so as to further improve their biocompatibility. Ni-free glass forming systems such as Zr–Al–Cu [9], Zr–Al–Co–(Cu) [10–13], Zr–Al–Co–Nb [14,15], Zr–Al–Co–Ag [16], Zr–Al–Cu–Fe [17], Zr–Al–Cu–Ag [18], Zr–Cu–Pd–Al–Nb [19], and Zr–Al–Cu–Fe–(Ti/Nb) [20] have been developed. Among the Ni-free Zr-based BMGs, Zr–Al–Co system BMGs are attractive due to the absence of copper, which is also a toxic element to cellular metabolism and may result in high cytotoxicity if released excessively during corrosion [7]. In addition, the Zr–Al–Co BMGs are spontaneously passivated with low passive current densities and wide passive potential regions in physiologically relevant environments, indicating their high corrosion resistance [13,15]. Although Co ions are known to be cytotoxic, the total mass of Co ions released by a unit surface area of glassy
⁎ Corresponding author. Tel.: + 86 10 8233 9705; fax: + 86 10 8231 4869. E-mail address: [email protected] (T. Zhang). 0022-3093/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jnoncrysol.2012.04.022
Zr56Al16Co28 alloy (0.3 ng/mm2) is significantly lower than the Co–Cr– Mo alloy (5.8 ng/mm 2) [13]. Moreover, the corrosion resistance of Zr–Al–Co BMGs can be further improved by the Nb addition due to the formation of highly protective passive film on the alloys' surface [14,15,21]. However, the addition of Nb to the Zr-based BMG shows a detrimental effect on their glass-forming ability, which may hinder their wide application [14,21]. It has been found in previous study that the addition of Ag to Zr-based glassy alloys can improve the GFA and mechanical properties [16,22]. By alloying appropriate amount of Ag in Zr53Al16Co31 alloy, the Zr53Al16(Co0.75Ag0.25)31 BMG with a critical diameter of 20 mm can be fabricated by copper mold casting. The Zr–Al–Co–Ag BMGs exhibit good mechanical biocompatibility with a combination of higher compressive yielding strength and lower Young's modulus compared to those of commercial metallic biomaterials such as Ti– 6Al–4V, 316 L stainless steel and Co–Cr–Mo alloys [16]. Meanwhile, from the viewpoint of biomedical applications, silver is a potent antibacterial agent with a very broad spectrum of activity, and the Ag-containing materials have proved their effectiveness in reducing infections [23]. Consequently, Ag-bearing Zr-based BMGs are considered to be more promising biomaterials. As a potential biomaterial, which interacts with in vivo environment, the corrosion resistance and the biocompatibility are two of the key properties, which require comprehensive examination. Thus, the critical issues of this work are (1) to investigate the effect of Ag addition to the Zr–Al–Co BMG on the corrosion behavior of alloys in a physiologically relevant environment; (2) to study the effect of Ag addition on the surface chemistry
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of Zr-based BMGs and propose mechanistic understanding of their corrosion behavior; (3) to study the in vitro biocompatibility of Zr-based BMGs and compare with that of Ti–6Al–4V alloy. All of the results provide the foundational information of this family of Ni and Cu-free Zr-based BMGs for further biomedical applications.
2. Experimental Master alloys with nominal compositions of Zr53Al16(Co1 − xAgx)31 (described in atomic percentage, x = 0, 0.1, 0.25) were prepared by arc melting the mixtures of pure Zr, Al, Co, and Ag metals under Tigettered high-purity argon atmosphere. From the master alloys, the rods of 3 mm in diameter and 50 mm in length and sheets with a size of 10 mm × 50 mm × 1 mm were prepared by injection copper mold casting under argon atmosphere. The glassy structure of the specimens was verified by a X-ray diffractometer (XRD; Bruker AXS D8) with Cu–Kα radiation for transverse cross-section of alloy rods and square surface of alloy sheets. The XRD is operating at reflection mode at 40 kV and 40 mA with a beam size of 1.5 mm × 10 mm. Corrosion behavior of the Zr53Al16(Co1 − xAgx)31 alloys were tested by corrosion rates tests and electrochemical measurements in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min. All samples for corrosion behavior were prepared to have a size of about ϕ 3 mm × 6 mm with two end-surfaces being polished to #2000. Corrosion rates were estimated from the weight loss after immersion in PBS at 37 °C for one week. At least three samples of each composition were tested to confirm reproductility. The surfaces of the samples before and after the weight loss measurements were observed by scanning electron microscopy (SEM, CS 3400) in backscattered (BS) mode. Electrochemical measurements were conducted in a three-electrode cell using a platinum counter electrode and a saturated calomel reference electrode (SCE). Prior to tests, the specimens were mechanically polished in cyclohexane with silicon carbide paper up to #2000, and then degreased in acetone, washed in distilled water, dried in air, and further exposed to air for 24 h for good reproducibility. Anodic polarization curves were measured at a potential sweep rate of 50 mV/min after open-circuit immersion for about 20 min when the open-circuit potential became almost steady. The anodic polarization curves were measured at least three times to confirm reproducibility. In order to characterize the composition and chemical states of elements on the passive films of BMGs, the specimens were immersed in PBS for 1 h after mechanical polishing and then taken out for X-ray photoelectron spectroscopy (XPS, ESCALab250) analysis using a photoelectron spectrometer with Al Kα radiation (hv = 1486.6 eV). The potential cytotoxicity of the BMGs was evaluated via a Mouse MC3T3-E1 pre-osteoblast (ATCC, USA) cell culture for one week followed by a WST-1 (a water-soluble tetrazolium salt) assay on triplicate samples (n = 3). All samples for WST-1 assay were prepared to have a size of about 10 mm × 10 mm × 1 mm with the square surface being well polished to #3000. This assay is based on the reduction of tetrazolium salts to formazan (a chromogenic by-product) by mitochondrial dehydrogenases. The resulting formazan can be quantified spectrophotometrically and correlated with the number of viable cells and metabolic activity. Cells were cultured on the substrates in 96-well plates in a final medium volume of 100 μl. The seeding density was 5 × 10 3 cell cm − 2. After 7-day incubation, 10 μl of cell proliferation reagent WST-1 (Roche Applied Science, USA) was added to each well and incubated at 37 °C in a 5 vol.% CO2 balanced air atmosphere for 4 h. Afterwards, the media were mixed thoroughly for 1 min on a shaker. The absorbance of the supernatants was measured against a background control blank at 440 nm, using a Wallac 1420 Victor 2 multilabel counter (Perkin Elmer, USA). Cells were dehydrated and fixed as described by Ref. [24]. Dry specimens were mounted on aluminum stubs, coated with gold in an SPI sputtering
device for 10 s at 20 Ma and examined using a scanning electron microscope (SEM, LEO 1525). 3. Results Fig. 1 shows the XRD patterns of transverse cross-section of ascast Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) rods with a diameter of 3 mm. The patterns exhibit a characteristic of board diffraction hump without any distinct crystalline peaks, indicating the glassy structure of the rod samples. The corrosion rate of all alloys in PBS at 37 °C was estimated from the weight loss after immersion in PBS for one week according to the following equation: Rcorr = 8.76 × 10 4 w/adt (mm per year). Where w refers to the weight loss in gram, a and d are the total surface area exposed in mm 2 and the density of the sample in g/cm 3, respectively, and t is the immersed time in hour. All specimens show no observable weight loss with an accuracy of 10 − 5 g of electronic balance after immersed in PBS at 37 °C for one week, indicating a high corrosion resistance of those BMGs in PBS. After immersed in PBS for one week, the surfaces of all BMGs were observed by SEM. Fig. 2(a) and (b) present the BS-SEM images of the surfaces for Zr53Al16(Co0.75Ag0.25)31 BMG exposed to air and immersed in PBS for one week, respectively. The BMG surfaces are homogeneous and no crystalline phase can be observed, which corresponds to the XRD results. No pitting corrosion can be seen on the alloy surface after the immersion. The SEM results of the immersed Zr53Al16Co31 and Zr53Al16(Co0.9Ag0.1)31 BMGs (no shown here) also show no pitting corrosion was occurred on alloy surface. The corrosion rates measurements and the SEM morphology results demonstrate that Zr–Al–Co–Ag BMGs exhibit good corrosion resistance in PBS. Fig. 3 shows anodic polarization curves of Zr53Al16(Co1 − xAgx)31 (x =0, 0.1, 0.25) BMGs in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min for 1 h prior to testing. The open-circuit potential increases from −500 mV to −390 mV with the increase in Ag content from x =0 to x =0.25. The alloys are spontaneously passivated, and their passive current densities remain lower than 10− 1 A/m2 before the occurrence of pitting corrosion, which is characterized by an abrupt rise in the current density. The pitting potential increases significantly from 30 mV to 230 mV with the increase in Ag content from x = 0 to x= 0.25. The electrochemical results indicate that partial replacement of Co by Ag is effective in improving the corrosion resistance of the glassy Zr–Al–Co alloy in PBS.
Fig. 1. XRD patterns of transverse cross-section of as-cast Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) rods with a diameter of 3 mm.
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Fig. 2. BS-SEM images of the surface for Zr53Al16(Co0.75Ag0.25)31 BMG (a) exposed to air and (b) immersed in PBS for one week, respectively.
To clarify the effects of Ag alloying on the corrosion resistance and other surface-related chemical characteristics of the BMGs, XPS analysis was performed for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens after the open-circuit immersion in PBS at 37 °C for 1 h with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min. Fig. 4 shows the XPS spectra for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens: (a) survey spectra and narrow scans for (b) Zr3d, (c)Al2p, (d) Co2p, and (e) Ag3d. The XPS survey spectra of Zr53Al16(Co0.9Ag0.1)31 and
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Zr53Al16(Co0.75Ag0.25)31 BMGs over the wide binding energy region showed peaks of zirconium, aluminum, cobalt, silver, oxygen, and carbon, while no peaks of silver was observed for Zr53Al16Co31 BMG. The C 1s spectrum showing a peak at around 285.2 eV is from contaminant carbon on the top surfaces of the specimens. The O 1s spectrum consists of peaks originating from oxygen in metal\O\metal bond, metal\OH bond and/or bound water [25]. The peaks of Zr 3d, Al 2p, Co 2p, and Ag 3d are composed of peaks corresponding to the species in the oxidized states in the surface films and the metallic states in the underlying alloy surfaces just below the surface films. Fig. 4(b–e) shows the narrow scans of Zr 3d, Al 2p, Co 2p, and Ag 3d spectra measured for Zr–Al– Co–Ag BMGs. The Zr 3d peaks consist of two doublets: the Zr 3d5/2 and Zr 3d3/2 peaks corresponding to the Zr4+ oxide state appeared at 182.3 and 184.7 eV, respectively, and those from the Zr0 metallic state located at 178.6 and 180.9 eV, respectively. The Al 2p spectrum consists of two peaks located at 71.6 and 74.1 eV correspond to the Al0 metallic state and Al 3+ oxide state, respectively. The Co 2p spectrum consists of two doublets: the peaks located at 779.8 and 795.0 eV correspond to the Co 2p3/2 and Co 2p1/2 electrons from the Co2+ oxide state, and those appeared at 777.9 and 792.3 eV correspond to the Co 2p3/2 and Co 2p1/2 electrons from Co0 metallic state. The Ag 3d spectrum consists of two peaks at 368.0 and 374.0 eV, which correspond to the Ag 3d5/2 and Ag 3d3/2 electrons originating from the Ag0 metallic state. Fig. 5 shows the cationic contents in the surface films for Zr53Al16 (Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs after immersion in PBS for 1 h. In the figure, superscripts “ox” represents the oxidized states of the species, “nominal” represents the nominal content in atomic percentage of the species. The XPS analysis for the immersed alloys shows that the surface films are enriched in Zr and Al, deficient in Co, and no Ag was detected in the surface films for all alloys. With increasing Ag content from x = 0 to x = 0.25, the concentration of Zr in the surface films on the BMGs immersed in PBS increases from 70.7% to 72.6%, while that of Al decreases from 26.9% to 25.2%. Therefore, the enhancement of corrosion resistance of Zr–Al–Co–Ag BMGs is attributed to the change in surface composition. The WST-1 assay results, corresponding to cell proliferation after 7day growth of MC3T3-E1 cell, are illustrated in Fig. 6. Ti–6Al–4V alloy, known as a widely used biomaterial based on its excellent biocompatibility and high corrosion resistance, was employed as a reference material. The absorbance of Zr53Al16Co31 and Zr53Al16(Co0.75Ag0.25)31 BMGs is comparably high with that of the Ti–6Al–4V alloy, indicating their high cell viability and proliferation activity. Fig. 7(a) and (b) presents the SEM images for the MC3T3-E1 cell morphology on the surface of Zr53Al16(Co0.75Ag0.25)31 BMG and Ti–6Al–4V alloy. The SEM images on the surface of the two alloys show good cell viability and proliferation activity of MC3T3-E1 cell. The inset images show that the MC3T3-E1 cells can closely adhere to and extend across the surface of those two alloys. This further confirms the good biocompatibility of the BMGs studied in this work. 4. Discussion
Fig. 3. Anodic polarization curves of Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) BMGs in PBS at 37 °C with a 4 vol.% O2/N2 gas mixture flowing at a rate of 50 mL/min for 1 h prior to testing.
It can be seen form Fig. 5 that the surface films of the immersed alloys are enriched in Zr and Al, deficient in Co, and no Ag was detected in the surface films for all alloys. It has been reported that preferentially oxidation of Zr and Al in the surface film of the Zr-based BMGs occurs when they are exposed to air [14]. The Standard Electrode Potential (SEP), which is evaluated relative to the standard oxidation of hydrogen gas, measures the ability of the metal atoms to get oxidized. The SEP is −1.66 V for the reaction Al3+ + 3e− ↔ Al(s), −1.45 V for the reaction Zr4+ + 4e− ↔ Zr(s), −0.28 V for the reaction Co2+ + 2e− ↔ Co(s), and 0.7996 V for the reaction Ag + + e − ↔ Ag(s) [26]. A lower SEP corresponds to an easier oxidization reaction. When the Zr–Al–Co–Ag alloys were exposed to air, Zr and Al are preferentially oxidation in the surfaces due to their lower SEPs compared to those of Co and Ag. The SEP of the reaction forming Ag+ is the highest, which
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Fig. 4. XPS spectra for Zr53Al16(Co1 − xAgx)31(x = 0, 0.1, 0.25) specimens: (a) survey spectra and narrow scans for (b) Zr3d, (c)Al2p, (d) Co2p, and (e) Ag3d.
can be responsible for the absence of Ag+ ions in the surface film. Similar results can be observed in the XPS studies on the Ti–Zr–Cu–Pd–Sn and Ni–Pd–P–B BMGs [27,28], which found that the noble metal Pd element stays completely in the underlying alloy surface as a result of its highest SEP (0.915 V) of the reaction forming Pd2+ among all the constituents in the alloy systems. Immersion in the corrosive solutions results in the change in the composition in the surface films of specimens. The corrosion behavior of the Zr–Al–Co–Nb BMGs showed that Zr is further concentrated while Al content decreases in the spontaneously passivated films on the alloys after immersion in chloride medium compared to those exposed to air [14]. Therefore, after immersion in
PBS for 1 h, the surface films of Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs are enriched in Zr and Al and largely deficient in Co and Ag. Meanwhile, with increasing Ag content from x = 0 to x = 0.25, the concentration of Zr in the surface films on the BMGs immersed in PBS increases from 70.7% to 72.6%, while that of Al decreases from 26.9% to 25.2%. It has been reported that the high corrosion resistance of Zr–Al–Co–Nb BMGs is attributed to the formation of Zr 4+- and Al 3+-enriched oxide surface film in the corrosive solutions. With increasing Zr content and decreasing Al content in the surface films, the corrosion resistance of those Zr-based BMGs in chloride medium enhanced [14]. Moreover, a direct comparison on the corrosion resistance
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Fig. 5. the cationic contents in the surface films for the Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs after open-circuit immersion in PBS for 1 h.
of pure Zr and pure Al in chloride medium revealed higher pitting resistance of Zr than that of Al, which indicates that Zr4+ oxide is more stable than Al3+ oxide [29]. Thus, the addition of Ag is favorable for the alloys to form a protective surface film with higher chemical stability resulting in its higher corrosion resistance in PBS. The WST-1 assay results showed that the BMGs exhibited good biocompatibility. In the Zr–Al–Co–(Ag) system, Co ions are recognized to be the most cytotoxic among the constituents [13,16]. The low cytotoxicity of Zr–Al–Co–Ag BMGs can partly be attributed to their high corrosion resistance. The corrosion behavior and biocompatibility of metallic glasses or commercial metallic biomaterials have been extensively studied, the results show that high corrosion resistance of alloys can decrease the ions release in corrosive solution, which may resulting in their good biocompatibility [30–34]. For example, the study of metal ions release during corrosion of FeCobased amorphous metallic materials show that the total metal ions release of the amorphous alloys in PBS solution decreases with increasing corrosion resistance of those alloys. The Fe3Co67Cr3Si15B12
Fig. 7. SEM images for the MC3T3-E1 cell morphology on the surface of (a) Zr53Al16 (Co0.75Ag0.25)31 BMG and (b) Ti–6Al–4V alloy.
exhibits the best corrosion behavior in PBS and the fewest mass of metal ions released [31]. It has been reported that nanocrystalline 304 stainless steel (SS) shows higher corrosion resistance than that of microcrystalline 304 SS in artificial saliva, and the released potentially toxic ions such as Ni, Cr and Fe ions into artificial saliva from nanocrystalline 304 SS samples are much fewer than that of microcrystalline 304 SS, which resulting in the good biocompatibility of nanocrystalline 304 SS [32]. In addition, surface modification such as sandblasting or laser surface treatment (LSM) can significantly improve the corrosion resistance of NiTi alloy and reduce the release of toxic Ni ions in physiologically relevant environments, and the surface modified NiTi alloys exhibit an improved cytocompatibility [33,34]. As a result, it can be presumed that with the increase in Ag content, the corrosion resistance of Zr53Al16(Co1 − xAgx)31 (x = 0, 0.1, 0.25) BMGs significantly improves, and fewer Co ions will be released to the solutions. Meanwhile, partial replacement of Co by Ag reduces the nominal Co content of the BMGs. Therefore, Co substitution with Ag is expected to further reduce the potential cytotoxicity of Zr–Al– Co BMGs, although no obvious improvement was observed based on our 7 day proliferation experiments. Future efforts will be made on further evaluations regarding the long term biocompatibility of Zr– Al–Co–Ag BMGs. 5. Conclusions
Fig. 6. WST-1 assay results of Zr53Al16Co31 and Zr53Al16(Co0.75Ag0.25)31 BMGs after MC3T3-E1 cell proliferation for 7 days in comparison of the Ti–6Al–4V alloy.
The electrochemical properties, surface chemistry and cytotoxicity of Zr–Al–Co–Ag BMGs were investigated. The BMGs were spontaneously passivated with a low passive current density of the order of 10 − 1 A/m 2 and a wide passive potential region, indicating their high corrosion resistance in PBS. The addition of Ag to the alloy enhanced the pitting corrosion resistance in PBS. XPS analysis results show that a Zr- and Al-enriched passive film is formed on the BMGs
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after immersion in the solution. The increase in Ag content of the alloys results in an increase in Zr concentration and a decrease in Al concentration of the passive film, which is responsible for the high corrosion resistance of the alloys. WST-1 assay results show that Zr53Al16(Co0.75Ag0.25)31 BMG exhibits comparable cell viability and proliferation activity with those of Ti–6Al–4V alloy, demonstrating its good biocompatibility. Addition of Ag to the Zr–Al–Co BMG improved its corrosion resistance in PBS, which facilitates the MC3T3-E1 cell viability and proliferation activity. The high corrosion resistance and low in vitro cytotoxicity of Zr–Al–Co–Ag BMGs suggest an initial biocompatibility for biomedical applications. Future efforts will be made on further evaluations regarding the biocompatibility of Zr– Al–Co–Ag BMGs, in order to achieve a thorough understanding on the suitability of these materials to serve as biomaterials. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 51161130526). References [1] W.H. Wang, C. Dong, C.H. Shek, Bulk metallic glasses, Mater. Sci. Eng. R 44 (2004) 45–89. [2] A.L. Greer, E. Ma, Bulk metallic glasses: at the cutting edge of metals research, MRS Bull. 32 (2007) 611–619. [3] A. Inoue, Bulk amorphous alloys: practical characteristics and applications, Trans Tech Publications Ltd, Switzerland, 1999. [4] L. Huang, D.C. Qiao, B.A. Green, P.K. Liaw, J.F. Wang, S.J. Pang, T. Zhang, Biocorrosion study on zirconium-based bulk-metallic glasses, Intermetallics 17 (2009) 195–199. [5] M.Z. Ma, R.P. Liu, Y. Xiao, D.C. Lou, L. Liu, Q. Wang, W.K. Wang, Wear resistance of Zr-based bulk metallic glass applied in bearing rollers, Mater. Sci. Eng. A 386 (2004) 326–330. [6] W.H. Peter, R.A. Buchanan, C.T. Liu, P.K. Liaw, The fatigue behavior of a zirconiumbased bulk metallic glass in vacuum and air, J. Non-Cryst. Solids 317 (2003) 187–192. [7] S. Buzzi, K. Jin, P.J. Uggowitzer, S. Tosatti, I. Gerber, J.F. Löffler, Cytotoxicity of Zr-based bulk metallic glasses, Intermetallics 14 (2006) 729–734. [8] A. Yamamoto, R. Honma, M. Sumita, Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells, J. Biomed. Mater. Res. 39 (1998) 331–340. [9] A. Inoue, D. Kawase, A.P. Tsai, T. Zhang, T. Masumoto, Stability and transformation to crystalline phases of amorphous Zr–Al–Cu alloys with significant supercooled liquid region, Mater. Sci. Eng. A 178 (1994) 255–263. [10] T. Zhang, A. Inoue, New glassy Zr–Al–Fe and Zr–Al–Co alloys with a large supercooled liquid region, Mater. Trans. JIM 43 (2002) 267–270. [11] T. Wada, T. Zhang, A. Inoue, Formation, thermal stability and mechanical properties in Zr–Al–Co bulk glassy alloys, Mater. Trans. JIM 43 (2002) 2843–2846. [12] T. Wada, T. Zhang, A. Inoue, Formation and high mechanical strength of bulk glassy alloys in Zr–Al–Co–Cu system, Mater. Trans. JIM 44 (2003) 1839–1844. [13] T. Wada, F.X. Qin, X.M. Wang, M. Yoshimura, A. Inoue, N. Sugiyama, R. Ito, N. Matsushita, Formation and bioactivation of Zr–Al–Co bulk metallic glasses, J. Mater. Res. 24 (2009) 2941–2948.
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