Bio-corrosion and Cytotoxicity Studies on Novel

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The bio-corrosion behavior of Zr- and Cu-based metallic glasses in different types of artificial body fluids such as artificial ... studied for biocompatibility so far have either Cu or Al ... Thus, we have embarked on synthesis of glassy alloy free ... counter electrode. ..... Cu60Zr20Ti20 glassy alloys in HBSS are given in Fig- ure 8.
Bio-corrosion and Cytotoxicity Studies on Novel Zr55Co30Ti15 and Cu60Zr20Ti20 Metallic Glasses S. VINCENT, A. DAIWILE, S.S. DEVI, M.J. KRAMER, M.F. BESSER, B.S. MURTY, and JATIN BHATT Metallic glasses are a potential and compatible implant candidate for biomedical applications. In the present investigation, a comparative study between novel Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses is carried out to evaluate in vitro biocompatibility using simulated body fluids. The bio-corrosion behavior of Zr- and Cu-based metallic glasses in different types of artificial body fluids such as artificial saliva solution, phosphate-buffered saline solution, artificial blood plasma solution, and Hank’s balanced saline solution is evaluated using potentiodynamic polarization studies at a constant body temperature of 310.15 K (37 C). Surface morphology of samples after bio-corrosion experiments was observed by scanning electron microscopy. In vitro cytotoxicity test on glassy alloys were performed using human osteosarcoma cell line as per 10993-5 guidelines from International Organization for Standardization. The comparative study between Zr- and Cu-based glassy alloys provides vital information about the effect of elemental composition on biocompatibility of metallic glasses. DOI: 10.1007/s11661-014-2574-9  The Minerals, Metals & Materials Society and ASM International 2014

I.

INTRODUCTION

NON crystalline nature in metallic glasses gives rise to remarkable combination of physical and chemical properties such as high yield strength, high hardness, excellent corrosion, and wear resistance. Low elastic modulus exhibited by metallic glasses, which inhibits stress shielding effect, is desirable for bio-implant applications.[1–3] Additionally, metallic glasses possess high strength-to-weight ratios which subsequently result in smaller implants and thus minimize the effect of foreign body response.[4,5] The chemical and structural homogeneity in metallic glasses and presence of single phase also favor biocompatibility. These properties are promising attributes for bio-implants especially for load-bearing applications. As a result, extensive studies on biological property evaluation of metallic glasses, especially on Zr-based systems are carried out.[6–8] Ni and Be are known to be cytotoxic and carcinogenic,[9] and the presence of such elements in metallic glasses may decrease the biocompatibility. Thus, the

S. VINCENT, Doctoral Student, and JATIN BHATT, Associate Professor, are with the Department of Metallurgical and Materials Engineering, Visvesvaraya National Institute of Technology, Nagpur 440010, India. Contact e-mail:[email protected] A. DAIWILE, Doctoral Student, and S.S. DEVI, Principal Scientist, are with the Environmental Health Division, National Environmental Engineering Research Institute, Nagpur 440020, India. M.J. KRAMER, Division Director, is with the Department of Materials Science and Engineering, Ames Laboratory, US Department of Energy, Iowa State University, Ames, IA 50011. M.F. BESSER, Assistant Scientist, is with the Ames Laboratory, US Department of Energy, Iowa State University. B.S. MURTY, Professor, is with the Department of Metallurgical and Materials Engineering, Indian Institute of Technology Madras, Chennai 600036, India. Manuscript submitted July 28, 2014. METALLURGICAL AND MATERIALS TRANSACTIONS A

emphasis is on synthesis of glassy alloys that are free from such cytotoxic elements. Accordingly, a series of Ni- and Be-free Zr-based and Mg-based glassy alloys were synthesized and studied for biocompatibility tests.[10–12] Liu and co-workers[13–18] developed a series of Ni-free glassy alloys such as Zr-Nb-Cu-Pd-Al, Zr-TiCu-Fe-Al, Zr-Nb-Cu-Fe-Al, and Zr-Co-Al-Ag to study their biocorrosion properties and cytotoxic effects. These Zr-based glassy alloys[13–18] are reported to exhibit excellent biocorrosion resistance in artificial body fluids in comparison with traditional bio-implant materials such as 316L stainless steel and Ti-6Al-4V alloys. Cell viability in Zr-based glassy alloys is also found to be better than traditional implant materials. Huang et al.[19] reported good corrosion resistance and better cell adhesion morphology of Zr-Cu-Al glassy alloy in comparison with biomedical Ti alloy. However, Zr-based glassy alloys that are extensively studied for biocompatibility so far have either Cu or Al content[13–19] in excess and it has been reported that excess of Cu and Al may result in biological toxicity.[20] Thus, we have embarked on synthesis of glassy alloy free from such cytotoxic elements which are common constituents in many metallic glasses. In the present investigation, a novel metallic glass composition Zr55Co30Ti15 is synthesized. The biocorrosion evaluation of melt-spun Zr55Co30Ti15 is carried out in various artificial body fluids, and it is compared with Cu60Zr20 Ti20 melt-spun glassy alloy. Earlier, corrosion behavior of Cu60Zr20Ti20 metallic glass has been evaluated in different environmental conditions such as acidic, neutral, and alkaline media.[21] It is important to determine biocompatibility of Cu60Zr20Ti20 and hence an attempt has been made in the present research work. In vitro cytotoxicity testing is carried out on both the glassy alloy samples according to 10993-5 specification from

ISO using human osteosarcoma (HOS) cell lines. The present work aims to provide an understanding through a detailed study on (i) corrosion resistance and surface morphology analysis in artificial body fluids and (ii) cell viability and cell morphology analysis of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys.

II.

EXPERIMENTAL

Alloy ingots with nominal chemical composition for Zr55Co30Ti15 and Cu60Zr20Ti20 (at. pct) are prepared by arc-melting pure mixtures of Zr, Cu, Co, and Ti (>99.9 wt pct) in a Ti-gettered argon atmosphere. Alloy ingots were re-melted several times to ensure chemical homogeneity. Zr55Co30Ti15 and Cu60Zr20Ti20 rapidly solidified glassy ribbon samples with cross sections (thickness 9 width) of 0.04 9 1.07 mm and 0.08 9 2.6 mm, respectively, were produced by melt spinning under argon flow on a Cu wheel with a moderate wheel speed of 20 m/s. X-ray diffraction (XRD) was performed using a PANalytical X’pert-Pro diffractometer with Cu-Ka (k = 0.154 nm) radiation on solid flat melt-spun ribbon surface in reflection mode with a beam size of 12 mm wide 9 5mm long at 40 kV and 40 mA. The corrosion behavior of Zr55Co30Ti15 and Cu60Zr20 Ti20 glassy ribbon samples is studied in various physiologically relevant environments by electrochemical polarization experiments using a VersaSTAT 3 potentiostat. This test was performed in a three-electrode cell configuration using a saturated calomel reference electrode (SCE) (USCE = 241 mV) and platinum gage counter electrode. Prior to electrochemical measurements, the specimens were ultrasonically degreased with acetone, rinsed with distilled water, and dried with Table I.

pulsed air. Tests were conducted in four different artificially simulated body fluid (SBF) conditions, namely, artificial blood plasma solution (ABP, pH 7.4), artificial saliva solution (ASS, pH 6.2), phosphate-buffered saline solution (PBS, pH 7.4), and Hank’s balanced saline solution (HBSS, pH 7.4) at a constant temperature maintained at 310.15 K (37 C). The compositions of the electrolytes are listed in Table I. All electrolytes were prepared from analytical reagent-grade chemicals using distilled water. Potentiodynamic polarization experiments were performed at a scan rate of 0.167 mV/s. The working electrode (WE) was exposed only to an area of 1 cm2, while the rest of the specimen was embedded in a thermoplastic resin to provide electrical isolation. The WE immersed in test solutions was allowed to attain a stable open circuit potential (~30 min). 500 mL of fresh test solution was used for every potentiodynamic polarization test. The polarization curves were measured at least three times to confirm reproducibility of the data. The surface morphologies of the ribbon samples before and after corrosion were examined using scanning electron microscopy (SEM) in back-scattered (BS) mode using a JEOL JSM-820. In vitro cytotoxicity evaluation on Zr55Co30Ti15 and Cu60Zr20Ti20 glassy ribbon samples is carried out according to International Organization for Standardization 10993-5[22] using HOS cell lines (CRL-1543). In this process, HOS cell lines were first cultured in minimum essential medium (MEM) supplemented with 10 pct fetal bovine serum, 100 U/mL penicillin, and 100 lg/mL streptomycin in a humidified atmosphere of 5 pct CO2 and 95 pct air at 310.15 K (37 C). The medium extracts were prepared as follows: the meltspun ribbon sample was first immersed in MEM and incubated at 310.15 K (37 C) in a humidified atmo-

Compositions (g/L) of Artificial Saliva Solution, Phosphate-Buffered Saline Solution, Artificial Blood Plasma Solution, and Hank’s Balanced Saline Solution Solutions

Composition (g/L) NaCl NaHCO3 NaH2PO4 KCl KSCN KH2PO4 Lactic Acid Na2HPO4Æ3H2O MgCl2Æ6H2O CaCl2 Na2SO4 CaCl2Æ2H2O MgSO4Æ7H2O Na2HPO4Æ7H2O Glucose

Artificial Saliva Solution (ASS, pH 6.2)

Phosphate-Buffered Saline Solution (PBS, pH 7.4)

Artificial Blood Plasma Solution (ABP, pH 7.4)

Hank’s Balanced Saline Solution (HBSS, pH 7.4)

1.5 1.5 0.5 — 0.5 — 0.9 — — — — — — — —

8.0 — 1.15 0.2 — 0.2 — — — — — — — — —

8.036 0.352 — 0.225 — — — 0.238 0.311 0.293 0.072 — — — —

8.0 0.35 — 0.4 — 0.06 — — — — — 0.19 0.2 0.09 1.0

METALLURGICAL AND MATERIALS TRANSACTIONS A

Cu60Zr20Ti20

Intesity (a.u)

Intesity (a.u)

Zr55Co30Ti15

20

30

40

50

2

60

70

80

90

20

30

40

50 60 2 (Degree)

Degree

(a)

70

80

90

(b)

Fig. 1—(a, b) XRD pattern of (a) Zr55Co30Ti15 and (b) Cu60Zr20Ti20 metallic glasses.

III.

RESULTS

Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses prepared by melt-spinning technique have been characterized by XRD as shown in Figures 1(a) and (b), respectively. The presence of broad diffused diffraction hump and absence of sharp crystalline peaks in both the XRD pattern confirm the amorphous structure of Zr55Co30Ti15 and Cu60Zr20Ti20 melt-spun alloys. A. Biocorrosion Studies The corrosion behavior of melt-spun Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys in different artificial SBF solutions is studied using potentiodynamic polarization experiments. Figure 2 shows the polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys in ASS. METALLURGICAL AND MATERIALS TRANSACTIONS A

0.1

Artificial Saliva Solution (ASS)

0.0 -0.1

Potential, E(V)

sphere of 5 pct CO2 and 95 pct air for 72 hour. The samples were removed from MEM medium after 72 hour of incubation, and extracts were then refrigerated at 277.15 K (4 C) before testing. 5000 cells/well were seeded in 96-well plates to measure in vitro cytotoxicity. After 24 hour, the medium was replaced with 200 lL of the extract, and the cells were allowed to grow in a humidified atmosphere with 5 pct CO2 at 310.15 K (37 C) for 1, 2, 3, and 4 days, respectively. The cell viability was investigated using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, 20 lL of MTT (5 mg/mL) was added to each well, and plate was incubated for 3 hour at 310.15 K (37 C) in a humid chamber away from light. Medium was removed after 3 hour of incubation, cells were resuspended in 200 lL of dimethyl sulfoxide, and the reading was taken at 550 nm using Tecan Infinite Pro 1000. 50,000 cells/well were seeded in a 6well plate to analyze cell morphology. Cells were allowed to adhere for 24 hour before the media were replaced by test extract. Cell morphology was analyzed using a Carl Zeiss Axiovert 40 inverted microscope for periods of 1 and 4 days.

Zr 55Co 30Ti 15 Cu 60Zr 20Ti 20

-0.2 -0.3 -0.4 -0.5 -0.6 -0.7 1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

1E-4

2

Current density, I (A/cm )

Fig. 2—The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses in artificial saliva solution (ASS) medium.

Tafel extrapolation of polarization curves is used to determine corrosion current density (icorr) and corrosion potential (Ecorr) values. The polarization curve for Zr55Co30Ti15 ribbon sample shows significant passive behavior in which the alloy resists further active dissolution process. In case of Cu60Zr20Ti20 ribbon, the current density increases to a high value after the Ecorr is reached, indicating high rate of dissolution. Also, no sign of passivity was observed in polarization curve of Cu60Zr20Ti20 glassy alloy. The values of icorr and Ecorr for both the melt-spun ribbon samples in ASS solution are shown in Table II. From Figure 2 and Table II, it is evident that Zr55Co30Ti15 glassy alloy exhibits icorr (9.8E 10 A/cm2) which is two orders of magnitude less in comparison with that of Cu60Zr20Ti20 sample (6.4E 8 A/cm2). The icorr value in E 10 A/cm2 clearly suggests an excellent corrosion resistance of Zr55Co30 Ti15 glassy alloy against ASS solution. Both the glassy alloys are found to exhibit similar Ecorr values in ASS medium (Table II).

Table II. The Corrosion Potential (Ecorr) and Corrosion Current Density (icorr) for Zr55Co30Ti15 and Cu60Zr20Ti20 Glassy Alloys in Different Simulated Body Fluids Type of SolutionMetallic Glassicorr (lA/cm2)Ecorr (V vs SCE) ASS PBS ABP HBSS

Zr55Co30Ti15 Cu60Zr20Ti20 Zr55Co30Ti15 Cu60Zr20Ti20 Zr55Co30Ti15 Cu60Zr20Ti20 Zr55Co30Ti15 Cu60Zr20Ti20

0.0009 0.0640 0.0076 0.0216 0.4930 0.0250 0.1770 0.0230

0.41 0.36 +0.46 0.12 0.23 0.30 0.24 0.31

However, the polarization curves demonstrate superior corrosion resistance of Zr55Co30Ti15 glassy alloy. The values of icorr and Ecorr in PBS medium determined from Figure 4 are shown in Table II. The icorr values of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys are found to be 7.6E 9 and 2.1E 8 A/cm2, respectively. Although both the alloys demonstrate very small value of icorr, Zr55Co30Ti15 exhibits one order of magnitude less in comparison to Cu60Zr20Ti20. This further suggests superior corrosion resistance of former alloy in comparison with latter. The Ecorr value of Zr55Co30Ti15 is positive (+0.46 V), while Cu60Zr20Ti20 exhibits negative Ecorr ( 0.12 V) which implies the lesser stability of latter alloy in comparison with former in PBS medium. The SEM results of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys after corrosion test in PBS are shown in Figures 5(a) and (b), respectively. The BS-SEM image of Zr55Co30Ti15 (Figure 5(a)) confirms that no damage has been caused to sample. The electrolytic ions in the solution only adhere to the surface. The passive film exhibits strong resistance to any type of chemical

Phosphate Buffered Saline Solution (PBS)

1.2 1.0

Cu 60Zr 20Ti 20 Zr 55Co 30Ti 15

0.8

Potential, E(V)

Further, to examine surface morphology of sample after corrosion, the glassy alloys were observed using SEM. The SEM image of Zr55Co30Ti15 and Cu60Zr20 Ti20 samples subjected to potentiodynamic polarization measurements in ASS medium are given in Figure 3(a) and (b), respectively. The back-scattered SEM (BSSEM) image in Figure 3(a) shows that electrolyte solution has only adsorbed to the surface of Zr55Co30Ti15 sample. No other chemical damage was observed on the sample surface, which again suggests superior corrosion resistance of Zr55Co30Ti15 metallic glass. The BS-SEM image of Cu60Zr20Ti20 shown in Figure 3(b) clearly reveals a smaller to larger size holes exhibiting pitting type of corrosion. The SEM results corroborate well with polarization curves (Figure 2) in which Zr55Co30Ti15 showed good resistance to dissolution process in ASS solution. Cu60Zr20Ti20 alloy undergoes high rate of dissolution as evident from SEM image (Figure 3(b)). The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys in PBS solution are shown in Figure 4. The polarization curve for Zr55Co30Ti15 exhibits large passive region, thus signifying high resistance of alloy to dissolution process. The polarization curve for Cu60Zr20Ti20 also exhibits considerable passive region before the alloy undergoes selective dissolution indicating good corrosion resistance of Cu60Zr20Ti20.

0.6 0.4 0.2 0.0 -0.2 -0.4 1E-12

1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

2

Current density, I (A/cm )

Fig. 4—The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses in phosphate-buffered saline (PBS) medium.

Fig. 3—(a, b): SEM micrographs of corroded surface for (a) Zr55Co30Ti15 and (b) Cu60Zr20Ti20 metallic glasses in ASS medium. METALLURGICAL AND MATERIALS TRANSACTIONS A

Fig. 5—(a, b) SEM micrographs of corroded surface for (a) Zr55Co30Ti15 and (b) Cu60Zr20Ti20 metallic glasses in PBS medium.

METALLURGICAL AND MATERIALS TRANSACTIONS A

0.0

Artificial Blood Plasma Solution (ABP) -0.1 -0.2

Potential, E(V)

damage to the surface. The BS-SEM image of Cu60Zr20 Ti20 (Figure 5(b)) demonstrates small pit formation on the surface. It should be noted that the severity by pitting in Cu60Zr20Ti20 alloy is much smaller in PBS in comparison with ASS media (Figure 3(b)). This can be correlated with polarization curves of Cu60Zr20Ti20 in ASS and PBS media (Figures 2 and 4). Cu60Zr20Ti20 in ASS does not display any passive region, and alloy readily undergoes dissolution and thus large pits are observed in SEM. While the passive layer formation on Cu60Zr20Ti20 in PBS may offer some resistance to dissolution and thus small pits are observed in Figure 5(b). Figure 6 shows polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 melt-spun alloys in ABP Solution. Zr55Co30Ti15 glassy alloy demonstrates a large passive region and thus possesses good resistance against dissolution process. While Cu60Zr20Ti20 glassy alloy does not show any passive region, current density increases to a very high value after Ecorr is reached. This hints that the sample undergoes high rate of dissolution process in ABP medium. The values of icorr and Ecorr for Zr55Co30Ti15 and Cu60Zr20Ti20 alloys were determined from Figure 6 and shown in Table II. Cu60Zr20Ti20 glassy alloy is shown to exhibit a lesser icorr value (2.5E 8A/cm2) in comparison with Zr55Co30 Ti15 alloy (4.9E 7 A/cm2). These findings suggest higher corrosion resistance of Cu60Zr20Ti20 than that of Zr55Co30Ti15 glassy alloy. However, Ecorr value of Zr55Co30Ti15 is less negative than that of Cu60Zr20Ti20 indicating higher stability of former alloy in comparison with the latter. The surface morphology of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys after polarization experiments in ABP solution is shown in Figure 7. The BSSEM image of Zr55Co30Ti15 alloy in Figure 7(a) shows that electrolytic ions adhered to the surface of the sample. Further, no damage or pit formation is observed, which demonstrate strong passive film formation and superior corrosion resistance in Zr55Co30Ti15 alloy. Cu60Zr20Ti20 alloy exhibits few numbers of larger pits as shown in Figure 7(b).

-0.3 -0.4 -0.5

Zr 55Co 30Ti 15 Cu 60Zr 20Ti 20

-0.6 -0.7 1E-9

1E-8

1E-7

1E-6

1E-5

Current density, I (A/cm2)

Fig. 6—The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses in artificial blood plasma (ABP) medium.

The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys in HBSS are given in Figure 8. Formation of large passive region can be observed in polarization curve of Zr55Co30Ti15 alloy signifying superior corrosion resistance of the sample. The current density increases to a high value once Ecorr is reached in case of Cu60Zr20Ti20 alloy, suggesting inability of sample to resist dissolution process. The values of icorr and Ecorr for Zr55Co30Ti15 and Cu60Zr20 Ti20 glassy alloys were determined from Figure 8 and shown in Table II. The icorr value of Cu60Zr20Ti20 (2.2E 8 A/cm2) is found to be less than that of Zr55Co30Ti15 (1.7E 7 A/cm2), indicating better corrosion resistance of former alloy than that of the latter. On the other hand, Zr55Co30Ti15 exhibits lesser negative Ecorr value than Cu60Zr20Ti20. The surface morphology observed by SEM is given in Figure 9. The BS-SEM image of Zr55Co30Ti15 in Figure 9(a) does not show pit formation, while larger pits are clearly observed in Cu60Zr20Ti20 (Figure 9(b)). Resistance to pit formation in Zr55Co30Ti15 alloy can be correlated to the formation

Fig. 7—(a, b) SEM micrographs of corroded surface for (a) Zr55Co30Ti15 and (b) Cu60Zr20Ti20 metallic glasses in ABP medium.

0.0

Hank's Balanced Saline Solution (HBSS)

-0.1

Potential, E(V)

-0.2 -0.3 -0.4

Zr 55Co 30Ti 15

-0.5

Cu 60Zr 20Ti 20

-0.6 -0.7 -0.8 1E-11

1E-10

1E-9

1E-8

1E-7

1E-6

1E-5

1E-4

1E-3

2

Current density, I (A/cm )

Fig. 8—The polarization curves for Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses in Hank’s balanced saline solution (HBSS) medium.

of strong passive regions as observed in polarization curve (Figure 8). The icorr values of some well-known biomaterials such as Ti-6Al-4V, Co-Cr-Mo, and 316L Stainless Steel alloys in PBS media at 310.15 K (37 C) as reported in[23] are found to be 3.9E 10, 3.3E 10, and 1.24E 9 A/cm2, respectively. It is interesting to note that Zr55Co30Ti15 also exhibits similar lower icorr (7.6E 9 A/cm2) value suggesting it to be a potential material for biomedical applications. Further, Ecorr values of Ti-6Al-4V, Co-Cr-Mo, and 316L Stainless Steel alloys in PBS media at 310.15 K (37 C)[23] are reported as 0.43, 0.44 and 0.22 V, respectively, whereas Zr55Co30Ti15 exhibits Ecorr value of +0.46 V signifying it to be more stable than existing biomaterials. B. Cytotoxicity Studies The in vitro cytotoxicity of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys assessed by evaluating the viability of HOS cells is given in Figure 10. The cells were cultured in the extracts for 1 to 4 days. As evident

from figure, Zr55Co30Ti15 exhibits higher percentage of cell viability in comparison with Cu60Zr20Ti20 alloy. This further demonstrates good biocompatibility of Zr55Co30Ti15 glassy alloy. It is also interesting to note that percentage of cell viability increases at 4th day after a slight decrease at 2nd and 3rd days. The morphology of HOS cells which were cultured on Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys after day 1 and day 4 of culture is given in Figure 11. For comparison, cell morphology of control is given in Figure 11(a). HOS cells cultured on Zr55Co30Ti15 alloy after day 1 (Figure 11(b)) and day 4 (Figure 11(c)) show no change in their morphology, indicating absence of cytotoxic effect on cells. This further validates the cytotoxicity results that Zr55Co30Ti15 alloy exhibits very high cell viability ratio. Further, cell morphology of HOS cells cultured on Cu60Zr20Ti20 alloy after day 1 and day 4 are given in Figures 11(d) and (e), respectively. Although cell viability ratio is found to decrease in case of Cu60Zr20Ti20 (Figure 10), no major changes in cell morphology were observed. Interestingly, in both the compositions, cell population is found to increase after day 4 of culture, which indicates good cell compatibility over the metallic glass surface. Similar observation is reflected in cell viability results obtained after day 4 of culture.

IV.

DISCUSSION

The corrosion behavior of Cu60Zr20Ti20 glassy alloy in SBF medium reveals pitting corrosion. On the other hand, Zr55Co30Ti15 metallic glass exhibits strong resistance to pit formation in all physiological environments. In general, metallic glasses demonstrate high resistance to localized pitting corrosion owing to their non crystalline structure which lack grain boundaries or surface defects which are preferential sites for corrosion. However, chemical heterogeneities, composition fluctuations, and possible presence of defects in cast sample can act as an initiation point for pitting corrosion. The SBF medium is composed of aggressive compounds METALLURGICAL AND MATERIALS TRANSACTIONS A

Fig. 9—(a, b) SEM micrographs of corroded surface for (a) Zr55Co30Ti15 and (b) Cu60Zr20Ti20 metallic glasses in HBSS medium.

Zr55Co30Ti15 Cu60Zr20Ti20

100

% Viability

80

60

40

20

0 Control

1

2

3

4

Time in culture (days)

Fig. 10—Cytotoxicity of HOS cells cultured in Zr55Co30Ti15 and Cu60Zr20Ti20 metallic glasses.

(Table I) involving chloride, phosphate, and sulfate ions, and it is well known that pitting corrosion accelerates in such corrosive atmosphere. Chlorideinduced pitting corrosion is reported to be more aggressive due to the presence of nanocrystallites in glassy matrix.[24,25] Earlier, transmission electron microscopy analysis on Cu60Zr20Ti20 melt-spun ribbon by present authors[26] detected the presence of nanocrystals (~2 to 5 nm) in glassy matrix. The presence of such nanocrystals acts as an initiation site and could be possible reason for pitting corrosion in Cu60Zr20Ti20 glassy alloy. The mechanism for pitting corrosion[21] is that chloride ions tend to be preferentially absorbed at the interface between the nanocrystals and glassy matrix. Pits are easily initiated at this interface and thus breaking passive film. Consequently, weak interfacial bond is created between glassy matrix and nanocrystals leading to detachment of former from latter, thereby resulting in pits formation. It is interesting to note that tendency to pit formation is METALLURGICAL AND MATERIALS TRANSACTIONS A

minimized for Cu60Zr20Ti20 in PBS medium (Figure 5(b)) in comparison with ASS, ABP, and HBSS media. This could be due to less number of aggressive ions present in PBS medium (Table I). This further indicates that the rate of pit formation gets accelerated in the presence of more aggressive ions such as chloride ions, resulting in deeper and larger pits. Interestingly, no such pit formation is observed in case of Zr55Co30Ti15 glassy alloy. This suggests that Zr55Co30Ti15 glassy ribbon sample is probably free from nanocrystals and chemical heterogeneities. In absence of such active sites, breaking of passive film becomes difficult, and thus Zr55Co30Ti15 glassy alloy exhibits superior resistance to pitting. The corrosion behavior of Zr55Co30Ti15 and Cu60Zr20 Ti20 can also be explained on the basis of selective dissolution of galvanically active metals. The standard equilibrium electrode potentials[27] with respect to normal hydrogen electrode for the Zr/Zr4+, Ti/Ti2+, Cu/ 1.529, 1.628, Cu2+, and Co/Co2+ couples are +0.337, and 0.28 V, respectively. In case of Cu60Zr20 Ti20, there exists a large electrochemical potential difference between Cu and Zr (Ti), which acts as a driving force for selective dissolution of less noble Zr/Ti metals. Further, the selective dissolution of less noble metals is enhanced by the presence of weak regions such as glass/crystal interface. In the case of Zr55Co30Ti15, the difference in electrochemical potential between Co and Zr (Ti) is less, and thus the driving force required for selective dissolution is inadequate. Evaluation of biocompatibility is important before any implant applications. Zr55Co30Ti15 glassy alloy exhibits good biocompatibility, since negligible cytotoxic effect is observed under examined conditions. Conversely, Cu60Zr20Ti20 glassy alloy exhibits moderate toxicity on 2nd and 3rd days with cell viability of 65 and 72 pct, respectively, which on 4th day of cell culture increases to 82 pct (Figure 10). It is well known that Zr, Ti, and Co are nontoxic metals with good biocompatibility.[28,29] Although Cu ions are essential nutrient, it is reported[20] that excessive Cu ions may lead to biological toxicity. The Cu ions released due to localized corrosion

20 µm

(a)

20 µm

(b)

20 µm

(c)

20 µm

(d)

20 µm

(e)

Fig. 11—(a through e) Cell morphology of HOS cells in (a) Control; Cultured in Zr55Co30Ti15 after (b) day 1 and (c) day 4; Cultured in Cu60Zr20Ti20 after (d) day 1 and (e) day 4.

and selective dissolution of active metals lead to cytotoxic effects, and hence Cu60Zr20Ti20 is reported to display less biocompatibility. The release of cytotoxic elements also depends on stability of metallic glass composition. The stability of Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys can also be explained from a thermodynamics point of view. Lower enthalpy of chemical mixing values among constituent elements lead

to stable metallic glass compositions.[30,31] The enthalpy of chemical mixing values for Zr55Co30Ti15 and Cu60Zr20Ti20 glassy alloys calculated using Gallego’s approach[32] are 24.6 and 13.5 kJ/mol, respectively. This indicates that Zr55Co30Ti15 is a thermodynamically more stable metallic glass composition in comparison with Cu60Zr20Ti20. Consequently, the release of metal ions to the cell culture and SBF media is less in the METALLURGICAL AND MATERIALS TRANSACTIONS A

former in comparison with the latter. Thus, the present study indicates better cell compatibility of Zr55Co30Ti15 glassy alloy under adopted physiological test environments.

V.

CONCLUSIONS

A novel Zr55Co30Ti15 metallic glass is found to exhibit excellent corrosion resistance in SBF conditions with no changes in surface morphology. Although, Cu60Zr20Ti20 shows good resistance to corrosion, it is prone to pitting corrosion due to the presence of nanocrystals in glassy matrix. Zr55Co30Ti15 also demonstrates low cytotoxic effects and thus can be a promising candidate for biomedical applications.

ACKNOWLEDGMENTS One of the authors, Jatin Bhatt, is thankful to INDO US Science and Technology forum for Fellowship award (IUSSTF Fellowship/16-2012) to carry out part of above research work at the Ames Laboratory (US-DOE), Iowa State University. This work was supported by the U.S. Department of Energy (DOE), Office of Basic Energy Science, Division of Materials Science and Engineering. The research was performed at the Ames Laboratory, which is operated for the U.S. DOE by Iowa State University under contract DE-AC02-07CH11358. The authors’ are also thankful to Technical Education Quality Improvement Program (TEQIP II), Government of India for providing financial assistance.

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