In this study, the electrochemical responses of the as-cast Zr-, Ti-, and TiZr-based amorphous metallic alloys and the annealed Zr- and TiZr-based amorphous metallic alloys will be of the main focus. The flow chart of experimental procedures is shown in Figure 3.1.
In this chart, various amorphous metallic ribbons are fabricated by melt spinning, suction casting and co-sputtering. First, the rapid electrochemical screening methods (cyclic voltammetry and i-t amperometric analyses) are carried out in SBF for selecting potential candidates for biomedical applications. The MTT assay was also used for evaluating the in vivo biocompatibility for as-cast Zr-, Ti-, and TiZr-based amorphous metallic alloys. The
XRD, SEM/EDS were operated for measuring the composition and structure of the as-cast amorphous alloys.
Moreover, the electrochemically kinetic parameters were determined by polarization measurement and EIS, in vitro biocompatibility was also evaluated by implantation, the surface composition and structure of passive layer were studied by XPS for newly developed Ti42Zr40Si15Ta3 and Ti40Zr40Si15Cu5 amorphous metallic alloys. Besides, after various degrees of heat treatment, nanocrystalline phases can be produced in the originally fully amorphous matrix of Zr53Cu30Ni9Al8 and Ti42Zr40Si15Ta3, and the XRD and DSC were used for phase identification and crystallinity. The TEM images were captured for the direct evidence of the nanocrystalline phase formation in the amorphous matrix of Ti42Zr40Si15Ta3 after annealing.
The electrochemical responses of partially nanocrystalline and fully amorphous metallic alloys are determined by potentiodynamic polarization analysis and EIS for providing information of bio-corrosion behavior.
3.1 The preparation of amorphous metallic alloy ribbons
3.1.1 Raw Materials
In this research, the main focus will be placed on the Zr-, Ti- and TiZr-based metallic glasses. But some other common Fe- or Mg-based metallic glasses are also included for comparison. Eleven metallic glass ribbons, with nominal compositions of Fe70B20Si10
Mg65Cu25Gd10, Mg67Cu25Y8, Zr53Cu30Ni9Al8, Zr61Cu17.5Ni10Al7.5Si4, Zr53Cu30Al8Pd4.5Nb4.5, Ti65Si15Ta10Zr10, Ti40Cu36Pd14Zr10, Ti45Cu35Zr20, Ti40Zr40Si15Cu5 and Ti42Zr40Si15Ta3 (all in atomic percent) were produced utilizing the standard melt spinning process. All the composition were started with pure elements of Fe (99.99 wt.%), Si (99.99 wt.%), B (99.9 wt.%), Mg (99.99 wt.%), Cu (99.999 wt.%), Gd (99.9 wt.%), Y (99.9 wt.%), Zr (99.9 wt.%), Cu (99.999 wt.%), Ni (99.9 wt.%), Al (99.9 wt.%), Pd (99.9 wt.%), Nb (99.999 wt.%), Ti (99.99 wt.%), and Ta (99.99 at.%).
The MG ribbons were prepared firstly by induction melting, followed by rapid quenching via single roller melt-spinning under argon atmosphere [151], resulting in alloy ribbon samples with about 0.1 mm in thickness and about 10 mm in width. One thin film metallic glass sample with the nominal compositions of Ta57Zr23Cu12Ti8 was also produced by co-sputtering various alloy metal targets on microscope glass slides, with base pressure below 5 × 10−7 torr [152]. In addition, for the fabrication of the Zr53Cu30Ni9Al8 (all in atomic percent) bulk metallic plate with 2 mm thickness, the pure element of Zr (99.9 wt% purity), Cu (99.999 wt%
purity), Ni (99.9 wt% purity), and Al (99.99 wt% purity) were heated to melt and mixed to an
remelted and suction cast through the water-cooled Cu mold to form the 2 × 5 cm2 Zr53Cu30Ni9Al8 amorphous plates with high rapid cooling capability.
3.1.2 Melt spinning technique
The melt spinning technique with a more rapid cooling capability is recognized as a convenient way to prepare amorphous ribbons. The melt spinning technique and amorphous ribbons were operated and fabricated by Prof. Jang’s group in National Central University, Kaohsiung, Taiwan. Figure 3.2 displays the setup of a single-roller melt spinning technique [153].
The first step of this process is to put the alloy ingots and the pure metal into a pure iron-crucible with a thin layer of boron nitride (BN) which is covered on the inner surface [154]. The BN layer is able to prevent the interaction between alloys and the iron die at higher temperatures and to enhance the flowing rate of the melting alloys when pouring [154].
Several times of purged noble gas step are necessary before the melting spinning process carries out. Then, the iron-crucible is placed in an induction coil with the argon atmosphere of 1.5 atm [154]. When these ingots and pure metal are melted completely, the melt is poured onto the surface of copper wheel with a higher rotation speed of 15 m/s [154]. The melt is then cooled momentarily to become a thin ribbon type measuring 10 mm in width and about 100 μm in thickness. If the cooling rate is fast enough, the specimen would be fully amorphous [154].
3.1.3 Heat treatment of amorphous metallic ribbons
Then the amorphous plates were sliced to 0.4×0.4 cm2 for heat treatment and potentiodynamic polarization measurements. Thermal analysis and the carefully controlled preparation of different degrees of partially crystalline nano-phases in the Zr-based and Ti-based amorphous alloys were carried out by Perkin-Elmer Diamond differential scanning calorimeter (DSC) at constant heating rates of 40 K/min. The Zr53Cu30Ni9Al8 plates and Ti42Zr40Si15Ta3 ribbons were annealed at 713 K for 21, 23 and 40 min and 818 K for 10 min and 20 min under a protective argon atmosphere. After heat treatment, the outer oxidative layers of specimens were polished by P4000-grit abrasive papers and cleaned with acetone and ethanol by sonicator for other measurements. The picture of Perkin-Elmer DSC and scheme of power compensation DSC are presented in Figure 3.3 and Figure 3.4, respectively.
3.2 Microstructure and phase identification
3.2.1 X-ray diffraction (XRD) and nanoindentation
The nature of the amorphous metallic ribbons, prepared by melt spinning, and the thin film metallic glasses, fabricated by the magnetic sputtering deposition, are all examined by X-ray diffraction (XRD). The D8 X-ray diffractometry with Cu K radiation ( = 1.5406 A。), operated at 40 kV and 40 mA, and equipped with 0.02 mm graphite monochrometer, is utilized. The ranges of the diffraction angle 2θ of the specimens were covered from 20° to 80°, and every 0.1 steps for 4 seconds. The Young’s modulus and hardness of the produced MG samples were measured by a MTS Nanoindentation XP system (Figure 3.5) equipped
3.2.2 Optical microscopy (OM) observations
The optical microscopy is used to examine the basic morphology of the samples at lower magnifications. There are eyepiece with 10x and objective lens with 2x, 10x, 20x and 50x.
The roughness and amount of scratches were surveyed. The dirt and oxide could also be founded by OM and weeded out before the further analysis.
3.2.3 Scanning electron microscopy (SEM) observations
The JEOL JSM-6330 scanning electron microscopy (SEM) is selected to observe the microstructure and morphology of the amorphous metallic alloys, and is also used to check whether the compositions of the metallic glasses are mixed homogeneously. Moreover, JEOL JSM-6330 scanning electron microscopy (SEM) equipped with energy dispersive spectrometry (EDS) was used to observe the morphology and to verify the composition of the produced metallic glasses before and after electrochemical tests. In addition, the mapping function of the EDS system can also confirm the uniform atomic distribution.
3.2.4 Transmission electron microscopy (TEM) observations
To examine the crystalline phases of annealed TiZr-based amorphous metallic alloys, the transmission electron microscopy (TEM) was carried out for this purpose. The applied instrument is the FEI E.O Tecnai F20 G2 MAT S-TWIN Field Emission Gun TEM. In addition, the TEM specimens were observed by this TEM with operated voltage of 200 kV.
Moreover, the High Resolution Electron Microscopy (HREM) function was used to take the
image of interface between nanocrystalline phases and amorphous matrix for the annealed TiZr-based amorphous metallic alloys.
Furthermore, the cross-sectional TEM foil is fabricated by standard process of SEIKO SMI 3050 dual FIB system for amorphous and nanocrystllized amorphous metallic alloys.
There are three parts for this dual FIB system, including one beam for second-electron image to observe surface of the specimen, another beam for Ga ion beam to complete the etching function and observe cross-section images and patterns, and OM to check the position of the specimen on the substrate-supported holder. The standard procedures for fabricating the TEM foil by FIB system can be described as follows (Figure 3.6):
(1) For preventing the implantation of Ga ion on specimens during the fabricating processes, the carbon film is deposited on the surface of the specimen for the protection.
(2) The TEM foil with the trapezoid shape is fabricated by the slope-etching processes on the top and bottom site of the interesting area.
(3) The TEM foil with high thickness is further thinned by Ga ion beam with lower energy to the thickness lower than 100 nm.
(4) The bottom cut and side cut is fulfilled by Ga ion etching and then the TEM foil can be moved to carbon-coated Cu grid.
3.3 Thermal analysis
By using differential scanning temperature (DSC), the glasses forming ability parameters of as-cast amorphous metallic alloys such as Tg, Tx, Tm, and Tl can be detected. In
Perkin-Elmer Diamond DSC differential scanning calorimeter (DSC), operated at a constant heating rate of 40 K/min and a heating range from 323 to 873 K. Therefore, Tg, Tx, Tm and Tl
of amorphous metallic ribbons are quantified, and these parameters could provide the basis for heat treatment.
3.4 Immersion test under SBF
The simulation body-fluid test of Fe70B20Si10 Mg65Cu25Gd10, Mg67Cu25Y8, Zr53Cu30Ni9Al8, and Zr61Cu17.5Ni10Al7.5Si4 amorphous metallic ribbons were characterized by immersing these MGs into a 10 mL physiological isotonic solutions (the Hank's solution, pH:
6.5 and 2.0) at 37 °C. The typical Hank's solution is composed of 0.137 M of NaCl, 5.4 mM of KCl, 0.25 mM of Na2HPO4, 0.44 mM of KH2PO4, 1.3 mM of CaCl2, 1.0 mM of MgSO4, 4.2 mM of NaHCO3. Instead of using the common simulation body-fluid of Kokubo's solution, Hank's balanced salt solution was used for in-vitro tests for the biocompatibility of biomaterials due to the lower concentration of Cl− ion (148.8 mMfor Kokubo's solution, 143.7 mM for Hank's solution and 103.3 mM for human plasma). As reported by the previous studies, the concentration of chlorine ion is the major factor which causes the corrosion of MG metals such that the Hank's solution was used in the present study [81,155]. The pH values were verified by the pH meter.
3.5 Electrochemical analysis
The electrochemical properties of the amorphous metallic alloys and cp Ti in the SBF and the culture medium were firstly evaluated using a commercial electrochemical analyzer (CHI 614 D, CH Instruments Inc., USA). The electrochemical measurement was conducted
in a three-electrode scheme. The amorphous metallic ribbons were cut into small pieces with the dimension of around 4 x 4 mm2. The specimens served as the working electrode for the EC measurement where sputtered platinum film with the area of around 5 x 5 mm2 was used as the counter electrode and the reference electrode was with a standard Ag/AgCl. The MG ribbon was immersed into a 40 ml Hank’s solution (pH: 6.5) at 310 K for cyclic voltammetry (CV), potential state and potentiodynamic polarization measurements.
The CV measurement was carried out with the scanning potential from -1.0 V to +1.0 V and the scan rate of 0.1 V/s. In addition, materials used in medical applications may contact the living cells and expose to the membrane potential of 75-80 mV. A potential state measurement with a small applied voltage of 80 mV for 30 minutes was used to mimic the possible electrochemical reaction between the MG ribbons and cell tissues. Before the potentiodynamic polarization measurements, the as-cast and annealed Zr53Cu30Ni9Al8, as-cast and annealed Ti42Zr40Si15Ta3,Ti40Zr40Si15Cu5, Ti45Cu35Zr20 amorphous metallic alloys, and cp Ti were stabilized in SBF for 7200 to 12500 s with the criterion of the variation for the open circuit potential (OCP) less than 2.0 mV in 5 min. The potential scans were started from -0.5 V to 2.0 V with a 0.33 mV/s scanning rate. The important electrochemical parameters including corrosion potential (Ecorr) and corrosion current (Icorr) were determined by the Tafel extrapolation method. The AC impedance test was carried out with an amplitude of 10 mV in open-circuit potential and the frequency range is 10−2 to 104 Hz. The picture of CHI 614D electrochemical work station and schematic diagram of three electrode system are shown in Figure 3.7 and Figure 3.8, respectively.
3.6 Cell viability test
The standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (50 μg MTT in100 μl in PBS buffer, with pH: value about 7.35 to 7.40) was used to observe cell viability experiment of the metallic glasses. The vitro cell culture test of specimen was investigated using pluripotent mesenchymal cells. Balb/c mouse bone marrow stem cells, D1 cells (ATCC), were incubated in a low glucose Dulbecco’s modified Eagle’s medium (DMEM). The DMEM is composed of 10.0% fetal bovine serum (FBS), 1.5 g/L sodium bicarbonate, 1.0% NEAA, 1.0% Vitamine C and 1.0% penicillin and streptomycin [35]. The initial concentration for D1 cells were 5×104 cells/ml and prepared in a 6-well cell culture dishes. The MTT test was operated at 37 °C in the humidified atmosphere with 5%
CO2. Prior to the test, the specimens, including the MGs and the pure Ti with the explosion area of 25 mm2, were sterilized with 75% alcohol.
All the specimens were immersed into a 5 mL cultured medium for 72 hr with 3 individual repeating tests. Furthermore, this study tried to analyze potential toxic ions released from the MGs sample during the potential state test. The released ions may cause the death of the incubated cells in the medium. In this regards, the MTT assay culture medium after the potential state tests were filtered with a 0.2 μm Minisart® NML syringe filter. The D1 cell viability was again evaluated by the MTT assay with the DMEM medium after the potential state test at 37 °C for 24 hours. Cell viability was determined by measuring the cellular reduction of MTT to the crystalline formazan products which was dissolved in 100 μl dimethylsulfoxide (DMSO), and the absorbance for the solution at 570 nm was measured [156].
3.7 In vivo test
The in vivo test is majorly operated for evaluating the biocompatibility of the Ti42Zr40Si15Ta3,Ti40Zr40Si15Cu5, and Ti45Cu35Zr20 amorphous metallic alloys. For the in vivo tests, 6 male New Zealand white rabbits with the weigh about 2.5 to 3.5 kilogramswere purchased from the Taiwan Livestock Research Institute (Tainan, Taiwan), following Taiwan required ethical procedures. The rabbits were kept on a 12:12 light-dark cycle (light on at AM 06:00) and housed in a temperature-controlled room (25±1˚C). One hour before the operation, the rabbits were IM injected with Cefazolin (1gm/kg, YungShin, Taiwan) for anti-bacterial andthe Atropin (0.3 mg/kg, TTY Biopharm, Taiwan) for analgesia. Surgical procedures were anesthetized by the anesthetic of mixing Ketamine (40 mg/kg, Ketalar, Parke-Davis, Taiwan) with Xylazine (10 mg/kg, Rompun, Bayer HealthCare, Germany).
All the MG ribbons were sliced into small pieces with the size of around 3 mm x 3 mm.
The surgical sites are located below the epiphyseal growth plate of each right leg. The 3 mm x 3 mm x 0.5 mm fractures were made by medical saw for implantation and the wounds were then stitched by surgical sutures. The rabbits were then taken X-rays images on the surgery legs with 4.2 kV and the exposure time 3.5 s (HP 9178 A and HP 9816S; Hewlett- Packard, Fort Collins, CO, USA). The rabbits were recovered with a careful attention and finallyscarified 1 month after treatment. The retrieved legs with implantation were soaked in a10% formalin prior to the micro-computed tomography (μ-CT) observation. All proximal tibiae were assessed by μ-CT (Skyscan 1076: Skyscan, Antwerp, Belgium) after the tissues were removed. Data were collected at every 0.5° rotation step through 180°. The scanning width was 34 mm, and the height was 17 mm. The voxel size was isotropic and fixed at 8.7 μm [157]. Furthermore, the C-reactive protein (CRP) assay was carried out by Union Clinical Laboratory for checking whether any inflammation reaction would occur.