Structural Analysis, Electrochemical Behavior, and Biocompatibility of Novel Quaternary Titanium...

Structural Analysis, Electrochemical Behavior,and Biocompatibility of Novel Quaternary TitaniumAlloy with near b Structure

MONICA POPA, JOSE MARIA CALDERON MORENO, CORA VASILESCU,SILVIU IULIAN DROB, ELENA IONELA NEACSU, ANDREJ COER,JULIJA HMELJAK, GREGOR ZERJAV, and INGRID MILOSEV

This article analyses the microstructure, electrochemical behavior, and biocompatibility of anovel Ti-20Nb-10Zr-5Ta alloy with low Young’s modulus (59 GPa) much closer to that ofbone, between 10 and 30 GPa, than Ti and other Ti alloys used as implant biomaterial. XRDand SEM measurements revealed a near b crystalline microstructure containing b phase matrixand secondary a phase, with a typical grain size of around 200 lm. The corrosion behavior inneutral Ringer solution evidenced: self-passivation behavior characterizing a very resistantpassive film; an easy passivation as a result of favorable influence of the alloying elements Nb,Zr, and Ta that participate with their passive oxides to the formation of the alloy passive film;low corrosion and ion release rates corresponding with very low toxicity. In MEM solution, thenovel alloy demonstrated very high corrosion resistance and no susceptibility to localized cor-rosion. Biocompatibility was evaluated on in vitro human osteoblast-like and human immor-talized pulmonary fibroblast cell (Wi-38) lines and the new Ti-20Nb-10Zr-5Ta alloy exhibitedno cytotoxicity. The new Ti-20Nb-10Zr5Ta alloy is a promising material for implants due tocombined properties of low elastic modulus, very low corrosion rate, and good biocompati-bility.

DOI: 10.1007/s11661-014-2254-9� The Minerals, Metals & Materials Society and ASM International 2014

I. INTRODUCTION

TITANIUM is themost usedmetal for implants due toits excellent corrosion resistance, biocompatibility, andmechanical properties.[1,2] Titanium alloys that contain Vand Al[3–5] may cause long-term problems, such as periph-eral neuropathy, osteomalacia, and Alzheimer’s dis-eases.[6,7] Also, their high modulus produced insufficientloading of the bone adjacent to the implant, the so-calledstress shielding phenomenon that can lead to the potentialbone resorption and eventual failure of the implant.[6–8]

Therefore, it is imperative to develop new alloys to satisfythe necessities of low toxicity and elastic modulus.

Recently, b-type titanium alloys were designed. Thesealloys contain non-toxic and non-allergic metals Nb, Zr,Ta, Mo, and Sn, also b-stabilizing elements.[9,10] More-over, the alloys with Ti, Nb, Zr, Sn, and Ta have lowYoung’s modulus, around 60 GPa, close to that of bone

(10 to 30 GPa).[11] The first b-type alloy with goodmechanical and compatibility properties was Ti-13Nb-13Zr.[12] Okazaki et al.[13,14] examined the corrosionresistance, mechanical properties, corrosion fatiguestrength, and cytocompatibility of new b-type Ti-15Sn-4Nb-4Ta-0.2Pd-0.2O alloy[13] and Ti-15Zr-4Nb-4Ta-0.2Pd-0.2O-0.05N alloy[14] that exhibited very goodproperties and biocompatibility. Two novel orthopedicalloys Ti-34Nb-9Zr-8Ta with Young’s modulus of89 GPa and Ti-13Mo-7Zr-3Fe with Young’s modulusof 86 GPa were studied by Barnejee et al.[15] concerningtheir microstructure and strengthening mechanisms. Ti-24Nb-4Zr-7.9Sn alloy subjected to microarc oxidationtreatment forms a porous surface with biologicallyactive bone-like apatite layer which may improve thebiological response of the implants.[16] Taddei et al.[17,18]

obtained Ti-35Nb-7Zr-5Ta alloy by powder metallurgy,by sintering, solution treatment, and aging; this alloypresented a homogeneous microstructure, very goodYoung’s modulus of 55 GPa, and high biocompatibility.In Japan the Ti-29Nb-13Ta-4.6Zr alloy[19–23] that evi-denced nobler behavior compared to Ti-15Mo alloy wasproduced;[19] its surface was covered by calcium phos-phate[20–22] to increase its bioactivity and bone bindingstrength. A novel near b Ti-5Zr-3Sn-5Mo-15Nb[24] wasdeveloped; this alloy possesses a low elastic modulus(69 GPa) and good mechanical properties; its surfacewas modified by microarc oxidation. Ti-15Zr-4Nb-4Taalloy[25] free from cytotoxic elements displays highmechanical strength and high corrosion resistance; by

MONICA POPA, JOSEMARIA CALDERONMORENO, CORAVASILESCU, and SILVIU IULIAN DROB, Senior Researchers, andELENA IONELA NEACSU, Researcher, are with Institute of PhysicalChemistry ‘‘Ilie Murgulescu’’, Romanian Academy, Spl. Independentei202, PO BOX 12-194, 060021 Bucharest, Romania. Contact e-mail:[email protected] ANDREJ COER, Professor, andJULIJA HMELJAK, Researcher, are with College of Health Care,University of Primorska, 6310 Izola, Slovenia. GREGOR ZERJAV,Ph.D. Student, and INGRID MILOSEV, Head of Department, ChiefScientist, are with Jozef Stefan Institute, Jamova c. 39, 1000 Ljubljana,Slovenia.

Manuscript submitted July 24, 2012.Article published online March 15, 2014

3130—VOLUME 45A, JUNE 2014 METALLURGICAL AND MATERIALS TRANSACTIONS A

calcium solution treatment, followed by heat and hotwater treatments was induced the apatite-forming abil-ity. The new Ti-15Mo-1Bi alloy showed no cytotoxicityin in vitro test and demonstrated superior ability toretain bone in the in vivo implantation experiment.[26]

Binary Ti-50Zr alloy[27,28] revealed very good mechan-ical properties and superior biological activity and couldbe used as bioalloy.

Corrosion resistance is a very important propertybecause the ions released from implant into the sur-rounding tissues can affect its biocompatibility andmechanical integrity and can produce undesirable reac-tions in body.[29] So, the evaluation of the corrosionresistance in simulated human fluids represents one of thefirst stages in the procedure for the evaluation of a newalloy as implant material.[29] The next stage is the in vitrobiocompatibility. A focus on ‘‘tissue-tailored’’ biocom-patibility testing has developed in recent years, withemphasis on selection of appropriate in vitro cell modelsfor biocompatibility testing protocols.[30] Althoughin vitro testing can explain only parts of the candidateimplant biomaterial-recipient tissue interaction, it is asuitable, affordable, and safe method for initial assess-ment of both safety and efficiency of the biomaterial.

The suitability of a material for implant use necessitateslow specific weight and elastic modulus, good mechanicalproperties, high corrosion resistance, and biocompatibil-ity.[29,31] Majority of the recent binary, ternary, andquaternary biocompatible beta Ti-based alloys do notsatisfy all these conditions. The novel Ti-20Nb-10Zr-5Taalloy contains only non-toxic elements and combines theconditions of low specific weight and Young’s modulus(59 GPa) with very good mechanical properties,[32] cor-rosion resistance, and biocompatibility; also, this alloywas designed to resist in complex conditions of the humanbody regarding the concentration and pH gradients of thephysiological fluids. This alloy has a near b structure thatconfers it innovative properties; so a microstructuralanalysis (by XRD and SEM) and electrochemical tests inbiofluids were necessary to determine their effect on thealloy corrosion resistance; also, in vitro biocompatibility(human osteoblast-like and fibroblast cell lines) of a newTi-20Nb-10Zr-5Ta alloy was studied.

II. MATERIALS AND METHODS

A. Alloy and Sample Obtaining

Ti-20Nb-10Zr-5Ta novel alloy was obtained as ingotsby vacuum semi-levitation melting in a high-frequency

induction furnace with a cold crucible, type Fives Celles.The alloy synthesis was performed in two steps, meltingand re-melting at 2273 K (2000 �C) with cooling stagesinside furnace. The following materials were used forcomparative measurements: Ti (99.6 pct, annealed,2 mm foil); Ti-6Al-4V alloy (annealed, 2 mm foil)supplied by Goodfellow; and Ti-6Al-7Nb samples cutfrom femoral component of the commercial hip pros-thesis (Plus Orthopedics, Smith&Nephew).The mechanical properties of the new alloy[32]

revealed lower Young’s modulus-E (close to that ofbone) and the appropriate ultimate tensile strength-UTS, 0.2 pct yield strength-0.2 pctYS (Table I) withCP-Ti and Ti-6Al-4V ELI, Ti-6Al-7Nb alloys (all in theas-cast state). Therefore, the mechanical properties ofthe new alloy are very proper for an implant material.

B. Alloy Structural Analysis

The structural analysis was carried out on disks thatwere mechanically polished with abrasive paper until2000 grit and with a 1 lm alumina suspension to mirrorsurface; then, the samples were chemically etched in68 pct glycerin, 16 pct HF, and 16 pct HNO3 solution.The alloy microstructure was studied using a EVO

LS10 scanning electron microscope (SEM), with second-ary electron (SE) imaging and with a Quanta 3D FEGSEM with back-scattered electron (BSE) imaging, oper-ated at an accelerating voltage of 20 kV. The structureand composition of the layer deposited on the alloysurface after 2000 immersion hours in Ringer solution ofpH 7.58 were investigated with a Quanta 3D FEG SEMoperated at accelerating voltage of 15 kV, equipped withan energy-dispersive X-ray (EDX) spectrometer.The phase identification of the new alloy was carried out

by X-ray diffractometry (XRD) with a Rigaku Ultima IVdiffractometer h-2h using CuKa k = 1.5406 A radiationover the range 10< 2h/deg< 90 at 40 kV and 30 mA.

C. Alloy XPS Analysis

X-ray photoelectron spectroscopy (XPS) surface ana-lysis of the as-prepared film and of the films depositedon the alloy surface after 2000 hours in neutral Ringersolution was performed on Quantera SXM equipmentwith a base pressure in the analysis chamber of1.33 9 10�7 Pa. The X-ray source was Al Ka radiation(1486.6 eV, monochromatized) and the overall energyresolution is estimated at 0.75 eV by the full width athalf maximum (FWHM) of the Au 4f7/2 line.

Table I. Mechanical Properties of the New Ti-20Nb-10Zr-5Ta Alloy in Comparison with Conventional Implant Materials

(All in the As-cast State)

Property CP-Ti* Ti-6Al-4V* Ti-6Al-7Nb* Ti-20Nb-10Zr-5Ta

E (GPa) 105 114 105 59UTS (MPa) 344 860 1000 883EL (pct) 20 15 12 6.280.2 Pct YS (MPa) 170 to 244 799 900 566

*ASM data base.

METALLURGICAL AND MATERIALS TRANSACTIONS A VOLUME 45A, JUNE 2014—3131

D. Alloy Electrochemical Behavior in PhysiologicalSolutions

Two different physiological solutions were used:neutral Ringer of pH 7.58 and minimum essentialmedium (MEM) of pH 6.9 to 7.1 (by addition of NaOHsolution).

Ringer solution composition was (g/L): NaCl-6.8;KCl-0.4; CaCl2-0.2; MgSO4Æ7H2O-0.2048; NaH2PO4ÆH2O-0.1438; NaHCO3-1; and glucose-1.

MEM with Earle’s balanced salts supplied by SAFCBiosciences contains (g/L): CaCl2-0.2; MgSO4-0.09767;KCl-0.4; NaCl-6.8; NaH2PO4ÆH2O-0.14; eight differentvitamins; 20 different non-essential amino acids; andother compounds.

Solutions were prepared using Milli-Q Direct water(resistivity of 18.2 MX cm at 298 K (25 �C).

The electrochemical techniques of cyclic potentiody-namic and linear polarization were used.

1. In neutral Ringer solutionDisks with diameter of 10 mm were cut from ingots

and then were polished with metallographic paper until2000 grit and with 1 lm aluminum oxide to mirrorsurface. Then, the electrodes were rinsed with Milli-QDirect water at 298 K (25 �C), ultrasonically degreasedin acetone and Milli-Q Direct water for 15 minutes, anddried in a stream of nitrogen. Electrochemical measure-ments were carried out after 30 minutes of stabilizationat the open circuit potential. The cyclic potentiodynamicpolarization was applied beginning from �0.5 to+4.0 V (vs saturated calomel electrode, SCE) using ascan rate of 1 mV/s. Voltalab 80 equipment with itsVoltaMaster 4 program were used. From voltammo-grams, the main electrochemical parameters were deter-mined: Ecorr—corrosion potential, like zero currentpotential; Ep—passivation potential at which the currentdensity is constant; |Ecorr � Ep| difference represents thetendency to passivation (low values characterize a good,easy passivation); DEp—passive potential range of theconstant current; and ip—passive current density. Thelinear polarization measurements (Tafel) were per-formed for a range of ±100 mV around the open circuitpotential, with a scan rate of 1 mV/s. The same Voltalab80 equipment with its VoltaMaster 4 program thatdelivered the values of the corrosion current densities(icorr) and rates (Vcorr), and polarization resistance (Rp)obtained from Tafel curves was used.

The total quantity of the ions (ng/cm2) released in thesolution was determined.[33]

2. In MEM solutionSamples were cut in the shape of disks with 15 mm

diameter. Preparation of the samples was realized bymechanical grinding under a stream of water succes-sively up to 4000-grit SiC papers. The samples werecleaned with acetone, ethanol, and double-rinsed Milli-Q water in an ultrasonic bath for 2 minutes, and finallydried in a stream of nitrogen. Measurements wereperformed in a three-electrode corrosion cell (volume250 mL, Autolab, Metrohm Autolab) at a temperatureof 37 ± 0.1 �C. A specimen embedded in a Teflon

holder, with an area of 0.785 cm2 exposed to thesolution, served as the working electrode. An Ag/AgClelectrode (0.197 V vs standard hydrogen electrode,SHE) was used as reference electrode and carbon rodsas counter electrode. Potentials in the text are givenrespective to this reference electrode. Electrochemicalexperiments were carried out with an Autolab PGSTAT302N potentiostat/galvanostat and controlled by Nova1.6 and Nova 1.7 software. Prior to measurements,samples were allowed to stabilize at the open circuitpotential, Eoc, for 1.5 hour. The stable quasi-steadystate potential reached at the end of the stabilizationperiod is denoted as the corrosion potential, Ecorr.Electrochemical measurements were carried out follow-ing stabilization. Linear polarization measurementswere applied in the potential range ±10 mV vs Ecorr,using a 0.1 mV/s potential scan rate. Values of polar-ization resistance, Rp, were deduced from the slope offitted current density vs potential lines, using Novasoftware. Cyclic polarization measurements were re-corded with 1 mV/s potential scan rate, starting at250 mV negative to Ecorr. The potential was thenincreased in anodic direction up to the pre-determinedswitching potential, Esw. For Ti-20Nb-10Zr-5Ta and Ti-6Al-7Nb alloys and Ti metal, Esw was +6.0 and +3.0 Vfor the Ti-6Al-4V alloy. The potential was then reversedand progressed in the cathodic direction to a potential atwhich the current density changes the polarity.For each material, measurements were performed at

least in duplicate and the average values of the corrosionparameters are reported.

E. Biological Tests

1. Cell cultureA human osteoblast-like cell line (HOS; ATCC no.

CRL-1543, kindly gifted by prof. Janja Marc, Univer-sity of Ljubljana, Slovenia) and a human immortalizedpulmonary fibroblast cell line (Wi-38 VA-13 subline2RA; ATCC no. CCL-75.1) were used for in vitrobiocompatibility experiments. Both cell lines were cul-tured in MEM supplemented with 10 pct fetal bovineserum and 15lg/ml gentamicin (all Gibco) at 310 K(37 �C) in a humidified atmosphere with 5 pct CO2.

2. BiocompatibilityFor biocompatibility assessment of the novel bioalloy

and comparing alloys, an extraction in MEM wasperformed,[34] with an extension of the extraction

Table II. Toxicity Grades and their Corresponding

Percentages of Proliferating Cells

Toxicity GradePercentage [Pct] of

Proliferating Cells (P)

0 81 to 1001 61 to 802 41 to 603 21 to 404 0 to 20


timeframe. Briefly, sterilized disks of all four metalswere immersed in serum-free MEM supplemented withantibiotics, for 25 days at 310 K (37 �C). Simulta-

neously, a sample of serum-free MEM without theaddition of metal was incubated in the same conditionsand used for unexposed controls. After 25 days, themetal disks were aseptically removed from the extractand 10 vol pct fetal bovine serum was added to eachvial. HOS and Wi-38 cells were seeded in wells of 12-welltissue culture plates (TPP, Trasadingen) at appropriatedensities and grown in either negative control mediumor metal extract media for 48, 96, or 144 hours.

3. Alamar Blue assayCell proliferation was evaluated using the fluores-

cence-based AlamarBlue� assay (Invitrogen) accordingto the manufacturer’s protocol. Briefly, 500 cells per wellin 90 lL fresh growth medium were seeded in black flat-bottom 96-well cell culture plates and incubated for daysat the above-mentioned growing conditions, followed byaddition of 10 lL per well of AlamarBlue reagent and asubsequent 2 hours incubation. Fluorescence was mea-sured with a GENios plate reader (Tecan) and relativenumbers of proliferating cells were calculated withuntreated cells used as reference. Percentages of prolif-erative cells as measured after 5 days (P) were used forcalculation of the cytotoxicity grades (Table II).

Fig. 2—Optical micrograph (a) and SEM micrographs in SE mode of (b) the as-casted and (c, d) chemically etched surfaces of the Ti-20Nb-10Zr-5Ta alloy.

Fig. 1—XRD pattern of Ti-20Nb-10Zr-5Ta alloy.


4. Clonogenic assayAfter treatment with extract or control media, cell

survival was evaluated using the clonogenic assay aspreviously described.[35] Briefly, HOS and Wi-38 cells(500 and 1000, respectively) were seeded on 6-cm Petridishes (TPP) in 4 mL fresh complete growth mediumand incubated for 7 to 12 days, followed by crystalviolet (Sigma) staining and manual counting of colonies.Only colonies with more than 50 cells were counted. Thesurvival fraction (SF) was determined by calculatingplating efficiencies (PE = ratio between coloniescounted and cells initially seeded) for all experimentsand control and subsequently normalizing the PEs ofexperiments to the PEs of unexposed controls.

All in vitro experiments were repeated three times.Student’s t test and One-Way ANOVA were used forstatistical analysis and statistical significance was set atp< 0.05.

III. RESULTS

A. Microstructure of the Novel Ti-20Nb-10Zr-5Ta Alloy

XRD measurements (Figure 1) determined a near bcrystalline structure consisting mainly of b phase (JCPDSNo. 44-1288), with a calculated lattice parameter of thecubic b phase of 3.268 A, and some minor content of aphase. The right inset in Figure 1 shows the region of themain b peak and the weak feature proving the presence ofresidual a phase alloy, the left inset shows the region72< 2h < 80—indicating the absence of omega phaseprecipitation in the b phase alloy. The casting dendritestructure with a superimposed polygonal structureappeared only in the superior part of the ingot due tothe recrystallization of the alloy in the time of the vacuumcooling in the levitation furnace.[32] The grain size of bphase matrix, around 200 lm, was determined fromoptical micrographs (Figure 2(a)). SEM of polishedsurfaces (Figure 2(b)) evidences the typical surface topog-raphy, residual porosity, and polishing scratches of theas-cast alloy, while chemically etched surfaces reveal thetwo-phase structure (Figure 2(c)). The preferentiallyetched areas do not seem to have a specific pattern, theyare intersecting freely the grains of the alloy; neitherpreferentially located at grain boundaries nor inside thegrains. Higher magnification SEM analysis (Figure 2(d))does not identify clearly the presence of ordered lamellarstructures at the boundaries of the b grains or precipitatesinside the grains. The alloy surface was thus studied usingBSE-SEM.

BSE-SEM micrographs can reveal the distribution ofstabilizing elements, thus ascertain if the microstructuralfeatures observed after differential etching of the alloysurface can be correlated with the distribution of a and bphases. Brighter areas reflect a higher content ofalloying elements Nb, Zr, and Ta, heavier than Ti.The darker areas with high concentration of Ti (a-stabilizer) indicate the localization of a-phase precipi-tates inside the b grains. BSE-SEM micrographs(Figure 3) show in fact a fairly homogeneous distribu-tion of the alloying elements, without sharp boundaries

or lamellar patterns; there are scattered darker zonesthat indicate the crystallization of precipitates of the aphase inside the b grains.

B. XPS Analysis for the As-prepared Filmon Ti-20Nb-10Zr-5Ta Alloy Surface

The deconvoluted XPS spectra of the as-prepared filmon the Ti-20Nb-10Zr-5Ta alloy surface exhibited[36]:the binding energies of Ti doublet peaks 2p1/2 and 2p3/2,which prove the existence of surface titanium as TiO2

(Figure 4(a)); binding energies of Nb doublet peaks 3d3/2

and3d5/2 showing surfaceniobiumasNb2O5 (Figure 4(b));Zr 3d doublet peaks 3d3/2 and 3d5/2 which reveal zirconiumasZrO2 (Figure 4(c)); Ta 4f peaks 4f5/2 and 4f7/2 which findsurface tantalum as Ta2O5 (Figure 4(d)); and O 1s peakscomposed of three contributions which represent oxygenas O2�, OH�, and hydrate or adsorbed water, respectively(Figure 4(e)).

Fig. 3—SEM micrographs in BSE mode of the Ti-20Nb-10Zr-5Taalloy.


C. Alloy Electrochemical Behavior

Alloy electrochemical behavior in neutral Ringer solu-tion was studied by cyclic potentiodynamic and linearpolarization method. From cyclic voltammograms(Figure 5) it is evidenced that the novel alloy self-passivated with a very large passive potential range(DEp) and very low passive current density (ip about1.2 lA/cm2) characterizing a very resistant passive film(Table III) in comparison with Ti-6Al-4V, Ti-6Al-7Nb,Ti-13Nb-13Zr[37,38] alloys, and Ti metal.

The deconvoluted XPS spectra for Ti-20Nb-10Zr-5Taalloy after 2000 immersion hours in Ringer solution ofpH 7.58 are presented in Figure 6. New elementsdeposited from Ringer solution, were detected[36]: Ca

2p (as Ca2+ ions, Figure 6(f)), P 2p (as P5+ phosphateions, Figure 6(g)), and O 1s (as O2� bond into lattice,Figure 6(e)), indicating the formation of a calciumphosphate surface layer. Elements from the alloy arealso detected at low intensity: Ti 2p as Ti4+ (TiO2,Figure 6(a)); Nb 3d as Nb5+ (Nb2O5, Figure 6(b)); andZr 3d as Zr4+ (ZrO2, Figure 6(c)); while Ta 4f featuresas Ta5+ (Ta2O5, Figure 6(d)) are masked by the O 2speak at around 23 eV.SEM images of Ti-20Nb-10Zr-5Ta alloy surface after

2000 immersion hours in Ringer solution of pH 7.58(Figures 7(a) and (b)) showed the growth of a layer withinter-grown, twinned crystals. Elemental analysis (byEDX) of the layer deposited on the alloy surface inRinger solution (Figure 7(c)) proved that this is mainly

Fig. 4—XPS deconvoluted spectra recorded for the as-prepared Ti-20Nb-10Zr-5Ta alloy: (a) Ti 2p; (b) Nb 3d; (c) Zr 3d; (d) Ta 4f; and (e) O 1s.


composed of Ca, P, and O; the component elements ofthe alloy (Ti, Nb, Zr, and Ta) are also present in spectra,confirming the XPS results (Figure 6).

From Table IV low corrosion rates in ‘‘PerfectStable’’ resistance class[33] for Ti-20Nb-10Zr-5Ta alloyand Ti-6Al-7Nb alloy, ‘‘Very Stable’’ resistance class forTi-6Al-4V alloy and Ti metal, and ‘‘Very Stable’’ and‘‘Stable’’ resistance class for Ti-13Nb-13Zr alloy can beobserved.[37–39] Also, the reduced ion release rates(corresponding with a very low toxicity) and high

polarization resistances (order hundreds of kX cm2)reveal resistant passive films for Ti-20Nb-10Zr-5Taalloy.Alloy electrochemical behavior in MEM solution

Table V summarizes the corrosion parameters obtainedby electrochemical measurements of various Ti-basedmaterials in MEM[40,41]: corrosion potential at the endof stabilization period (Ecorr

OCP), polarization resistance(Rp), corrosion potential determined by the Tafelanalysis (Ecorr

PD ), and corrosion current density deter-mined by the Tafel analysis (icorr

PD ).Cyclic potentiodynamic polarization curves for Ti-

20Nb-10Zr-5Ta, Ti-6Al-7Nb, and Ti-6Al-4V alloys; andTi metal recorded in MEM solution are presented inFigure 5(b).

D. Alloy Biocompatibility

The overall cell proliferation, as measured with theAlamar Blue assay for Wi-38 and HOS cells exposed 48,96, and 144 hours to metal extracts, together withestablished toxicity grades, is reported in Table VI.Microscopical inspections of adherent cells after differ-ent incubation periods with the metal extracts are shownin Figures 8 and 9. The survival fraction (SF) values ofWi-38 and HOS cells incubated with all four metalextract media for 48, 96, and 144 hours are presented inFigure 9.

IV. DISCUSSION

XRD (Figure 1) and SEM (Figure 2) measurementsfor Ti-2-Nb-10Zr-5Ta alloy determined a near b crys-talline structure consisting mainly of b phase (80 pct),with a calculated lattice parameter of the cubic b phaseof 3.268 A, and some minor content of a phase (20 pct).XPS analysis (Figure 4) showed that the native film

on this alloy surface is formed from the protective TiO2,Nb2O5, ZrO2, and Ta2O5 oxides.Electrochemical studies in neutral Ringer solution

(Figure 5(a)) revealed that the corrosion (Ecorr) andpassive (Ep) potentials for Ti-20Nb-10Zr-5Ta alloy havemore electropositive values than those for Ti, Ti-6Al-4V,Ti-6Al-7Nb, and Ti-13Nb-13Zr[37,38] alloys (Table III),due to the favorable influence of the alloying elements.The passive current density for Ti-20Nb-10Zr-5Ta alloyis lower than that for Ti, Ti-6Al-4V, Ti-6Al-7Nb, andTi-13Nb-13Zr[37,38] (Table III) denoting a more resistantpassive state. The increase of the current density at

Fig. 5—Cyclic polarization curves recorded for the novel Ti-20Nb-10Zr-5Ta alloy, commercial Ti-6Al-4V and Ti-6Al-7Nb alloys, andTi metal in: (a) neutral Ringer solution; (b) minimum essential med-ium (MEM) at 310 K (37 �C).

Table III. Main Corrosion Parameters in Neutral Ringer Solution at 310 K (37 �C)

Biomaterial Ecorr (mV) Ep (mV) |Ecorr � Ep| (mV) DEp (mV) ip (lA/cm2)

Ti-20Nb-10Zr-5Ta �185 �150 35 >4000 1.2Ti-6Al-4V �250 �200 50 >4000 2.5Ti-6Al-7Nb �250 �200 50 >4000 1.8Ti-13Nb-13Zr[37] �600 — — >3000 7.54Ti-13Nb-13Zr[38] �350 — — >2000 1.01Ti �400 �350 50 >4000 9.0


Fig. 6—XPS deconvoluted spectra for: (a) Ti 2p; (b) Nb 3d; (c) Zr 3d; (d) Ta 4f; (e) O 1s, (f) Ca 2p; and (g) P 2p, recorded on Ti-20Nb-10Zr-5Ta alloy surface after 2000 immersion hours in Ringer solution of pH 7.58.


about +1.8 V may be due to the change in the structureof passivating oxide film.[33,42] The tendency to passiv-ation (|Ecorr � Ep|) has very low values (about 35 mV)

showing that the alloy is easily passivated as a result ofthe positive effects of the alloying elements Nb, Zr, andTa that participate with their passive oxides Nb2O5,ZrO2, and Ta2O5 to the formation of the alloy passivefilm.[14] This was confirmed using XPS analysis(Figure 6).After 2000 immersion hours in neutral Ringer solution,

a layer of calcium phosphate is deposited on the alloysurface; this fact was demonstrated by XPS spectra thatshowed the presence of calcium (Figure 6(f)), phospho-rous (Figure 6(g)), and oxygen ions (bond into lattice,Figure 6(e)) on the alloy surface. The layer with inter-grown, twinned crystals (SEM observations) reached thethickness more than a hundred microns on some zones,due to the growth of some macro-crystals (platelets)perpendicularly on the surface (Figure 7(a)).The detailedstudy of the primary layer (near the alloy surface) revealedthat the first deposited layer is a continuous coatingformed of rounded particles with dimensions around100 nm (Figure 7(b)). The EDX analysis (Figure 7(c))exhibited the formation of the phosphate layer andconfirmed the XPS results.In neutral Ringer solution, comparing with Ti, Ti-6Al-

4V, Ti-6Al-7Nb, and Ti-13Nb-13Zr[37,39] alloys (Table IV),lower corrosion current densities (0.058 lA/cm2 for newalloy comparing with 0.091 lA/cm2 for Ti-6Al-4V alloy,0.087 lA/cm2 forTi-6Al-7Nballoy and0.095 lA/cm2 forTi, 1.77 lA/cm2[37] or about 5 lA/cm2[39] for Ti-13Nb-13Zr alloy) and rates were obtained for Ti-20Nb-10Zr-5Ta alloy proving the enhancement of the protectiveproperties by alloying with Nb, Zr, and Ta.In MEM solution, the open circuit potentials, Eoc, are

in the range from �0.38 V for Ti-6Al-7Nb alloy to�0.16 V for Ti metal (Table V). Ti and all three alloysshow high Rp values indicating high corrosion resistanceof these materials in MEM. The values of corrosionpotential Ecorr

PD are more electropositive for novel alloythan those for Ti-6Al-4V, Ti-6Al-7Nb, and Ti-13Nb-13Zr[40,41] alloys and are little more negative than that ofTi metal Table V). The values of corrosion currentdensity range from 0.016 lA/cm2 for Ti-13Nb-13Zralloy,[40] to 59 lA/cm2 for Ti metal, to 127 lA/cm2 forTi-20Nb-10Zr-5Ta alloy (Table V).Cyclic potentiodynamic polarization curves recorded

for Ti-20Nb-10Zr-5Ta alloy in MEM solution(Figure 5(b)) show that, starting from the Ecorr, theanodic current density increases in the Tafel region untilis interrupted at approximately +0.1 V, when theincrease ceases and the first anodic plateau A isestablished where the current density is independenton the electrode potential. Thus, the passive range isestablished without an active/passive peak like in Ringersolution (Figure 5(a)). XPS analysis performed on novelTi-20Nb-10Zr-5Ta alloy confirms that a spontaneouslyoxide film is also formed (Figure 4) similarly as forcommercial alloys. The anodic plateau A is establishedat slightly more negative potentials for Ti-based alloys(+0.1 V) than for Ti metal (+0.3 V) and extends up toapproximately +1.2 V being characterized by a currentdensity value of about 9 lA/cm2 for Ti-20Nb-10Zr-5Taand about of 5 lA/cm2 for Ti-13Nb-13Zr alloy.[41] AtE>+1.2 V, the current density started to increase and

Fig. 7—SEM micrographs (a, b) and EDX spectrum (c) recorded onTi-20Nb-10Zr-5Ta alloy surface after 2000 immersion hours in Ring-er solution of pH 7.58.


the second current density plateau B is formed. Com-pared to the first anodic plateau, the values of thepassive current densities are higher reaching 19 lA/cm2

for Ti-20Nb-10Zr-5Ta alloy and 25 lA/cm2 for Ti-13Nb-13Zr alloy.[41] These two anodic plateaus in thepolarization curves are related to the change in compo-sition of the passive oxide layer formed at the surface ofTi metal and its alloys[43,44] i.e., the first anodic plateau

A corresponds to the formation of TiO2 containing asmall amount of sub-oxides (TiO and Ti2O3) while inrange of the plateau B, sub-oxides diminished and TiO2

is the predominant oxide.When the sweep is reversed at Esw, the current density

decreases and reaches the values of E0corr, which is closeto the value of E0corr in the forward sweep. As Ti metaland Ti-based commercial alloys, novel Ti-20Nb-10Zr-5Ta

Table IV. Corrosion and Ion Release Rates in Neutral Ringer Solution at 310 K (37 �C)

Biomaterial Time (h) icorr (lA/cm2) Vcorr (lm/year) Resistance Class Ion Release (ng/cm2) Rp (kX/cm2)

Ti-20Nb-10Zr-5Ta 0 0.058 0.638 PS 64.82 250.331000 0.069 0.759 PS 77.11 238.122000 0.110 1.210 VS 122.94 197.84

Ti-6Al-4V 0 0.091 1.001 VS 101.70 218.721000 0.101 1.111 VS 102.62 193.652000 0.263 2.893 VS 293.93 142.24

Ti-6Al-7Nb 0 0.087 0.957 PS 97.23 221.341000 0.092 1.012 VS 102.82 205.662000 0.155 1.705 VS 173.23 154.25

Ti-13Nb-13Zr[37] 0 1.77 — S — —Ti-13Nb-13Zr[38] 0 0.12 — VS — —Ti-13Nb-13Zr[39] 0 5 — S — —Ti 0 0.095 1.045 VS 106.17 189.32

1000 0.132 1.452 VS 147.52 164.152000 0.321 3.531 VS 358.75 116.22

PS: perfect stable; VS: very stable.

Table V. Corrosion Parameters of Various Ti-Based Materials in MEM

Material EcorrOCP (mV) Rp (kX/cm2) Ecorr

PD (mV) icorrPD (lA/cm2)

Ti-20Nb-10Zr-5Ta �267 175 �268 162.6Ti-6Al-4V �302 222 �302 46.8Ti-6Al-7Nb �381 330 �365 62.4Ti-13Nb-13Zr[40] — — �627 0.016Ti-13Nb-13Zr[41] — — �700 —Ti �159 249 �151 59.4

Table VI. Comparison of Cell Proliferation Percentages (P) of WI-38 and HOS Cells and Cytotoxicity Grades of Extract Media

SampleIncubation withMetal Extract (h)

P Pct Toxicity Grade

Wi-38 HOS Wi-38 HOS

Unexposed control 48 100 100 reference96 100 100 reference144 100 100 reference

Ti-20Nb-10Zr-5Ta 48 92 100 0 096 100 97 0 0144 95 88 0 0

Ti-6Al-4V 48 92 100 0 096 100 100 0 0144 100 100 0 0

Ti-6Al-7Nb 48 100 100 0 096 100 91 0 0144 84 82 0 0

Ti 48 91 100 0 096 100 100 0 0144 100 80 0 1


alloy is not susceptible to localized pitting corrosion inMEM (Figure 5(b)) and also not in neutral Ringersolution (Figure 5(a)), despite that both solutions arerich in chloride ions. The potential scan was reversed at+6.0 V for Ti metal, Ti-20Nb-10Zr-5Ta, and Ti-6Al-7Nb alloys. The current density remains independent ofthe potential to even more positive potentials, i.e., up to+8.5 V for Ti-6Al-7Nb alloy, +10 V for Ti-20Nb-10Zr-5Ta alloy, and +10.8 V for Ti metal (results notshown).

Concerning the cell proliferation, compared to thecontrol medium, the Alamar Blue assay for Wi-38 cellsshowed no reduction in cell proliferation (p> 0.05) forTi-20Nb-10Zr-5Ta, Ti-6Al-4V, and Ti extracts, exceptfor prolonged incubation (144 hours) with Ti-6Al-Nbextract, which significantly reduced the percentage ofproliferating Wi-38 cells (p = 0.002). Notwithstandingthe statistical significance, proliferation reductioncaused by the Ti-6Al-Nb extract was not large enoughto be regarded as grade 1 toxicity, since P was 84 pct(see Table VI).

The Alamar Blue assay for HOS cells revealed nosignificant differences in cell proliferation between eitherTi-20Nb-10Zr-5Ta and Ti-6Al-4V extracts-treated oruntreated cells (p< 0.05), except for prolonged incuba-tion (144 hours) with Ti and Ti-6Al-Nb extracts, which

caused a significant reduction in the percentage ofproliferating cells (p = 0.023 and p = 0.026, respec-tively). HOS proliferation reduction produced by the Ti-6Al-Nb extract was not large enough to be regarded asgrade 1 toxicity, since the observed P was 82 pct (seeTable VI).The cell proliferation percentage was 95 pct for Wi-38

and 88 pct for HOS cells after 144 incubation hours inthe new alloy extract; the toxicity was 0.Microscopical studies, immediately after incubation

with metal extracts did not show any apparent mor-phological changes. After 48 hours of cell incubation,the Wi-38 cells presented mainly an elongated morphol-ogy for Ti-20Nb-10Zr-5Ta extract (Figure 8). On theother hand, the HOS cells adopted the same elongatedmorphology beginning with 48 hours incubation forcontrol, Ti-20Nb-10Zr-5Ta, and Ti extracts (Figure 9).The clonogenic assay for HOS cells (Figure 10)

partially confirmed the results of Alamar Blue assay,since it showed no significant differences in survivalfraction (SF) between either metal extract-treated oruntreated cells (p< 0.05), except for prolonged incuba-tion (144 hours) with Ti-6Al-7Nb, which statisticallysignificantly reduced SF of HOS cells (p = 0.03). As forWi-38 cells (Figure 10), no significant differences in SFwere observed between either metal extract and controls,

Fig. 8—Phase-contrast images of adherent Wi-38 cells after 48, 96, and 144 h incubation with metal extracts.


Fig. 9—Phase-contrast images of adherent HOS cells after 48, 96, and 144 h incubation with metal extracts.

Fig. 10—Survival fractions of Wi-38 and HOS cells incubated with metal extract media for 48, 96, and 144 h.


regardless of incubation duration (p> 0.05), with theexception of a slightly reduced SF of cells treated withTi-6Al-4V extract for 96 hours (p = 0.023).

V. CONCLUSIONS

The Ti-20Nb-10Zr-5Ta alloy revealed a near b crys-talline microstructure containing mainly b phase andsome minor a phase. The Young’s modulus (59 GPa) isadvantageous for implant applications, being muchcloser to that of bone, between 10 and 30 GPa, thanTi (CP-Ti: 105 GPa) and other Ti alloys used as animplant material (Ti-6Al-4V ELI: 114 GPa).

The corrosion behavior in neutral Ringer solutiondetermined: self-passivation behavior characterizing avery resistant passive film; easy passivation as a result ofthe beneficial influence of the alloying elements Nb, Zr,and Ta that participate with their passive oxides Nb2O5,ZrO2, and Ta2O5 to the formation of the alloy passivefilm; low corrosion and ion release rates correspondingwith a very low toxicity.

In MEM solution the novel Ti-20Nb-10Zr-5Ta alloyyields electrochemical characteristic very similar to Timetal and Ti-based commercial alloys, indicating a highcorrosion resistance and no susceptibility to localizedpitting corrosion.

Biocompatibility tests of proliferation of fibroblast(Wi-38) and osteoblast-like human cell (HOS) linesdemonstrated no cytotoxicity for the new Ti-20Nb-10Zr-5Ta alloy extract. Microscopical inspection of theadherent cells showed that both Wi-38 and HOS cellsadopted an elongated morphology after 48 hours incu-bation. No significant differences between the survivalfraction of Wi-38 and HOS cells incubated in new alloyextract and controls were observed.

All results revealed that the new near b-type Ti-20Nb-10Zr-5Ta alloy combines a low elastic modulus withvery low ion release rate in biofluids and excellentbiocompatibility. Therefore, the new alloy is a verypromising material for long-term implants.

ACKNOWLEDGMENTS

This work was supported by European transnationalMNT-ERA Net project—SURFUNCTI. Also, sup-port of the EU (ERDF) and Romanian Governmentinfrastructure POS-CCE O 2.2.1 project INFRA-NANOCHEM—No. 19/2009 is gratefully acknowl-edged. The financial support by the Ministry of HigherEducation, Science and Technology, Republic ofSlovenia is acknowledged.

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