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Vol. 8. Issue 5.
Pages 4108-4114 (September - October 2019)
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Vol. 8. Issue 5.
Pages 4108-4114 (September - October 2019)
Original Article
DOI: 10.1016/j.jmrt.2019.07.020
Open Access
Preparation, structural and microstructural characterization of Ti-25Ta-10Zr alloy for biomedical applications
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Fernanda de Freitas Quadrosa,b, Pedro Akira Bazaglia Kurodaa,b, Karolyne dos Santos Jorge Sousaa,b, Tatiani Ayako Goto Donatoa,b, Carlos Roberto Grandinia,b,
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carlos.r.grandini@unesp.br

Corresponding author.
a UNESP — Univ Estadual Paulista, Laboratório de Anelasticidade e Biomateriais, 17.033-360, Bauru, SP, Brazil
b IBTN-Br — Institute of Biomaterials, Tribocorrosion and Nanomedicine — Brazilian Branch, 17.033-360, Bauru, SP, Brazil
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Tables (2)
Table 1. Chemical composition of Ti-25Ta-10Zr alloy.
Table 2. Phases percentage and lattice parameters, for the samples of Ti-25Ta-10Zr alloy, obtained by Rietveld’s Method, after each processing condition.
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Abstract

Titanium alloys are widely used in the biomedical field due to their good corrosion resistance, high mechanical strength/density ratio, low elastic modulus, and good biocompatibility. With the addition of β-stabilizers elements (such as tantalum), the decrease of the elastic modulus and excellent corrosion resistance are obtained. Zirconium despite being a neutral element appends a good corrosion resistance to titanium alloys. In this paper, Ti-25Ta-10Zr alloy was prepared and characterized aiming biomedical applications. Structural and microstructural analysis corroborated each other and showed the presence of characteristic peaks of three crystalline structures, hexagonal (α phase) and orthorhombic (α’’ phase), dissolved in a matrix with body-centered cubic crystalline structure (β phase). In the indirect cytotoxicity test, no cytotoxic effects were observed on the produced alloy.

Keywords:
Ti alloys
Microstructure
Biomaterials
Full Text
1Introduction

Titanium uses in medicine started after the mid-20th century when titanium alloys began to replace stainless steel and Co–Cr alloys in biomedical applications [1,2]. Below 882 °C, titanium has a hexagonal compact (hcp) crystalline structure, also known as α phase and, above such temperature has body-centered cubic (bcc) crystalline structure, also known as β phase [3]. The α phase is characterized by having a good mechanical resistance and β phase by presenting low elastic modulus and high resistance to corrosion and wear [4].

The first titanium alloys produced were characterized as being of α-type, developed for the aerospace industry [5]. Then α + β type alloys have gained prominence due to its properties [6] and, it can be cited as an example, the Ti-6Al-4V alloy widely used as biomaterial [7], however, research reports that aluminum and vanadium ions when present in the bloodstream can cause toxic reactions and neurological problems to the patient as the Alzheimer’s disease [8].

Tantalum is a β-stabilizer element and has a high elastic modulus, high mechanical strength and high hardness due to its extremely strong chemical bonds, resulting in a high melting temperature [9]. Mainly due to good corrosion resistance and its biocompatibility, tantalum can be used in the biomedical field [10], but its production cost is very high [11].

Zirconium belongs to the same family of titanium on the Periodic Table and can assist in corrosion resistance, mechanical strength, biocompatibility [12]. Although zirconium is considered a neutral element in binary Ti-alloys, however, in the presence of another beta stabilizer element (as tantalum, for example), it improves the retention of this phase and decreases the temperature of the α’ martensitic transformation [13–17]. The allotropic transformation of zirconium occurs at 862 °C [18]. Below this temperature, its crystalline structure is hexagonal compact (α phase) and above such temperature, the crystalline structure is body-centered cubic (β phase).

Currently, β-type titanium alloys have been produced, by presenting good biocompatibility, good mechanical strength/density ratio, and high corrosion resistance, besides low elastic modulus [19]. Such alloys have been produced mainly with the addition of molybdenum, zirconium, tantalum, and niobium, which are elements that do not exhibit cytotoxic effects in the human body [20–23].

The alloys developed with these elements have advantageous properties to be used in the orthopedic area, such as the Ti-20Nb-20Ta-20Zr-20Mo and Ti-29Nb-13Ta-4.6Zr alloys. The high entropy Ti-20Nb-20Ta-20Zr-20Mo alloy, which constitutes a new class of structural materials, presents high biocompatibility when compared to commercially pure titanium, in addition to the hardness and yield stresses superior to the Ti-6Al-4V alloy [24]. Likewise, the Ti-29Nb-13Ta-4.6Zr alloy also has excellent properties, but its mechanical durability is unsatisfactory to be used as a biomedical implant. To reverse this result, the fatigue of the material is improved by the modification of the surface through the formation of a nanostructured gradient under the sample [25].

The objective of this paper is to presents the preparation and the structural and microstructural characterization of the Ti-25Ta-10Zr alloy, as well as verify the feasibility of this sample in the biomedical area.

2Materials and methods

After the separation of the precursors, commercially pure titanium (99.8% purity, Sandinox); zirconium (99.8% purity Aldrich) and tantalum (99.9% purity, Goodfellow), the melting of alloy Ti-25Ta-10Zr was held in an arc-furnace, with water-cooled copper crucible, non-consumable tungsten electrode and argon controlled atmosphere [13,14,26,27]. To ensure a good homogeneity and eliminate the possibility of containing tantalum aggregated or segregated in the sample, the melting was held by ten times. Then, the sample was subjected to homogenization heat treatment with a heating rate of 10 °C/min up to 1000 °C, in which remained at this temperature for 24 h and then cooled slowly in the furnace [16,27].

Chemical characterization was obtained by dissolution in acidic medium, followed by the detection of elements in an inductively coupled plasma optical emission spectrometry (ICP-OES) equipment (Varian, Vista model) and energy dispersive spectroscopy (EDS) technique using a scanning electron microscopy (SEM) equipment (Carls Zeiss, EVO-015 model, with Oxford INCA probe). To confirm the stoichiometry of the material, density measurements were carried out following the Archimedes’ Principle. In this method, the samples were weighed in water and in the air, and then the density of the material was obtained [28].

The alloy structure was analyzed by X-ray diffraction measurements, with the diffractograms submitted to the Rietveld’s structural refinement. Diffraction patterns were obtained on a Rigaku D/Max-2100 PC model diffractometer, using the powder method, Cu-Kα radiation (λ = 1.544 ??), 20 mA current and potential of 40 kV, the scan rate of 0.02 degrees/min, 10°–100°, in the fixed time mode. From the GSAS [29] software, with EXPEGUI interface [30] and crystallographic database sheets of the International Centre for Diffraction Data (ICDD), it was made the refinement of the diffractograms, obtaining the lattice parameters and the quantification of the phases. The crystallographic data of the α’’ phase have been entered manually based on the results suggested by Banumathy et al. [31].

The crystalline microstructure was obtained by optical and electronic microscopy. To obtain the images, it was used an Olympus optical microscope, BX51M model and a Carl Zeiss SEM, model EVO-015.

Cells were obtained according to research protocols approved by the Ethical Committee for Animal Research of the University of São Paulo of Bauru, Brazil (Protocol #004/2016). Osteogenic cells were isolated by sequential trypsin/collagenase digestion of calvaria bone from newborn (2-day-old) Wistar rats, as previously described by Donato et al. [32].

The obtained cells were cultured in α-minimum essential medium (α-MEM) (FBS, Cultilab, Campinas, Brazil) containing 10% fetal bovine serum (FBS, Cultilab) and 1% penicillin (Cultilab), and incubated at a density of 110 cells/mm². 24 h after plating, the cell cultures were supplemented with 50 µg/mL ascorbic acid (Sigma, St. Louis, MO, USA) and 10 mM β-glycerophosphate (Sigma). The cells were incubated under standard cell culture conditions (37 °C, 95% humidity and 5% CO2) and the medium was changed every 2–3 days [33–35].

A standard colorimetric assay — 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT assay, Sigma) was used to estimate cell viability and proliferation [36,37]. The evaluation of indirect cell viability of the Ti-25Ta-10Zr alloy was done by MTT assay at 48 h. For indirect cytotoxicity test, the extracts of the alloy were obtained – 1 g of alloy/10 mL medium at 48 h – rat calvaria derived osteogenic cells were grown on the microplates and the culture medium was substituted by the obtained extracts. The tests were made using 96 wells microplates (Corning). The polystyrene from the microplate was used as the negative control (no cytotoxicity), while a solution of α-MEM, 10% of FBS, and 1% of phenol was the positive control (cytotoxicity). After this time, then routinely processed for MTT assay [35]. The optical density (OD) of the wells was determined using a plate reader at a test wavelength of 640 nm in a SpectraMax Plus microplate reader (Molecular Devices).

For the morphology analysis, the osteogenic cells were on the glass coverslip and the culture medium was substituted by the obtained extracts of the alloy. After 48 h, the cells were fixed and were then routinely processed for SEM [34]. Specimens were mounted onto aluminum substrates, sputtered with gold, and examined in a Carl Zeiss EVO LS-15 model microscopy. The polystyrene was used as the negative control (no cytotoxicity), while a solution of α-MEM, 10% of FBS, and 1% of phenol was the positive control (cytotoxicity).

3Results and discussion

Table 1 shows the chemical composition of the alloy using the ICP-OES technique. The values found are near the proposed composition, which shows that the stoichiometry of the alloy was respected [38].

Table 1.

Chemical composition of Ti-25Ta-10Zr alloy.

  Al  Cr  Cu  Fe  Hf  Mn  Mo  Ni  Si  Ta  Zr  Ti 
wt%  0.05  0.02  0.64  0.03  0.04  0.01  0.01  0.01  0.08  23.9  11.3  Balance 

Fig. 1 shows an image of the EDS mapping of the elements present in a sample of Ti-25Ta-10Zr alloy, where the red points represent titanium, the green points represent tantalum and the blue points represent zirconium. A homogeneous distribution, without the presence of agglomerates or segregates, can be observed. The experimental value of density, obtained by Archimedes’ Principle, was (5.72 ± 0.01) g/cm3. Comparing with the theoretical value, 5.73 g/cm3, it can be observed an excellent agreement, reinforcing that the stoichiometry was respected.

Fig. 1.

EDS mapping for Ti-25Ta-10Zr alloy. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)

(0.19MB).

Fig. 2 shows a comparison of X-ray diffractograms from the alloy after melting and after homogenization heat treatment. It can be observed that the alloy presented the α’/α’’ martensitic phases, with a hexagonal and orthorhombic crystalline structure, respectively, plus a few peaks of β phase, with a body-centered cubic crystalline structure. Tantalum is a strongly β-stabilizer, and its presence makes zirconium no neutral and passes the acts also in the stabilizing of the β phase [39,40]. The homogenization heat treatment causes changes in intensity and in the width of the peaks, which may be a result of changes in lattice parameters, crystallite size, microstrain and phase percentage [41].

Fig. 2.

X-ray diffraction patterns for Ti-15Ta-10Zr alloy, as cast and after homogenization heat treatment, compared with cp-Ti, cp-Ta, and cp-Zr.

(0.18MB).

Fig. 3 shows the X-ray diffractograms, analyzed by Rietveld’s method, for each of the studied conditions. The black line indicates the experimental data of the diffractogram; the red line indicates the intensity calculated by refinement; the green line shows the background, and the blue line shows the residue, which is the difference between the experimental and calculated intensity. The graph shows a good agreement between experimental and calculated intensities, which indicates a good refinement of the data. The merit parameters of the refinement are chi-squared (χ2) equal to 1.090 for the as-cast condition and 1.107 for sample after homogenization heat treatment; R-factor (RF2) is equal to 7.67% and 11.02%, respectively, for the same conditions mentioned above. The results show low χ2 values, which indicates a good refinement of the data. The χ2 must be equal to 1 for a perfect refinement, but values below 5 already reflect an optimized refinement. For RF², the accepted values are around 10% [42,43].

Fig. 3.

X-ray diffractograms of Ti-25Ta-10Zr alloys analyzed by Rietveld’s Method in the (a) as-cast and (b) after homogenization heat treatment conditions. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)

(0.27MB).

In Table 2 are presented the phases quantities and the lattice parameters, for Ti-25Ta-10Zr alloys, obtained by Rietveld’s Method, after each processing condition. The lattice parameters (a, b and c) of α’ phase are dilated compared to titanium due to the addition of tantalum and zirconium, due to these elements have larger atomic radius than titanium, and zirconium has the same hexagonal structure [9]. Homogenization heat treatment promoted the transformation of α’’ phase for the α phase; the treatment was carried out at a temperature above the titanium β-transus and cooled slowly giving the necessary conditions for the metastable phase α’’ turn to less energy phase α [44]. With respect to lattice parameters of α’ phase, the homogenization heat treatment promoted an increase in the unit cell due to thermal expansion [45,46].

Table 2.

Phases percentage and lattice parameters, for the samples of Ti-25Ta-10Zr alloy, obtained by Rietveld’s Method, after each processing condition.

Phase (%)Lattice parameter (??)
α'  α’’  β 
As cast–  –  2.883 (4)  2.883 (4)  4.733 (1) 
–  64  –  2.996 (1)  5.126 (2)  4.738 (1) 
–  –  32  3.309 (1)  3.309 (1)  3.309 (1) 
Heat treated65  –  –  2.976 (1)  2.976 (1)  4.729 (1) 
–  –  –  –  – 
–  –  35  3.303 (1)  3.303 (1)  3.303 (1) 
cp — Ti [28]  –  –  –  2.951  2.951  4.684 

Fig. 4 shows the optical and the scanning electronic micrographs, both for as-cast and after homogenization heat treatment conditions, for the Ti-25Ta-10Zr alloy. Such results corroborate X-ray diffractograms results. The alloy presented a structure composed of needles that are typical of structures of phases α’ (thicker needles), α’’ (finer needles) and grain boundary characteristic of β-type alloys [2]. Homogenization heat treatment promoted the growth of acicular structures and of the grains.

Fig. 4.

Optical (left) and SEM (right) micrographs of Ti-15Ta-10Zr alloy in the (a) as cast and (b) after homogenization heat treatment conditions.

(0.56MB).

Fig. 5 shows the hardness values of the alloy and makes a comparison with cp-Ti and Ti-6Al-4V alloy [47]. It was observed that the Ti-25Ta-10Zr alloy presents values of hardness significantly higher than cp-Ti and Ti-6Al-4V alloy. This occurs due to the presence of the α’’ phase and solid solution hardening caused by the addition of tantalum and zirconium elements that hinder the dislocations motion and, consequently, the microstructure of the alloy is influenced from the deformation of the crystalline lattice.

Fig. 5.

Vickers microhardness for Ti-25Ta-10Zr, compared with cp-Ti and Ti-6Al-4V.

(0.25MB).

It was observed that the alloy presents values of hardness significantly higher than the pure titanium, being a result of the hardening in solid solution caused by the addition of tantalum and zirconium that possess atomic mass superior to the titanium.

The results of the indirect cytotoxicity test are presented in Fig. 6 and showed that the extracts of Ti-25Ta-10Zr no inhibit the proliferation of osteogenic cells and the number of viable cells was similar to the negative control (no cytotoxicity) and higher to the positive control (cytotoxicity).

Fig. 6.

Indirect cytotoxicity test in primary culture of osteogenic cells cultured for 48 h on Ti-25Ta-10Zr alloy.

(0.1MB).

The date observed by SEM (Fig. 7) corroborate the indirect MTT test. The cell morphology was preserved even after being in contact with the extract of Ti-25Ta-10Zr, showing the same of the negative control (no cytotoxicity), contrasting with the positive control (cytotoxicity — phenol solution) cell morphology. The extract of alloy did not interfere in the cellular adhesion, that was being well adhered and numerous and, long cellular processes on the glass, as well as cell mitosis process. These characteristics were not seen in positive control [34,36]. This information suggests that Ti-25Ta-10Zr alloy may be interesting material to an application as biomaterials.

Fig. 7.

SEM in primary culture of osteogenic cells cultured for 48 h with extract of Ti-25Ta-10Zr alloy. (A) Negative Control (NC) — glass; (B) extract of Ti-25Ta-10Zr; (C) Positive Control (PC) — 1% phenol solution. Arrowhead: cells in mitosis process and cytoplasmatic processes.

(0.13MB).
4Conclusions

As the obtained results of chemical composition, density measurements, EDS and mapping indicated, the Ti-25Ta-10Zr alloy was melted in a homogeneous way, preserving the proposed stoichiometry.

Structural analysis showed a coexistence of α’ phase (hexagonal compact), α’’ (orthorhombic) and β (body-centered cubic). By means of the Rietveld’s method, it was possible to quantify the fraction of these phases in the alloy and observed the variations in the lattice parameters caused by the addition of zirconium and tantalum.

In the microstructural analysis, the presence of intra-grain needles was observed, characteristic of martensític phases α’ and α’’, in agreement with the x-ray diffractogram.

The hardness values were above the commercially pure titanium and Ti-6Al-4V alloy, whereby the continuous solution hardening of the alloy.

In Ti-25Ta-10Zr alloy were not present any indirect cytotoxic for primary culture of osteogenic cells. To the contrary, the alloy presented perfectly cellular division, maintaining the morphology, indicating good integration between materials — cells, characteristics desirable to a biomaterial.

The present study showed Ti-25Ta-10Zr alloy becomes attractive material for use as a biomaterial.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank Brazilian agencies CNPq (Grants #307.279/2013-8, #157.509/2015-0, #400.705/2015-0, and #122.484/2016-9) and FAPESP (Grants #2015/09.480-0 and #2015/25.562-7) for their financial support.

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