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Original Article
DOI: 10.1016/j.jmrt.2019.11.051
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Available online 1 December 2019
On the mechanical biocompatibility of Ti-15Zr-based alloys for potential use as load-bearing implants
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134
D.R.N. Correaa,b,
Corresponding author
diego.correa@ifsp.edu.br

Corresponding author.
, L.A. Rochab,c, T.A.G. Donatob,c, K.S.J. Sousac, C.R. Grandinib,c, C.R.M. Afonsod, H. Doie, Y. Tsutsumie,f, T. Hanawae
a IFSP - Federal Institute of Education, Science, and Technology, Grupo de Pesquisa em Materiais Metálicos Avançados, 18095-410, Sorocaba, SP, Brazil
b IBTN/BR – Institute of Biomaterials, Tribocorrosion, and Nanomedicine, Brazilian Branch, 17.033-360, Bauru, SP, Brazil
c UNESP – Univ Estadual Paulista, Laboratório de Anelasticidade e Biomateriais, 17.033-360, Bauru, SP, Brazil
d UFSCar – University of São Carlos, Department of Materials Engineering, 13565-905, São Carlos, SP, Brazil
e Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, 101-0062, Tokyo, Japan
f Research Center for Structural Materials, National Institute for Materials Science, Tsukuba, 305-0047, Ibaraki, Japan
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Table 1. Chemical composition of the samples employed in this study (wt%).
Table 2. Gas analysis of the samples employed in this study (ppm).
Table 3. Mechanical properties comparison of some commercial metallic biomaterials.
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Abstract

This study evaluated the mechanical properties and cytocompatibility of recently developed Ti-15Zr-based alloys with Mo addition for potential use as load-bearing implants. The phase composition and microstructure were changed by the alloying elements, being the β phase fully retained on the Ti-15Zr-10Mo and Ti-15Zr-15Mo samples. The TEM analysis showed that a small quantity of ω phase was precipitated on the samples with a high amount of Mo. Regarding the mechanical properties, the Ti-15Zr-10Mo sample presented high mechanical strength and large elongation (854 ± 63 MPa and 18.7 ± 2.8 %). However, the Ti-15Zr-15Mo sample exhibited better mechanical compatibility, due to its combination of low Young’s modulus (75 ± 1 GPa) and high Vickers microhardness (346 ± 4 HV). Some dimple-type structures found along the fractured surface confirmed the ductile behavior of these samples. The MTT test indicated non-cytotoxic effects of all samples when in contact with osteoblastic cells (p < 0.05). The wettability values of the samples were adequate for biomedical applications. The mechanical properties of the Ti-15Zr-15Mo sample were better than some commercial metallic biomaterials, which highlights its great potential for use as load-bearing implants.

Keywords:
Ti-Zr-Mo alloy
Mechanical properties
Load-Bearing implant
Biomaterial.
Full Text
1Introduction

Load-bearing implants are commonly used in total joint replacements of damaged bone tissues. During their life span, the implants should support several cyclic biomechanical loads and be able to restore the functions of the human bone [1]. Hip, knee and shoulder joints are hard tissues that frequently suffer damages due to their complex structure and critical work conditions [2]. Joint degeneration diseases, such as arthrosis and arthritis, are natural health problems related to aging. It is believed that 90 % of the population over 40 years is suffering from some degenerative diseases nowadays [3]. As the average life expectancy of the world population is still growing, the amount of elderly people is increasing considerably [4]. Therefore, the demand for load-bearing implants in hard tissue replacements will tend to rise consequently.

Titanium (Ti) and its alloys have been constantly employed as dental and orthopedical implants due to their high strength-to-density ratio, excellent corrosion resistance and recognized biocompatibility [1,3]. A considerable part of these biomedical Ti alloys is used in load-bearing implants, as an alternative to 316 L stainless steel and Co-Cr-Mo alloys, because of their relative low Young’s modulus [2,5]. The combination of high mechanical strength and low Young’s modulus have been sought in novel Ti-based alloys, once it ensures superior support of biomechanical loads for long-term, avoiding stress shielding effect and revision surgeries. In special, the stress shielding effect has been one of the main drawbacks of metallic implants, once it is caused by Young’s modulus mismatch between the implant and adjacent bone tissues, conducting bone resorption and osteopenia. These requirements have been fulfilled by β-type Ti-based alloys, with bcc crystalline structure, instead of α and α + β alloys [6,7]. Moreover, the development of novel biomedical Ti-based alloys have been designed with Al- and V- free composition, once it has been reported that the ion releasing from the biomedical Ti-6Al-4 V alloy was responsible for adverse tissue reactions and neurological disorders. Therefore, novel biomedical β-type Ti-based alloys have been manufactured with the addition of non-cytotoxic alloying elements, such as Mo, Zr, Nb, Ta and Sn [3,8].

Ti-15Zr-based alloys with Mo addition have been recently developed in order to achieve low Young’s modulus and non-cytotoxic chemical composition [9]. Zirconium (Zr) presents similar chemical properties than Ti, which can improve mechanical strength, corrosion resistance and biocompatibility of the solid solution [10,11]. Molybdenum (Mo) is a strong β-stabilizer, which tends to decrease Young’s modulus, still maintaining high mechanical strength [12,13]. Zr composition has been set up from our previous study with binary Ti-Zr alloys [14]. In other studies [15,16], we found that the above-mentioned Ti-Zr-Mo alloys have suited low Young’s modulus and adequate tribocorrosion resistance for biomedical applications. However, an investigation of the tensile properties is still required for a profound evaluation of their potential use as load-bearing implants.

In this sense, this paper aims to evaluate the tensile properties and cytocompatibility of the recently developed Ti-15Zr-xMo (x = 0, 5, 10 and 15 wt%) alloys for biomedical applications, in special for load-bearing implants. Ti-15Zr-based alloys were produced in an argon arc-melting furnace and molded in a centrifugal casting machine. The mechanical properties were evaluated by tensile tests, Young’s modulus and Vickers microhardness measurements, being the results correlated with the corresponding phase composition and microstructure, which were analyzed by XRD, SEM and TEM imaging. Cytocompatibility and cell proliferation were evaluated by MTT colorimetric assay.

2Materials and methods2.1Sample preparation

Commercially pure Ti (CP-Ti Grade 2; Sigma-Aldrich Co., USA), pure Zr (99.80 %; Sigma-Aldrich Co., USA) and pure Mo (99.95 %; Goodfellow Ltd., USA) were melted in an arc-melting furnace (AD TAC 501D, Diavac Ltd, Japan), producing ingots of Ti-15Zr-xMo (0, 5, 10 and 15 wt%) alloys, with a mass of 60 g each. Each ingot was re-melted for 10 times to ensure homogeneity. The chemical composition and interstitial content of the samples are shown in Tables 1 and 2. The metallic content was measured by ICP-AES equipment (Shimadzu Scientific Instruments, Japan), C content by LECO CSLS600 instrument (LECO Inc., Japan), and the other interstitial elements (N, H, and O) by LECO TCH-600 instrument (LECO Inc., Japan). Subsequently, the ingots were molded in a rod shape by a centrifugal casting machine (MSE-50TMD-Z, Yoshida Cast Kogyo, Japan). The samples for tensile tests had dimensions of 5 × 56 mm, while for Young’s modulus measurements 3 × 52 mm, and for Vickers microhardness 5 × 1.5 mm. The samples for tensile tests were previously machined in a dumbbell shape (gauge dimensions: 3 × 18 mm) using Lathe CNC equipment (LB EX 3000 EX-II, Japan).

Table 1.

Chemical composition of the samples employed in this study (wt%).

  Ti  Zr  Mo  Fe  Cr  Sn  Hf 
Ti-15Zr  Bal.  15.0  <0.01  0.02  0.01  <0.01  <0.01  0.01 
Ti-15Zr-5Mo  Bal.  15.0  5.31  0.02  0.01  0.03  <0.01  0.03 
Ti-15Zr-10Mo  Bal.  14.8  10.5  0.02  0.01  0.06  <0.01  0.02 
Ti-15Zr-15Mo  Bal.  15.2  15.7  0.02  0.01  0.09  <0.01  0.02 
Table 2.

Gas analysis of the samples employed in this study (ppm).

 
Ti-15Zr  65  1045  15 
Ti-15Zr-5Mo  23  968  25 
Ti-15Zr-10Mo  20  957  47 
Ti-15Zr-15Mo  20  856  53 
2.2Microstructural characterization

The phase composition was examined by X-ray diffraction (XRD) using a Bruker instrument (D8 Discover, Japan), Ni-filtered, with CuKα radiation (λ =0.1544 nm), current of 40 mA, and voltage of 40 kV, with a continuous step of 0.08° per s−1. Microstructural analysis was carried out by laser scanning microscopy (LSM), in a Olympus microscope (LEXT OL4000, Japan), scanning electron microscopy (SEM), with a Hitachi equipment (S-3400NX, Hitachi High-Technologies Corp., Japan), and transmission electron microscopy (TEM), in a Tecnai equipment (G2 F20 HRTEM, FEI, Corp., USA). The standard metallographic procedures were composed of grinding the surface with SiC abrasive paper up to #1000 grid and polishing in alumina (0.25 μm) and diamond (0.10 μm) colloidal solutions. The microstructures were revealed by etching the surfaces with an H2O, HNO3 and HF solution (85:10:5) for 10 s. The TEM samples were prepared by ion polishing in a PIPS equipment (Model 691, Gatan Corp., USA).

2.3Mechanical characterization

The tensile test was carried out in uniaxial mode, at room temperature, with a strain rate of 1.67 × 10−5 s-1, in a universal testing machine (Shimadzu AG-2000B, Japan). The strain data were acquired with a non-contact optical strain gauge. The tests were performed in triplicate for each sample. The yielding tensile stress (YTS), ultimate tensile stress (UTS), and elongation were obtained from the stress-strain curves. The surface fracture analysis was done by SEM imaging. The Young’s modulus values were measured by the free resonance vibration method (JE-RT3, Nippon Techno-Plus Co. Ltd., Japan). The Vickers microhardness was measured in a Shimadzu HMV-1 equipment with 0.300 kgf (2.942 N) for 15 s. The average values were obtained after 10 measurements for each sample. The elastic admissible strain was calculated by the YTS to Young’s modulus ratio and the specific strength by the YTS to density ratio. The density values were previously obtained from the Archimedes’ principle.

2.4Biological characterization

The MC3T3-E1 pre-osteoblastic cells (ATCC, Rockville, MD, USA) were grown on minimum essential medium (α-MEM, Invitrogen, USA) and supplemented with 10 % fetal bovine serum (FBS) and 1 % gentamicin (Invitrogen, USA). The cells were seeded at 2 × 104 cells per well and incubated under standard cell culture conditions (37 °C, 95 % humidity and 5 % CO2). The medium was replaced after 2 days. A standard MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to estimate cell viability and proliferation [17,18]. The indirect cell viability was obtained by the MTT assay after 72 h, being the extracts collected at 1 g/mL from the medium after 48 h. The cells were grown in microplates (96 wells, USA) with the obtained extracts. A polystyrene plate was used as a negative control, while a solution of α-MEM, 10 % FBS, and 1 % phenol was set up as positive control [17,19]. A plate reader (SpectraMax Plus microplate reader) at 640 nm measured the optical density of the wells. The test was made in triplicate with n = 8.

The cells were inserted on the glass coverslip and the culture medium was replaced by the obtained extracts of the samples. After 72 h, the cells were processed for scanning electron microscopy (SEM). The samples were fixed on aluminum substrates, sputtered with gold, and examined by SEM imaging (EVO LS15, Carl Zeiss, USA) [17,19]. Contact angle measurements were acquired in a Ramé-Hart 100-00 goniometer (Ramé-Hart Instrument Co., USA), with droplets of distilled water (30 μL). The average values were taken after 10 measurements from 3 distinct droplets along the surfaces. The statistical analysis of the MTT results was performed by one-way analysis of variance (ANOVA), with a post hoc Tukey test, at the level of significance p < 0.05.

3Results and discussion

The phase composition of the samples is shown in Fig. 1. The Ti-15Zr sample exhibited diffracted peaks from the hcp (martensitic α’ phase) crystalline structure, whilst the Ti-15Zr-5Mo sample showed a biphasic composition composed of diffracted peaks from the orthorhombic (martensitic α” phase) and bcc (β phase) crystalline structures. The Ti-15Zr-10Mo and Ti-15Zr-15Mo samples presented only diffracted peaks from bcc crystalline structure, indicating that the amount of alloying elements was enough to retain the metastable β phase. In fact, earlier studies have reported the effect of Zr addition on the decay of the martensite start temperature (Ms), as well as the effect of Mo on the β-transus temperature [10,20]. The cell lattice parameters were obtained by the Bragg’s law, by using the highest intense (101¯0) α’ phase and (110) β phase peaks. The Ti-15Zr sample displayed values (a =0.2998 nm and c =0.4894 nm) higher than metallic α-Ti (a =0.2950 nm and c =0.4685 nm), due to the larger atomic radius of Zr (0.159 nm) in comparison to Ti (0.145 nm) [21]. On the other hand, the β phase cell lattice parameter slightly decreased in the Ti-15Zr-5Mo (0.3398 nm), Ti-15Zr-10Mo (0.3323 nm) and Ti-15Zr-15Mo (0.3316 nm) samples which were related to the smaller atomic radius of Mo (0.136 nm) [21].

Fig. 1.

XRD patterns of the Ti-15Zr-based samples.

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The microstructural features of the samples are displayed in Fig. 2. The Ti-15Zr sample exhibited needle-like structures typical of the martensitic α’ phase, while the Ti-15Zr-5Mo sample presented grain boundaries of the β phase with thin acicular structures of α” phase along the intergranular region. The Ti-15Zr-10Mo and Ti-15Zr-15Mo samples showed only equiaxial β phase grains with average size around 100 μm. The results indicated that the Mo content was effective to modify the microstructure of the samples through its β-stabilizing effect [13]. However, it is possible to observe a secondary β-stabilizing effect from the Zr content, once the β phase starts to be retained on binary Ti-Mo alloys only at 9 wt% [12]. Earlier studies have already observed this β-stabilizer action of the Zr when alloyed in multi-component Ti-based alloys [22].

Fig. 2.

Microstructure of the Ti-15Zr-based samples: OM (onset) and SEM (inset).

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The TEM images of the samples are presented in Figs. 3–6, with the correspondent bright field (BF) and dark field (DF) images, and the selected area electron diffraction pattern (SAED). The Fig. 3a and 3b show a general and inward view of the α’ phase lamella in the Ti-15Zr sample. The respective SAED patterns exhibited diffracted spots on the [ 101¯0 ]α’-Ti and [ 101¯2 ]α’-Ti zone axis, confirming the presence of only α’ phase on the microstructure. In Fig. 4a and 4b have displayed a view of the α” phase needles dispersed along with the β phase matrix in the Ti-15Zr-5Mo sample. The SAED pattern showed orientation relationships of [001]β-Ti/[001]α’’-Ti and [ 11¯3 ]β-Ti/[ 11¯3 ]α’’-Ti between the zone axis respectively, which ensures the biphasic composition of the microstructure. Regarding the Ti-15Zr-10Mo sample (Fig. 5a and 5b), the BF and DF images exhibited a general view of the intergranular region of the β phase with dispersion of some nanometric ω phase particles, with size less than 50 nm. The respective SAED pattern on the [111]β-Ti zone axis presented some mild streaks of ω phase between the β phase spots. Similarly, the Ti-15Zr-15Mo sample (Fig. 6a and 6b) exhibited the same dispersion of ω phase particles through the β phase matrix. The correspondent SAED pattern exhibited stained ω phase spots, with an orientation relationship of [ 112¯0 ]ω-Ti/[ 11¯0 ]β-Ti between the zone axis. As it is well known, the metastable ω phase is retained on Ti alloys with high amount of β-stabilizer elements, being formed after proper heat treatment (athermal or isothermal) and/or cold working [23,24]. As the studied samples were in the as-cast condition, it could be presumed that the detected ω phase was formed by athermal precipitation during the centrifugal casting cooling. It is worth to point out that its nanometric size turns difficult its identification by conventional XRD and SEM measurements. This amount of ω phase on the samples can affect directly their mechanical properties once it is the hardest Ti phase, as already observed in earlier studies [8,23].

Fig. 3.

TEM analysis of the Ti-15Zr sample: (a) general view and (b) inward view of the α’ phase lamella (BF image + SAED pattern).

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Fig. 4.

TEM analysis of the Ti-15Zr-5Mo sample: BF image and SAED pattern of α”+β phases on the (a) [001]β-Ti/[001]α’’-Ti and (b) [ 11¯3 ]β-Ti/[ 11¯3 ] α’’-Ti zone axis.

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Fig. 5.

TEM analysis of the Ti-15Zr-10Mo sample: (a) BF and (b) DF images of β + ω phases and the respective SAED pattern.

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Fig. 6.

TEM analysis of the Ti-15Zr-15Mo sample: (a) BF and (b) DF images of β + ω phases and the respective SAED pattern.

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The representative stress-strain curve of the samples is compared in Fig. 7. All samples presented a similar behavior. It was impossible to notice any brittle fracture on the curves. As can be seen, remarkable UTS values were observed in all samples. The Ti-15Zr-5Mo sample presented the highest UTS value, whilst Ti-15Zr-10Mo presented the largest elongation. The distinct tensile properties of the samples were a result of the different deformation modes of the Ti phases, once it can be changed from twinning to slip along the α → β transformation or during the metastable phase precipitation [25]. The fractured surface images of the samples after tensile tests are depicted in Fig. 8. It was possible to observe typical dimple-type structures in all fractured surfaces. The quantity and size of the dimples slightly changed with the composition, having the Ti-15Zr-5Mo and Ti-15Zr-10Mo samples exhibited deeper and larger dimples than the other ones. It was not identified intergranular fracturing or cleavage-like facets produced by the brittle fracture on the images, besides the small amount of ω phase precipitated in the Ti-15Zr-10Mo and Ti-15Zr-15Mo samples. All samples presented rough surface fractures, which confirms a ductile behavior, according to previous reports from the literature [26]. The tensile properties of the samples are compared in Fig. 9. The Ti-15Zr-5Mo sample exhibited the highest UTS and YTS values (1274 ± 62 MPa and 1053 ± 65 MPa), although its elongation was the lowest (8.3 ± 1.8 %), possibly as a result of the α” phase precipitation on the β matrix, which can block the dislocation movements during the plastic deformation [6,8]. As observed in other Ti-based alloys, the combination of phase precipitation and solid solution hardening can affect directly the mechanical properties [27]. The Ti-15Zr-10Mo (854 ± 63 MPa and 18.7 ± 2.8 %) and Ti-15Zr-15Mo (933 ± 34 MPa and 10.3 ± 1.9 %) samples presented better combination of YTS and elongation values than the Ti-15Zr (555 ± 95 MPa and 13.8 ± 0.8 %) sample, which could be related to the distinct deformation mechanisms along the hcp and bcc crystalline structures [6,8]. It was possible to notice that the low quantity of ω phase affected the mechanical strength of Ti-15Zr-10Mo and Ti-15Zr-15Mo samples, but did not largely decrease their elongation values. Zhou et al. [28,29] found an interesting effect of Zr addition on the suppression of ω phase precipitation in the ternary Ti-Zr-Mo alloys. The authors used this fact to develop novel Ti-based alloys, with changeable Young’s modulus, for applications as spinal fixation devices and removal implants.

Fig. 7.

Representative stress-strain curve of the samples.

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Fig. 8.

Fractured surface after tensile tests.

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Fig. 9.

Tensile properties of the Ti-15Zr-based samples.

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The Young’s modulus and Vickers microhardness values of the samples are compared in Fig. 10. The Young’s modulus values exhibited a gradual reduction due to the β phase precipitation, which has the lowest value between the Ti phases [3]. The Ti-15Zr-15Mo sample presented the lowest Young’s modulus value (69 ± 1 GPa), which was remarkable below the CP-Ti (105 ± 1 GPa). Regarding the Vickers microhardness, all studied samples presented higher values than the CP-Ti (201 ± 2.8 HV), because of the solution solid strengthening. As it is well-known, Zr and Mo elements act as hardeners when added to Ti’s solid solution [10,12]. The Ti-15Zr-5Mo sample exhibited the highest Vickers microhardness value (611 ± 12 HV), which can be a result of the phase precipitation strengthening effect that occurred along its biphasic structure (α” + β) [9]. In a similar way, the precipitates of ω phase did not produce a considerable embrittlement effect in the Ti-15Zr-10Mo and Ti-15Zr-15Mo samples, once Young’s modulus and Vickers microhardness remained lower than the other ones. Young’s modulus and Vickers microhardness behavior were quite similar to our previous study conducted on hot-rolled samples [15].

Fig. 10.

Young’s modulus and Vickers microhardness of the Ti-15Zr-based samples.

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A comparison of the mechanical properties between the studied samples and some commercial metallic biomaterials are presented in Table 3. In this table are depicted the values of SS 316 L (stainless steel), Co-Cr-Mo alloy, CP-Ti (grade 2), Ti-6Al-4 V ELI (Ti-64), Ti-13Nb-13Zr (Ti-1313) and Ti-15Mo, which are commonly used in the manufacturing of biomedical implants [30,31]. In addition, it is also presented the values of some recent developed Ti alloys targeted for biomedical implants, such as Ti-35.5Nb-7.3Zr-5.7Ta (TNZT), Ti-12Mo-6Zr-2Fe (TMZF) and the binary Ti-Nb and Ti-Ta alloys [32]. In Table 3 is presented the mechanical properties together with the elastic admissible strain and specific strength, which are important factors to consider along with the design and processing of load-bearing implants [31]. As can be seen, Ti-15Zr-15Mo displayed higher YTS and UTS than most commercial materials, with elongation comparable to TNZT, Ti-6Al-4 V, and Ti-13Nb-13Zr alloys. The admissible elastic strain and specific strength values of this sample assured its excellent mechanical performance (1.3 % and 179 MPa/g.cm−3), which is comparable only with Ti-6Al-4 V (0.8 % and 198 MPa/g.cm−3), TNZT, and TMZF (1.2–1.4 %). The elastic admissible value of the Ti-15Zr-15Mo sample is almost the same as those obtained by Ozan et al. [31] in the Ti-Zr-Nb alloys, where it was found that high values could safeguard the adjacent bone tissues from stress shielding effect and biomechanical failures. This finding indicates that the developed sample had great mechanical biocompatibility for use as biomedical implants. Further studies about the fatigue behavior of these samples are welcome to evaluate their mechanical properties under dynamic loads [7].

Table 3.

Mechanical properties comparison of some commercial metallic biomaterials.

  Young’s modulus (GPa)  YTS (MPa)  UTS (MPa)  Elongation(%)  Elastic admissible strain (%)  Specific strength(kPa. m3/kg)  Ref. 
F15 CoCrMo  240  450  900-1540  0.2  54  [8,26] 
F138 SS 316 L  200  190-690  540-1000  12-40  0.1-0.4  24-86  [8,26] 
F67 CP Ti  105  275  345  20  0.3  61  [8,26] 
F1108 Ti-64  110  875  965  10-15  0.8  198  [8,26] 
F1713 Ti-1313  79-84  836-908  973-1037  10-16  1.0–1.1  160-174  [8,26] 
F2066 Ti-15Mo  78  544  874  21  0.7  109  [8,26] 
TNZT  55-66  800  830  10  1.2-1.4  –  [30,31] 
TMZF  74-85  1000-1060  1060-1100  18-22  1.2-1.4  –  [30,31] 
Ti-(10-80)Nb  65-93  760-930  900-1030  10  0.8-1.4  –  [30,31] 
Ti-(70-80)Ta  80-100  350-600  600-650  10-25  0.4-0.8  –  [30,31] 
Ti-15Zr  103 ± 4  555 ± 95  905 ± 47  13.8 ± 0.8  0.5 ± 0.2  121 ± 2  This study 
Ti-15Zr-5Mo  79 ± 2  1053 ± 65  1274 ± 62  8.3 ± 1.8  0.9 ± 0.1  157 ± 1  This study 
Ti-15Zr-10Mo  74 ± 1  854 ± 63  1002 ± 18  18.7 ± 2.8  1.0 ± 0.1  168 ± 1  This study 
Ti-15Zr-15Mo  69 ± 1  933 ± 34  1015 ± 42  10.3 ± 1.9  1.3 ± 0.2  179 ± 1  This study 

In the indirect cytotoxicity test (Fig. 11), it can be noted that the number of viable cells in the presence of the extracts was high when compared to the positive control, which is also similar to the negative control. The statistical analysis (p < 0.05) indicated a significant difference between all the samples and the positive control (*). When compared to the negative control, only the Ti-15Zr-10Mo sample exhibited a significant difference (**). As this test evaluates the effects of particles and ions released into the culture medium by the samples, it can be concluded that the studied samples did not liberate any toxic component and neither affected the growth and cell viability during the studied time [33]. In Fig. 12 is possible to observe that the cells presented a similar morphology in all studied extracts, being quite different from the positive control (1 % phenol solution). It was possible to notice that the extracts did not interfere in the cell adhesion and morphology, having the cells exhibited a central flattened cell body on the glass with numerous cellular processes, as well as in the negative control. These aspects are far different from the positive control, where the cells displayed a damaged cytoplasm without any cellular processes [17,34]. Regarding the contact angle measurements, the Ti-15Zr-15Mo (78.9° ± 0.1) sample presented the lowest value between the samples (Ti-15Zr: 86.7° ± 0.1; Ti-15Zr-5Mo: 97.2° ± 0.1; Ti-15Zr-10Mo: 88.6° ± 0.1), and it was comparable to the commercial biomaterials CP-Ti (73.5° ± 0.1) and Ti-64 (72.8° ± 0.1). As the contact angle has some influence on the proper cell viability (adhesion, differentiation, and proliferation) of the metallic surface [35], it can be concluded that the surface of the Ti-15Zr-15Mo sample could be a suitable substrate for the interaction of bone cells with the implant. Further studies on the biological response of the Ti-15Zr-15Mo sample are welcome to evaluate in-depth its interaction with tissues and cells.

Fig. 11.

Indirect cytotoxicity test of the Ti-15Zr-based alloys after 72 h (n = 8).

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Fig. 12.

SEM images of cultured cells (72 h): A) Negative control; B) Ti-15Zr; C) Ti-15Zr-5Mo; D) Ti-15Zr-10Mo; E) Ti-15Zr-15Mo and F) Positive control.

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4Conclusion

In this study, Ti-15Zr-based alloys with different amounts of Mo were produced for use as load-bearing implants, such as total joint replacements. The phase composition and microstructure were dependent on the alloying elements, being the Mo content of 5 wt% enough to precipitate the β phase. The Ti-15Zr-10Mo and Ti-15Zr-15Mo samples, composed by the β phase and small quantity of ω phase, presented similar mechanical strength, but distinct elongation and Young’s modulus values. All samples exhibited dimples-type structures along their fractured surfaces, which indicated a characteristic ductile behavior. Regarding a possible biomedical application, the Ti-15Zr-15Mo sample showed better mechanical biocompatibility than most commercial metallic materials, without any evidence of cytotoxicity on osteoblastic cells and proper wettability values. Therefore, the designed alloy could be suitable to mitigate the stress shielding effect, support the biomechanical loads of the body, and increase the lifespan of the implant without inducing cytotoxic effects.

Conflict of interest statement

I would like to inform that all authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript.

Acknowledgments

The authors acknowledge Ph.D. Livia Sottovia and Professor Nilson C. Cruz, from the Laboratory of Technological Plasmas (UNESP – Campus Sorocaba) for the contact angle measurements. The research was supported by the Brazilian funding agencies National Council for Scientific and Technological Development (CNPq), grants #207417/2015-6, #400.705/2015-0, and #308.204/2017-4, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grants #2010/20440-7, #2015/00851-6, and #2015/25.562-7, and also by the Japan Agency for Medical Research and Development (AMED), International Collaborative Research Program: Strategic International Research Cooperative Program (SICP), No. 16jm0310021h0004 and The Light Metal Educational Foundation, Inc. The authors also thank the reviewers for valuable comments.

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