In vivo osteoconductivity of surface modified Ti-29Nb-13Ta-4.6Zr alloy with low dissolution of toxic trace elements
In vivo osteoconductivity of surface modified Ti-29Nb-13Ta-4.6Zr alloy with low dissolution of toxic trace elements
Eri Takematsu 0 1
Kimihiro Noguchi 1
Kensuke Kuroda 1
Toshiyuki Ikoma 1
Mitsuo Niinomi 1
Nobuhiro Matsushita 1
0 Department of Biomedical Engineering, University of Texas at Austin , Austin, Texas , United States of America, 2 Department of Mathematics, Western Washington University , Bellingham, Washington , United States of America, 3 Institute of Materials and Systems for Sustainability, Nagoya University, Chikusa , Nagoya , Japan , 4 Department of Materials Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology , Meguro, Tokyo , Japan , 5 Institute for Materials Research, Tohoku University, Aoba-ku, Sendai, Japan, 6 Department of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University , Suita, Osaka , Japan
1 Editor: MarÂõa Angeles PeÂrez, Universidad de Zaragoza , SPAIN
Simulated Body Fluid (SBF) has served as a useful standard to check the bioactivity of implant materials for years. However, it is not perfectly able to imitate human serum; sometimes disparities between the SBF test and animal test were confirmed. Therefore, to ensure the reliability of the results of the SBF test obtained from our previous study, an animal study was performed to check osteoconductivity of surface modified implant materials. Three types of solution processes, hydrothermal (H), electrochemical (E), and hydrothermal-electrochemical (HE), were performed on the Ti-29Nb-13Ta-4.6Zr alloy (TNTZ) to improve its bioactivity, and their bioactivities were measured in vivo using bone-implant contacts (BICs). BICs of the HE- and H-treated samples were significantly higher than that of the control. Metal ion diffusion towards the bone was also evaluated to examine the adverse effect of metal ions. No metal ion diffusion was observed, indicating the safety of our solution processed implant materials.
Data Availability Statement: All relevant data are
within the paper.
Funding: This research was funded by a grant from
the Collaboration Research Project of Advanced
Materials Development and Integration of Novel
Structured Metallic and Inorganic Materials
supported by MEXT as well as the collaborative
research of the Institute for Materials Research at
Tohoku University (http://www.msl.titech.ac.jp/
For the development of a new implant material, its bioactivity is a primary concern. To
evaluate bioactivity in a simple and inexpensive way, the simulated body fluid (SBF) test was
developed by Kokubo et al. [
]. This test assesses bioactivity of a sample through the formation of
apatite on the sample. Since this is an easy and simple test, much research has relied on this
test for checking bioactivity of samples. For example, NaOH-CaCl2-heat-water treated
titanium alloy induced apatite formation in one day, implying its possible usage for implant
]. However, several research groups have reported that the results of the SBF tests and
animal tests contradict each other [2±5]. A recent review about the relationship between the
SBF test and in vivo test highlighted that 8 out of 33 reports displayed different bioactivity
results between the SBF test and in vivo test [
]. These disagreements are possibly due to
differences between SBF and actual animal serum. In particular, SBF is different from human blood
serum in terms of three important factors: i) the absence of proteins, ii) the presence of TRIS
buffer in the SBF solution, and iii) the irregular carbonate content [
]. Human serum is
composed of proteins that play important roles in osteointegration and carbonate, which acts as a
pH buffer in serum. Thus, it is very important to consider these factors when checking the
bioactivity of implant materials to minimize disparities with animal tests.
In our previous study, the SBF tests were conducted on a surface modified
Ti-29Nb-13Ta4.6-Zr (TNTZ) alloy [
]. This alloy is newly developed alloy for biomedical use due to its ideal
Young's modulus close to human bone and its high biocompatibility [
To improve its biocompatibility, three types of surface modification processes were
performed in the previous study: hydrothermal (H), electrochemical (E), and
hydrothermal-electrochemical (HE) processes [
]. One of the reasons for employing these solution processes is
that they enable us to create metal oxides on the surface containing a lot of sodium ion, which
facilitates the formation of apatite, leading to higher osteoconductivity. A second advantage is
that they are environmentally friendly processes without using an energy consuming vacuum
system. A third advantage is that it has good mechanical properties. In our previous study,
these solution-processed TNTZ had a sufficient adhesive strength to be used as a biomaterial
if we control the processing time properly [
]. In addition to those advantages, HE process
has been known to create a fast-growing thin ceramic layer for several applications [
Therefore, it is also expected that fabrication time is much reduced than other processes such
as the NaOH-CaCl2-heat-water treatment for the application of implant material.
Our previous report revealed that the order of apatite inductivity for the three processes
was HE > H > E without impairing adhesive strength between alloy surface and apatite layer
]. However, these results were obtained without considering the in vivo environment, in
which blood flow and proteins such as collagen exist. Therefore, in this paper, bioactivity of
the TNTZ alloy surface modified by the three processes mentioned above was evaluated in vivo
based on bone-implant contact (BIC). Moreover, we conducted a non-parametric statistical
analysis on the BIC data of the TNTZ samples to provide a new insight into the treatment
effects of these solution processes. It is noted that the statistical analysis presented in this paper
is applicable to a wide range of biomedical studies where only small numbers of observations
are available. Lastly, in addition to the BIC measurements, electron dispersive x-ray
spectrometer (EDX) mapping was also performed to observe metal ion diffusion towards the bone
region since metal ions were reported to cause bone resorption .
Materials and method
Material and sample pretreatment
The material used in this study was a forged TNTZ bar. The forged TNTZ bar was solutionized
at 1073 K for 3.6 ks followed by water quenching. TNTZ samples with a thickness of 5 mm
and a diameter of 2 mm were machined from the forged TNTZ bar subjected to solution
treatment. The TNTZ samples were then degreased prior to the experiment by sonication in
acetone and distilled water (Millipore Milli-Q, Merck Millipore; Germany) for 10 min each and
air-dried at ambient temperature.
H, E, and HE processes were then performed on the TNTZ samples. All the samples were
treated with 5 M NaOH solution containing 0.17 wt% of NH4F for 1 h. For the H and HE
processes, the hydrothermal temperature was set at 90ÊC, and for the E and HE processes, the
applied current density was set to 15 mA/cm2 [
]. All of the treated TNTZ samples were then
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stored in 5X PBS solution (1 to 2 dilution of 10X PBS, Thermo Fisher Scientific, Japan) at
room temperature to retain the surface hydrophilicity until they were implanted [
The TNTZ samples subjected to each solution treatment mentioned above and untreated
control were implanted in the tibia metaphysis of seven-week old Sprague Dawley rats (Charles
River Japan, Yokohama) weighing 330±360 g. Bioactivities were evaluated from new bone
formation. Before surgery, all the TNTZ samples were cleaned with distilled water. Rats were kept
in quarantine for seven days. Prior to surgery, the rats were anesthetized with pentobarbital
(25±30 mg/kg), and the operation region was shaved and cleaned with povidone-iodine
solution and ethanol. Holes with a diameter of 0.5 mm and a length of 2 mm were created at the
middle of the tibia metaphysis using a slow speed rotary drill, and then the TNTZ samples
were inserted in the hole. To prevent festering of the operation site, Penicillin G was
administered. Then, the skin was sutured and cleaned with povidone-iodine solution.
After surgery, the rats were kept individually in polycarbonate cages and reared for two
weeks. No complications or ill effects were observed while rearing. Rats were then sacrificed
by exsanguination being anesthetized with 2±3% isoflurane and the implanted TNTZ samples
with surrounding tissues were explanted. The TNTZ samples were fixed in 10% neutral
buffered formalin solution followed by rinsing with distilled water. Then, the rinsed TNTZ
samples were dehydrated in a graded series of ethanol, followed by acetone, and embedded in
methylmethacrylate. Subsequently to polymerization, each TNTZ sample was mechanically
sliced to be 20 μm thick using a microcutting machine (BS-3000N, EXAKT GmbH, Germany)
and a microgrinding machine (MG-4000N, EXAKT GmbH, Germany). The sliced sample
was then stained with toluidine blue and observed by light microscopy to determine BIC. This
experiment was carried out at the laboratory of Hamri Co., Ltd. (Ibaraki, Japan), which has
been approved by the Association for the Assessment and Accreditation of Laboratory Animal
Care (AAALAC) International. The experimental protocol was approved by the Institutional
Animal Care and Use Committee (IACUC) of Hamri Co., Ltd. (Ibaraki, Japan).
Measurement of BIC
BIC was determined by the linear measurement of direct bone contact with the implant
surface, which was calculated using the following formula:
BIC The sum of the length of the bone formation on the implant surface
Tota l inserted implant length
BIC was measured separately in cancellous and cortical bone regions using a fluorescence
microscope (Olympus BX-51-33PH-SP, Japan) (n = 3-6/group). Statistical analysis was then
performed to determine the treatment effects of different solution processes.
Statistical analysis was performed on the BIC data of the TNTZ samples with and without each
alkali solution treatment in the cancellous bone region and is detailed below in the following
Outlier detection. Due to small sample sizes, outlier detection was performed by R using
Hampel's identifier [
]; the statistic is commonly referred to as the modified Z-score [
Hampel's identifier utilizes robust measures of center (median) and scale (median absolute
deviation), and hence it is a more reliable metric compared to the raw Z-score or methods
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based on sample mean and sample standard deviation, especially for small sample sizes. Any
observation exceeding the cut-off value of 3.5, as recommended by Iglewicz and Hoaglin
], was declared as an outlier in our study.
Pairwise comparisons of treatments. With such small sample sizes, it was not possible
to assume any underlying distribution of the data. Therefore, a robust non-parametric
rankbased multiple contrast testing procedure [
] was performed using the nparcomp package in
]. The procedure above assessed the overall significance and pairwise comparison results
at the same time without any contradiction based on the concept of simultaneous test
]. It is robust to non-normal distributions, small or unequal sample sizes, and unequal
variances while maintaining a high power [
]. It is important to note that non-parametric
procedures utilize relative effects rather than means to compare samples.
Effect sizes. Effect sizes, which are used to supplement the results from statistical tests, are
reported to provide a more intuitive understanding of the magnitude of differences between
samples. To facilitate the understanding of its magnitude, we employed the Hasselblad-Hedges
effect size, which is useful for relative effects [
]. Specifically, the formula is:
p1; p2 1:81 ln
where p1 and p2 are the relative effects of the two samples being compared. the
HasselbladHedges effect size allowed us to interpret the magnitude of effects easily due to its similarity to
one of the most widely used effect sizes called Cohen's d. Specifically, the common cut-off
values of 0.2, 0.5, and 0.8 in absolute values were respectively used as small, medium, and large
effects in this study. Statistical significance was determined at p < 0.05, where p denotes the
Analysis of metal ion dissolution
The interface region of bone and TNTZ on the sliced implanted sample was observed using
a scanning electron microscopy (SEM) (Hitachi S 450, Japan) with an acceleration voltage of
15 kV. To confirm any metal ion diffusion from the TNTZ alloy into surrounding tissue, the
interface region of the bone and TNTZ alloy on the sliced implant was analyzed using an
energy-dispersive X-ray spectrometer (EDX, JEOL JSM-5410LV, Japan). Then, elemental
mappings of Ca, Ti, Nb, Ta, and Zr were conducted. For surface chemistry analysis, X-ray
photoelectron spectroscopy (XPS) was performed in a PerkinElmer 5500MT spectrometer. XPS
data were acquired using Al Kα X rays with pass energy 8 kV. Sputter cleaning was done with
Ar+ sputter gun. MaltiPack software was used for the analysis.
Results and discussion
BIC. No significant differences were observed in the cortical bone region, while
significant differences were detected in the cancellous bone region between samples-Box plots of
BICs of the TNTZ samples without the alkali solution treatment (referred to as raw) and with
each alkali solution treatment in the cortical bone and cancellous bone region are shown in Fig
1. The bottom and top of the box show the first and third quartiles (bottom 25% and top 25%),
respectively, and the middle bold line in the box shows the median. The top and bottom
horizontal lines of the vertical dotted lines for each box exhibit the minimum and maximum values
of each sample, respectively.
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Fig 1. Box plots of BICs of TNTZ samples without (raw), and with H, E, and HE processes (H, E, and HE, respectively) in the (a) cortical and (b) cancellous bone
In the cortical bone region, there were only minor differences in the BIC among the TNTZ
samples without and with alkali solution treatments, and the median of each TNTZ sample
was between 20±50%. On the other hand, BIC in the cancellous bone region showed clear
differences among the TNTZ samples with and without alkali solution treatments. The TNTZ
samples subjected to the HE, H, and E processes had much higher median values of 78, 62, and
51%, respectively, than the raw TNTZ sample (15%). Such a different tendency in the BIC
values between the cortical and cancellous bone regions may arise from blood flow differences in
these two regions. It is widely known that the blood flow is much faster in the cancellous bone
than in the cortical bone [
]. Therefore, healing of the cancellous bone is faster than that of
the cortical bone. Moreover, the implanted period of the TNTZ samples in this study was two
weeks. Thus, it is difficult to observe the differences in BICs of the TNTZ samples in the
cortical bone region where the healing was relatively slow. For the reasons mentioned above,
analyzing BIC in the cancellous bone was appropriate for evaluating the difference in the
osteoconductivity among the TNTZ samples with and without each alkali solution treatment.
Statistical comparisons. Pairwise and effect size comparisons clearly show the difference
in each treatment- No outlier was detected using Hampel's identifier. As such, all of the
observations were used in the analysis. Specifically, there were 5, 5, 6, and 3 observations from the
TNTZ samples subjected to H, E, and HE processes, and the raw TNTZ sample for the
cancellous bones, respectively.
Table 1 summarizes the adjusted p-values for the all-pairwise differences in the BICs of
the cancellous bones. The p-values were adjusted appropriately for multiplicity using the
multivariate t-distribution, and statistical significance was determined at p < 0.05. In particular,
statistically significant evidence of alkali solution treatment effects was found when the TNTZ
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sample subjected to the H process (H) and the raw TNTZ sample (Raw) were compared, and
also when the TNTZ sample subjected to the HE process (HE) and the raw TNTZ sample
(Raw) were compared.
A question that arose from these findings was if there was any practical difference between
the TNTZ samples subjected to H and E, HE and E, and H and HE. Even though the pairwise
comparisons above revealed no statistically significant difference, this may be due to the low
power of detecting any statistically significant difference for very small sample sizes. As a
remedial measure, the Hasselblad-Hedges effect size was also calculated for measuring the practical
magnitude of pairwise differences. Table 2 summarizes the Hasselblad-Hedges effect size for
the cancellous bones. In the table, both the effect sizes measured by C(p1, p2) and the
corresponding magnitude of effect for the six all-pairwise comparisons were reported. Several
medium and large effects were observed by using the effect size C(p1, p2). For the cancellous
bones, the difference between the TNTZ samples subjected to H and Raw, E and HE, E and
Raw, and HE and Raw, were considered either medium or large.
The analysis above suggested advantages of some methods, which were not clear from the
statistical tests. In particular, the effect size of -0.681 for the pair E and HE showed a medium
advantage of HE over E. Moreover, the comparison between H and HE resulted in the effect
size of -0.224, indicating a small advantage of HE over H. From these observations, some
advantage of the HE treatment over H and E was suggested. In addition, the ones that showed
statistically significant differences (H vs. Raw and HE vs. Raw) had large effects (1.191 and
1.416, respectively), implying substantial advantages of the H and HE treatments over the
untreated TNTZ sample.
Diffusion of alloying elements. No alloying elements diffused into bone- Figs 2±4 show
EDX mapping on the surface of the TNTZ sample subjected to each alkali solution treatment.
Ca indicates the bone region, and Ti, Nb, Ta, and Zr indicate the alloying elements. Metal ions
were under the detection limit in the bone region, implying that there was less metal ion
diffusion towards the bone region. The bone directly connected with the TNTZ samples subjected
to H and HE as shown in Figs 2 and 4. However in Fig 3 (E), only a small part of the bone
connected with the TNTZ sample subjected to E. Therefore, EDX mapping revealed poor
osteoconductivity of the TNTZ sample subjected to E.
Fig 2. EDX mappings of Ca, Ti, Nb, Ta and Zr at interface between the TNTZ sample subjected to H and bone along with SEM image.
BIC. In the cancellous bone region, statistical analysis of BIC indicated that the TNTZ
samples subjected to the H and HE processes had significantly higher BIC values than that of
the raw TNTZ sample (p < 0.01) while the BIC value of the TNTZ sample subjected to the E
process was not significantly different from that of the raw TNTZ sample. These differences
could be attributed to the chemical composition of the sample surface. Our previous
experimental results suggested that the sample surface with higher Nb oxide is likely to inhibit apatite
formation, and a higher amount of Nb was found on the surface of the TNTZ subjected to the
E process [
]. In this study, it was also confirmed by XPS that a higher amount of Nb was
found on the surface of the TNTZ sample subjected to the E process; a lower amount of Nb
was found on the surfaces of the TNTZ samples subjected to the H and HE processes. Table 3
shows the Nb/Ti ratio of the TNTZ sample subjected to the E process is more than twice
as much as those of the TNTZ samples subjected to the H and HE processes. Our previous
] has confirmed that Nb on the surface of the TNTZ sample exists as Nb oxide. The
reason why Nb oxide is likely to exist on the surface of the TNTZ sample has not been
determined yet. A previous report [
] revealed that surface oxides were mainly Na-contained
amorphous titanium oxides, where Ti4+ ion was coordinated with oxygen and stable with some Na+
ions. When the TNTZ samples were subjected to the H and HE processes, their surfaces were
mainly composed of the Na-contained amorphous titanium oxide. However, for the TNTZ
sample subjected to the E process, Nb oxides took up spaces where Na-containing amorphous
titanium oxides existed, as shown in Fig 5. Here, the Nb oxide does not contain Na+. As a
result, the total amount of Na+ ions decreases. Therefore, the TNTZ sample subjected to the E
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Fig 3. EDX mappings of Ca, Ti, Nb, Ta and Zr at interface between the TNTZ sample subjected to E and bone along with SEM image.
process contained less Na+ ions on its surface, which releases less Na+ ions in blood plasma,
resulting in a less negatively charged surface as compared to those of the TNTZ samples
subjected to the H and HE processes. As a result, the surfaces of the TNTZ samples subjected to
the H and HE processes were very much negatively charged and could attract Ca2+ ions easily.
Once Ca2+ ion is adsorbed on the surface, fibronectin, which is considered as an important cell
adhesion molecule, binds with the surface via a Ca2+ site by its carboxyl group [
Fibronectin adsorbs more on the surfaces of the TNTZ samples subjected to the H and HE processes,
leading to higher osteoblast cell adsorption and a higher BIC value. The tendency of the BIC
values matched with that of the in vitro experiment, which analyzed osteoconductivity using
an SBF immersion test [
]. According to this study, apatite inductivity was the highest for the
TNTZ sample subjected to the HE process, followed by the H and E processes. This tendency
corresponded with the results from the in vivo experiment and supported the usefulness of the
immersion test in SBF solution as an indicator of osteoconductivity.
Alloying elements. Several studies showed adverse effects of the Ti4+ ions release on bone
remodeling. Mine et al. suggested that the Ti ion inhibited the differentiation of osteoblasts
and altered the ratio of RANK ligand and osteoprotegerin (OPG) gene expression, which
caused bone resorption at the interface of dental implant and tissues [
]. Other reports
showed that only a small amount of Ti altered the behavior of macrophage-like RAW264 cells
to enhance phagocytosis, which caused oxidative stress and inflammation [
]. Therefore, it is
imperative to investigate any metal ions diffusion towards the bone region. EDX mapping
results showed metal ion diffusions towards bone region that is less than a detection limit,
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Fig 4. EDX mappings of Ca, Ti, Nb, Ta and Zr at interface between the TNTZ sample subjected to HE and bone along with SEM image.
which indicates that there is no or little amount of metal ion diffused to bone. This suggests
the possible usage of alkali treated TNTZ as a safe implant material. The reason why the TNTZ
samples subjected to alkali solution treatment showed good corrosion resistance was
speculated because the fabricated oxide layer was a thicker amorphous layer. The previous report by
Mizutani revealed that a thick oxide layer prevented the dissolution of any metal ion [
thicknesses of the surface layers fabricated on the TNTZ samples subjected to the E, H, and
HE processes were 0.65, 0.78, and 2.26 μm, respectively. Such thick oxide layers may prevent
metal ion diffusion as earlier stated. Moreover, it can be said that amorphous passive layers
have a good corrosion resistance because they hardly contain grain boundaries or structural
]. According to the previous report , fabricated oxide layers are composed of
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Fig 5. Schematic illustration of the position of oxides. Left: when treated by the H and HE method. Right: when treated by the E method.
an amorphous phase, which may serve as a good passive layer. From these factors, little of no
metal ion diffusion was observed on the TNTZ sample subjected to alkali solution treatment.
However, this is a bulk level study, therefore further study will be needed to scrutinize the
cytotoxic effect of diffusion elements from TNTZ.
In summary, the bioactivity of TNTZ, whose surface was modified by alkali solution
treatments (H, E, and HE processes), was evaluated in vivo based on the bone-implant contact
(BIC) test and deemed as safe biomaterials. The HE process, in particular, is a very promising
surface modification process for TNTZ as it results in high osteoconductivity and corrosion
resistance. Specifically, the degree of osteoconductivity in vivo matched with the results from
the in vitro experiment [
], supporting the usefulness of the immersion test in SBF solution as
an indicator of osteoconductivity in this study. Also, our statistical analysis of the BIC data
using recent non-parametric hypothesis tests and the Hasselblad-Hedges effect size empirically
support the conclusion. It is noted that the statistical analysis presented in this paper could be
highly applicable to other studies in biomedical research where small sample sizes are
prevalent. The present results suggest that HE treated TNTZ alloy may be helpful for developing
novel orthopedic implant due to its great osteoconductivity and corrosion resistance.
This work has been partly supported by the project of Creation of Life Innovation Materials
for Interdisciplinary and International Researcher Development, MEXT, Japan. The authors
gratefully acknowledge the support from the collaborative research of Institute for Materials
Research at Tohoku University. The authors also thank Prof. Katsumata of Tokyo University
of Science for helpful advice and Dr. Seki of Hamri Co., Ltd for conducting animal tests.
Conceptualization: Toshiyuki Ikoma, Nobuhiro Matsushita.
Data curation: Eri Takematsu.
Formal analysis: Kimihiro Noguchi.
Funding acquisition: Nobuhiro Matsushita.
Investigation: Eri Takematsu, Nobuhiro Matsushita.
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Methodology: Kimihiro Noguchi, Nobuhiro Matsushita.
Project administration: Nobuhiro Matsushita.
Supervision: Mitsuo Niinomi, Nobuhiro Matsushita.
Validation: Kimihiro Noguchi, Kensuke Kuroda, Toshiyuki Ikoma.
Writing ± original draft: Eri Takematsu.
Writing ± review & editing: Kimihiro Noguchi, Kensuke Kuroda, Toshiyuki Ikoma, Mitsuo
Niinomi, Nobuhiro Matsushita.
11 / 12
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