Colloids and Surfaces B: Biointerfaces 189 (2020) 110889

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Biocompatibility, osseointegration, antibacterial and mechanical properties T

of nanocrystalline Ti-Cu alloy as a new orthopedic material
S. Moniri Javadhesaria, S. Alipourb, M.R. Akbarpourc,*
a
Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
b
Department of Materials Science and Engineering, Sharif University of Technology, P.O. Box 11365-9466, Azadi Ave., Tehran, Iran
c
Department of Materials Engineering, Faculty of Engineering, University of Maragheh, Maragheh, P.O. Box 83111-55181, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: The demands for high-performance biomaterials are driving the development of new metallic alloys with im-
Biomaterial proved mechanical and biological responses. In this study, a nanocrystalline Ti-Cu intermetallic alloy was
Ti-Cu alloy prepared by a powder metallurgy route, and its application as an orthopedic material was evaluated by the
Antibacterial activity microstructural, mechanical, corrosion, antibacterial, cytotoxicity and osseointegration examinations.
Biocompatibility
Microstructural characterization revealed the formation of TiCu and Ti2Cu3 as major phases with 23 nm grain
Osseointegration
size in the structure of the alloy. The synthesized alloy exhibited ultra-high hardness of 10 GPa, acceptable
toughness of 8.14 MPam1/2, a ∼98 % anti-bacterial rate against S. aureus and E. coli, excellent cell viability to
MG-63 osteosarcoma cells, and high osteoblast formation rate, which indicate a great potential of this alloy for
biomedical application.

1. Introduction sputtering, sol-gel, alloying, chemical vapor deposition, and electro-

Nowadays, metallic biomaterials are applied in various parts of the
human body, such as implants replacing shoulders, knee, hips and
elbow, as well as in orodental structures [1,2]. Among the various
metallic biomaterials, those used as load bearing orthopedic implants,
should possess excellent biocompatibility, high wear resistance in the
body, a combination of high strength and low Young's modulus, high
resistance to fatigue and corrosion, high ductility and low cytotoxicity
[3]. The common metals used as biomaterials, especially in bone and
teeth, are 316L stainless steel, cobalt-chromium alloys, commercial
pure titanium and titanium-based alloys such as Ti-6Al-4 V, Ti-Nb-Zr,
and Ti-Nb-Ta-Mo [3–5]. In the meantime, titanium alloy, a lower elastic
modulus closer to that of human bone, as well as high strength, ex-
cellent corrosion resistance, and biocompatibility is considered as one
of the ideal choices for orthopedic and dental implants in the medical
industry compared to other materials [3,6]. Considering that the for-
mation of bacterial biofilms on the implant surface [7], low ability of
bone tissue growth on the surface, and poor wear resistance are the
main challenges of titanium alloys in the field of biomaterials, the de-
velopment of titanium alloys by changing chemical composition seems
to be necessary. However, to improve the antibacterial properties of
titanium and its alloys, various metals such as silver and copper have
been added to the alloy surface by methods such as ion implantation,
chemical methods [8]. Ti-Cu alloys, due to lower price of copper than
silver and the low toxicity of Cu2 + ions in the body compared to Ag+,
are considered as one of the new titanium alloys in the field of ortho-
pedics and dentistry [8]. In a study by Zhang et al. [9], titanium alloys
with 2, 3, and 4 wt% Cu were produced through casting, solid solution
and aging process and their antibacterial, mechanical, and bio-corro-
sion properties were examined. The obtained microstructure included
the alpha titanium matrix with dispersed Ti2Cu precipitates. Their re-
sults indicated that the increase in the amount of copper element sig-
nificantly increased the antibacterial rate to a maximum of 92.57 %
after 24 h of culture in S. aureus for Ti-4% wt Cu sample. In addition,
the uniform distribution of Ti2Cu fine precipitates in the titanium ma-
trix due to the application of T6 thermal treatment on the bulk samples
was a major factor in the improvement of antibacterial behavior along
with mechanical and anti-corrosion properties. In another study [10],
the cytotoxicity of Ti-Cu alloys with 2, 5, 10, and 25 wt% Cu was ex-
amined by CCK8 assessment. Their results revealed very good bio-
compatibility of these Ti-Cu alloys. In addition, increasing the amount
of Cu up to 25 wt% in the Ti-Cu alloy had no effect on cell proliferation
and differentiation. On the other hand, copper ion is one of the trace
elements that is essential for human health and plays an effective role in
the formation of various enzymes such as cytochrome c oxidase and
superoxide dismutase [10]. Also, the need for this ion during pregnancy

⁎
Corresponding author.
E-mail address: akbarpour@maragheh.ac.ir (M.R. Akbarpour).

https://doi.org/10.1016/j.colsurfb.2020.110889
Received 28 August 2019; Received in revised form 18 February 2020; Accepted 20 February 2020
Available online 20 February 2020
0927-7765/ © 2020 Elsevier B.V. All rights reserved.
S. Moniri Javadhesari, et al.
and lactation is vital for the growth and development of the fetus and
neonates [11–14]. However, the high amounts of copper ions through
cyclic reactions of oxidation/reduction during the conversion of Cu (I)
to Cu (II) as well as the production of ROSs can cause cytotoxicity to
cells and lead to their death [15]. In addition, large amounts of copper
ions in the human body can cause diseases such as stomach upset,
nausea, and diarrhea [16,17]. It is ascertained that the formation of
intermetallic compounds in the alloy not only improves the strength
and hardness of Ti-Cu alloys [18,19], it also may lead to a much lower
ion release in the body compared to Ti-Cu solid solutions and copper-
rich phases. Therefore, the development of Ti-Cu alloy microstructure
to achieve simultaneous mechanical, antibacterial, and biocompat-
ibility properties by increasing intermetallic compounds in Ti-Cu
system is of interest. This solution eliminates concerns about the toxi-
city of copper ions in the body and makes possible the addition of high
amount of Cu alloying element to Ti to achieve high antibacterial ef-
fects in the alloy, which avoids inflammation after orthopedic surgeries.
Furthermore, researches show that the surface grain size of the mate-
rials is an important factor for bone formation ability; therefore na-
nostructured materials show higher bone formation efficiency than
micro-grained materials [5,20]. Thus, in the current study, a nanos-
tructured Ti-Cu alloy containing 57.03 wt% Cu with a completely in-
termetallic structure was synthesized and its microstructure, mechan-
ical properties, antibacterial behavior, cell viability and osteoblast
formation ability were studied.
2. Materials and methods
Elemental copper powder with particle size less than 20 μm and
purity of 99.7 % and titanium powder with particle size less than 70 μm
and purity of 99.5 % were used in this study. High-energy ball milling
of Ti-57.03 wt% Cu powder mixture was performed in a planetary ball
mill (Retsch 400MA) at 300 RPM. Milling was carried out in a high-
chromium carbon hardened steel vial under Ar atmosphere with a ball
to powder weight ratio (BPR) of 10:1 at room temperature for 30 h.
0.5 wt% stearic acid was added to the mixture to accelerate the milling
process. The milled powder was compacted under the pressure of 1 GPa.
Then the compacts were sintered at 900 ᴼC for 1 h under 10−4 to 10−5
Torr vacuum.
X-ray diffraction (XRD) patterns (using a Philips diffractometer)
with CuKα radiation) was applied for microstructural and phase ana-
lysis. The average grain size and microstrain of samples were de-
termined from broadening of the XRD peaks using Scherer equation.
Scanning electron microscopes (SEM, Philips XL30 and Mira3 XMU)
equipped with EDS were used for observations of powder morphology
and microstructure of sintered samples. Transmission electron micro-
scopy (TEM, FEG Philips CM200) was also used for microstructural
analysis of the samples. Vickers microhardness of the samples was
measured by a Vickers indenter (QV-1000DAT) at load of 30 kg and a
dwell time of 15 s.
The osteoblast MG-63 cells obtained from National Cell Bank of Iran
(NCBI; Pasteur institute) were cultured in Dulbecco’s Modification of
Eagles Medium (DMEM; Gibco, Scotland) containing 100 μg/mL peni-
cillin (Sigma–Aldrich), 100 μg/mL streptomycin (Sigma–Aldrich), and
10 % fetal bovine serum (FBS; Gibco, Scotland) in a humidified atmo-
sphere of 5% CO2 at 37 °C. The culture medium was refreshed every 2
days. The Ti and TiCu samples were cut into 10 mm diameter round
discs (3 discs for each sample) and sterilized by immersing in ethanol
70 % and PBS. In order to assess the cell adhesion on samples, 2 × 104
cells/ml were seeded in a 6 well culture plate containing discs. Cell
culture continued for 2, 4 and 7 days. Then, the culture medium was
removed and samples were fixed with 4% paraformaldehyde. Finally,
cells were stained by Hoechst 33258 for 15 min and studied under the
Olympus fluorescent microscope by a filter of 460–490 nm wave-
lengths.
The viability of cultured MG-63 cells was examined using indirect 3-
2
Colloids and Surfaces B: Biointerfaces 189 (2020) 110889
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma, USA) assessment. Briefly, 1 mL of culture medium was added to
each sample (5 mg) and incubated at 37 °C for 2, 4 and 7 days. 104
cells/ml were seeded on a 96-well plate and incubated at 37 °C for 24 h.
Then, the culture medium was exchanged with the 2, 4 and 7-day ex-
tract of samples and incubation continued for 24 h. Finally, the medium
was removed and 100 μL of MTT reagent (0.5 mg/mL in PBS) was
added to each well and incubated at 37 °C for 4 h. After 4 h, 100 μL of
DMSO was added to each well to dissolve formazan crystals completely
and the absorbance was read at 545 nm using an ELISA Reader (Stat
Fax-2100, Miami, FL, USA).
Alizarin red staining was applied to stain the calcium mineral de-
posits to visualize the extent of the apatite deposition. 2 × 104 cells/ml
were seeded on the surface of samples for 7 days. Then, the culture
medium was removed and three drops of 2% Alizarin red (pH 4.2 with
10 % ammonium hydroxide) were added to the coated samples and left
for 5 min. The excess of dye was removed by rinsing in distilled water
and drying by blotting paper. The samples were observed under the
light microscope (Axiophot, Zeiss, Germany) and five images were re-
corded from random areas on each sample.
At the next step, 10 mm diameter discs of Ti and Ti-Cu alloy were
prepared and assessed for antibacterial properties. The specimen’s
surfaces were grinded mechanically with emery papers down to 5000
grade, and then polished with 1 μm wet polishing diamond paste. Then,
the samples were washed with acetone, deionized water, and ethanol.
At the end, the samples were sterilized by autoclaving for 20 min before
antibacterial experiments. Briefly, 0.5 McFarland suspensions of E. coli
and S. aureus were prepared and diluted 100 times to decrease the
number of colony forming units (CFU). Then, the alloy discs were
placed at the bottom of a culture plate and 10 μl of each bacterial
suspension was added to the surface of each disc. Discs were covered by
sterilized microscopic cover glasses and incubated at 37 °C, 5% CO2 and
95 % relative humidity for 24 h. Finally, each alloy disc along with
cover glass was transferred to a test tube containing 5 ml of Luria-
Bertani (LB) medium, mixed carefully and 10 μl of each resulted sus-
pension was uniformly spread on a LB-agar plate. The plates were in-
cubated at 37℃ for 24 h and then the number of colonies was counted.
The same procedure was performed on a microscopic cover glass as a
control. Cu2+ ion release from Ti-Cu samples was measured in a 0.9 %
NaCl solution at 37 ± 1 °C according to ISO 10993-12 at 2, 4 and 7
days’ intervals using Inductively Coupled Plasma Mass Spectrometry
(ICP-MS). Pure titanium was used as a control sample for all of the
experiments.
Corrosion resistance of the Ti and Ti-Cu samples was measured by
using Potantiodynamic polarization test that was recorded in simulated
body fluid (SBF) solution at 25 °C with a scan rate of 10 mV.s−1. For
corrosion test disk samples with 6 mm diameter (surface
area = 28.26 mm2) were used. Before the tests, the samples were
grinded with emery papers down to 3000 grade.
3. Results and discussion
3.1. Microstructure characterization
Ti-57.03 wt% Cu powder mixture was mechanically milled for 30 h.
Then the milled powder was successfully sintered at the temperature of
900 ᴼC under vacuum. Fig. 1a shows a high magnification SEM-BSE
micrograph of the sintered sample. The figure reveals complicated
phase structure formed during sintering process. The co-existence of
TiCu, TiCu4, Ti2Cu3, and Ti2Cu phases in the Ti-Cu alloy is confirmed by
EDS analysis of different regions in BSE image. EDS analysis of the main
phases in the structure marked with points A and B are shown in
Fig. 1b, c. It is found that TiCu and Ti2Cu3 are the phases with high area
fraction in the bulk Ti-Cu alloy prepared by vacuum sintering. The
formation of TiCu4, TiCu2 and Ti2Cu phases as minor phases also was
revealed. The formation of these phases is the result of solid-state
S. Moniri Javadhesari, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110889

Fig. 1. (a) BSE-SEM micrograph, (b, c) EDS analysis of points A and B, (d) XRD pattern of the synthesized alloy.

diffusion reactions which are thermodynamically favorable in this (TEM) was used. Fig. 2a shows TEM image of the sintered Ti-Cu alloy.
system. Fig. 1d shows the XRD pattern of the sintered sample. From the The figure confirms nanocrystalline structure of the alloy. The average
figure, it can be seen that the sample contains the dominant phases of grain size of 23.5 ± 2.1 nm was obtained by intercept method for the
TiCu and and Ti2Cu3, and minor phases of TiCu4, TiCu2 and Ti2Cu, alloy, which is slightly higher than the value obtained by XRD. Fig. 2b
which have formed during sintering at high temperature. The compli- also represents selected area diffraction pattern of the alloy. The figure
cated intermetallic phase structure formed during sintering of this alloy reveals the presence of TiCu and Ti2Cu3 phase in the microstructure in
has been reported in previous works [21,22]. line with the findings obtained from SEM and XRD investigations.
In recent years, various studies have been conducted on the pro-
duction of bulk Ti alloys with different amounts of 2, 3, 4, 5, 10 and
3.2. Mechanical properties
25 wt% copper through casting or powder metallurgy methods possess
[9,10]. For example, in a study by Zhang et al. [9], titanium alloy
Vickers micro-hardness measurements were performed on polished
samples with 2, 3 and 4 wt% of copper were produced through casting
surface of the alloy. The hardness value of the alloy was ≈10 GPa
method followed by T4 and T6thermal cycles. The microstructure of the
(1020 ± 12 HV), which is very high value for Ti-Cu alloys. The hard-
produced samples was in the form of lamellar eutectoid with the alpha
ness value obtained for TiCu alloy prepared by vacuum sintering in this
titanium matrix and Ti2Cu intermetallic precipitates. In another study
work is about 50 % higher than the value reported by Shon et al. [22]
by Dong et al. [18], bulk titanium alloy with 5 and 25 wt% Cu were
for TiCu prepared by high energy milling and high frequency induction
produced by mechanical alloying and spark plasma sintering. The mi-
heating.
crostructure of the samples produced in their study included the ultra-
The high micro-hardness of the alloy can be attributed to the pre-
fine grained titanium matrix with Ti2Cu intermetallic precipitates.
sence of large amounts of TiCu and Ti2Cu3 intermetallic phases, as well
However, in the present study, the obtained microstructure is com-
as to the nano-size grains [23,24]. Fig. 3 displays the micro-hardness
pletely a multiphase intermetallic structure. Table 1 summarizes the
values of the synthesized alloy in this study compared to other ortho-
microstructural features of the sintered Ti-Cu alloy. Grain size and
pedic implant materials. The micro-hardness values for commercial ti-
microstrain of the sample was calculated using Williamson-Hall
tanium [25], Ti-6Al-4V [26], NiTi alloy [27], 316L stainless steel [28],
method.
Ti-Cu [9], Ti-Nb-Zr [4], Ti-Ag [28], Ti-Ta-Zr-Nb [27], and MP35N [29]
For detailed study of the sample, transmission electron microscopy
were 134, 500, 318, 172, 262, 660, 264, 433 and 238 Vickers,

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S. Moniri Javadhesari, et al.
Table 1
Microstructural features of the sintered Ti-Cu at 900 ᴼC.
Density (g/cm3) Crystallite Size (nm) (calculated from XRD results)
6.1 19.5
Fig. 2. TEM micrograph and selected area diffraction pattern of the alloy.
Fig. 3. Hardness of the synthesized alloy in comparison to other biomedical
materials.
respectively. As it is clear, the synthesized Ti-Cu alloy in this study, with
an ultrahigh hardness of over 1020 HV, could be a very good candidate
for hard and wear resistant orthopedic implant materials.
Generally, when measuring micro-hardness with sufficiently large
loads, median cracks generate around the indent on the surface of the
sample. Measuring the length of these cracks can be a criterion for the
prediction of material fracture toughness, which is presented by Nihara
et al. [30] according to Eq. (1).
c influenced by the microstructure of the alloy [35,36]. In a study by Mao
KIC = 0. 023 ( )−3/2 . HV . a1/2
a (1)
In this formula, KIC is the fracture toughness, c the length of the crack
trace measured from the center of the indentation in mm, a is half of the
average length of two indent diagonals in mm and Hv is the Vickers
hardness. By placing c, a, and Hv values in Eq. (1), the value of the
fracture toughness of the Ti-Cu alloy sample was 8.14 ± 0.15 MPam1/
2 This tendency can be attributed to the nature of the bonds between
. This amount of toughness was the average of 10 different measure-
ments and is considered to be quite acceptable for a very hard material
for wear resistant applications such as hard orthopedic implants.
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Colloids and Surfaces B: Biointerfaces 189 (2020) 110889
Microstrain (%) Phases Microhardness (HV)
0.290 TiCu, Ti2Cu3, Ti2Cu, TiCu4 1020 ± 12
3.3. Antibacterial behavior
Antibacterial behavior of Ti and Ti-Cu alloy against gram positive S.
aureus and gram negative E.coli showed high antibacterial behavior of
Ti-Cu alloy compared to Ti (Fig. 4a), assessed by colony counting for
24 h direct contact cultures. In order to compare the antibacterial
properties of the samples, the antibacterial rate was calculated. The
antibacterial rate (% R) shows the difference between the number of
colonies formed on control plate and the sample (Ncontrol-Nsample) di-
vided by the number of colonies in the control sample (Ncontrol) mul-
tiplied by 100, as shown in Eq. (2) [31]:
R (%)= (Ncontrol-Nsample)/Ncontrol×100 (2)
Fig. 4b represents the antibacterial rate of pure titanium and Ti-Cu
alloy. Antibacterial rate of Ti-Cu alloy against E. coli and S. aureus was
96.45 % and 97.99 %, respectively. Considering the recommended
R ≥ 90 % as good antibacterial property of a material [36], it can be
deduced that the synthesized Ti-Cu alloy is a strong antibacterial ma-
terial. Previous studies on the antibacterial behavior of metal implants
including copper, copper alloys, copper-modified surface, and copper
containing alloys have demonstrated that the release of Cu2+ ions is the
main reason for their anti-bacterial properties [31,32]. It has been re-
ported that the released Cu2+ ions make contact with outer membrane
of bacteria during nutrients supplement or due to presence of electro-
static loads on bacterial surface. The presence of porins in this layer of
the bacterial membrane allows the Cu2+ ions cross into the bacterial
cell [33]. The entry of Cu2+ ions into the cell produces intercellular
ROSs such as superoxide radical anion (O2−), hydrogen peroxide
(H2O2), and highly reactive hydroxyl radicals (OH). These highly re-
active species react with biologically important molecules such as li-
pids, proteins, and nucleic acid, leading to oxidative damage or death of
organism [34]. In general, the behavior of metal ion release is usually
et al. [37], the anti-bacterial behavior of Ti-6Al-4V titanium alloy was
compared with Cu-containing Ti-6Al-4V-5 alloy. The microstructure
obtained for the Ti-6Al-4V-5Cu alloy had a titanium base, containing
interstitial copper atoms and Ti2Cu intermetallic phase. They claimed
that copper atoms, when interstitially placed in titanium base, tend to
be released as Cu2+ ions compared to Ti2Cu intermetallic compound.
titanium and copper in the structure of the synthesized alloy. In other
words, the nature of the bond between copper and titanium is metallic
S. Moniri Javadhesari, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110889

Fig. 4. (a) Colony counting of E. coli and S. aureus cultured on the surface of Ti/ Ti-Cu alloy for 24 h, (b) Antibacterial rate of Ti and TiCu samples against S.aureus and
E.coli at 24 h incubation time, and (c) amount of Cu2+ release from the Ti-Cu sample in the 0.9 % NaCl solution with different immersion time at 37 °C.

in Ti(Cu) solid solution, but in the intermetallic compounds, the nature
of atomic bonds changes to ionic and covalent. Since covalent and ionic
bonds between titanium and copper are much stronger than metallic
bonds [38], they have much less tendency to release copper atoms as
Cu2+ ions.
Since the microstructure of the synthesized alloy is completely in-
termetallic, it can be argued that the Ti-Cu alloy bulk sample has a very
low tendency to release the copper element as ion. To examine this, the
release of Cu2+ ions from the Ti-Cu alloy was measured by ICP test.
Fig. 4c displays the total release of Cu2+ ions from Ti-Cu alloy after 2, 4,
and 7 days of immersion in a 0.9 % NaCl solution at 37 ± 1 °C. ICP
results showed that very low amounts of ions are released from the
alloy and this amount raised gently over time. The total amount of
released Cu2+ ions were 6.7, 9.3 and 11 ppb after 2, 4 and 7 days of
immersion, respectively. In general, the antibacterial activity of the
materials containing metallic ions can be interpreted by ion releasing or
contact sterilization/killing mechanisms [39]. When copper atoms are
interstitially dissolved in the titanium matrix, Cu2+ ions releasing will
be the dominant antibacterial mechanism. But in Ti-Cu intermetallic
compounds, the antibacterial property is mainly due to contact ster-
ilization [39]. Contact sterilization means that bacteria are damaged
when contacted with a phase containing copper atoms and bacteria
adhesion as well as formation of bacterial biofilms on the implant
surface are not allowed [40]. Due to the fact that the Minimum In-
hibitory Concentration (MIC) of Cu2+ ion for has been reported
448 × 109 ppb and 256 × 109 ppb against S. aureus and E. coli bacteria,
respectively [41]; therefore, due to the very low amount of released
Cu2+ ions (less than 12 ppb), it can be argued that the main

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S. Moniri Javadhesari, et al.
Fig. 5. (a) Fluorescent images of MG-63 cells cultured on the surface of Ti and Ti-Cu alloy for 2, 4, and 7 days after staining with DAPI (blue), and (b) the viability of
MG-63 cells cultured in the 2, 4, and 7 days extract media. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
contribution of the antibacterial property of this alloy is related to the
contact sterilization mechanism.
3.4. Biocompatibility assay
In order to investigate biocompatibility of synthesized alloys, MG-
63 osteoblast cells were cultured on the surface of the samples for 2, 4
and 7 days. The growth rate and viability of the cells was evaluated by
DAPI staining (Fig. 5a). The figure indicates that by prolonging the
culture time from 2 to 4 and then 7 days, the number of dead cells is
increased, which can be attributed to the release of Ti4+ and Cu2+ ions
over time and their interaction with the cells. The results for Ti and Ti-
Cu alloy showed no significant difference in growth rate and the
number of dead osteoblast cells. Thus, it can be deduced that the pre-
sence of copper in the Ti-Cu alloy in the form of intermetallic phases
does not have significant influence on the number of dead cells.
MTT assay was used to measure the cell death rate of Ti-Cu alloy in
comparison with pure titanium. The basis of this method is the reduc-
tion of MTT (dimethylthiazole-2 and 5-diphenyl tetrazolium bromide)
6
Colloids and Surfaces B: Biointerfaces 189 (2020) 110889
to formazan dye by mitochondrial enzymes of living cells. As Fig. 5b
displays, viability rate of the cells cultured in the extract medium of
pure titanium and Ti-Cu alloy is close to each other and shows a slight
decrease by prolonging the time of immersion for preparation of extract
medium (p < 0.05). According to the standard [42], the viability rate
of the Ti-Cu sample is within the range of zero grade (90–100 %) of
biomaterials. This means that the synthesized Ti-Cu alloy does not have
cell toxicity and has a good biocompatibility to MG-63 cells. The lower
viability rate of the cells cultured in the extract media of the samples in
comparison to control group can be due to the release of metallic ions,
i.e. Ti4+ in pure titanium and Ti4+ and Cu2+ ions in Ti-Cu alloy ex-
tracts, and their subsequent effects on MG-63 cells.
In general, copper ion is one of the trace elements that is essential
for human health and plays an effective role in the formation of various
enzymes in the body [10]. However, large amounts of copper ions can
cause toxicity to cells and their death, which leads to diseases such as
stomach upset, nausea, and diarrhea. As discussed earlier, due to the
intermetallic microstructure of Ti-Cu alloy in this study (Fig. 1) and its
very low tendency to release copper ions (maximum 11 ppb in 7 days),
S. Moniri Javadhesari, et al. Colloids and Surfaces B: Biointerfaces 189 (2020) 110889

Fig. 6. Osteoblast formation on the surface of a

Ti-Cu alloy and pure titanium, (a) optical
image of the surface of implants stained by
alizarin red, and (b) the absorbance values of
calcium mineral deposits measured at 612 nm
by ELISA. (For interpretation of the references
to colour in this figure legend, the reader is
referred to the web version of this article.)

firstly, it can be declared that this amount of copper ions released is materials from the protein absorption stage to bone proliferation has
much lower than the determined range in standards [16]. Secondly, due been reported by L. Zhang et al. [43]. As is apparent, a considerable
to the very low amount of the released ions, it does not have a sig- amount of osteogenesis will occur for nanostructured Ti-Cu alloy with
nificant effect on the cell death, and therefore the viability rate is close an average grain size of 19.5 nm due to the aforementioned reasons.
to that of the pure titanium.
3.6. Corrosion resistance
3.5. Osseointegration
Fig. 7 shows potantiodynamic polarization curves recorded for the
Ti and Ti-Cu samples in simulated body fluid (SBF) solution at 25 °C
An important factor for designing orthopedic implant materials is
with a scan rate of 10 mV.s−1. The corrosion parameters such as cor-
enhanced osseointegration between synthesized materials and bone
rosion potential (Ecorr) and corrosion current density (icorr) were ex-
tissue [43]. To examine the amount of osseointegration, cells were
tracted using Tafel extrapolation and gathered in Table 2. It can be seen
cultured on the surface of the samples for 7 days, and then treated by
that the polarization curve of Ti–Cu sample has shifted to higher cur-
alizarin red to stain calcium mineral deposits and phosphorus. Fig. 6
rent density while more positive potential, indicating that corrosion
shows calcium deposits formation on the surface of the samples,
tendency of Ti-Cu sample is lower than cp-Ti. But, as the corrosion rate
marked with dark brown areas. Also, the absorbance values of the cells
is proportional to corrosion current (corrosion current density),
cultured on the Ti-Cu alloy and pure titanium at 612 nm were measured
to be 0.32 and 0.30, respectively. The Alizarin red staining results in-
dicate significant amounts of calcium and phosphorous deposits on Ti-
Cu alloy compared to pure titanium, which can be attributed to the
release of Cu2+ ions in the Ti-Cu alloy sample as well as to its nanos-
tructural properties. In a study by Wang et al. [44], the presence of
copper element in the implant’s microstructure, as well as the release of
Cu2+ ions, led to acceleration of inflammation and apoptosis of implant
surrounding tissues in the early stages of implantation, which resulted
in the acceleration of osteogenesis and bone healing process. However,
one of the most important and essential factors affecting cell response
and osseointegration on the implant material is the grain size of the
implant surface [43]. In addition to dimensional similarity of nanos-
tructured materials to bone and cartilage tissue, they possess unique
surface properties such as surface topography, surface chemistry, sur-
face wettability, and surface energy due to a significant increase in
surface area and roughness compared to conventional micro-grained
materials. These unique surface features of nanostructured materials
lead to increased absorption of proteins such as fibronectin, vitronectin,
and laminin with nano-dimension (20−80 nm in length and 2−3 nm in
diameter) and their placement on the implant surface [45–47]. The Fig. 7. Potantiodynamic polarization plots drawn after 2 h immersion in SBF
stages of osseointegration on nanostructured materials and common solution at 25 °C.

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S. Moniri Javadhesari, et al.
Table 2
Electrochemical parameters extracted from the potantiodynamic polarization
plots in Fig. x.
Sample Ecorr (mV) Ic (μA cm−2)
Ti −736 1.329
Ti-Cu −447.295 4.675
therefore Ti-Cu alloy shows higher corrosion rate in comparison to cp-
Ti. In fact, the passivation region, resulted by the presence of naturally
formed TiO2 thin layer on the surface of Ti sample, is disappeared in Ti-
Cu sample and leads to increase in the corrosion current density more
than 3 times compared with cp-Ti sample. Decrease in corrosion re-
sistance of Ti with the addition of high amount of Cu has been reported
in previous works [48].
4. Conclusions
Nanocrystalline Ti-50 at% Cu alloy as a new biomedical alloy was
produced. The alloy showed nanocrystalline structure with 23 nm
average grain size and included mainly TiCu and Ti2Cu3 phases. The
produced alloy showed excellent mechanical properties; i.e. ultra-high
microhardness of 10 GPa and acceptable toughness of 8.14 MPam1/2.
Biological studies revealed very good biocompatibility of Ti-Cu
alloy as that of pure Ti sample. The Ti–Cu alloy showed antibacterial
activity against E. coli and S. aureus and also stimulated osteoblast
formation. Based on the Strong antibacterial activity and good bio-
compatibility, the Ti–Cu alloy can reduce bacterial infection during
implant surgeries and have a potential application as a new orthopaedic
material. However, further work needs to be done to reveal the bio-
compatibility in vivo.
Data availability
The raw/processed data required to reproduce these findings cannot
be shared at this time as the data also forms part of an ongoing study.
Data will be made available on request.
CRediT authorship contribution statement
S. Moniri Javadhesari: Investigation, Methodology, Validation,
Project administration, Writing - review & editing, Writing - original
draft. S. Alipour: Visualization, Investigation, Writing - original draft.
M.R. Akbarpour: Supervision, Conceptualization, Data curation,
Methodology, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
This Paper is Published as Part of a Research Project Supported by
the University of Maragheh Research Affairs Office.
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