Acta Biomaterialia 106 (2020) 410–427

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Full length article

A biodegradable Zn-1Cu-0.1Ti alloy with antibacterial properties for

orthopedic applications
Jixing Lin a,b,1, Xian Tong a,b,1, Zimu Shi c, Dechuang Zhang b, Lishu Zhang d, Kun Wang a,
Aiping Wei a, Lufan Jin a, Jianguo Lin b, Yuncang Li e, Cuie Wen e,∗
a
Department of Material Engineering, Zhejiang Industry & Trade Vocational College, Wenzhou 325003, PR China
b
School of Materials Science and Engineering, Xiangtan University, Xiangtan 411105, PR China
c
Key Laboratory of Materials Physics, Institute of Solid State Physics, Chinese Academy of Sciences, Hefei 230031, PR China
d
Institute of Advanced Materials for Nano-Bio Applications, School of Ophthalmology & Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou
325027, PR China
e
School of Engineering, RMIT University, Melbourne, Victoria 3001, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Zinc (Zn) alloys are receiving increasing attention in the field of biodegradable implant materials due to
Received 17 September 2019 their unique combination of suitable biodegradability and good biological functionalities. However, the
Revised 19 January 2020
currently existing industrial Zn alloys are not necessarily biocompatible, nor sufficiently mechanically
Accepted 11 February 2020
strong and wear-resistant. In this study, a Zn–1Cu–0.1Ti alloy is developed with enhanced mechanical
Available online 14 February 2020
strength, corrosion wear property, biocompatibility, and antibacterial ability for biodegradable implant
Keywords: material applications. HR and HR + CR were performed on the as-cast alloy and its microstructure, me-
Antibacterial ability chanical properties, frictional and wear behaviors, corrosion resistance, in vitro cytocompatibility, and an-
Biodegradable metal tibacterial ability were systematically assessed. The microstructures of the Zn–1Cu–0.1Ti alloy after differ-
Friction and wear ent deformation conditions included a η-Zn phase, a ε -CuZn5 phase, and an intermetallic phase of TiZn16 .
In vitro biocompatibility The HR+CR sample of Zn–1Cu–0.1Ti exhibited a yield strength of 204.2 MPa, an ultimate tensile strength
Zn alloy
of 249.9 MPa, and an elongation of 75.2%; significantly higher than those of the HR alloy and the AC al-
loy. The degradation rate in Hanks’ solution was 0.029 mm/y for the AC alloy, 0.032 mm/y for the HR+CR
alloy, and 0.034 mm/y for the HR alloy. The HR Zn–1Cu–0.1Ti alloy showed the best wear resistance, fol-
lowed by the AC alloy and the alloy after HR + CR. The extract of the AC Zn–1Cu–0.1Ti alloy showed over
80% cell viability with MC3T3-E1 pre-osteoblast and MG-63 osteosarcoma cells at a concentration of ≤
25%. The as-cast Zn–1Cu–0.1Ti alloy showed good blood compatibility and antibacterial ability.

Statement of Significance

This work repots a Zn–1Cu–0.1Ti alloy with enhanced mechanical strength, corrosion wear property, bio-
compatibility, and antibacterial ability for biodegradable implant applications. Our findings showed that
Zn–1Cu–0.1Ti after hot-rolling plus cold-rolling exhibited a yield strength of 204.2 MPa, an ultimate ten-
sile strength of 249.9 MPa, an elongation of 75.2%, and a degradation rate of 0.032 mm/y in Hanks’ Solu-
tion. The hot-rolled Zn–1Cu–0.1Ti showed the best wear resistance. The extract of the as-cast alloy at a
concentration of ≤ 25% showed over 80% cell viability with MC3T3-E1 and MG-63 cells. The Zn–1Cu–0.1Ti
alloy showed good hemocompatibility and antibacterial ability.
© 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

With the acceleration of the aging process in human soci-

ety, the hazards of senile osteoporosis are becoming more dan-
gerous and the risk of osteoporosis-induced skeletal fractures in
∗
Corresponding author. these patients is increasing [1]. Osteoporosis is listed as the sec-
E-mail address: cuie.wen@rmit.edu.au (C. Wen). ond most harmful disease to human health by the World Health
1
Joint first author.

https://doi.org/10.1016/j.actbio.2020.02.017
1742-7061/© 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427 411

Organization (WHO) [2]. In addition, the incidence of osteoporosis Table 1

Chemical composition of Zn–1Cu–0.1Ti alloy.
appears to be occurring at younger ages. At present, fracture fixa-
tion materials mainly consist of non-biodegradable metals such as Alloy Cu Ti Impurity Zn
stainless steels, cobalt (Co)-based alloys, and titanium (Ti) alloys, Zn–1Cu–0.1Ti 1.06 0.102 0.069 98.77
which generally remain in the body permanently or need follow-
up surgery to remove them after fracture healing, which brings un-
necessary pain and economic burden to patients and society [3,4].
tibacterial property for S. aureus. Leone et al. [23] reported that the
Thus, the use of biodegradable metal materials for bone fracture
addition of Ti to Zn alloys refined the matrix grains, thus result-
fixation is highly desirable. In the past, the research direction of
ing in enhanced mechanical properties. Simultaneously adding low
biodegradable materials mainly focused on magnesium (Mg) alloys
concentrations of Cu and Ti to Zn alloys improved their strength,
[5], iron (Fe) alloys [6], polymers [7], and bioceramics [8]. However,
hardness, and creep resistance, the latter also a highly desired ma-
due to the low ultimate tensile strength of polymer materials and
terial property for implant application [22]. However, most of the
the low toughness of bioceramic parts [7,8], these materials do not
existing Zn alloys still have inadequate material properties (e.g.,
meet the mechanical property requirement for bone fracture fixa-
mechanical, corrosion, biological, and antibacterial properties) for
tion. On the contrary, metallic materials possess good mechanical
implant material applications.
properties such as high strength and toughness, which could be
Although patient antibiotic prophylaxis and sterilization of im-
utilized for bone fixation; therefore, the development of biodegrad-
plants are generally performed before implantation, the failure rate
able metal materials for bone tissue engineering applications has
of implantation due to bacterial infection at the initial stage is still
become a hot research topic [9,10]. Research on biodegradable Mg,
as high as 4∼6% because once a bacterial infection occurs at the
Fe, and zinc (Zn)-based alloys is receiving increasing attention and
site of implantation, it is challenging to treat [27]. Postoperative
has achieved substantial advances in terms of their mechanical
infections with implants are considered a significant challenge in
properties and biocompatibility in recent years. Nonetheless, both
implant surgery. Therefore, an implant material with intrinsic an-
Mg and Fe alloys have certain disadvantages; for instance, most
tibacterial ability is highly desirable. Ren et al. [28] systematically
Mg alloys exhibit high degradation rates and Fe alloys degrade too
investigated a variety of Cu-containing medical metals such as
slowly in the physiological environment [11]. However, it is worth
stainless steels, Ti alloys, and Co-based alloys, and demonstrated
noting that MgYREZr alloy devices have already been successfully
the good antibacterial ability of these materials stemming from
commercialized such as Bioabsorbable interference screws (MAG-
the durable and broad-spectrum antibacterial characteristics of
NEZIX®, and Magmaris by BIOTRONIK), but Zn-based biodegrad-
Cu ions. Therefore, Cu-containing Zn alloys can be expected to be
able materials are still in the development stage [12]. On the other
promising implant materials with intrinsic antibacterial ability.
hand, Zn and its alloys have moderate corrosion rates between
After implantation, there is always a certain degree of micro-
those of Mg-based and Fe-based alloys.
movement at the bone-implant interface, resulting in wear de-
Zn is an essential trace element in the human body [13]. As a
bris which may cause adverse tissue responses. Moreover, interface
component of more than 300 kinds of enzymes in the body, Zn
damage to implants may also occur due to wear and chemical cor-
participates in a variety of biological activities such as maintain-
rosion in the physiological environment. Therefore, the corrosion
ing human immune function, growth, and development, promot-
and micro-abrasive wear behaviors of implant materials should be
ing insulin secretion, and enhancing memory [14–16]. More im-
investigated. The corrosion wear performances of stainless steels
portantly, Zn can also improve bone tissue development and have
[29], Ti alloys [30,31], Co–Cr–Mo alloy [32], and Mg alloy [33] have
an osteoinductive effect on bone tissue [17]. Hence, Zn and its al-
been reported in recent years. However, the corrosion wear behav-
loys can provide potential biological functions as implant materi-
ior of Zn alloys is still rarely reported.
als for medical applications such as bone fixation and cardiovas-
In this study, we developed a new Zn–1Cu–0.1Ti alloy for
cular stents. However, pure Zn has extremely low yield strength
potential biodegradable implant applications. Plastic deformation
(∼29.3 MPa) and elongation (∼1.2%) in its as-cast condition due
processes including hot-rolling and hot-rolling followed by cold-
to its hexagonal close-packed (hcp) crystal structure [18]. To date,
rolling were performed on the as-cast alloy, and its microstruc-
approaches for effectively improving the mechanical properties of
ture, mechanical properties, corrosion resistance, friction and wear
pure Zn include plastic deformation and the addition of alloying el-
behaviors in both Hanks’ solution and a dry-sliding friction envi-
ements such as germanium (Ge) [18], Mg [19,20], calcium (Ca) [19],
ronment, antibacterial ability, and in vitro cytotoxicity after various
strontium (Sr) [19], copper (Cu) [21,22], and Ti [23]. Venezuela
plastic deformation processes were systematically investigated.
and Dargusch [24] comprehensively reviewed the influence of al-
loying and fabrication techniques on the mechanical properties,
biodegradability and biocompatibility of Zn alloys, and suggested 2. Experimental procedure
that the wrought Zn-xMg alloy (x < 1.5%) exhibits the best com-
bination of mechanical properties, biodegradability, and biocom- 2.1. Material preparation
patibility. Li et al. [25] reviewed the mechanical aspects of indus-
trial and biomedical Zn and its alloys and recommended that more A Zn alloy with a nominal composition of Zn–1Cu–0.1Ti (wt.%
mechanical property tests are needed to evaluate the mechanical hereafter) was prepared by casting using pure Zn (purity 99.995%,
properties of biodegradable Zn-based metals such as creeping re- Huludao Zinc Industry Co. Ltd., China), pure Cu (99.99%, Jiangxi
sistance, corrosion fatigue, and stability of mechanical properties Copper Co. Ltd., China), and a Zn–2Ti master alloy (Hunan High
during storage and service. Hernández-Escobar et al. [26] reviewed Broad New Material Co. Ltd., China) as starting materials. The raw
the most recent progress on the development of absorbable Zn- materials were melted in a graphite crucible at 550–600 °C un-
based alloys for biomedical implant applications and suggested to der the protection of argon (Ar) gas and then cast into a square
re-evaluate the mechanical requirements and define the standard mold which was preheated to 250 °C. The chemical composition
specifications of the absorbable metal implant, which are indepen- of the as-cast Zn–1Cu–0.1Ti alloy is listed in Table 1, as mea-
dent with those of the permanent implants. Tang et al. [21] inves- sured by X-ray fluorescence (XRF; S4 Pioneer, Bruker, Germany).
tigated a series of biodegradable Zn–x Cu (x = 1, 2, 3, 4 wt.%) alloys An as-cast ingot with dimensions of 85 mm × 85 mm × 20 mm
and reported a good elongation of ∼50.6% for a Zn–4Cu alloy, along was annealed at 340 °C for 10 h for homogenization, followed by
with acceptable cytotoxicity for human endothelial cells and an- cooling to room temperature in air. Plates with dimensions of
412 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
60 mm × 20 mm × 10 mm were cut from the alloy ingot for hot-
rolling and hot-rolling plus cold rolling using electrical discharge
machining (EDM). The alloy plates were heated to 250 °C and held
for 5 min before each hot-rolling process. The as-cast Zn–1Cu–0.1Ti
alloy plates were divided into two groups for plastic deformation:
the first group of plates was hot-rolled to a total reduction of 80%
in thickness at 0.5 mm reduction per pass; and the second group
of plates was hot-rolled to a total reduction of 40% in thickness at
0.5 mm reduction per pass and then air-cooled to room tempera-
ture, followed by cold-rolling to a total reduction of 40% in thick-
ness at 0.2 mm reduction per pass with room temperature of 25 °C.
Both groups of plate samples exhibited a final thickness of 2 mm.
2.2. Microstructure characterization
The microstructures of the as-cast, hot-rolled, and hot-rolled
plus cold-rolled (denoted AC, HR, and HR+CR, respectively here-
after) Zn–1Cu–0.1Ti alloy samples were characterized by optical
microscopy (OM; DM2500C, Leica, Germany). All samples were
polished and etched with a 0.5 vol.% HNO3 ethanol solution. The
microstructures of the HR and HR+CR samples were observed in
the rolling direction. X-ray diffraction analysis (XRD; D8 ADVANCE,
Bruker, Germany) with Cu–Kα radiation was performed to identify
the phases of the alloy samples, and the scanning spectra were
recorded over a 2θ range from 10 ° to 90 ° at a step size of
0.02 °s−1 . Scanning electron microscopy (SEM; Pro-X FEI, Phenom,
Netherlands) along with energy dispersive spectrometry (EDS; X-
Max, Oxford, England) were carried out to evaluate the microstruc-
tures and chemical compositions of the phases of the alloy samples
at 15 keV.
2.3. Mechanical testing
The tensile properties of the AC, HR, and HR+CR samples of
Zn–1Cu–0.1Ti were carried out using a universal testing machine
(Instron-3369, USA) at a strain rate of 1 mm/min at room tempera-
ture. The dimensions of the tensile samples were prepared accord-
ing to ASTM E8/E8M-08 [34] and the samples after deformation
were cut by EDM along the rolling direction for microstructural ob-
servation. The fracture surfaces of the samples after tensile testing
were observed by SEM. The hardness of the AC, HR, and HR+CR
alloy samples was measured using a Vickers microhardness tester
(MicroMet 60 0 0, Buehler, USA) with a load of 10 0 gf and a holding
time of 15 s.
2.4. Electrochemical testing
The corrosion behaviors of the AC, HR, and HR+CR samples
of Zn–1Cu–0.1Ti in Hanks’ solution [35] were investigated by po-
tentiodynamic polarization testing and electrochemical impedance
spectroscopy (EIS) testing with a pH of 7.40 at 37 °C using an
electrochemical workstation with a three-electrode electrochemi-
cal cell (ParStat 2273, Princeton Applied Research, USA). The sam-
ples were prepared by grinding with SiC paper up to 20 0 0 grit
and polishing to 0.5 μm with diamond paste. During electrochem-
ical testing, samples with an exposed area of 0.5 cm2 were set as
the working electrode, a platinum plate electrode as the counter
electrode, and a saturated calomel electrode (SCE) as the reference
electrode. The potential polarization tests were performed over a
scan range from −800 mV to 500 mV relative to the open circuit
potential (OCP) at a potential scan rate of 1 mV s–1 . EIS measure-
ments were carried out with a disturbance frequency ranging from
100 kHz to 0.01 Hz and a perturbation signal-amplitude of 10 mV
at OCP. The corrosion rate of the samples was calculated based on
ASTM G102-89 [36]. All procedures were done at least in triplicate
samples.
2.5. Immersion testing
The degradation rates of the AC, HR, and HR+CR samples of Zn–
1Cu–0.1Ti were measured by immersion tests. disk samples with
dimensions of diameter 8 mm and thickness 2 mm were cut by
EDM and polished up to 20 0 0 grit. Immersion tests were carried
out by soaking the samples in Hanks’ solution for 14 and 30 d at
37 °C, where the ratio of specimen surface area to solution volume
was 1 cm2 per 20 ml. The morphology and composition of the cor-
rosion products on the sample surfaces after immersion tests were
characterized by SEM and EDS. Ultrasonic cleaning using a mixed
solution of CrO3 (200 g/L) and AgNO3 (10 g/L) was used to remove
the corrosion products after immersion tests. The weights of the
samples before and after immersion tests were measured by an
electronic balance with an accuracy of 0.1 mg (Sartorius BSA124S-
CW, Germany) and the degradation rate was calculated based on
ASTM G31-72 [37]. Measurements were performed at least in trip-
licate. The phases of the corrosion products of HR Zn–1Cu–0.1Ti
alloy after immersion in Hanks’ solution for 30 d were analyzed
by a micro-area XRD diffractometer (micro-XRD; D/Max Rapid IIR,
Rigaku, Japan) at a voltage of 40 kV and a current of 150 mA with
Cu–Kα radiation at a step size of 0.1 s−1 . The XRD data were over a
2θ range of 0–90 °. Micro-Fourier transform infrared spectroscopy
(micro-FTIR; Nicolet 6700, Thermo Fisher, USA) with a wave range
of 40 0 0–650 cm−1 at a resolution of 4 cm−1 was performed to
identify the functional groups in the corrosion products. X-ray pho-
toelectron spectroscopy (XPS; Thermo Fisher Scientific, USA) with
an Al–Kα source at a step size of 1 eV was used to analyze the
chemistry of the corrosion products. High-resolution narrow scans
at a step size of 0.1 eV were performed to determine the binding
states of Zn 2p, O 1 s, Ca 2p, and C 1 s of the corrosion products.
2.6. Friction and wear testing
The friction and wear behaviors of the AC, HR, and HR+CR sam-
ples of Zn–1Cu–0.1Ti were investigated using a high-speed recip-
rocating friction tester (HSR-2M; Lanzhou Zhongke Kaihua Tech-
nology Development Co., China). For comparison, as-cast pure Zn
(hereafter denoted AC pure Zn) was also tested under the same
conditions. The tests were carried out under a wear load of 200 gf,
a friction stroke length of 5 mm, a friction frequency of 1 Hz, a test-
ing time of 10 min, and a friction pair of ZrO2 with a diameter of
3 mm. Disk samples having 15 mm diameter and 10 mm thickness
were cut by EDM and mounted with epoxy resin, and their sur-
faces were polished to a roughness (Ra) of 0.5 μm using diamond
paste before wear testing. Both sample surfaces and the friction
pair were cleaned using ethanol before friction and wear testing.
Both dry-sliding wear tests and corrosion wear tests in a liquid lu-
brication environment were performed on the wear-testing sam-
ples. The corrosion wear tests were carried out by immersing the
samples in a sink containing 50 ml Hanks’ solution at a constant
temperature of 37 °C, and the dry-sliding friction wear tests were
performed at room temperature. The weights of the samples before
and after the friction and wear tests were measured using an elec-
tronic balance to determine the weight loss. The surface morphol-
ogy and roughness after friction and wear tests were characterized
by SEM and a 3D-contour testing software.
2.7. Surface contact angle testing
The surface contact angle of the AC, HR, and HR+CR samples of
Zn–1Cu–0.1Ti and AC pure Zn was measured using a contact angle
goniometer (OCA15EC, Data Physics, Germany) at room tempera-
ture. Alloy samples with dimensions of diameter 8 mm and thick-
ness 2 mm were ground with 20 0 0 grit sandpaper. A 4 μl droplet
of deionized water dyed with ink was used as the testing medium.
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Three points were randomly selected on each sample for testing,
and the average value was taken as the result of surface contact
angle.
2.8. Hemolysis testing
Hemolysis tests of the AC, HR, and HR+CR samples of Zn–1Cu–
0.1Ti and AC pure Zn were carried out according to ISO 10993-
4: 2002 [38]. Extracts of the alloy samples were prepared using
0.9% normal saline solution at an extraction ratio of 5 g/10 ml. The
hemolysis test procedures were carried out under a strict protocol
at Wenzhou Medical University under the guidance of its Ethics
Committee, which gave ethics approval beforehand. Fresh blood
from healthy humans (12 ml) had 0.4 ml of 20 g/l potassium ox-
alate reagent added and the solution washed with 30 ml of nor-
mal saline three times, then centrifuged to obtain red blood cells.
Finally, normal saline was used to configure a 16% red blood cell
suspension. The metal extract (4 ml) was incubated for testing, and
the deionized water and 0.9% sodium chloride solution were used
as the positive and negative controls, respectively. All centrifuge
tubes were placed in a water bath at 37 °C for 30 min, then 0.2 ml
of the 16% red blood cell suspension was added to each centrifuge
tube and all tubes were continually cultured at 37 °C for 60 min.
The samples were centrifuged at 10 0 0 r/min for 5 min at 25 °C un-
til erythrocytes precipitated, then 200 μl of the supernatant was
pipetted into a 96-well plate. The absorbance values were mea-
sured by a microplate photometer (Multiskan MK3, Thermo Scien-
tific, USA) at a wavelength of 545 nm, and the hemolysis percent-
age was calculated according to the following formula:
Hemolysis percentage
 
= absorbanceexperimental group − absorbancenegative group /
 
absorbancepositive group − absorbancenegative group ×100%
2.9. Cytotoxicity evaluation
The in vitro tests were performed by both indirect and di-
rect contact assays using MC3T3-E1 pre-osteoblast cells (mouse
cell lines, NIH/3T3, GDC030) and MG-63 osteosarcoma cells (hu-
man cell lines, MG-63, GDC074) (purchased from cell bank of Chi-
nese Academy of Sciences, Shanghai, China). Cells were cultured
in alpha-minimum essential medium (α -MEM; 0.1 mM, Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS;
Lonsera; Cat Number: S711-001S) and antibiotics (100 units/ml
penicillin and 100 μg/ml streptomycin; Aladdin; Cat Number:
PH1513) in a humidified atmosphere of 5% CO2 at 37 °C for 72 h.
disk samples of as-cast Zn–1Cu–0.1Ti and pure Zn with dimensions
of diameter 8 mm and thickness 2 mm were prepared for the cyto-
toxicity assessment. The disk samples were ultrasonically cleaned
in analytically pure ethanol with a concentration of 99.7% and ster-
ilized in ultraviolet (UV) radiation before testing.
For the direct cell assay, MC3T3-E1 cell droplets
(3 × 104 cells/well) were seeded on the surfaces of the disk
samples and cultured at 37 °C in 95% relative humidity with 5%
CO2 for 24 h and 48 h, respectively. To testing the reducibility of
the metal disk, blank absorbance was measured at 450 nm in the
culture medium and CCK-8 solution without cells but with metal
samples in the same cultural environment. If the absorbance is
minimal, it means that CCK-8 solution has not been reduced and
can be directly added for testing. Before the CCK-8 assay, the origi-
nal culture medium was discarded to avoid the interference of the
reduction effect from metal samples. The cells were washed twice
with fresh culture medium, and then finally replaced with culture
medium. For the indirect cell assay, the extracts were prepared
by immersing the disk samples of as-cast Zn–1Cu–0.1Ti and pure
Zn in the culture medium for 72 h at a ratio of 1.25 cm2 ml−1
413
according to ISO 10993-5: 2009 [39]. The MC3T3-E1 or MG-63
cells were seeded in a 96-well plate with 5 × 103 cells per well
and cultured for 24 h. The original medium was replaced with the
diluted extracts of different concentrations of 100%, 25%, and 12.5%
and then incubated for 1, 3, and 5 d for evaluation of cell viability.
Both the cell assays were done at least in triplicate.
After incubation for the corresponding time for both direct and
indirect cell assays, 10 μl CCK-8 solution (Dojindo, Japan) was
added to each well and then incubated for 2 h. Optical density was
measured at the wavelength of 450 nm using a microplate reader
(Gen5, Biotek, USA). The cell viability of the MC3T3-E1 and MG-63
cells of the pure Zn and Zn–1Cu–0.1Ti was calculated according to
the following formula [39]:
Cell viability (% ) = ( (ODt − ODblank )/(ODnc − ODblank ) )×100%
(2)
where ODt is the absorbance value of the experimental group;
ODnc is the absorbance value of the negative control group; and
ODblank is the absorbance value of the α -MEM medium.
2.10. Antibacterial evaluation
The antibacterial properties of as-cast Zn–1Cu–0.1Ti and pure
Zn were evaluated by the inhibition zone diameter (IZD) method
according to DIN EN ISO 20645-2004 [40]. Staphylococcus aureus
(S. aureus) (CMCC26003) was placed into 5 ml of medium and
shaken for 24 h in a constant temperature shaker, then the bac-
terial solution was diluted to a concentration of 1 × 108 CFU/ml
with phosphate-buffered saline (PBS) buffer. Then Luria-Bertani
(LB) broth powder (10 g/l peptone, 5 g/l yeast extract powder,
10 g/l sodium chloride) was added to 950 ml distilled water, then
adjusted to pH 7.0∼7.2 with 0.1 mol/l NaOH solution after en-
(1) tirely dissolving and stirring all contents, and then made up to
a volume of 10 0 0 ml with distilled water. The medium solution
and the pure Zn and Zn–1Cu–0.1Ti alloy disk samples (diame-
ter 8 mm × thickness 2 mm) were autoclaved at 121 °C for 40 min
and sterilized by UV sterilization on a sterile bench for 30 min.
Agar powder (1.5 g per 100 ml of the medium) was added to the
medium, and then autoclaved for 30 min after heating and dissolv-
ing. The medium solution (20 ml) was poured into a culture dish at
a temperature of 45 °C and UV-sterilized for 30 min to prepare an
agar medium plate. The alloy sample was placed in the center of
the plate and 500 μl of the bacterial suspension was evenly spread
on the surface of the agar medium with a pipette. At least five
times duplicates were measured for statistical analysis. The IZD
values were observed and measured after incubation at 37 °C for
24 h, and the antibacterial properties were calculated according to
the formula given by [40]:
H = (D − d )/2 (3)
where H is the inhibition zone in mm, D is the total diameter of
the specimen and inhibition zone in mm, and d is the diameter of
the specimen in mm. When H is equal to or larger than 1 mm and
there is no growth of bacteria, the antibacterial property is good;
when H is equal to 0 mm and there are regions with some bacteria,
the antibacterial property is limited; and when H is equal to 0 mm
and there are regions with many bacteria, there is no antibacterial
property.
2.11. Statistical analysis
In this study, the experimental results are expressed as means
± standard deviations. One-way analysis of variance (V17.0, SPSS,
USA) was applied to determine the statistical significance of the
differences observed between groups. P < 0.05 was accepted as
statistically significant.
414 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Fig. 1. XRD patterns of AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti.
3. Results
3.1. Microstructure of Zn–1Cu–0.1Ti
Fig. 1 shows the XRD patterns of the AC, HR, and HR+CR sam-
ples of Zn–1Cu–0.1Ti. It can be seen that all of the samples con-
tain an η-Zn phase (the matrix), an intermetallic compound TiZn16
phase, and an ε -CuZn5 phase. Owing to the small additions of Cu
and Ti in the Zn–1Cu–0.1Ti alloy, the intensity of the diffraction
peaks of the ε -CuZn5 phase and the TiZn16 phase in the XRD pat-
terns of the AC and HR Zn–1Cu–0.1Ti samples is weak, and all the
diffraction peaks of the η-Zn matrix phase show similar intensities.
As for the HR+CR sample, the η-Zn phase shows significantly en-
hanced intensity for the (004) peak but decreased intensity for the
other peaks; and the ε -CuZn5 phase shows increased peak inten-
sities and its main peak shows an intensity similar to that of the
main peak (101) of the η-Zn phase.
Fig. 2 shows the optical microstructures of the AC, HR, and
HR+CR samples of Zn–1Cu–0.1Ti. It can be seen that the mi-
crostructure of the AC sample consists mainly of a white irregu-
lar η-Zn matrix phase with an average size of 53.9 ± 7.6 μm and a
short gray rod or granular eutectic intermetallic compound TiZn16
phase (Fig. 2(a)). Most of the eutectic TiZn16 precipitates are dis-
tributed along the grain boundaries, with a small amount of the
TiZn16 phase distributed in the grain matrix of the η-Zn phase. As
for the microstructure of the HR sample, the η-Zn matrix phase
and eutectic TiZn16 precipitates are elongated along the rolling di-
rection, and the TiZn16 phase shows the morphology of fibers. The
size of the η-Zn phase is increased as compared to the AC sam-
ple, and the average size reaches 84.4 ± 12.5 μm. Also, many cellu-
lar crystal grains with inconspicuous grain boundaries appear in
Fig. 2. Microstructures of Zn–1Cu–0.1Ti alloy samples: (a) AC; (b) HR; and (c) HR + CR.
Table 2
EDS analysis results of different spots in Zn–1Cu–
0.1Ti alloy samples.
Spot no. Zn (at.%) Cu (at.%) Ti (at.%)
Spot 1 90.4 ± 0.3 0.4 ± 0.2 9.2 ± 0.2
Spot 2 92.3 ± 1.4 0.4 ± 0.1 7.3 ± 1.3
Spot 3 97.0 ± 0.4 0.9 ± 0.1 2.1 ± 0.3
Spot 4 98.6 ± 0.1 1.3 ± 0.0 0.1 ± 0.1
Spot 5 88.6 ± 0.1 7.7 ± 0.1 3.7 ± 0.0
Spot 6 87.0 ± 0.4 13.0 ± 0.4 0.00
the η-Zn phase (as marked in Fig. 2(b)), indicating that incom-
plete dynamic recrystallization and grain growth occurred during
hot rolling. The HR+CR sample shows clear processing flow lines
and black ε -CuZn5 particles distributed in the matrix along the
rolling direction (Fig. 2(c)). In addition, the ε -CuZn5 phase is re-
fined compared to that of the HR sample.
Fig. 3 shows SEM images of the AC, HR, and HR+CR samples
of Zn–1Cu–0.1Ti. The EDS analysis results for the different micro
spots in Fig. 3 are shown in Table 2. From the SEM images and
the corresponding EDS data, the short rod-like and granular phases
corresponding to spots 1, 2, and 3 in Fig. 3(a) are mainly com-
posed of Zn, Ti, and a small amount of Cu. The amount of Ti in
the three spots varies significantly and its relative atomic content
is 9.2 ± 3.2% for spot 1, 7.3 ± 1.3% for spot 2, and 2.1 ± 0.3% for spot
3, respectively. Because the particle size of the phase in spot 3 is
very small, a portion of the electron beam was irradiated onto the
Zn matrix during EDS analysis, resulting in a higher Zn content in
this spot. According to the XRD patterns and the Zn–Ti phase dia-
gram [41,42], it can be suggested that the short rod-like and gran-
ular Ti-rich phase is the TiZn16 phase. Based on the Zn–Ti phase
diagram, the solid solubility of Ti in a Zn matrix is very small (only
0.0 07∼0.015 wt.% at 30 0 °C [42]). When the Ti content is higher
than 0.21 wt.% and the temperature reaches about 418.6 °C, a eutec-
tic reaction occurs in the Zn–1Cu–0.1Ti alloy and the TiZn16 phase
forms with an excess of Ti and Zn, which is distributed on the
grain boundary and inside of the grains. At the same time, due
to the faster solidification of the Zn–1Cu–0.1Ti alloy, the Ti un-
derwent an unequilibrium eutectic reaction, resulting in entirely
different Ti contents in the different precipitation phase, concen-
trated in local areas such as eutectic branches and forming severe
segregation. Spot 4 is a Zn-rich phase that contains small amounts
of Cu and Ti, and can be inferred to be an η matrix phase. The
rod-shaped phase shown in spot 5 (Fig. 3(b)) is mainly composed
of Zn, Cu, and Ti, and the relative atomic contents of Cu and Ti
are 7.7 ± 0.1% and 3.7 ± 0.0%, respectively. The atomic contents of
these two elements are similar to those reported in the ternary in-
termetallic compound of Cu2 TiZn22 by Zermout et al. [43]. It can
be deduced that the phase in spot 5 is Cu2 TiZn22 . Since the con-
centration of the Cu2 TiZn22 phase is small, there are no significant
diffraction peaks observable in the XRD spectra. The finer granular
phase of spot 6 (in Fig. 3(c)) has only Zn and Cu elements, and the
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427 415

Fig. 3. SEM images of Zn–1Cu–0.1Ti alloy samples: (a) AC; (b) HR; and (c) HR + CR.

atomic fraction of Cu was 13.0 ± 0.4%. These atomic ratios are close
to those of the CuZn5 phase. Based on the results from XRD, EDS,
and the Zn–Cu phase diagram [44], it can be concluded that this
intermetallic phase is CuZn5 .
3.2. Mechanical properties of Zn–1Cu–0.1Ti
Fig. 4 shows the tensile stress-strain curves, yield strength
(σ ys ), ultimate tensile strength (σ uts ), elongation (ε ), hardness,
and fracture surfaces of the AC, HR, and HR+CR samples of the
Zn–1Cu–0.1Ti alloy. The σ uts , σ ys , ε , and hardness values were
92.4 ± 4.4 MPa, 86.1 ± 2.6 MPa, 1.4 ± 0.8%, and 72.6 ± 0.6 HV, re-
spectively for the AC sample; 205.7 ± 5.5 MPa, 175.4 ± 3.8 MPa,
39.2 ± 1.4%, and 70.6 ± 1.8 HV, respectively for the HR sample; and
249.9 ± 3.8 MPa, 204.2 ± 4.3 MPa, 75.2 ± 1.9%, and 56.4 ± 0.8 HV,
respectively for the HR+CR sample (Fig. 4(b)). The σ uts and ε
of the HR+CR sample increased by 21.5% and 91.8%, respectively
as compared to those of the HR sample. It can be seen that the
HR+CR sample exhibits extraordinarily high ductility with an
ε = 75.2 ± 1.9%. The SEM images of the fracture surfaces of the AC,
HR, and HR+CR samples are shown in Fig. 4(c)–(e). The fracture
surface of the AC sample shows a morphology of cleavage fractures
with river patterns and cleavage steps, and some small dimples
in localized areas (Fig. 4(c)). It can be inferred that the AC sample

Fig. 4. Mechanical properties and fracture surfaces of Zn–1Cu–0.1Ti alloys (a) tensile stress-strain curves; (b) tensile yield strength, ultimate strength, elongation, and hard-
ness; (c) SEM image of AC fracture surface; (d) SEM image of HR fracture surface; and (e) SEM image of HR+CR fracture surface.
416 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427

exhibits a mixed mode of fracture with brittle cleavages and a
few ductile dimples. In the HR and HR+CR samples, the fracture
surfaces are homogeneously distributed with round, equiaxed
dimples of different sizes and depths. The fracture mode of these
samples is typical ductile microvoid coalescence, indicating that
thermomechanical processes can significantly improve the duc-
tility of the Zn–1Cu–0.1Ti alloy. Furthermore, the dimples on the
fracture surfaces of the HR+CR sample are denser, deeper, and
larger compared to those of the HR sample and the area occupied
by the dimples is larger than that of HR sample, indicating a better
plastic deformation ability.
3.3. Corrosion behavior of Zn–1Cu–0.1Ti
Fig. 5 shows the corrosion behaviors of the AC, HR, and
HR+CR samples of Zn–1Cu–0.1Ti in Hanks’ solution at 37 °C.
The OCP and potentiodynamic polarization curves of the sam-
ples are shown in Fig. 5(a) and (b), respectively. The corre-
sponding corrosion performance indicators obtained by Tafel re-
gion extrapolation are listed in Table 3. The OCP curves of the
AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti reach a steady
fluctuation state after immersion for 1200s, and the OCP val-
ues of the samples in descending order are AC > HR+CR

Fig. 5. Corrosion behaviors of AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti in Hanks’ solution: (a) OCP curves; (b) potentiodynamic polarization curves; (c) Nyquist plots;
(d) Bode impedance modulus diagrams; (e) Bode phase angle diagrams; and (f) degradation rate after immersion for 14 d and 30 d.
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Table 3
Electrochemical performance parameters of AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti in Hanks’ solution.
Sample Corrosion potential (Ecorr ), V vs. SCE
AC −1.025 ± 0.264
HR −1.123 ± 0.185
HR + CR −1.100 ± 0.201
Table 4
Fitted results of EIS spectra for AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti in Hanks’ solution.
Samples R s ( ) R f ( ) CPEf (μF)
AC 1.6 ± 0.2 2667.2 ± 3.1 19.8 ± 0.4
HR 0.9 ± 0.2 467.4 ± 2.3 80.0 ± 0.7
HR+CR 1.4 ± 0.3 1629.0 ± 3.8 13.1 ± 0.2
> HR. From the potentiodynamic polarization tests, the corro-
sion potential, corrosion current density, and corrosion rate are
−1.025 ± 0.264 V, 21.5 ± 0.4 μA/cm2 , and 315 ± 6 μm/y for the AC
sample; −1.123 ± 0.185 V, 111.2 ± 0.9 μA/cm2 , and 1628 ± 13 μm/y
for the HR sample; and −1.100 ± 0.201 V, 67.7 ± 0.5 μA/cm2 , and
991 ± 7 μm/y for the HR+CR sample, respectively. The AC sample
shows the lowest current density and corrosion rate, and high-
est self-corrosion potential, and the HR sample shows the highest
current density and corrosion rate, and the lowest self-corrosion
potential. The self-corrosion potential results are consistent with
the OCP results. Furthermore, there is a narrow passivation re-
gion on the anodic polarization curve for all the samples, with a
breakdown potential of −0.739 V, −0.826 V, and −0.821 V for AC,
HR, and HR+CR, respectively. In addition, the blunt current den-
sity in ascending order is 0.0587 mA/cm2 for AC  0.1557 mA/cm2
for HR + CR < 0.4140 mA/cm2 for HR. The current density of
the HR sample is significantly higher than those of the AC and
HR + CR samples. The blunt current density and the magnitude
of the breakdown potential are related to the stability of the passi-
vation films on the metals. A lower blunt current density and more
positive breakdown potential in the passivation state correspond to
a lower dissolution rate of the metal and more stable passivation
film. According to the magnitude of the breakdown potential and
the blunt current density, the passivation film of the AC sample
shows the highest stability among the three samples. Therefore, it
can be concluded that the corrosion resistance of the samples in
descending order is AC > HR + CR > HR.
Fig. 5(c) shows the Nyquist plots of the AC, HR, and HR+CR
samples of Zn–1Cu–0.1Ti. Table 4 lists the fitted results of EIS
spectra for AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti. It can
be seen that there are two different capacitive arcs in both the
low-frequency region and the high-frequency region in the studied
samples. The semicircular capacitance arc in the high-frequency
region is related to the charge transfer in the corrosion process,
and the semicircular capacitance arc in the low-frequency region
is associated with the diffusion process of the passivation layer
[45,46]. The AC sample shows the largest capacitive arc radius and
the HR sample shows the smallest. An equivalent circuit consisting
of the resistance (R) and constant phase element (CPE) is shown
in Fig. 5(c), where Rct and CPEd1 are the electric double layer re-
sistance and capacitance between the electrode surface and solu-
tion, Rf and CPEf are the resistance and capacitance of the corro-
sion products on the sample surface, respectively, and Rs is the
solution resistance. As shown in Table 4, the impedance of the
samples in descending order is AC > HR+CR > HR and a higher
impedance value indicates a stronger corrosion resistance. Fig. 5(d)
and Fig. 5(e) show the Bode impedance modulus and phase angle
diagrams of the AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti. In
the low-frequency region and intermediate-frequency region (0.01
∼ 100 Hz), the impedance modulus of the samples in descending
417
Corrosion current density (Icorr ) (μA/cm2 ) Corrosion rate (Vcorr ) (μm/y)
21.5 ± 0.4 315 ± 6
111.2 ± 0.9 1628 ± 13
67.7 ± 0.5 991 ± 7
n1 Rct () CPEd1 (μF) n2 Vcorr (μm/y) Chi square
0.68 ± 0.10 1980.4 ± 3.3 1166.7 ± 3.9 0.63 ± 0.14 263 ± 1 5.33 × 10−3
0.54 ± 0.07 1097.2 ± 5.6 1098.7 ± 3.9 0.73 ± 0.05 475 ± 5 5.15 × 10−3
0.82 ± 0.08 1426.1 ± 2.5 219.8 ± 0.7 0.77 ± 0.08 365 ± 1 8.29 × 10−3
order is AC > HR + CR > HR and the HR sample showed a signif-
icantly lower value than those of both AC and HR + CR samples.
Meanwhile, the phase angles of all the samples were less than or
equal to 60° in this frequency region; while the phase angles of
AC and HR+CR samples were higher than 60° in high-frequency
region, which confirmed the presence of a stable, compact film
of corrosion products of the sample surface [47]. The Vcorr of the
samples determined by the EIS spectra via Stern-Geary method is
263 ± 1 μm/y for the AC sample, 475 ± 5 μm/y for the HR sample,
and 365 ± 1 μm/y for the HR+CR sample, which is similar to the
trend of Vcorr measured by the polarization test. Based on the re-
sult of EIS spectra, the corrosion resistance of the samples in de-
scending order is AC > HR + CR > HR, which is consistent with
the potentiodynamic polarization results.
Fig. 5(f) shows the average degradation rate of the AC, HR, and
HR + CR samples of Zn–1Cu–0.1Ti after immersion in Hanks’ so-
lution for 14 d and 30 d It can be seen that the degradation rate
after immersion for 14 d is 0.094 ± 0.010 mm/y for the AC sam-
ple, 0.149 ± 0.012 mm/y for the HR sample, and 0.113 ± 0.015 mm/y
for the HR+CR sample, and the degradation rate after im-
mersion for 30 d was 0.029 ± 0.022 mm/y for the AC sample,
0.034 ± 0.018 mm/y for the HR sample, and 0.032 ± 0.011 mm/y
for the HR+CR sample. The degradation rate of all three sam-
ples significantly decreased with increasing immersion time. Also,
the AC sample shows the lowest degradation rate compared with
the HR+CR and HR samples for immersion for both 14 d and
30 d In addition, the HR sample shows the lowest corrosion re-
sistance with the highest degradation rate. The degradation rate
of Zn–1Cu–0.1Ti after immersion in Hanks’ solution for 30 d is
similar to that of the extruded Zn–1Cu alloy in c-SBF solution
(0.033 mm/y) reported by Tang et al. [21]. Based on the results
from polarization, EIS, and immersion tests, the corrosion resis-
tance performance of the Zn–1Cu–0.1Ti samples in descending or-
der is AC > HR + CR > HR.
Fig. 6 shows the SEM images of the surface morphologies and
cross-sections of the AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti
after immersion in Hanks’ solution for 14 d and 30 d As can be
seen, all the sample surfaces had granular or massive corrosion
products deposited after the immersion tests. After immersion in
Hanks’ solution for 14 d and 30 d, the overall thickness of the
corrosion products on the sample surfaces in descending order is
HR > HR + CR > AC; similarly, the HR sample surface showed the
greatest amount of corrosion products with an overall thickness of
13.68 ± 0.39 μm and 31.96 ± 3.20 μm after immersion for 14 d and
30 d, respectively. Also, the amount and overall thickness of corro-
sion products on the sample surfaces after immersion for 30 d in-
creased as compared to immersion for 14 d. Also, the amount and
overall thickness of corrosion products on the sample surfaces af-
ter immersion for 30 d increased compared to immersion for 14 d.
It is worth noting that there are no visible cracks or partial spalling
418 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Fig. 6. Surface morphologies and cross-sections of AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti after immersion in Hanks’ solution for 14 d and 30 d.
in the corrosion surfaces, indicating that the corrosion products
on the sample surfaces of Zn–1Cu–0.1Ti provide a protective effect
which prevents further corrosion.
Fig. 7 shows the EDS, XRD, FTIR, and XPS analysis results for
the corrosion products on the HR-processed Zn–1Cu–0.1Ti after im-
mersion in Hanks’ solution for 30 d. It can be seen that the corro-
sion products are mainly composed of C, O, P, Zn, Ca, and small
amounts of Mg and Cu, as analyzed by EDS (Fig. 7(a)). The XRD
results reveal that the corrosion products consist of a Zn matrix,
ZnO, and ZnCO3 (Fig. 7(b)). In the FTIR spectrum of the corro-
sion products (Fig. 7(c)), vibration absorption peaks are observed at
waves of approximately 2956, 1539, 1421, 1064, and 864 cm−1 , re-
spectively. According to the results reported in [48–51], a vibration
absorption peak at a wave in the range 1050–1180 cm−1 belongs
to PO34− ; in the ranges of 830–890 cm−1 , 1390–1500 cm−1 , and
1540–1620 cm−1 belongs to CO23− ; and in the range of 2920 cm−1
belongs to HPO24− . It can be concluded that PO34− , CO23− , and
HPO24− co-exist in the corrosion products of the HR sample after
immersion in Hanks’ solution for 30 d. The XPS spectrum of the
corrosion products on the HR-processed Zn–1Cu–0.1Ti after immer-
sion in Hanks’ solution for 30 d is shown in Fig. 7(d). Four distinct
peaks can be observed in the XPS spectrum, corresponding to the
signal absorption peaks of the main elements including Zn, Ca, O,
and C, respectively. An XPS high-resolution narrow scan was per-
formed on the main elements in the full spectrum and the results
are shown in Fig. 7(e). The high-resolution narrow scanning spec-
tra show values of 1044.8 and 1021.8 eV for Zn 2p1; 531.8 eV for O
1 s; 347.4 eV for Ca 2p; and 285.0 eV for C 1 s. Furthermore, there
are C KLL broad peaks, Na 1 s, and Na KLL peaks in the full spec-
trum (Fig. 7(d)). The Na peak might be derived from the deposition
of solute in Hanks’ solution. Based on the XPS data, it can be de-
duced that the main components of the corrosion products on the
HR sample are ZnO and ZnCO3 .
Combining the EDS, XRD, FTIR, and XPS results, it can be in-
ferred that the corrosion products of the HR-processed Zn–1Cu–
0.1Ti sample are mainly ZnO, ZnCO3 , and Zn3 (PO4 )2 ; and also pos-
sibly ZnHPO4 because Hanks’ solution contains anions of HPO24− .
Drelich et al. [52] reported that the corrosion products of pure Zn
wires implanted in vivo over long-term surveillance were mainly
ZnO, ZnCO3 , and Zn3 (PO4 )2 , and their experimental results indi-
cated that pure Zn could be safely degraded in vivo within a time
frame of approximately 1∼2 years.
3.4. Friction and wear behavior of Zn–1Cu–0.1Ti
Fig. 8 shows the friction behaviors and Brinell hardness of the
AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti and compared to
AC pure Zn. The friction coefficient curves for Zn–1Cu–0.1Ti and
pure Zn during dry-sliding wear and corrosive wear testing in
Hanks’ solution are shown in Fig. 8(a). It can be seen that the fric-
tion coefficient of all the samples is relatively stable at the ini-
tial stage of wear under dry sliding, and then increases rapidly
with sharp fluctuations with increasing wear time. The friction co-
efficient of the AC pure Zn fluctuates between 0.695 and 1.541.
The friction coefficient of the AC Zn–1Cu–0.1Ti fluctuates between
0.581 to 2.405; that of the HR and HR+CR Zn–1Cu–0.1Ti fluctuates
between 0.587∼2.237 and 0.527∼2.411, respectively. Under friction
and wear conditions with solution lubrication, the friction coeffi-
cients for all the samples show a similar trend, fluctuating signifi-
cantly in the initial stage of wear and then tending to become sta-
ble after 5 min.
The Brinell hardness of the AC sample of pure Zn and AC, HR,
and HR+CR samples of Zn–1Cu–0.1Ti is shown in Fig. 8(b). The
Brinell hardness of the studied samples was 28. 7 ± 0.1 HB for
AC pure Zn, 71. 3 ± 0.3 HB for AC Zn–1Cu–0.1Ti, 70. 4 ± 0.2 HB
for HR Zn–1Cu–0.1Ti and 56. 2 ± 0.2 HB for HR+CR Zn–1Cu–0.1Ti,
respectively. It can be seen that the Brinell hardness of the stud-
ied samples showed the same trend relative to the results of the
Vickers hardness shown in Fig. 4(b).
Based on the Brinell hardness results of the studied samples,
it can be concluded that the wear loss of AC pure Zn and HR+CR
Zn–1Cu–0.1Ti is higher than that of AC and HR Zn–1Cu–0.1Ti, as
shown in Fig. 8(c).
The friction coefficient, wear loss, and surface roughness of the
AC sample of pure Zn and the AC, HR, and HR+CR samples of
Zn–1Cu–0.1Ti after dry-sliding wear and corrosive wear testing in
Hanks’ solution are shown in Fig. 8(c). The AC pure Zn shows a
friction coefficient of 0. 741 ± 0.037, a wear loss of 2. 41 ± 0.05 mg,
and a surface roughness of 1. 02 ± 0.05 μm; while the AC Zn–1Cu–
0.1Ti shows a friction coefficient of 1. 039 ± 0.024, a wear loss
of 1. 87 ± 0.09 mg, and a surface roughness of 1. 19 ± 0.08 μm.
After thermomechanical processes, the friction coefficient, wear
loss, and surface roughness of the HR and HR+CR samples of Zn–
1Cu–0.1Ti are 0. 796 ± 0.031, 1. 49 ± 0.03 mg, 0. 96 ± 0.08 μm,
and 0. 731 ± 0.052, 2. 02 ± 0.11 mg, 0. 94 ± 0.06 μm, respectively.
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427 419

Fig. 7. EDS, XRD, FTIR, and XPS analysis results for corrosion products of HR samples of Zn–1Cu–0.1Ti after immersion in Hanks’ solution for 30 d: (a) EDS elemental
composition; (b) XRD spectra; (c) FTIR spectra; (d) XPS full spectrum; and (e) XPS high-resolution narrow scan results for Zn 2p, O 1 s, Ca 2p, and C 1 s.
420 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427

Fig. 8. Friction behaviors and Brinell hardness of AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti and AC pure Zn: (a) friction coefficient curves in dry-sliding wear and
corrosive wear testing in Hanks’ solution; (b) Brinell hardness; and (c) friction coefficient, wear loss and surface roughness after wear testing.

Under the dry friction and wear conditions, the friction coefficient,
wear loss, and surface roughness of the samples are much higher
than those in the liquid lubrication environment. In addition, all
the studied alloy samples show a long, stable plateau in the liq-
uid lubrication environment. It can be concluded that the AC, HR,
and HR+CR samples of Zn–1Cu–0.1Ti and AC pure Zn exhibit bet-
ter wear resistance in the Hanks’ solution environment than in the
dry-sliding condition, and the HR sample of Zn–1Cu–0.1Ti shows
much better wear resistance than the AC and HR+CR samples in
the Hanks’ solution environment.
Fig. 9 shows the SEM images of the worn surface morphologies
of the AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti and AC pure

Fig. 9. Surface morphologies of AC, HR, and HR + CR samples of Zn–1Cu–0.1Ti and AC pure Zn after dry-sliding wear and corrosive wear tests.
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Fig. 10. Hemolysis percentages of AC pure Zn and Zn–1Cu–0.1Ti samples.
Zn after dry-sliding wear and corrosive wear testing in Hanks’ so-
lution. The worn surface of the samples exhibit many plowed fur-
rows under dry-sliding friction which were wide and deep, and
parallel to the sliding direction. The edges of the plowed furrows
show visible plastic deformation and some pits are formed in lo-
calized areas due to the metal falling off. After friction and wear in
Hanks’ solution, all the samples show smooth worn surfaces, and
there are shallow and narrow plowed furrows. The wear mech-
anism consists mainly of abrasive wear and almost no adhesive
wear. In addition, there is a mixture of corrosion and wear debris
on the outer edges of the wear scars. The wear mechanism dur-
ing dry-sliding wear consists of adhesive wear and plastic deforma-
tion, and the wear mechanism under solution lubrication consists
of plowing and slightly abrasive wear. In addition, the width of the
grinding surface after dry-sliding wear and wet lubrication wear
in descending order is AC pure Zn > HR+CR Zn–1Cu–0.1Ti > AC
Zn–1Cu–0.1Ti > HR Zn–1Cu–0.1Ti.
3.5. Hemolysis percentages of Zn–1Cu–0.1Ti
Fig. 10 shows the hemolysis percentages of the AC samples of
Zn–1Cu–0.1Ti compared to the AC pure Zn. It can be seen that the
hemolysis percentages of red blood cells are 2. 44 ± 0.08% and
3. 62 ± 0.14% for the extracts of pure Zn and Zn–1Cu–0.1Ti al-
loy, respectively. According to the requirements for the hemolysis
rate of ISO 10993-4: 2002 [38], the hemolysis percentages of clini-
cally applicable implant materials should less than 5%. The hemol-
ysis percentages of both these materials are far below 5% and no
hemolysis occurs. The hemolysis results indicate that the Zn–1Cu–
Fig. 11. Contact angles and cell survival rates of AC Zn and Zn–1Cu–0.1Ti surfaces: (a) contact angle of pure Zn; (b) contact angle of Zn–1Cu–0.1Ti; and (c) survival rate of
MC3T3-E1 cells after culturing for 24 and 48 h.
421
0.1Ti alloy possesses good blood compatibility and meets the re-
quirements for the hemolysis rate of implant materials.
3.6. Cytotoxicity of Zn–1Cu–0.1Ti
The chemical composition, wettability, topography, and rough-
ness of biomaterial surfaces can affect cell compatibility [53].
Fig. 11 shows the contact angle and cell survival rate of the AC
pure Zn and Zn–1Cu–0.1Ti surfaces. It can be seen that the contact
angles of the AC pure Zn and Zn–1Cu–0.1Ti are 62. 4 ± 0.9° and
63. 7 ± 2.6°, respectively (Fig. 11(a) and (b)). This result indicates
hydrophilicity and there is no noticeable difference between the
contact angles of both surfaces. Solař et al. [54] reported that hy-
drophilic surfaces have better cell adhesion than hydrophobic sur-
faces, and Li et al. [55] reported that the cells tend to adhere onto
surfaces with a water contact angle of approximately 70°. There-
fore, the surface contact angles of both pure Zn and Zn–1Cu–0.1Ti
should be beneficial to cell adhesion and attachment.
The survival rates of MC3T3-E1 pre-osteoblast cells after cul-
turing on AC pure Zn and Zn–1Cu–0.1Ti for 24 and 48 h are
shown in Fig. 11(c). As can be seen, the survival rate of MC3T3-
E1 cells after culturing for 24 h is 73. 6 ± 1.0% for AC pure Zn and
44. 3 ± 3.3% for AC Zn–1Cu–0.1Ti. When the culturing time reaches
48 h, the survival rate decreases to 55. 5 ± 8.4% for AC pure Zn and
13. 9 ± 0.6% for AC Zn–1Cu–0.1Ti. The survival rates of the cells af-
ter culturing for 24 and 48 h are lower than 75%, showing a cyto-
toxicity grade of 2–4 as defined by ISO 10993-5 [39].
Fig. 12 shows the ion concentrations of the extracts of AC pure
Zn and Zn–1Cu–0.1Ti and the cell viability of MC3T3-E1 and MG-
63 cells after culturing with different concentrations of extracts
for 1, 3, and 5 d. It can be seen that the Zn ion concentration
in the extract of AC pure Zn is 14. 16 ± 0.26 μg/ml and the Zn,
Cu, and Ti ion concentrations in the extract of Zn–1Cu–0.1Ti are
33. 97 ± 2.11 μg/ml, 14. 2 ± 0.9 ng/ml, and 0. 4 ± 0.2 ng/ml, re-
spectively (Fig. 12(a)). The undiluted extracts (i.e., 100% concentra-
tion) of AC pure Zn and Zn–1Cu–0.1Ti show low cell viability for
both MC3T3-E1 and MG-63 cells at all culturing time points of 1,
3, and 5 d (Fig. 12(b)). The cell viability is lower than 55% and
the extracts are substantially cytotoxic due to the high metal ion
concentrations. When the concentration of the extracts of both AC
pure Zn and Zn–1Cu–0.1Ti is diluted to 25%, the corresponding cell
viability significantly increases after culturing for all time points
(Fig. 12(c)). The cell viability of MC3T3-E1 cells is 64. 48 ± 1.02%
for pure Zn and 82. 74 ± 0.34% for Zn–1Cu–0.1Ti after culturing
for 1 d The cell viability of MG-63 cells is 87. 30 ± 1.98% for pure
Zn and 65. 45 ± 0.42% for Zn–1Cu–0.1Ti after culturing for 1 d Af-
ter further diluting the extracts of AC pure Zn and Zn–1Cu–0.1Ti to
12.5% concentration, the cell viability of both MC3T3-E1 and MG-
63 cells exceeds 90% after culturing for 1 d, indicating good in vitro
cytocompatibility and non-toxicity. The cell viability with different
422 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Fig. 12. (a) Ion concentrations of extracts of AC pure Zn and Zn–1Cu–0.1Ti; cell viability of MC3T3-E1 and MG-63 cells after culturing with extracts of AC pure Zn and
Zn–1Cu–0.1Ti for 1, 3, and 5 d at concentrations of: (b) 100%; (c) 25%; and (d) 12.5%.
extract concentrations shows a similar trend after culturing for 3
and 5 d, and the cell viability of MC3T3-E1 and MG-63 cells in-
creases with the dilution of the extracts. Furthermore, the cell via-
bility of MC3T3-E1 and MG-63 cells cultured with the extracts for
1, 3, and 5 d shows no significant difference between AC pure Zn
and Zn–1Cu–0.1Ti.
3.7. Antibacterial property of Zn–1Cu–0.1ti
Fig. 13 shows the antibacterial effect of AC pure Zn and Zn–
1Cu–0.1Ti after co-culturing with S. aureus for 24 h at 37 °C. A bac-
teriostatic ring appears around the samples of pure Zn and Zn–
1Cu–0.1Ti, and the value of IZD (H) is 6. 19 ± 0.42 mm for pure Zn
and 6. 99 ± 0.33 mm for Zn–1Cu–0.1Ti. The Zn–1Cu–0.1Ti exhibits
a good antibacterial effect and its IZD is higher than that of pure
Zn, and the differences between the two groups were statistically
significant (P < 0.05).
4. Discussion
4.1. Microstructures and mechanical properties of Zn–1Cu–0.1Ti
The AC Zn–1Cu–0.1Ti exhibits σ uts = 92.4 MPa, σ ys = 86.1 MPa,
ε = 1.4%, and hardness of 72.6 HV, respectively, significantly higher
than those of AC pure Zn (σ uts = 33.6 MPa, σ ys = 29.3 MPa,
ε = 1.2%, and hardness = 37.9 HV [18]), indicating that the ad-
dition of Cu and Ti can effectively improve the tensile properties
and hardness of the Zn alloy. Strengthening of Zn–1Cu–0.1Ti alloy
by adding the alloying elements of Cu and Ti occurred through var-
ious mechanisms including Hall-Petch strengthening, solid-solution
strengthening, and precipitation hardening. Firstly, due to the ad-
dition of the Cu and Ti alloying elements, the η-Zn matrix phase in
the AC Zn–1Cu–0.1Ti showed an average grain size of ∼54 μm, sig-
nificantly less than that of the AC pure Zn (∼600 μm). The grain
size of the AC Zn–1Cu–0.1Ti is over 11 times finer than that of
the AC pure Zn. It is well known that metals of finer grain size
exhibit greater yield strength based on the Hall-Petch strengthen-
ing mechanism (that is, σ y = σ o + k d−1/2 , where σ y is the yield
stress, σ o is a materials constant for starting stress for dislocation
movement (or resistance of the lattice to dislocation motion), k is
the strengthening coefficient (a constant unique to each material),
and d is the grain diameter) [56–57]. Secondly, the addition of the
Cu element resulted in solid-solution strengthening of the Zn alloy
matrix. Some of the Cu atoms and the η-Zn matrix phase formed
a solid-solution phase because the difference of their atomic radii
is less than 15%, causing lattice strains on surrounding host atoms
which provide resistance for dislocation movement, leading to en-
hanced strength and hardness relative to those of the pure Zn [58].
Third, the remaining Cu and Ti formed fine and reticular second
phases of ε -CuZn5 and TiZn16 distributed along the grain bound-
aries or inside the grains. These second phases resulted in precip-
itation hardening since the particles resist the dislocation motion
and developing the bow around the second particles, which led to
increased hardness of the Zn alloy.
As for the HR sample of Zn–1Cu–0.1Ti, deformation heat ac-
cumulated during the cyclical heating and deformation of the
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
Fig. 13. Antibacterial effect of AC pure Zn and Zn–1Cu–0.1Ti after co-culturing with S. aureus for 24 h: (a) image of pure Zn; (b) image of Zn–1Cu–0.1Ti; and (c) inhibition
zone diameter (IZD) of pure Zn and Zn–1Cu–0.1Ti.
hot-rolling process, which caused localized dynamic recoveries and
recrystallization due to the low recrystallization temperature of the
alloy. This led to a significant dynamic softening of the alloy and
thus the hardness of the HR sample decreased slightly and the
elongation increased notably as compared to the AC sample, while
the fracture mode changed from a brittle cleavage fracture to a
ductile dimple fracture.
In the case of the HR+CR sample of Zn–1Cu–0.1Ti, more ε -
CuZn5 particles precipitated during the hot-rolling and cold-rolling
processes in the alloy, which resulted in a reduced concentra-
tion of Cu in the η-Zn matrix phase, weakening the solid-solution
strengthening of the Cu. This caused a significant decrease in hard-
ness. Moreover, the hard and brittle ε -CuZn5 particles that precip-
itated were crushed into fine particles during the hot rolling and
cold rolling. These fine ε -CuZn5 particles homogeneously dispersed
in the η-Zn matrix, leading to significantly increased strength and
ductility. Therefore, the HR+CR sample of Zn–1Cu–0.1Ti exhibits
the best overall mechanical properties among the three samples.
4.2. Corrosion behavior of Zn–1Cu–0.1Ti
The standard electrode potential is −0.763 V for Zn, 0.342 V for
Cu, and −1.630 V for Ti [56,59]. Therefore, alloying with Cu can be
expected to increase the corrosion potential of a Zn alloy due to
its more positive standard electrode potential than that of the Zn.
However, the corrosion potential of the η-Zn phase matrix is lim-
ited due to the low content of Cu. Both Cu and Ti mainly form
the second phases of ε -CuZn5 , TiZn16 , and probably Cu2 TiZn22 and
other precipitation phases with Zn. In general, second phases have
a dual effect on the corrosion behavior of the relevant material
[60]. Firstly, they cause galvanic corrosion. The second phase and
the matrix phase of a Zn alloy will form a micro-battery cell due
423
to the difference in their corrosion potentials. The precipitated
phase serves as the cathode electrode and a potential difference
is formed between the precipitated particles and the matrix phase,
causing galvanic corrosion which accelerates the intergranular cor-
rosion of the Zn alloy. Secondly, they exert a barrier effect. When
the second phase of a Zn alloy is distributed in the matrix and
forms a continuous network, it can act as a corrosion barrier for
the Zn matrix, preventing the corrosion from proceeding fully and
thereby slowing the corrosion of the Zn alloy.
The microstructure of the AC Zn–1Cu–0.1Ti is composed of an
η-Zn phase and the second phases of TiZn16 and ε-CuZn5 . These
two second phases are mainly distributed along the grain bound-
aries and form a network in the matrix, and only some of them are
distributed inside the grains, so the network of the second phases
can act as a barrier and hinder corrosion. As for the HR sample of
Zn–1Cu–0.1Ti, the TiZn16 intermetallic phase of the network in the
AC alloy was crushed and formed a fiber-like or dispersed gran-
ular structure along the rolling direction, resulting in a weaken-
ing of the barrier effect. In such cases galvanic corrosion plays a
major role, resulting in an accelerated corrosion rate of the alloy
with a decreased corrosion resistance compared to the AC alloy. Af-
ter HR processes, the coarse skeletal ε -CuZn5 phase of the HR+CR
Zn–1Cu–0.1Ti is refined and dispersed in the matrix, resulting in
a significant reduction of the active cathode region and weakened
galvanic corrosion, therefore exhibiting better corrosion resistance.
According to Ren et al. [61], the degradation process of pure
Zn and Zn–Mg–Zr alloys in SBF can be expressed by the reaction
between Zn and water:
Zn + H2 O → ZnO + 2H+ + 2e− (4)
When the immersion time is short (14 d), the corrosion prod-
uct adheres to the surface of the sample in a point-like manner
424 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
without forming a dense corrosion layer. It forms galvanic corro-
sion with the η-Zn phase matrix in which the η-Zn phase acts
as the anode and the corrosion product as the cathode. This leads
to high degradation of all the samples of AC, HR, and HR+CR Zn–
1Cu–0.1Ti. With a prolonged immersion time (30 d), the corrosion
product layer gradually thickens and becomes denser, which can
prevent the Cl− ions in the environment from entering the ma-
trix and so prevent further corrosion to a certain extent, thereby
slowing the corrosion rate and protecting the substrate of the al-
loy. Therefore, the studied alloy samples show a lower degradation
rate after immersion in Hanks’ solution for 30 d.
4.3. Friction and wear behavior of Zn–1Cu–0.1Ti
As can be seen from Fig. 8(c), the AC sample of pure Zn and the
HR+CR sample of Zn–1Cu–0.1Ti shows a lower friction coefficient
and higher wear loss and surface roughness than the AC and HR
Zn–1Cu–0.1Ti. This is because the hardness of both samples was
considerably lower than that of the AC and HR Zn–1Cu–0.1Ti.
According to the Holm-Archard equation [62], the wear loss Q
can be calculated using the formula given by:
Q = kw/H
where k is the wear factor, w is the applied load, and H is the
hardness of the worn surface. When the applied load is a constant
200 gf (∼2 N), the wear rate is inversely proportional to the hard-
ness. According to the macro-hardness data of Brinell hardness for
the samples shown in Fig. 8(b), it can be concluded that the wear
loss of the AC pure Zn and HR+CR Zn–1Cu–0.1Ti is higher than
that of the AC and HR Zn–1Cu–0.1Ti, indicating the same trend as
shown in Fig. 8(c).
The AC sample of pure Zn and the AC, HR, and HR+CR sam-
ples of Zn–1Cu–0.1Ti exhibit superior wear resistance in the liquid
environment of Hanks’ solution than in the dry-sliding wear envi-
ronment. This is because the Hanks’ solution acts in three ways to
affect the friction and wear of the samples during corrosive wear.
Firstly, it provides a lubrication effect on the samples during the
corrosive-wear process and the solution acts as a lubricant. The
hard abrasive particles falling off the surface of the samples are
washed away by the solution, so these hard particles can no longer
abrade the sample. The wear mechanism under the lubrication of
Hanks’ solution is therefore slight abrasive wear, which is a type
of two-body wear and the change of friction coefficient during the
wear process is relatively smooth and stable. Secondly, the Hanks’
solution provides cooling to the process. At a constant temperature
of 37 °C, Hanks’ solution has a good cooling effect on the friction
surface, which can take away the heat generated by the friction
process over time and reduce the tendency of the alloy to undergo
adhesive wear. Thirdly, although the Hanks’ solution has a corro-
sive effect on the Zn alloy, Zn alloys possess much better corrosion
resistance than Mg alloys and so the corrosion effect on the Zn al-
loy is insignificant in a short wear period. Therefore, the medium
of the Hanks’ solution does not aggravate the wear on the Zn al-
loy, unlike the aggravated wear on Mg alloys in SBF reported by
Liu et al. [63].
In the dry-sliding friction process, since the hardness of the
friction pair is relatively high, the hardness difference between the
test samples and the friction pair is relatively significant. So as the
wear time is considerably lengthened, the surface temperature of
the alloy continuously increases due to the accumulation of friction
heat, resulting in partially softened surfaces of the metal samples
which leads to the adhesive wear and plastic deformation. In ad-
dition, the partially peeled protective layers exists in the form of
solid debris between the friction pair and samples, and the abrad-
ing effect of the debris on the samples causes plowed furrows
whose depth and width increase with testing time. The amount
of wear debris that is not easily discharged increases with testing
time and the obstruction effect on the friction pair is enhanced, re-
sulting in severe abrasive wear on the surface of the samples. The
friction coefficient of the samples in dry-sliding wear increases and
fluctuates substantially, indicating a type of three-body wear.
In addition, there is a clear running-off period in the case of
dry-sliding wear, while the running-in period is short or even ab-
sent in the case of wet friction and wear with Hanks’ solution.
This is mainly because dry friction wear is a type of three-body
wear and its main wear mechanism is a fatigue layer undergoing
fatigue fracture due to the multiple plastic deformation, while the
wet friction and wear is a type of two-body abrasive wear, which
does not require a long running-in period, and so the stable pe-
riod becomes longer. Therefore, for the abovementioned reasons,
the friction coefficient, wear loss, and surface roughness of all the
samples under the dry friction condition are significantly higher
than those under the wet friction and wear environment of Hanks’
solution.
4.4. Hemolytic properties, cytocompatibility, and antimicrobial ability
of Zn–1Cu–0.1Ti
(5)
New biodegradable implant materials for bone fixation not only
need to exhibit suitable biodegradability and mechanical prop-
erties, but also require good biocompatibility and even biofunc-
tionalities such as antibacterial ability. The recommended daily
intakes of Zn, Cu, and Ti ions for adults are about 12∼16 mg,
0.9∼1.2 mg, and 0.3∼2 mg, respectively and excessive intake of
these metal ions causes systemic toxicity [49,64–66]. Most of the
Zn, Cu, and Ti ions absorbed into the systemic circulation are ex-
creted through feces and urine. As shown in Fig. 5(f), the degra-
dation rate of HR Zn–1Cu–0.1Ti after immersion in Hanks’ solu-
tion for 30 d is 0.032 mm/y. The release efficiency of each kind
of ion after implantation can be estimated according to the cor-
responding degradation rate, and the corrosion rate can be cal-
culated to be 0.016 mg/cm2 /day. Assuming that the dimensions of
a screw implant are 2.5 mm × 18 mm with a surface area of ap-
proximately 2.0 cm2 [67], it can be estimated that the ion release
rates of Zn, Cu, and Ti are 32.4 μg/d, 0.33 μg/d, and 0.03 μg/d, re-
spectively, which is far below the recommended daily intake levels
of these elements. In addition, Bian et al. [49] reported that the
local ion concentrations of Mg and Ge surrounding the implant in
vivo were diluted and much lower than those of the in vitro testing
experimental results due to the circulation system. In addition, be-
cause the corrosion products of the Zn–1Cu–0.1Ti alloy in Hanks’
solution are mainly composed of ZnO, ZnCO3 , and ZnPO3 , these
products are similar to the degradation products of the Mg-based
alloy implants reported by Witte et al. [68], which were nontoxic
and non-irritating to the human body. It can be concluded that the
degradation products of the Zn–1Cu–0.1Ti alloy can be excreted by
the physiological system and should not cause systemic toxicity.
When the AC pure Zn and Zn–1Cu–0.1Ti samples are directly
cultured with MC3T3-E1 and MG-63 cells, both samples show low
cell viability, indicating that the surfaces of both materials produce
certain toxic effects. This is because the concentration of the metal
ions released is higher than the threshold of the ions required for
cell proliferation, resulting in a decrease in the proliferation capac-
ity of the MC3T3-E1 cells. In addition, the Zn–1Cu–0.1Ti alloy ex-
hibited lower cell activity than pure Zn, mainly because its degra-
dation rate (314.6 μm/y) is faster than that of pure Zn (156.7 μm/y
for AC pure Zn obtained from potentiodynamic polarization tests
in Hanks’ solution [18]). The excess metal ions are released from
the surface of the sample during cell culturing, which causes pro-
found cell death. With culture time increasing, the concentration
of metal ions in the culture solution gradually increases, resulting
in a continuous decrease in the survival rate of MC3T3-E1 cells. In
 J. Lin, X. Tong and Z. Shi et al. / Acta Biomaterialia 106 (2020) 410–427
response to this, many researchers [20,21] proposed using diluted
alloy extracts to evaluate the in vitro cytotoxicity of metallic bio-
materials.
To assess the effect of extract concentration on in vitro cyto-
compatibility tests, Wang et al. [69] recommended that cytotoxic-
ity be assessed using a diluted extract method, which use a 6∼10
times dilution of extracts for in vitro cytotoxicity testing. Dambatta
et al. [70] found that osteoblast cells were highly active when cul-
tured in extracts of a Zn–3Mg alloy with a Zn ion concentration
below 0.5 mg/ml (0.1 and 0.5 mg/l), but more toxic when cultured
in extracts with ion concentrations of 1.0 mg/ml and 2.0 mg/ml.
Furthermore, when osteoblast cells were cultured in alloy extracts
with Zn ion concentrations of 0.1 and 0.5 mg/ml for 1, 3, and 7 d,
the relative activity of cells increased to the maximum and then
decreased with the elongation of culture time. Ma et al. [71] also
found that extracts with low concentrations of Zn ions promoted
cell proliferation and growth, but extracts with high concentrations
of Zn ions inhibited cell activity. For the difference of cell viabil-
ity between the MC3T3 and MG63 cells, it may be because the
cell proliferation and tolerance of the both cell lines for Zn ions
with different level were different. This kind of phenomenon has
also been reported by other researchers, for example, Shen et al.
[72] reported that the HOS cells showed a significant reduction in
cell viability and induced cytotoxicity at a higher extract concen-
tration; while the MG63 cells had a slight negative impact on cell
viability at the same level of extract.
In this study, the relative cell viability of both MC3T3-E1 and
MG-63 cells increases as the concentration of the extract decreases
(Fig. 12). Moreover, the results indicate that a low concentration
extract (≤ 25%) of Zn–1Cu–0.1Ti shows no apparent toxic effect on
MC3T3-E1 cells.
For the effect of metal ions on antibacterial activity, Sukhodub
et al. [73] systematically studied the antibacterial ability of metal
ions and reported that the metal ion sterilization rate from high
to low was: Ag+ , Cu2+ , Zn2+ , Mg2+ . Among these metal ions, Zn
ions have a good antibacterial ability when they reach a certain
concentration and can kill various bacteria and fungi. Wahab et al.
[74] reported that a concentration of ≥ 11 mM of Zn acetate had
a good killing effect on S. aureus. Cu and its alloys also had good
bacteria-killing effects on pathogenic bacteria such as Escherichia
coli, Mycobacterium tuberculosis, and methicillin-resistant S. au-
reus [75–77]. Tang et al. [21] reported that the antibacterial proper-
ties of Zn–xCu (x = 1, 2, 3, 4%) alloys increased with an increase in
Cu content. The antibacterial testing in this study shows that the
Zn–1Cu–0.1Ti alloy has a good antibacterial ability which is better
than that of pure Zn, as indicated by a higher IZD (Fig. 13).
5. Conclusions
This Zn–1Cu–0.1Ti alloy has been developed as a biodegradable
Zn-based alloy for bone implant applications. It achieves enhanced
mechanical properties (both strength and ductility) and antibacte-
rial ability through alloying Zn with Ti and Cu. Thermomechanical
processes of HR and HR+CR were performed on the AC Zn–1Cu–
0.1Ti alloy to further boost the mechanical properties. The main
conclusions are as follows:
1. The microstructure of the AC Zn–1Cu–0.1Ti alloy was composed
of a η-Zn matrix phase, an intermetallic compound TiZn16
phase, and an ε -CuZn5 phase. Most of the intermetallic com-
pound TiZn16 phase was distributed along the grain boundaries
of the alloy. The alloy after HR exhibited elongated η-Zn, TiZn16 ,
and Cu2 TiZn22 phases along the rolling direction. The alloy after
HR+CR exhibited visible processing flow lines and finely dis-
persed ε -CuZn5 particles in the η-Zn matrix along the rolling
direction.
425
2. The HR+CR sample of the Zn–1Cu–0.1Ti alloy exhibited the
highest tensile properties among the three conditions of AC,
HR, and HR+CR, with a yield strength of 204.2 MPa, an ulti-
mate strength of 249.9 MPa, and an elongation of 75.2%; these
are 2.7 times the yield strength, 2.4 times the ultimate ten-
sile strength, and 53.7 times the elongation of the AC alloy. The
fracture mode of the Zn–1Cu–0.1Ti alloy was transformed from
brittle fractures with mixed fracture characteristics of river pat-
terns, cleavage steps, and a few small dimples in localized ar-
eas in the AC alloy to ductile fractures with homogeneously
distributed, round, equiaxed dimples in the HR alloy and the
HR+CR alloy. The dimples on the fracture surface of the HR+CR
alloy were deeper, larger, and denser compared to those of the
HR alloy. The HR+CR alloy showed a hardness of 56.4 HV, lower
than those of the HR alloy (70.6 HV) and the AC alloy (72.6 HV).
3. Potentiodynamic polarization tests on Zn–1Cu–0.1Ti showed
that the corrosion potential, corrosion current density, and cor-
rosion rate were −1.025 V, 21.5 μA/cm2 , and 315 μm/y for the
AC alloy; −1.123 V, 111.2 μA/cm2 , and 1628 μm/y for the HR al-
loy; and −1.100 V, 67.7 μA/cm2 , and 991 μm/y for the HR+CR
alloy. The degradation rate of Zn–1Cu–0.1Ti after immersion
in Hanks’ solution for 30 d was 0.029 mm/y, 0.032 mm/y, and
0. 034 ± 0.018 mm/y for the AC, HR and HR+CR alloys, re-
spectively. The corrosion products consisted of Zn(CO)3 , ZnO,
Zn3 (PO4 )2 , and also probably ZnHPO4 .
4. The AC, HR, and HR+CR samples of Zn–1Cu–0.1Ti and the AC
pure Zn exhibited better wear performances in the liquid lubri-
cation environment of Hanks’ solution than in the dry-sliding
wear condition, and the HR sample showed significantly better
wear resistance among the samples. The wear mechanisms of
the dry-sliding wear and corrosion wear were three-body abra-
sive wear and two-body abrasive wear, respectively.
5. The extract of AC Zn–1Cu–0.1Ti alloy at a concentration ≤ 25%
showed no significant cytotoxicity to MC3T3-E1 and MG-63
cells. The AC Zn–1Cu–0.1Ti alloy exhibited good blood compat-
ibility and antibacterial property.
Overall, based on the testing results for mechanical proper-
ties, corrosion and corrosive-wear property, degradation behavior,
biocompatibility, and antibacterial ability, the Zn–1Cu–0.1Ti alloy
is a promising candidate for bone fracture fixation applications
such as bone screws and plates of biodegradable implants due to
its markedly enhanced mechanical properties, wear resistance, an-
tibacterial ability, and suitable degradation rate.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
This work was supported by the Wenzhou Science and Technol-
ogy Bureau through the project G20180027 and 2018ZG008. CW
and YL also acknowledge the financial support for this research
by the Australian Research Council (ARC) through the Discovery
Project DP170102557 and Future Fellowship FT160100252. JL ac-
knowledges the support for this research by Hunan Provincial Nat-
ural Science Foundation of China through the Grant 2016JC2005.
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