Acta Biomaterialia 6 (2010) 626–640

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

Research on an Mg–Zn alloy as a degradable biomaterial

Shaoxiang Zhang a, Xiaonong Zhang a,c,*, Changli Zhao a, Jianan Li a, Yang Song a, Chaoying Xie a,
Hairong Tao b, Yan Zhang b, Yaohua He b, Yao Jiang b, Yujun Bian c
a
State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China
b
Orthopaedic Department of the 6th People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, People’s Republic of China
c
Shanghai Origin Material and Medical Technology Co. Ltd., Shanghai 200240, People’s Republic of China

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

Article history: In this study a binary Mg–Zn magnesium alloy was researched as a degradable biomedical material. An
Received 17 December 2008 Mg–Zn alloy fabricated with high-purity raw materials and using a clean melting process had very low
Received in revised form 1 June 2009 levels of impurities. After solid solution treatment and hot working the grain size of the Mg–Zn alloy
Accepted 11 June 2009
was finer and a uniform single phase was gained. The mechanical properties of this Mg–Zn alloy were
Available online 21 June 2009
suitable for implant applications, i.e. the tensile strength and elongation achieved were 279.5 MPa
and 18.8%, respectively.
Keywords:
The results of in vitro degradation experiments including electrochemical measurements and immer-
Mg–Zn alloy
Degradation
sion tests revealed that the zinc could elevate the corrosion potential of Mg in simulated body fluid (SBF)
Mechanical properties and reduce the degradation rate. The corrosion products on the surface of Mg–Zn were hydroxyapatite
Cytotoxicity (HA) and other Mg/Ca phosphates in SBF. In addition, the influence caused by in vitro degradation on
In vivo biocompatibility mechanical properties was studied, and the results showed that the bending strength of Mg–Zn alloy
dropped sharply in the earlier stage of degradation, while smoothly during the later period.
The in vitro cytotoxicity of Mg–Zn was examined. The result 0–1 grade revealed that the Mg–Zn alloy
was harmless to L-929 cells. For in vivo experiments, Mg–Zn rods were implanted into the femoral shaft
of rabbits. The radiographs illustrated that the magnesium alloy could be gradually absorbed in vivo at
about 2.32 mm/yr degradation rate obtained by weight loss method. Hematoxylin and eosin (HE) stained
section around Mg–Zn rods suggested that there were newly formed bone surrounding the implant.
HE stained tissue (containing heart, liver, kidney and spleen tissues) and the biochemical measure-
ments, including serum magnesium, serum creatinine (CREA), blood urea nitrogen (BUN), glutamic-pyru-
vic transaminase (GPT) and creatine kinase (CK) proved that the in vivo degradation of Mg–Zn did not
harm the important organs. Moreover, no adverse effects of hydrogen generated by degradation had been
observed and also no negative effects caused by the release of zinc were detected. These results suggested
that the novel Mg–Zn binary alloy had good biocompatibility in vivo.
Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Due to their very low corrosion potential, magnesium and its
alloys are susceptible to dissolution in aqueous solutions, particu-
larly in those containing chloride ion electrolytes [1,2]. Making use
of the corrodible properties of magnesium alloys, in recent years
biomaterials engineers have become interested in developing mag-
nesium-based biodegradable medical devices [3–18].
As shown in Table 1, various magnesium alloys have been re-
searched as biodegradable materials and some of them have
shown biocompatibility. For example, compared with poly-96L/
* Corresponding author. Address: State Key Laboratory of Metal Matrix Compos-
ites, School of Materials Science and Engineering, Shanghai Jiao Tong University, No.
800 DongChun Road, Shanghai 200240, People’s Republic of China. Tel./fax: +86 21
3420 2759.
E-mail address: xnzhang@sjtu.edu.cn (X. Zhang).
4D-lactide, the magnesium alloys AZ31 and AZ91 enhanced the
osteogenesis response and increase newly formed bone [4]. Heub-
lein et al. reported that the alloy AE21 gradually degraded in pig ar-
tery and might be a promising cardiovascular implant material [3].
A more encouraging indication is that a biodegradable magnesium
stent has been used in clinical experiments [18].
However, it should be noted that most of the reported biomed-
ical magnesium alloys contain aluminum and/or rare earth (RE)
elements. It is well known that Al is harmful to neurons [19] and
osteoblasts [20] and also associated with dementia and Alzhei-
mer’s disease [19]. The administration of RE (Pr, Ce, Y, etc.) could
lead to hepatotoxicity [21]. Excessive yttrium ions (Y3+) have been
shown to change the expression of some rat genes and to have ad-
verse effects on DNA transcription factors [22]. Consequently, Al
and RE are unsuitable alloying elements for biomedical magnesium
materials, particularly when they are above certain levels. This has

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.actbio.2009.06.028
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
Table 1
The nominal chemical compositions of Mg alloys researched as degradable biomaterials.
Alloy type Aluminum (wt.%) Rare earth element
AE21 2 1 wt.% (Ce, Pr, Nd)
AZ31 3 1
AZ91 9 1
WE43 3 wt.% (Nd, Ce, Dy), 4 wt.% (Y)
LAE442 4 2 wt.% (Ce, La, Nd, Pr)
AM60B 6 0.07
Mg–Ca
Mg–Zn–Y 0.36–1.54 wt.% Y
Mg–Mn–Zn
led to a demand for the development of a novel biodegradable
magnesium alloy without Al, RE or other harmful elements.
With the purpose of searching for suitable alloying elements for
biomedical magnesium alloys, Song [12] explored in vitro corro-
sion rates of several magnesium alloys, pointing out that Ca, Mn
and Zn could be appropriate candidates. Further investigations
demonstrated that Mg–Ca [6] and Mg–Mn–Zn [16] alloys gradually
degraded within bone and had good biocompatibility both in vitro
and in vivo. Moreover, the degradation process did not raise the
serum Mg2+ level and no kidney disorders were observed.
Zinc is one of the most abundant nutritionally essential ele-
ments in the human body [23], and has basic safety for biomedical
applications. Furthermore, zinc can improve the corrosion resis-
tance and mechanical properties of magnesium alloys. For exam-
ple, the corrosion rate of magnesium could be reduced by
increasing the mass fraction of zinc in magnesium [24]. Moreover,
zinc can effectively strengthen magnesium [12,25] through a solid
solution hardening mechanism [25].
According to the Mg–Zn binary phase diagram [26] the maximum
solubility of Zn in Mg is 6.2 wt.% (i.e. 2.5 at.%) at 325 °C. In this paper a
patented Mg–6 wt.% Zn alloy (Mg–6Zn) [27] was developed with the
aim of making a novel magnesium alloy with good biocompatibility,
moderate degradation rate and good mechanical properties. Analy-
sis and evaluation of the in vitro and in vivo degradation of this Mg–
6Zn alloy are also presented in this paper.
2. Materials and methods
2.1. Materials
Magnesium alloys with a nominal composition of Mg–6 wt.% Zn
were prepared using high-purity magnesium (P99.99%) and zinc
(P99.9999%) ingots. Melting was carried out at 700–750 °C in a
high-purity graphite crucible. After about 30 min holding and stir-
ring, the melt was cast into a steel mold at about 700 °C. A protec-
tive cover gas (99.99% purity argon) was employed throughout the
melting and casting processes. The as cast ingots of Mg–6Zn alloy
were solid solution treated at about 350 °C for 2 h, followed by
quenching in water. Finally, the heat-treated alloy sample was
hot extruded at about 250 °C with an extrusion ratio of 8:1.
Disk samples for the in vitro degradation and cytotoxicity
experiments, having a diameter of 11.3 mm (so that the cross-sec-
tion area was 1 cm2) and a height of 2.0 mm, were machined from
the extruded Mg–6Zn rods. Rectangular samples with the dimen-
sions 5  5  30 mm were made and used for three-point bending
testing. Cylindrical rods with a diameter of 4.5 mm and a length of
10 mm were used in the animal implant experiments. All samples
were ground with SiC paper up to 1200 grit and polished with
1 lm diamond paste, followed by ultrasonic cleaning in acetone,
ethanol and distilled water. Before the in vitro cytotoxicity and
in vivo degradation experiments all samples were sterilized with
29 kGy of 60Co radiation.
627
Zinc (wt.%) Other References
[3]
[4]
[4,5,8,10]
[4]
4 wt.% Li [4,5]
0.33 wt.% Mn [14]
<2 wt.% Ca [6,7,17]
2 [11]
1 1.2 wt.% Mn [16]
2.2. Composition and microstructure characterization
The chemical compositions of the Mg–6Zn alloy was deter-
mined by inductively coupled plasma-atomic emission spectrome-
try (ICP-AES) (Iris Advantage 1000). The microstructures of the as
cast, heat-treated and extruded samples were characterized by
optical microscopy. X-ray diffraction analysis (XRD) (Rigaku
D/MAX255) was used to examine the phase of the as cast, heat-
treated and extruded Mg–6Zn.
2.3. Mechanical properties
Tension and compression tests were carried out with a MTS
810.22 universal testing machine, according to ASTM E8-04 and
ASTM E9-89a (2000) [28,29]. The tensile samples had a gauge length
of 35 mm and the compressive samples had diameter of 6 mm and a
height of 9 mm. A crosshead speed of 0.5 mm min1 was used.
2.4. In vitro degradation tests
In order to evaluate the in vitro degradation properties, electro-
chemical measurements and immersion tests were performed in a
simulated body fluid (SBF), containing 6.800 g l1 NaCl, 0.200 g l1
CaCl2, 0.400 g l1 KCl, 0.100 g l1 MgSO4, 2.200 g l1 NaHCO3,
0.126 g l1 Na2HPO4 and 0.026 g l1 NaH2PO4 [30]. The pH value
of SBF was adjusted with HCl and NaOH solution to 7.44 and the
temperature was maintained at 37 ± 0.5 °C. Pure Mg (>99.99%)
was also tested as a counterpart.
2.4.1. Electrochemical measurements
Electrochemical measurements were performed using a three
electrode system (PARSTATÒ 2273). A saturated calomel electrode
(SCE) and a platinum mesh were used as the reference and counter
electrodes, respectively. Potentiodynamic polarization curves were
measured at a scan rate of 1 mV s1. The corrosion rates of the
samples were calculated by extrapolating the polarization curve
according to ASTM-G102-89 [31]. Electrochemical impedance
spectroscopy (EIS) analysis was also performed at open circuit po-
tential with a perturbing signal of 5 mV. The frequency varied
from100 to 1 MHz. All of the EIS results were fitted and analyzed
using ZsimpWin 3.10 Echem software.
2.4.2. Immersion tests
Immersion tests were carried out in conformation with ASTM-
G31-72 [32] (the ratio of surface area to solution volume was
1 cm2:20 ml). Samples were removed after 3 and 30 days of
immersion, rinsed with distilled water and dried at room temper-
ature. Then the surface morphology after immersion was observed
using scanning electron microscopy (SEM) with an energy disper-
sive spectrometer (EDS). XRD (Rigaku, D/MAX255) was used to
determine the phase of corrosion products on the Mg–6Zn surface.
Lastly, the samples were cleaned with 180 g l1 chromic acid to re-
move the corrosion products and the degradation rates (in units of
628 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
mm year1) were obtained according to ASTM-G31-72. The corro-
sion rate is given by Eq. (1) [32]:
Corrosion rate ¼ ðK  WÞ  ðA  T  DÞ ð1Þ
where the coefficient K = 8.76  104, W is the weight loss (g), A is
the sample area exposed to solution (cm2), T is the exposure time
(h) and D is the density of the material (g cm–3).
The pH value of the solution was also recorded during the
immersion tests (PHS-3C pH meter, Lei-ci, Shanghai).
2.5. In vitro loss of mechanical integrity
Magnesium alloys have shown very good properties for orthopedic
applications [13]. However, the loss of mechanical integrity during
degradation may inhibit clinical realization [33]. To evaluate the influ-
ence of in vitro degradation on the mechanical properties rectangular
samples with the dimensions 5  5  30 mm were weighted to an
accuracy of 0.1 mg and then immersed in physiological saline [0.9%
NaCl, Baxter Healthcare (Shanghai) Co. Ltd.]. The samples were taken
out, cleaned with ethanol and distilled water, dried in warm air and
weighed each day to monitor the mass change. The immersion tests
continued until the weight loss percentage [weight loss percent-
age = (Wbefore immersion  Wafter immersion)  Wbefore immersion, where W
means weight] reached about 5%, 10% and 20%, respectively. Finally
three-point bending tests with a support span of 25 mm were carried
out with a MTS 810.22 universal testing machine to observe the influ-
ence of weight loss on mechanical integrity. The morphology of the
fracture surface was examined by SEM (JEOL JSM 6460).
2.6. Cytotoxicity assessments
L-929 cells were cultured in Dulbecco’s modified Eagle’s med-
ium (DMEM) (Gibco), supplemented with 10% fetal bovine serum
(FBS) in a humidified incubator at 95% relative humidity and 5%
CO2 at 37 °C. Cytotoxicity was determined by indirect contact. Ex-
tracts were prepared according to ISO 10993-5: 1999 [34]. The
extraction media were serially diluted to 50 and 10% concentration
after 72 h incubation in a humidified atmosphere with 5% CO2 at
37 °C. The DMEM medium acted as a negative control while DMEM
medium containing 0.64% phenol as a positive control. Cells were
incubated in 96-well flat bottomed cell culture plates at
2.5  104 cells ml1 medium in each well and incubated for 24 h
to allow cell attachment. Then the medium was replaced by
100 ll extraction medium. After incubation in a humidified atmo-
sphere for 2, 4 and 7 days cell morphology was observed by optical
microscopy (Nikon ELWD 0.3 inverted microscope). 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma) was then dissolved in phosphate-buffered saline (PBS) at
a concentration of 5 mg ml1. After that 20 ll MTT was added to
each well and the samples were incubated for 4 h (37 °C, 5% CO2,
95% relative humidity). Subsequently, 150 ml formazan solution
was added to each well and optical density (OD) measurements
were conducted at 490 nm using a Wellscan MK3 spectrophotom-
eter (Labsystem). The cell relative growth rate (RGR) was calcu-
lated according to the following formula:
RGR ¼ ODtest =ODnegative  100% ð2Þ
2.7. In vivo degradation and biocompatibility
2.7.1. Surgery
Animal tests were approved by the Ethnics Committee of the
6th Hospital of Shanghai Jiao Tong University. The in vivo degrada-
tion experiments were performed in the animal laboratory of the
hospital. A total of 12 adult New Zealand rabbits (6 females) with
a body weight of 2.0–2.5 kg were used. The rabbits were ran-
domly divided into two groups. In the experimental group one
Mg–6Zn rod was implanted into the femoral shaft of each rabbit.
In the control group each rabbit had a hole drilled in the femoral
shaft, without any implantation. Before implantation a dose of
30 mg kg1 sodium pentobarbital (Shanghai Xinya pharmaceutics
Ltd.) was administered by intravenous injection. The femoral re-
gion was cleaned with physiological saline and 75% ethanol. All
animals received a subcutaneous injection of penicillin as an
anti-inflammatory.
2.7.2. Biochemical tests
During the experiments 5 ml blood samples were taken from
the helix vessel of the rabbits before operation and at 3, 15, 21
and 28 days after surgery. The blood biochemical tests, including
for serum magnesium, serum creatinine (CREA), blood urea nitro-
gen (BUN), glutamic-pyruvic transaminase (GPT) and creatine
kinase (CK), were performed with a Hitachi 7600-020 automatic
biochemical analyzer.
2.7.3. Radiographic and histological evaluation
Radiographs were used to observe the in vivo degradation
process during the experiments. Six and 18 weeks post-implanta-
tion bone samples around the Mg–6Zn rod were fixed in 4%
formaldehyde and then embedded in methyl methacrylate. Histo-
logical evaluation was performed on hematoxylin and eosin (HE)
stained sections. The HE stained method was also used to ob-
serve the tissues around the hydrogen gas bubble 3 weeks after
implantation.
In addition, tetracycline labeling hard tissue fluorescence was
used to observe the interaction between bone and implants. Mean-
while, heart, kidney, spleen and liver tissue from the rabbits were
also inspected with HE staining to verify whether degradation of
the magnesium alloy harmed these important visceral organs.
2.7.4. In vivo degradation characterization
The rabbits were killed 14 weeks post-implantation. The
implanted rods were retrieved and weighed and the weight loss
corrosion rate calculated according to ASTM-G31-72. The surface
morphology of the implants was also observed using SEM and EDS.
2.8. Statistical analysis
The two samples t-test was used to determine whether any sig-
nificant difference existed in the cytotoxicity and blood biochemi-
cal experiments. The statistical significance was defined as P < 0.05.
3. Results
3.1. Microstructure and mechanical properties
Fig. 1 illustrates the optical microstructure and XRD results for
the as cast, heat-treated and extruded Mg–6Zn alloys. The micro-
structure of extruded pure Mg is also displayed in Fig. 1d.
It can be seen from Fig. 1a that there were two main phases in
the as cast samples, i.e. the matrix a phase and the second phase c-
MgZn, precipitating along the grain boundary [26]. After solid solu-
tion treatment the c phase disappeared (Fig. 1b) and the alloy had
a supersaturated single phase microstructure. With such a single
phase microstructure the material was much easier to process by
hot working. The grain size of the extruded samples (Fig. 1c) was
much finer than that of the as cast and heat-treated ones, indicat-
ing that hot working had refined the microstructure and thus
should improve the mechanical properties. Neither precipitates
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640 629

Fig. 1. Microstructures of (a) as cast, (b) heat-treated, (c) extruded Mg–6Zn alloy and (d) extruded Mg and (e) X-ray diffraction results for the Mg–6Zn alloy.

Table 2
Chemical composition of the Mg–6Zn alloy.

Material Chemical composition (wt.%)

Fe Si Ni Cu Al Mn Zn Mg
Mg–6Zn 0.0038 0.0016 0.0005 0.0005 0.0085 0.0004 5.6210 Balance

nor bulk impurities were observed, suggesting a uniform micro-
structure in the extruded samples (Fig. 1c). In addition, the grain
size of the extruded pure Mg was similar to that of the extruded
Mg–6Zn alloy (Fig. 1d). The XRD results in Fig. 1e also demonstrate
that the c-MgZn peaks can be clearly identified in the as cast sam-
ple, while after heat treatment and extrusion the c-MgZn peaks
disappeared in the XRD results, which is consistent with the optical
microstructures in Fig. 1a–c.
The chemical composition of the Mg–6Zn alloy obtained by ICP-
AES is listed in Table 2. The impurity content of the Mg–6Zn alloy
630 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640

Table 3
Mechanical properties of the extruded Mg–6Zn alloy.

Alloy Modulus (GPa) Yield strength (MPa) Tensile strength (MPa) Elongation (%) Compression strength (MPa)
Mg–Zn 42.3 ± 0.1 169.5 ± 3.6 279.5 ± 2.3 18.8 ± 0.8 433.7 ± 1.4
Mg–Ca [6,17] 239.63 ± 7.21 10.63 ± 0.64 273.2 ± 6.1
Mg–Mn–Zn [38] 44 246 280 20a
a
Deduced from Fig. 4 in Zhang et al. [38].

Fig. 2. Electrochemical measurements (a) polarization curves, (b) Nyquist plots, (c) Bode phase plots and (d) the equivalent circuit for fitting Nyquist plots.

Table 4
In vitro and in vivo degradation rates of Mg and the Mg–6Zn alloy.

Material In vitro (mm year1) In vivo (mm year1)

Electrochemical measurements Immersion tests 14 weeks
3 days 30 days

Mg 0.20 0.43 ± 0.04 0.10 ± 0.07

Mg–6Zn 0.16 0.20 ± 0.05 0.07 ± 0.02 2.32 ± 0.11

was very low, ensuring better degradation properties and
biocompatibility.
Table 3 summarizes the mechanical properties of the ex-
truded Mg–6Zn in comparison with the reported properties of
Mg–Ca and Mg–Mn–Zn alloys [6,17]. It should be noted that
the Mg–6Zn alloy demonstrated higher ultimate strength in
tension and compression and higher elongation than the
extruded Mg–Ca alloy. Although the Mg–6Zn alloy investigated
in this study had a much coarser grain size than that reported
for the Mg–Mn–Ca alloy (>20 lm for the former and <9 lm for
the latter), both alloys showed comparable mechanical
properties.
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640 631

Fig. 3. Surface morphology after immersion of (a) Mg for 3 days, (b) Mg–6Zn for 3 days, (c) Mg for 30 days and (d) Mg–Zn for 30 days and (e) the EDS spectrum of the
rectangular area in (b) and (f) EDS of the rectangular area in (d).

3.2. In vitro degradation tests
3.2.1. Electrochemical measurements
Fig. 2 shows typical polarization curves for the Mg–6Zn alloy
and pure Mg. Current plateaus with different breakdown potentials
were observed in the anodic parts of the curves (Fig. 2a), indicating
the existence of a protective film on the surface. The corrosion
rates obtained from the polarization curves are listed in Table 4.
It can be seen that the rate of electrochemical degradation of the
Mg–6Zn alloy is slower than that of pure Mg. The corrosion poten-
tial (Ecorr) and breakdown potential (Ept) are also shown in Fig. 2a.
The Ecorr value of the Mg–6Zn alloy is greater than that of Mg, indi-
632 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640

cating that the Mg–6Zn alloy is less susceptible to corrosion. The

addition of Zn also increased the Ept value (Fig. 2a, Ept1 > Ept2), sug-
gesting that the corrosion layers on the Mg–6Zn sample were more
protective than those on the pure Mg sample.
Fig. 2b and c presents Nyquist plots and Bode phase plots,
respectively. It can be deduced from the Bode phase plots that an
equivalent circuit with two time constants is reasonable. The
equivalent circuit is shown in Fig. 2d, and the polarization resis-
tance Rp (Rp = Rpo + Rct, the higher the Rp, the lower the corrosion
rate [35]) of Mg and Mg–6Zn are shown in Fig. 2b. The Rp of Mg–
6Zn is higher than that of Mg, which is in consistent with the Ecorr
data and electrochemical rates.

3.2.2. Immersion experiments

The degradation rates of the pure Mg and the Mg–6Zn alloy after 3
and 30 days immersion are listed in Table 4. The Mg–6Zn alloy de-
graded more slowly than pure Mg, which is in good agreement with
the electrochemical results. Fig. 3a–d illustrates the surface mor-
phologies and Fig. 3e–f shows the EDS results for the surface corro- Fig. 5. The pH of SBF during 72 h immersion.
sion products on the Mg–6Zn alloy. As shown in Fig. 3a and b, both
pure Mg and the Mg–6Zn alloy sample experienced pitting corrosion
and were covered with partially protective corrosion products.
A number of cracks were observed on the surface of the samples
after 30 days immersion (Fig. 3c and d). The EDS results (Fig. 3e and
f) reveal that the surface corrosion products (rectangular area in
Fig. 3b and d) were rich in O, Mg, P and Ca. The XRD results suggest
that magnesium hydroxide [Mg(OH)2] and hydroxyapatite (HA)
were precipitated on the Mg–6Zn surface (Fig. 4). Furthermore,
Kuwahara et al. [36] pointed out that the corrosion products on
the surface of Mg immersed in Hank’s solution might be amor-
phous (Ca0.86Mg0.14)10(PO4)6(OH)2, a rather complicated com-
pound. In view of the similar ion concentrations in the SBF used
in this study to those in Hank’s solution, there might be some
amorphous phosphates containing magnesium/calcium, as Kuwa-
hara indicated. In fact, a strong background and broadened peaks
can be observed in Fig. 4, which might be due to the presence of
amorphous corrosion products.
Fig. 5 illustrates the pH variation over the first 72 h of the
immersion tests. It can be seen that the pH rose rapidly, i.e. from
7.44 to 8 in 2 h. After 20 h the pH had stabilized. At the end of Fig. 6. The deterioration in bending strength during in vitro degradation.
the immersion tests (after 30 days) the pH was 9.22 for pure Mg
and 9.32 for the Mg–6Zn alloy.
Hydrogen evolution showed a similar trend to the pH value. In
the early stage of immersion both pure Mg and the Mg–6Zn alloy

Fig. 7. The corrosion surface and corrosion holes formed on the samples with 12%
weight loss.

reacted with SBF acutely and a rapid generation of bubbles was ob-
served, indicating a fast rate of hydrogen evolution. After 48 h
Fig. 4. X-ray diffraction pattern of Mg–6Zn immersed in SBF for 30 days. immersion, however, fewer bubbles appeared, suggesting that
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
Fig. 8. Morphology of the fracture section after a three-point bending test: (a) extruded Mg–6Zn alloy; (b) after 5.8% weight loss; (c) after 11.9% weight loss; and (d) after 18%
weight loss.
the reaction had slowed down and that the rate of hydrogen evo-
lution had decreased.
It should be noted that the degradation rate measured after
30 days immersion was lower than that measured after 3 days
(Table 4), because the corrosion films, including HA and other phos-
phates, had a protective effect and hence retarded further
degradation.
3.3. In vitro loss of mechanical integrity
Fig. 6 demonstrates the influence of in vitro degradation on the
bending strength of the Mg–6Zn alloy. As shown in Fig. 6, the
bending strength decreased rapidly in the early stage of degrada-
tion. After that the bending strength deteriorated continuously as
the percentage weight loss increased. This may be due to surface
defects such as corrosion holes formed during degradation, as
shown in Fig. 7.
Some dimples were observed on the fracture surface (Fig. 8),
which indicates ductile fracture characteristics of the Mg–6Zn
alloy during the degradation process. The bending tests showed
that degradation undermined the bending strength, although the
fracture mode was still ductile during degradation.
3.4. Cytotoxicity assessments
Fig. 9 shows the morphologies of L-929 cells cultured in differ-
ent extracts after 7 days incubation. Fig. 10 shows the RGR of L-929
cells after 2, 4 and 7 days incubation. It can be seen from Fig. 9 that
the cell morphologies in different extracts were normal and
633
healthy, similar to that of the negative control. There was no signif-
icant difference between the RGR of cells in the extracts and those
in the negative control. According to ISO 10993-5: 1999 [34] the
cytotoxicity of these extracts was Grade 0–1. In other words, the
Mg–6Zn alloy has a level of biosafety suitable for in cellular
applications.
3.5. In vivo degradation and biocompatibility
3.5.1. Biochemical tests
After implantation of the Mg–6Zn rods no rabbits displayed
inflammation and there were no unexpected deaths. The variations
in serum magnesium, CREA, BUN, GPT and CK are shown in Fig. 11.
There were no significant differences (P > 0.05) in these biochemi-
cal indicators before and after operation, which indicated that deg-
radation of the Mg–6Zn implants did not raise serum Mg2+ levels
and did not affect kidney and liver function either. The tests indi-
cated good biocompatibility of the Mg–6Zn alloy in vivo.
3.5.2. Viscera histology
Fig. 12 shows HE stained slices of heart, liver, kidney and spleen.
It can be seen that the tissues were normal, which is in good agree-
ment of the biomechanical tests (Fig. 11) and suggests good bio-
compatibility of the Mg–6Zn alloy in vivo.
3.5.3. Radiographic evaluation
Fig. 13a and b shows the radiographs 3 and 12 weeks post-oper-
ation. The Mg–6Zn implant started to degrade in the first 3 weeks,
as is evident from the observation that the edge of the implant be-
634 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
Fig. 9. L-929 cell morphology after 7 days incubation: (a) negative; (b) 10% extraction; (c) 50% extraction; and (d) 100% extraction.
Fig. 10. RGR of L-929 cells after 2, 4 and 7 days incubation. *P > 0.05.
came fuzzy and subcutaneous bubbles appeared (Fig. 13a). No ad-
verse effects due to these gas bubbles were observed in the rabbits
and they disappeared after 6 weeks without any intervention,
which is in agreement with the literature [4,6]. After 12 weeks
the implant was too blurry to be recognized (Fig. 13b). The radio-
graphs offer evidence that the implant gradually degraded within
the bone. In fact, when the rabbits were killed 14 weeks after
implantation an obvious irregular shaped hole could be observed
in the femora and most of the implant had been absorbed
(Fig. 13c).
3.5.4. Bone tissue histology
New bone formation as well as a gap between the residual im-
plant and the surrounding bone tissues can be observed in Fig. 14,
which shows the tetracycline labeling fluorescence results at
14 weeks. In comparison, the femora of rabbits in the control group
had already healed after 3 weeks and the hole had disappeared.
Fig. 15 demonstrates the bone tissue response to the Mg–6Zn
implants 6 and 18 weeks post-implantation. There are newly
formed trabecular and osteoblasts (black arrow in Fig. 15a) at
6 weeks and at 18 weeks more new bone can be found (black ar-
row in Fig. 15b), indicating that the hole in the femur gradually
healed during degradation of the Mg–6Zn alloy.
The tissues around the gas bubble 3 weeks after implantation
are illustrated in Fig. 16, composed of two layers, an inner compact
one and outer loose connective tissue. No obvious inflammatory
response can be observed in Fig. 16. It is hypothesized that the
hydrogen gas diffuses into the surrounding tissues [8] and thus
the bubble had disappeared at 6 weeks.
3.5.5. In vivo degradation characterization
After the rabbits were killed 14 weeks post-implantation the
residual Mg–6Zn rod material was weighed. It was found that
the residual weight was only 13% of the original weight, i.e. 87%
of the implant had been degraded. The in vivo degradation rate ob-
served in this study was faster than in previous reports. For exam-
ple, a Mg–Mn–Zn alloy showed 54% degradation 18 weeks post-
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640 635

Fig. 11. Blood biochemical indicators: (a) serum magnesium; (b) CREA; (c) BUN; (d) GPT; and (e) CK. The blue dashed lines in (a–e) represent the level before surgery.

operation [16]. The in vivo corrosion rate according to Eq. (1) was
2.32 mm year1 (also listed in Table 4).
The surface morphology of the residual rod material 14 weeks
after implantation is shown in Fig. 17a, showing a rough surface
covered with a thick layer of corrosion products. The corrosion
products contained large amounts of O, Mg, P, Ca, Na and Zn
(Fig. 17b). It can be seen that the in vivo degradation morphology
is different from the in vitro morphology and the compositions are
also slightly different, suggesting dissimilar degradation behaviors
of this magnesium alloy in vitro and in vivo.
4. Discussion
4.1. Mechanical properties
Zinc is a common alloying element for magnesium and has a
solution hardening effect on magnesium alloys [25]. Although
the maximum solubility drops to 1.6 wt.% (i.e. 0.6 at.%) at
25 °C in the equilibrium state [26], a solid solution treatment
can be carried out to obtain a supersaturated solution to avoid
precipitation of the c-MgZn phase. In this way a uniform micro-
636 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
Fig. 12. HE stained slices of (a) heart, (b) liver, (c) kidney and (d) spleen. Scale bar 100 lm.
structure can be obtained (Fig. 1) and the corrosion process can
be slowed, due to the homogeneity. Moreover, the mechanical
properties can also be enhanced by solution hardening. For
example, Table 3 suggests that zinc can effectively improve the
mechanical properties.
Hot working (hot extrusion in this paper) can also refine the
grain size and hence increase the yield strength and tensile
strength according to the Hall–Petch relationship [11]. Compared
with degradable polymeric materials such as l-HA/PLLA 50/50
(tensile strength 103 MPa, Young’s modulus 12.3 GPa) [37], the
Mg–6Zn alloy has improved mechanical properties. The elastic
modulus of the magnesium alloy in this paper (42.3 GPa) is also
closer to that of human femur bone (15–20 GPa [38]) than that
of polymers and titanium alloys.
However, the mechanical properties will deteriorate gradu-
ally during the degradation process, as shown in Fig. 6. In this re-
gard, the degradation process must be precisely controlled so
that the mechanical integrity meets requirements before the bone
has healed. Nevertheless, long-term in vivo deterioration of
mechanical integrity needs to be investigated in detail.
4.2. The degradation process
Magnesium in aqueous solution (for instance SBF) dissolves
according to the following equations [1]:
anodic reaction : Mg ! Mg2þ þ 2e
cathodic reaction : 2H2 O þ 2e ! H2 þ 2OH 
Mg2þ þ 2OH ! MgðOHÞ2
The existence of chloride ions (Cl) transforms Mg(OH)2 into
soluble MgCl2 [6], resulting in excess OH ions in the solution.
Eventually the pH will rise (shown in Fig. 5). In fact, even though
the bulk solution has a pH value as low as 4, the local pH near
the surface of the Mg could be >10 [39]. As a result, if the solution
contains ions such as PO43, Ca2+, etc. HA [Ca10(PO4)6(OH)2] is
likely to nucleate and grow on the magnesium surface due to the
supersaturated condition at high pH [40]. This phenomenon ex-
plains the detection of HA by XRD in this study (Fig. 4).
Moreover, when Mg2+ ions dissolve into the solution, phos-
phates containing Mg/Ca form and tightly attach to the matrix. In
general, the reaction among HnPO4(3–n)– (representing various
phosphate ions, where n = 0, 1 or 2), Ca2+ and Mg2+ could be de-
scribed primarily as:
þ Ca2þ þ Mg2þ þ OH ! Mgx Cay ðPO4 Þz
ð3nÞ
Hn PO4
ðinsoluble productsÞ ð6Þ
Taking the excess OH into consideration, some complicated
compounds [represented by MgxCay(PO4)z(OH)] might precipitate
on the surface (Fig. 3). Previous studies [4,16] have shown that
the corrosion layer containing such magnesium-substituted cal-
cium phosphate compounds on Mg can promote osteoinductivity
and osteoconductivity, predicting good biocompatibility of
magnesium.
It is difficult to determine the in vivo degradation rate precisely
ð3Þ because the circumstances in vivo are quite complicated. The Mg–
ð4Þ 6Zn implant had been degraded by 87% 14 weeks post-operation
and the corrosion rate was 2.32 mm year1 according to Eq. (1).
ð5Þ Compared with previous investigations (Table 5), it seems that
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640 637

Fig. 13. Radiographs (a) 3 weeks post-operation, (b) 12 weeks post-operation and (c) macroscopic photograph 14 weeks post-operation, with an irregular shaped hole in the
femur (arrow).

the Mg–6Zn in the present study degraded slightly faster. Several

Fig. 14. Tetracycline labeling 14 weeks post-operation.
methods can be used to regulate the degradation rate, for instance
calcium phosphate coating and anodic oxidation.
Zinc is able to elevate the corrosion potential of magnesium alloys
and improve the corrosion resistance [41], which is in agreement
with this study (Table 4). In addition, zinc has beneficial effects on
the corrosion film and can reduce the effect of impurities once the
tolerance limits have been exceeded [42]. Furthermore, it has been
found that zinc can also elevate the charge transfer resistance of
magnesium and thus reduce the corrosion rate [43]. The degradation
rates of the Mg–6Zn alloy (0.20 ± 0.05 for 3 days and 0.07 ± 0.02 for
30 days) are slower than those of pure Mg (0.43 ± 0.04 for 3 days and
0.10 ± 0.07 for 30 days).
For both the Mg–6Zn alloy and pure Mg the degradation
rates for 30 days were lower than those for 3 days, owing to
the protective layer on the surface (shown in Fig. 3a–d).
During the early stage of immersion in SBF Mg and the Mg–
6Zn alloy degraded quickly, accompanied by the rapid formation
638 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640

Fig. 15. HE stained bone surrounding Mg–Zn rods: 6 weeks (a) and 18 weeks (b) post-implantation. Scale bar 500 lm.

Fig. 16. HE stained tissues around the gas bubble 3 weeks after implantation.

of an insoluble protective corrosion layer, which retarded

degradation.
It should be noted that the compositions and the morphologies
of the in vitro and in vivo degradation products are different. This
is mainly due to the different corrosive environments. The in vitro
testing solution was SBF, containing inorganic ions such as Cl,
H2PO4 and Ca2+. Thus, the corrosion products were mineral-like
and the corrosion layer could be clearly observed, which included
HA, Mg(OH)2 and other amorphous magnesium-substituted cal-
cium phosphates. However, there were numbers of organic compo-
nents in the in vivo environment, such as proteins and cells. Rettig
et al. found that albumin influenced the corrosion process of mag-
nesium alloys in SBF [44]. As a consequence, the in vivo degrada-
tion products are quite complex. The composition of the in vivo
degradation products in Fig. 17b shows a high nitrogen level in
the corrosion layer, indicating the possible adsorption and/or adhe-
sion of proteins, cells or other tissue fragments to the magnesium
Fig. 17. Surface morphology and composition of the corrosion products of the
alloy.
implant after 14 weeks degradation: (a) SEM; and (b) EDS of framed area in (a).
The different morphologies and compositions in vitro and
in vivo suggest that degradation of the magnesium alloy will be
greatly influenced by the surrounding environments. Thus, if the
magnesium alloy is used in different parts of the body (e.g. in fe- behaviors must be taken into consideration, otherwise the rate of
mur marrow or within muscle) possible different degradation degradation may be estimate incorrectly.
 S. Zhang et al. / Acta Biomaterialia 6 (2010) 626–640
Table 5
Comparison of in vivo degradation rates.
Percent Degradation rate
degradation (mm year1)
Mg–6Zn 87% after 14 weeks 2.32
Mg–Ca 1.27a
AZ91D 3.516  104
LAE442 1.205  104
Mg–Mn–Zn 54% after 18 weeks
a
The Mg–Ca degradation rate was deduced from
(2.28 mg mm2 year1).
4.3. Biocompatibility
The in vitro cytotoxicity of Mg–6Zn was found to be Grade 0–1,
indicating that the Mg–6Zn alloy is safe as an implantable material.
There was a gap between the bone and the residual implant
14 weeks post-operation (Fig. 14). This may be due to the rapid
degradation and rapidly elevated ambient pH. As a consequence,
osteoblasts were unable to proliferate and adhere very well. Never-
theless, there was still some newly formed bone surrounding the
implant despite the fast degradation, and the amount of new bone
increased from 6 to 18 weeks post-operation (Fig. 15). Zreiqat et al.
[45] found that magnesium ions could enhance the adhesion of hu-
man bone-derived cells (HBDC) and increased the levels of a5b1-
and b1-integrin receptors. It has been reported that zinc can also
promote bone formation [46]. Therefore, it is postulated that the
Mg–6Zn alloy has good biocompatibility in bone.
In addition, the corrosion layer on the Mg–6Zn alloy contained
Mg, Ca and P, which could reinforce osteoblast activity. Hence, we
come to the conclusion that rapid degradation of the Mg–6Zn alloy
was not harmful to adjacent bone tissues. A previous study [9] also
confirmed that fast degrading AZ91D scaffolds induced extended
peri-implant bone remodeling with good biocompatibility.
The excess magnesium produced by degradation can be ex-
creted by the kidneys, as indicated in Fig. 11a, with no kidney, liver
or heart disorders being observed during degradation (Figs. 11 and
12), and no adverse effects of bubbles were observed. These results
also imply good in vivo biocompatibility of the Mg–6Zn alloy.
Nevertheless, the mechanism of hydrogen absorption and
whether the hydrogen can be metabolized or will accumulate in cer-
tain organs are still unknown and need further detailed investiga-
tion. In addition, it can be seen from Fig. 16 that there are dual
connective tissues around the bubble, which may affect the adhesion
of cells and be one reason for the gap between the residual implant
and the surrounding bone, as shown in Fig. 14. If the degradation rate
is slow enough it is possible to avoid bubble formation, as the hydro-
gen can diffuse into the surrounding tissues [8].
Zinc is an essential element for humans, is absolutely necessary
and is non-toxic except at extreme exposures. The human require-
ment for zinc is estimated to be 15 mg day1 [23]. The degradation
rate of Mg–6Zn within rabbit femora in this study was
2.32 mm year1, i.e. 1.14 mg cm2 day1. The zinc content of
the Mg–6Zn alloy in this study was 5.621 wt.% (Table 2), so the zinc
release rate will be 0.0641 mg cm2 day1. For an implant with
the dimensions 4.5  10 mm (cylindrical, with a surface area of
1.732 cm2), the zinc release rate would be about 0.11 mg day1,
which is much lower than the suggested intake of 15 mg day1
for adults. Besides, the released zinc could be absorbed by the sur-
rounding tissues [16] and excreted through the gastrointestinal
route and the kidney [23]. Therefore, it is proposed that the zinc re-
lease during degradation is safe.
In summary, even though the Mg–6Zn alloy degraded rapidly
in vivo, there was still newly formed bone surrounding the im-
plants and no disorders were found in heart, liver, kidney and
spleen, suggesting good biocompatibility. Furthermore, in order
639
to adjust the degradation rate, surface modification technologies
(e.g. electrodeposition of HA coating, spray coating with PLGA,
Reference etc.) are presently being applied to the Mg–6Zn alloy by our group.
Present study
[6] 5. Conclusions
[5]
[5]
In this paper a binary Mg–6 wt.% Zn alloy was investigated as a
[16]
biomedical degradable magnesium. The following conclusions can
Li et al. [6] be drawn.
The Mg–6Zn alloy fabricated with high-purity raw materials
and using a clean melting process had very low contents of impu-
rities. After solid solution treatment and hot working the grain size
of Mg–6Zn alloy was refined and a uniform single phase was
obtained.
The mechanical properties of the Mg–6Zn alloy were suitable
for implant applications, i.e. the tensile strength and elongation
achieved were 279.5 MPa and 18.8%, respectively. Degradation
of the Mg–6Zn alloy resulted in a deterioration in the mechanical
properties and mechanical integrity was rapidly lost in the early
stages of corrosion, but more slowly in the later stages of in vitro
degradation.
Zinc elevated the corrosion potential of the magnesium alloy
and the in vitro degradation rate of the Mg–6Zn alloy was slower
than that of high-purity magnesium in SBF. A protective layer of
HA and other Mg/Ca phosphates formed on the surface of Mg–
6Zn when immersed in SBF.
In vitro cytotoxicity to L-929 cells implies that the Mg–6Zn alloy
is safe for cellular applications, with a cytotoxicity grade of 0–1.
Animal implant experiments indicated that the Mg–6Zn alloy
gradually degraded within the femoral shaft, with a degradation
rate of 2.32 mm year1. Subcutaneous hydrogen gas bubbles pro-
duced by degradation disappeared 6 weeks after implantation
without discernable adverse effects. It was found that the bubble
was enveloped by two layers of connective tissue. Owing to the rel-
atively rapid degradation, a gap between the implant and sur-
rounding bone tissues was observed, however, there was still
newly formed trabeculae and osteoblasts.
According to the biochemical indicators and the HE stained sec-
tions there were no disorders of the heart, kidney, liver and spleen
and no negative effects of zinc release were observed, indicating
that the binary Mg–6Zn alloy had good biocompatibility in vivo.
Acknowledgements
The authors are grateful for the supports from the National Nat-
ural Science Foundation of China (No. 30772182), Shanghai Jiao
Tong University Interdisciplinary Research Grants (Grant No.
YG2007MS26 and YG2009MS53) and the 863 High-tech Plan of
China (No.2009AA03Z424).
Appendix A. Figures with essential colour discrimination
Certain figures in this article, particularly Figs. 1–7 and 11–17, are
difficult to interpret in black and white. The full colour images can be
found in the on-line version, at doi:10.1016/j.actbio.2009.06.028.
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