Acta Biomaterialia 64 (2017) 421–436

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Acta Biomaterialia
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Full length article

Development of magnesium-based biodegradable metals with dietary

trace element germanium as orthopaedic implant applications
Dong Bian a, Weirui Zhou a, Jiuxu Deng b, Yang Liu a, Wenting Li a, Xiao Chu c, Peng Xiu d, Hong Cai d,
Yuhui Kou b, Baoguo Jiang b,⇑⇑, Yufeng Zheng a,⇑
a
Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China
b
Department of Trauma and Orthopedics, People’s Hospital Peking University, Beijing 100044, China
c
Department of Orthopedics, Guangdong Key Lab of Orthopaedic Technology and Implant Materials, Guangzhou General Hospital of Guangzhou Military Command,
Guangzhou 510010, China
d
Department of Orthopedics, Peking University Third Hospital, Beijing 100191, China

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

Article history: From the perspective of element biosafety and dietetics, the ideal alloying elements for magnesium
Received 19 June 2017 should be those which are essential to or naturally presented in human body. Element germanium is a
Received in revised form 13 September unique metalloid in the carbon group, chemically similar to its group neighbors, Si and Sn. It is a dietary
2017
trace element that naturally presents in human body. Physiological role of Ge is still unanswered, but it
Accepted 3 October 2017
Available online 4 October 2017
might be necessary to ensure normal functioning of the body. In present study, novel magnesium alloys
with dietary trace element Ge were developed. Feasibility of those alloys to be used as orthopaedic
implant applications was systematically evaluated. Mg-Ge alloys consisted of a-Mg matrix and eutectic
Keywords:
Mg-Ge alloy
phases (a-Mg + Mg2Ge). Mechanical properties of Mg-Ge alloys were comparable to current Mg-Ca,
Biodegradable metals Mg-Zn and Mg-Sr biodegradable metals. As-rolled Mg-3Ge alloy exhibited outstanding corrosion resis-
Degradation tance in vitro (0.02 mm/y, electrochemical) with decent corrosion rate in vivo (0.6 mm/y, in rabbit tibia).
In vivo New bone could directly lay down onto the implant and grew along its surface. After 3 months, bone and
Osseointegration implant were closely integrated, indicating well osseointegration being obtained. Generally, this is a
pioneering study on the in vitro and in vivo performances of novel Mg-Ge based biodegradable metals,
and will benefit the future development of this alloy system.

Statement of Significance

The ideal alloying elements for magnesium-based biodegradable metals should be those which are essen-
tial to or naturally presented in human body. Element germanium is a unique metalloid in the carbon
group. It is a dietary trace element that naturally presents in human body. In present study, feasibility
of Mg-Ge alloys to be utilized as orthopedic applications was systematically investigated, mainly focusing
on the microstructure, mechanical property, corrosion behavior and biocompatibility. Our findings
showed that Mg-3Ge alloy exhibited superior corrosion resistance to current Mg-Ca, Mg-Zn and Mg-Sr
alloys with favorable biocompatibility. This is a pioneering study on the in vitro & in vivo performances
of Mg-Ge biodegradable metals, and will benefit the future development of this alloy system.
Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction attracted great attention since the early 2000 s [1–4]. The ideal
Mg-based biodegradable metals are expected to corrode gradu-
Crystalline magnesium (Mg) and magnesium-based alloys ally in vivo, with an appropriate host response, then dissolve
specifically designed for biodegradable implant applications have completely upon fulfilling the mission to assist with the tissue
healing [5]. During degradation, elements in the implant matrix
will release in the form of metal ions and then they participate
⇑ Corresponding author.
in tissue healing positively, neutrally or negatively, depending on
⇑⇑ Co-corresponding author.
the dose and releasing rate. Therefore, besides magnesium, the
E-mail addresses: jiangbaoguo@vip.sina.com (B. Jiang), yfzheng@pku.edu.cn
(Y. Zheng). major components of Mg-based biodegradable metals should

https://doi.org/10.1016/j.actbio.2017.10.004
1742-7061/Ó 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
422 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
be preferably essential elements or trace elements in human
body.
Many kinds of essential/possibly essential elements found in
human body could be used as alloying elements to improve
in vitro and in vivo performances of Mg-based biomaterials, includ-
ing corrosion control, mechanical improvement, biological regula-
tion, etc. Till now, many kinds of biomedical Mg-based alloys with
essential/possibly essential elements addition have been devel-
oped, including binary Mg-Ca [6–8], Mg-Zn [9,10], Mg-Mn [11],
Mg-Fe [12], Mg-Cu [13,14], Mg-Sr [15–18], Mg-Si [19], Mg-Sn
[20–23], Mg-Li [24] and multicomponent Mg alloys based on these
binary alloys, Mg-Zn-Ca [25] and Mg-Si-Ca-Zn [26], etc.
Germanium (Ge) is a lustrous, hard, grayish-white metalloid in
the carbon group, chemically similar to its group neighbors, Si and
Sn. It is usually used as a semiconductor in transistors and various
other electronic devices. Ge presents in human body and it is a
dietary trace element which might be necessary to ensure normal
functioning of the body [27]. For the general population, the major
source of Ge is from food. Ge presents in practically all food and
only in minute amounts [28]. The estimated average dietary intake
of Ge in humans is in the range of 0.4–3.4 mg per day. Ge normal-
izes many physiological functions such as lowering of high blood
pressure in humans, restoring deviant blood characteristics to their
normal range including pH, glucose, the minerals sodium, potas-
sium, etc. [29]. Many pharmacological effects of some organic ger-
manium compounds have been investigated. They include
antitumor, anticancer, anti-inflammation, antioxidation, radiopro-
tection and pro-immune functions [27]. Organic germanium
appears to be remarkably safe while relatively high doses of ger-
manium dioxide and other inorganic germanium compounds can
cause severe poisoning including fatal cases [28,30]. Animal stud-
ies have already showed promising results, despite sufficient clin-
ical trials are still needed [28].
From the viewpoint of material science, limited information
about Mg-Ge alloys are related to thermodynamic characters
[31,32], and the effects of Ge on the microstructure and mechan-
ical property [33,34]. To the authors’ knowledge, no report on
the corrosion behavior, in vitro or in vivo biocompatibility of
Mg-Ge alloys can be found. Recently, Liu et al. [33] reported
their experimental results on the binary Mg-Ge alloys. They
found that microstructures of Mg-Ge binary alloys consisted of
two components: a-Mg dendrites and a-Mg + Mg2Ge eutectic
phase. Grain size of a-Mg dendrites was effectively refined with
Ge addition and tensile strengths of Mg-Ge binary alloys were
enhanced while elongations were reduced. Benefits of Ge on
thermal stability and mechanical property of Mg have also been
verified in another work [34]. Even though no referable literature
of Ge on Mg corrosion can be found, effects of this intrinsic met-
alloid/semimetallic element on Mg degradation is really interest-
ing. The continuously net-like second phase (a-Mg + Mg2Ge) in
Mg-Ge alloys, as reported in previous work [33,34], might act
as a barrier during corrosion [35].
In the present study, binary Mg-xGe (x = 1.5, 2.5 and 3 wt%)
alloys were designed and fabricated through traditional casting.
The Ge content range (1.5–3 wt%) was determined by referring to
Refs. [33] and [34], as the strength was not further improved when
Ge content exceeded 3.4 wt% while significantly impaired plastic-
ity occurred. For biosafety purpose, Ge content should also be lim-
ited in a proper low range. To further improve mechanical
property, these binary alloys were hot rolled into thin plates. Their
feasibility to be utilized as orthopedic applications was systemati-
cally investigated, mainly focusing on the microstructure, mechan-
ical property, corrosion behavior, in vitro cytocompatibility and
in vivo biocompatibility.
2. Experimental details
2.1. Material preparation
Mg-Ge alloys were melted and casted by using commercial
magnesium (99.95 wt%) and commercial pure Ge powder
(99.9 wt%) under the protection of a mixed gas atmosphere of
SF6 (1 vol%) and CO2 (99 vol%). Briefly, Mg and Ge powders were
well blended in a mixer, then melted at 700–800 °C for 30 min
before casting. The ingots were machined into 5 mm thick plates
and homogenized at 400 °C for 3 h before rolling. Afterwards, they
were hot rolled into thin plates with a final thickness of 2.5 mm at
a rolling reduction of 0.2 mm each pass. The plate was annealed at
400 °C for 10 min at each rolling interval. The actual Ge contents in
the Mg-xGe alloys were 1.38 ± 0.30, 2.53 ± 0.21 and 3.26 ± 0.20 wt
%, respectively. Specimens for microstructural characterization,
corrosion test and in vitro biocompatibility evaluation were cut
into 10  10  2 mm3 disks. Cylindrical pins with a diameter of
2.2 mm and length of 10 mm were machined parallel to the rolling
direction for in vivo test. Samples were mechanically grounded
with SiC sandpaper up to 2000 grit, ultrasonically cleaned in ace-
tone and absolute ethanol, then dried in hot air. For cell assays,
samples were sterilized under ultraviolet radiation for 2 h before
use. Pins for animal test were separately encapsulated and steril-
ized under Co60 c ray radiation at 25 KGy.
2.2. Microstructural characterization
Specimens for microstructural observation were polished into
mirror-like surface with 5 lm diamond polishing paste. After-
wards, they were etched in 4% HNO3/alcohol solution and observed
under an optical microscope (Olympus BX51M, Japan). Average
grain size and phase proportion were calculated in ImageJ software
(ImageJ 1.43 u, USA) by analyzing microstructural images (n = 5).
An X-ray diffractometer (XRD, Rigaku DMAX 2400, Japan) was
employed to identify the constituent phases by using Cu Ka radia-
tion at a scan rate of 4°/min operated at 40 kV and 100 mA.
2.3. Tensile test
Tensile specimens were machined parallel to the rolling direc-
tion according to ASTM-E8-04 [36]. Tensile tests were performed
at a crosshead speed of 1 mm/min on a universal material testing
machine (Instron 5969, USA) at room temperature. An average of
at least three parallel samples was taken for each group.
2.4. Electrochemical test
A traditional three-electrode cell system was used for electro-
chemical test with a platinum foil as counter electrode (CE), a sat-
urated calomel electrode (SCE) as reference electrode.
Experimental samples with an exposed area of 1 cm2 acted as
working electrode. Measurements were carried out in Hank’s solu-
tion [37], on an electrochemical workstation (CHI660C, China).
Open circuit potential (OCP) was continuously monitored for
3600 s and then electrochemical impedance spectroscopy (EIS)
was measured from 100 kHz to 10 mHz using a 5 mV amplitude
perturbation. Potentiodynamic polarization tests were performed
at a scanning rate of 1 mV/s and the initial potential was 300 mV
below the corrosion potential (Ecorr). Electrochemical parameters
and corrosion rates were calculated according to ASTM-G102-89
[38]. Three duplicate samples were taken for each material for sta-
tistical analysis.
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
2.5. Immersion test
Immersion tests were carried out in Hank’s solution at
37 ± 1 °C according to ASTM-G31-72 [39], with an exposure ratio
of 20 mL/cm2. The volume of emerged hydrogen was recorded by
using a calibrated burette in accordance with Ref. [35]. The corre-
sponding corrosion rate was calculated based on the following
equation [40]: pH = 2.279VH (PH: corrosion rate, mm/y; VH: hydro-
gen evolution rate, mL/cm2/d). After immersion for 3 and 20 days,
samples were removed out of the solution, gently rinsed with
distilled water, and dried in open air. Surface morphology as well
as cross section morphology of the corroded samples was observed
under an environmental scanning electron microscope (ESEM, FEI
Quanta-200F, the Netherlands), operating in backscattering
electron mode. Surface chemistry of the corroded samples was
determined by using an imaging X-ray photoelectron spectrometer
(XPS, Axis Ultra, Kratos Analytical Ltd.) with Al Ka radiation. High
resolution narrow scanning was performed to determine the
binding states of Mg 2p, O 1s, Ca 2p, P 2p, C 1s, and Na 1s. Fourier
transform infrared spectroscopy (FTIR, Nicolet iS 50, Thermo
Scientific) was utilized to identify the functional groups in the
corrosion products. The spectrum was recorded from 4000 to
500 cm 1. Constituent phases in the corrosion product layer were
determined by using an X-ray diffractometer (XRD, X’Pert Pro
MPD). The test was performed by using a Mono-Capillary PreFix
Module operated at 45 kV and 40 mA.
2.6. Cytotoxicity
Cytotoxicity of the experimental materials was evaluated by
indirect cell assay, in which human osteoblast-like MG63 cell line
was used. Cells were cultured in minimum essential medium
(MEM) supplied with 10% fetal bovine serum (FBS), 100 U/mL peni-
cillin and 100 lg/mg streptomycin at 37 °C in a humidified atmo-
sphere with 5% CO2. Alloy extracts were prepared by using a
serum-free MEM with an extraction ratio of 1 cm2/mL under stan-
dard cell culture condition. After incubation for 72 h, the super-
natant fluid was withdrawn, centrifuged and stored at 4 °C
before use. Ion concentrations and pH values of the extracts were
measured by inductively coupled plasma atomic emission spec-
troscopy (ICP-AES, Leeman) and a pH meter (PB-10, sartorius),
respectively.
Methylthiazol tetrazolium (MTT) assay was adopted to evaluate
the cytotoxicity of the alloy extracts according to ISO 10993-
5:2009(E) [41]. In brief, cells were seeded onto 96 well culture
plate at a density of 3  103 cells per 100 lL. After incubation for
24 h to allow attachment, the medium was replaced by alloy
extracts with normal MEM as control. After 1, 3 and 5 days’ incuba-
tion, 10 lL MTT was added to each well and another 4 h was kept.
Afterwards, 100 lL formazan solubilizing solution (10% SDS in
0.01 M HCl) was added into each well and left overnight in the
incubator. Then, spectrophotometric absorbance of each well was
measured by using a microplate reader (Bio-Rad 680) at 570 nm
with a reference wavelength of 630 nm.
2.7. Alkaline phosphate activity measurement
Alkaline phosphate activity (ALP) of MG63 cells was determined
by hydrolysis of p-nitrophenyl phosphate (p-Npp) using a test kit
(Nanjing Jiancheng Bioengineering Institute, China). Briefly, after
5-day cultivation with extracts, cells were rinsed with a phosphate
buffer solution (PBS, pH 7.4) for three times and then lysed in 0.1%
Triton X-100 by freezing and thawing for 60 min at 37 °C. 30 lL of
the resulting cell lysates was then transferred to each well of a new
96 well plate, cultured with 50 lL carbonated buffer solution (pH
= 10) and 50 lL substrate solution (4-amino-antipyrine) for 15 min
423
at 37 °C. Afterwards, 150 lL of chromogenic agent (potassium fer-
ricyanide) was added into the mixed solution. The spectrophoto-
metric absorbance was measured at 520 nm by a microplate
reader (Bio-RAD680). For normalization, total protein content
was determined using a Bicinchoninic Acid (BCA) protein assay
kit (Beijing Biosea Biotechnology, China). The ALP activity was
expressed and normalized by the total protein content (U/gprot).
2.8. Animal test
2.8.1. Animal model and surgery
Based on the in vitro performances of Mg-xGe alloys, as-rolled
Mg-3Ge with the best mechanical properties and superior corro-
sion resistance was chosen to perform in vivo test. Twelve female
New Zealand rabbits (2–2.5 kg) were provided by Laboratory Ani-
mal Center of Peking University People’s Hospital (animal use per-
mit No.: SYXK (jing) 2011-0010). Animals were kept in separated
cages and health condition was verified by preoperative observa-
tion 1 week before operation. Three animals were set as normal
control and they were sacrificed at 3 months. The remaining nine
animals all received implantation. Briefly, animals were anaes-
thetized with 30 mg/kg pentobarbital sodium by ear vein injection.
Operation sites beneath the bilateral knee-joint were shaved and
sterilized followed by decortication. A pre-drilled hole, 2.2 mm in
diameter, was made on lower edge of the lateral tibial plateau by
using an orthopaedic drill. An as-rolled Mg-3Ge pin was inserted
into the predrilled hole. Then, 4  104 U penicillin was dropped
in the incision site before suturing. 40  104 U penicillin was
intramuscular injected in case of postoperative infection and this
injection lasted for 3 days. The anesthetic, surgical and
post-operative care protocols were examined by and fulfilled the
requirements of the Ethics Committee of Peking University
People’s Hospital.
2.8.2. Postoperative observation, micro-CT, and histological evaluation
All animals were clinically examined for general condition and
in particular for significant signs of lameness, infection, subcuta-
neous emphysema formation and loss of appetite. Three animals
were sacrificed by over-dosage anesthesia at each time point
post-operation (1, 2 and 3 months), respectively. Rabbit tibias were
retrieved and the implantation section (1.5 cm) was truncated for
micro-CT analysis and histological analysis. Bones were scanned in
a self-built micro-CT device at a spatial resolution of 35 lm. Three-
dimensional (3D) reconstruction was performed in Amira software
(Amira 5.4.1, Visage Imaging). Retrieved bones were fixed in 10%
formalin and dehydrated in gradient ethanol/distilled water mix-
tures. Then, they were embedded in methacrylate and sectioned
into 150 lm slices on a EXAKT system (EXAKT Apparatebau,
Norderstedt, Germany). The hard tissue sections were further
grounded to 50–80 lm before staining with hematoxylin-eosin
(H&E) or toluidine blue (Guge Biological Technology Co., Ltd.).
Details about the hard tissue slicing process could be found in
our previous work [42]. Animal liver and kidney were also
retrieved for pathological examination by conventional H&E
staining.
2.9. Statistical analysis
Data in this work were expressed as means ± standard
deviation. Statistical analysis was performed with SPSS 18.0
software (SPSS Inc., Chicago, USA). Differences between groups
were analyzed using one-way analysis of variance (ANOVA)
followed by Tukey test. The statistical significance was defined as
d
p < 0.05.
424 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
3. Results
3.1. Microstructure
Fig. 1 shows the optical microstructures, average grain size and
corresponding XRD patterns of experimental materials. All of the
alloys were composed of two phases, a-Mg and intermetallic com-
pound Mg2Ge with face centered cubic crystal system, as shown in
Fig. 1(c). As-cast Mg-Ge alloys consisted of dendrites of a-Mg
matrix (bright white dendrites) and eutectic phases (a-Mg + Mg2
Ge, brown rod-like/network shaped) at the interdendritic regions,
as shown in Fig. 1(a). Area fraction of the eutectic phases increased
from 17.0 ± 1.3% in Mg-1.5Ge to 24.3 ± 2.9% in Mg-2.5Ge and then
to 33.9 ± 2.5% of Mg-3Ge. Meanwhile, average size of the eutectic
phase continuously increased with increasing Ge addition. The
a-Mg dendrites in Mg-2.5Ge and Mg-3Ge were significantly
Fig. 1. (a) Microstructures of as-cast and as-rolled Mg-Ge alloys; (b) average size of the a-Mg matrix and eutectic phase,
experimental alloys.
refined compared to Mg-1.5Ge, as shown in Fig. 1(b). Microstruc-
tures of the as-rolled Mg-Ge alloys were much more uniform com-
pared to their as-cast counterparts. Area fractions of the eutectic
phases in the as-rolled Mg-Ge alloys (16.2 ± 1.1%, 25.1 ± 2.3% and
35.9 ± 4.3%) were similar to their as-cast counterparts. Besides,
Eutectic phases were significantly refined after rolling.
3.2. Mechanical property
Fig. 2 displays the tensile mechanical behaviors of as-cast and
as-rolled Mg-Ge alloys. Tensile yield strengths (YS) and ultimate
tensile strengths (UTS) of the as-cast Mg-Ge alloys were in the
range of 36.6–51.4 MPa and 116.4–143.2 MPa, respectively.
As-cast Mg-2.5Ge and Mg-3Ge exhibited higher strengths and
elongations than as-cast Mg-1.5Ge. After rolling, YSs and UTSs of
Mg-Ge alloys were significantly improved, however, elongations
d
p < 0.05 and * p < 0.01; (c) XRD patterns of
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436 425

Fig. 2. (a) Tensile stress-strain curves of experimental Mg-Ge alloys; (b) tensile yield strengths, ultimate tensile strengths and elongations of Mg-Ge alloys.

of Mg-1.5Ge and Mg-2.5Ge were impaired. Significantly improved
YS, UTS and elongation were found in as-rolled Mg-3Ge as they
were improved to 175.1 ± 5.1 MPa, 236.4 ± 0.7 MPa and 17.7 ± 2.5
%, respectively.
3.3. Corrosion behavior
OCP, EIS and Potentiodynamic polarization results were dis-
played in Fig. 3(a)–(c) and corresponding electrochemical parame-
ters were listed in Table 1. OCP values of all the experimental
materials could generally stabilize after immersion for 1000 s.
OCP curves of as-cast Mg-1.5Ge and as-rolled Mg-1.5Ge fluctuated
frequently compared to the remaining ones, implying that it was
difficult to reach a steady state for the formation and dissolution
of the corrosion product film at the electrode/solution interface.
For potentiodynamic polarization curves, the cathodic polarization
current reflected the severity of hydrogen evolution reaction on
platinum electrode while the anodic branch represented dissolu-
tion of magnesium. As-cast Mg-2.5Ge, as-cast Mg-3Ge and as-
rolled Mg-3Ge exhibited lower cathodic current density compared
to the remaining alloys. A little potential increase above the Ecorr
would cause a soaring increase in anodic current of Mg-1.5Ge.
The increase on the current of the anodic branches was reduced
for the samples with higher Ge content (2.5 and 3 wt%) when
applying the same voltage disturbance above the Ecorr, implying
inhibited anodic reaction. The highest corrosion current density
was found in as-cast Mg-1.5Ge and it was more than 2 orders of
magnitude of the lowest one, as-rolled Mg-3Ge. Among as-cast
Mg-Ge alloys, corrosion current density significantly decreased
with Ge content reaching 2.5 wt%. After hot rolling, reduced

Fig. 3. (a) OCP curves of Mg-Ge alloys in Hank’s solution; (b) potentiodynamic polarization curves of Mg-Ge alloys in Hank’s solution; (c) Nyquist plots of Mg-Ge alloys; (d)
volume of evolved hydrogen during immersion; (e) magnesium ion released into Hank’s solution; (f) Ge release after immersion.
426 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
Table 1
Corrosion rates of Mg-Ge alloys in Hank’s solution.
Alloy Ecorr (V)
as-cast Mg-1.5Ge 1.456
as-cast Mg-2.5Ge 1.603
as-cast Mg-3.0Ge 1.532
as-rolled Mg-1.5Ge 1.532
as-rolled Mg-2.5Ge 1.542
as-rolled Mg-3.0Ge 1.536
Ecorr: corrosion potential; icorr: corrosion current density; vcorr: corrosion rate calculated from polarization plots by using Tafel region extrapolation; PH: corrosion rate
calculated from hydrogen evolution.
Fig. 4. Surface morphologies (a) and cross section morphologies (b) of Mg-Ge alloys after immersion in Hank’s solution for 3 and 20 days. CP in (b) means corrosion products.
corrosion current densities were revealed for Mg-1.5Ge, while
intensified corrosion was happened in Mg-2.5Ge. As-cast Mg-3Ge
and as-rolled Mg-3Ge owned the largest loop in the Nyquist plots,
demonstrating their superior corrosion resistance among the
alloys. Meanwhile, as-cast Mg-1.5Ge exhibited the smallest loop,
indicating its poor corrosion resistance. This finding was in agree-
ment with the potentiodynamic polarization results.
Evolved hydrogen during immersion test was revealed in Fig. 3
(d). At the initial stage of immersion (0–50 h), no much difference
was found among the materials, all showing a low hydrogen evo-
lution rate. Thereafter, as-cast Mg-1.5Ge kept being corroded at a
relatively high corrosion rate level throughout the whole immer-
sion period. However, other alloys maintained a low hydrogen evo-
lution trend and hydrogen volume gradually stabilized. Except for
as-cast and as-rolled Mg-1.5Ge, volume of generated hydrogen was
all well below 10 mL/cm2 after 20 days. As-cast Mg-2.5Ge, as-cast
Mg-3Ge and as-rolled Mg-3Ge exhibited the slowest hydrogen
evolution rate, suggesting their superior corrosion resistance.
Fig. 3(e) and (f) present released ion concentrations of Mg and
Ge in Hank’s solution at different immersion durations. Continuous
dissolution of Mg and alloying element Ge into Hank’s solution was
found as their concentrations increased persistently with pro-
longed immersion time. Mg and Ge concentrations in Hank’s solu-
tion decreased with increased Ge content in the alloys, suggesting
the positive effect of Ge on corrosion resistance. Hot rolling could
further improve corrosion resistance as as-rolled Mg-Ge alloys
icorr (lA/cm2) vcorr (mm/y) PH (mm/y)
188.2 4.32 5.06
1.2 0.03 0.16
0.9 0.02 0.22
35.6 0.82 1.25
13.6 0.31 0.58
0.7 0.02 0.13
exhibited less dissolved Mg and Ge ions than their as-cast counter-
parts at day 10 and 20. Results of ion release were well consistent
with the hydrogen evolution behaviors and all the corrosion tests
demonstrated that as-rolled Mg-3Ge exhibited the best corrosion
resistance.
Fig. 4(a) shows typical corrosion morphologies of Mg-Ge alloys
after immersion in Hank’s solution for different durations. A corro-
sion product layer was formed on sample surface with some white
clusters/particles deposition. Some microcracks emerged on the
sample surface as a result of the shrinkage of corrosion product
layer during dehydration. Severely localized corrosion was
observed on as-cast Mg-1.5Ge, which was consistent with its
highest corrosion rate among all the alloys. Except for as-cast
Mg-1.5Ge, corrosion product layers on the remaining alloys were
relatively uniform and compact. Judging from the cross-section
morphology in Fig. 4(b), differences in the corrosion behaviors
could be easy to tell. As-cast Mg-1.5Ge and as-rolled Mg-1.5Ge
both suffered severely localized corrosion. A thick corrosion pro-
duct layer with holes and cracks was observed above those deep
corrosion sites. With increasing Ge addition, localized corrosion
was suppressed as the boundary of the Mg-Ge matrix became more
regular and smoother. Thickness of the corrosion product layer
decreased with the increase of added Ge, suggesting improvement
in the corrosion resistance. The thin corrosion product layer on
as-cast and as-rolled Mg-3Ge could be hardly distinguished from
the Mg matrix and surrounding resin. This layer was closely
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436 427

Fig. 5. (a) FTIR spectra of as-rolled Mg-Ge alloys after immersion in Hank’s solution for 20 days; (b) XRD patterns after 20-d-immersion; (c) XPS analysis of the corrosion
products; (d) typical high resolution narrow scan results of Mg 2p, O 1s, Ca 2p, P 2p, C 1s and Na 1s among the corroded Mg-Ge alloys.

adherent to the Mg-3Ge matrix and could possibly provide protec-
tion to the matrix.
Fig. 5 shows the XPS, FTIR and XRD results of the corrosion
products on Mg-Ge alloys. Since the results of as-cast Mg-Ge alloys
were similar to their as-rolled counterparts, so only the as-rolled
results were listed here. XPS results revealed that the corrosion
products were mainly composed of Mg, O, Ca, P, C and limited
Na, as shown in Fig. 5(c). Ge was not detected mainly because of
its minor content in this layer. FTIR indicated the presence of
H2O, OH , CO23 , PO34 and possible HPO24 , as depicted in Fig. 5(a)
[43,44]. Further XRD analysis confirmed the existence of Mg
(OH)2 in the corrosion products. Apparent diffraction peaks of
CaCO3 were only detected on the Mg-1.5Ge sample. Due to a thick
corrosion product layer on Mg-1.5Ge, strong peaks from Mg(OH)2
could be detected with minor signals from the a-Mg matrix. High
resolution narrow scan of XPS also confirmed the presence of
CaCO3 on Mg-2.5Ge and Mg-3Ge, since one Ca 2p3/2 peak assigned
to 347.4 eV, accompanied by C 1s at 289.7 eV and O 1s at 531.6 eV,
was detected [45]. No obvious CaCO3 signals on the XRD patterns
of Mg-2.5Ge and Mg-3Ge should be ascribed to their limited
amounts. Single Mg 2p peak at 50.4 eV along with O 1s at 532.9
eV and C 1s at 289.7, as well as the presence of CO23 proved by
FTIR, indicated possible presence of MgCO3 or hydrated MgCO3
[46]. P showed a single peak at 133.4 eV. This peak was related
to PO34 or HPO24 , as also proved in FTIR. Generally, corrosion prod-
ucts of Mg-Ge alloys were mainly composed of Mg(OH)2 accompa-
nied with small amount of CaCO3. Besides, a small quantity of
carbonates, phosphates or their complex compounds might also
exist.
3.4. Cytotoxicity and ALP activity
Fig. 6(a) and (b) present pH values and ion concentrations of the
extract media. Due to the buffer agent in MEM, no much difference
was found in the pH values among all the extracts. Distinctive ion
releasing behaviors observed in Hank’s solution were not observed
in MEM. Magnesium concentrations in various extracts were no
longer apparently different. On the contrary, they seemed to be
close to each other. Magnesium concentrations fluctuated between
132 lg/mL and 160 lg/mL, while Ge concentrations were in the
range of 1.8–3.8 lg/mL. Ge concentrations in as-rolled Mg-2.5Ge
and as-rolled Mg-3Ge extracts were apparently higher than the
remaining extracts.
Fig. 6(c) and (d) show the MG63 cell viability cultured in Mg-Ge
alloy extracts and ALP activity after culturing for 5 days. According
to ISO 10993-5, cytotoxicity of biomaterials should be Grade 0 or
428 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
Fig. 6. (a) pH values and (b) ion concentrations of the extract media; (c) MG63 cell viability and (d) ALP activity to Mg-Ge extracts.
Grade 1, which means cell viability should exceed 80% [41]. Except
for as-rolled Mg-3Ge, the remaining alloys all satisfied this crite-
rion, exhibiting good cytocompatibility. Cell viability in as-rolled
Mg-3Ge extract was significantly lower than other extracts during
the whole culture period. After 3 days, cell viability of as-rolled
Mg-3Ge was already lower than 80%, and further impaired viability
(47.9 ± 3.6%) was found at day 5. Cell viability of as-rolled Mg-Ge
alloys was lower than the as-cast ones, mainly due to the higher
Mg and Ge concentrations in their extracts. Compared to control,
increased ALP activity was observed for MG63 cells treated with
Mg-Ge extracts. As-cast Mg-1.5Ge owned the highest ALP concen-
tration among all the experimental materials. Among the Mg-Ge
alloys in as-rolled condition, as-rolled Mg-3Ge exhibited the high-
est ALP concentration.
3.5. In vivo performance
Animals received implantation all survived and no obvious
signs of lameness and loss of appetite were observed. No infection
was found on wounds or peri-implant tissues through autopsy and
micro-CT examination.
3.5.1. Micro-CT analysis
In vivo degradation of Mg-Ge implants and osseointegration
between implants and surrounding bones were characterized
through micro-CT assessment, as depicted in Fig. 7. Generally,
implantation of Mg-Ge pin did not cause observable bone damage
and normal bone morphology was observed, as revealed from the
3D reconstruction (Fig. 7(a) and (b)).
Continuous degradation of the implant could be observed and
no obvious signs of localized severe corrosion was found. In the
first two months, a few gas bubbles in quite limited size could be
detected in medullary cavity of rabbit tibia, as marked in green
color in Fig. 7(b). Up to 3 months, the previously existed gas bub-
bles around the implant could be no longer observed. During the
whole implantation period, Mg-Ge implant maintained intact, as
shown in Fig. 8 (a). Surface morphology of the Mg-Ge implant
became rougher as time went on. Some mild and shallow corrosion
pits could be observed on implant surface after 3 months, implying
the appearance of minorly localized corrosion. Residual implant
volume calculated from 3D reconstruction is presented in Fig. 8
(b). At the initial stage after implantation (1 mon), the implant
suffered a fast degradation as implant volume significantly
reduced with a volume loss of 15.4 ± 5.9%. Mitigated degradation
was observed in the subsequent 2 months and the implant could
maintain 75.6 ± 3.2% of its original volume after 3 months.
Bone conductive property of Mg-Ge implant was also proved in
the micro-CT assessment as newly formed bones could directly
attach to the material surface and stretched along implant surface,
as clearly revealed in Fig. 7(c). Qualitatively, continuous
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
Fig. 7. Micro-CT analysis of implant degradation and bone-implant interfaces. (a) 3D reconstruction of bone and implant; (b) 3D reconstruction with different parts rendered
with different colors, semitransparent gray for bone, orange for Mg-Ge implant, green for gas bubble; (c) 2D slice.
osseointegration and strengthened bonding between bone and
implant could be observed from the 2D and 3D reconstructions.
At 1 month, both ends of the implant were embed in the cortical
bone, showing fast osseointegration. However, weak bonding
could be recognized as some voids could be found between
implant and bone (not completely integrated). In the following
month (2 months), two end faces of the implant were gradually
covered by newly formed bones, but still not directly contacted.
After 3 months, bone and implant were well integrated as the
implant was tightly sealed by cortical bones. In addition, cortical
thickness surrounded the implant was thicker than that far from
the implant, as revealed in Fig. 7(c). Bone-implant contact ratio
(BIC) can quantitatively reflect the osseointegration status. In this
study, BIC was calculated as the proportion of bone-implant
directly contacted area to the total implant surface in Amira soft-
ware. Continuously increased BIC value suggested enhanced
osseointegration and bone-implant bonding, as shown in Fig. 8(c).
3.5.2. Histological analysis
Fig. 9 shows tissue responses adjacent to the Mg-Ge implant
after 1, 2 and 3 months with normal tissue as control. Clearly, con-
tinuous degradation of the Mg-Ge implant could be observed since
the implant surface became much rougher with time. Generally,
the implant went through a macroscopic uniform corrosion mode
without severely localized corrosion. No abnormality was found
with bone tissues surrounding the implant, showing good histo-
compatibility. No microscopic differences were noted in the tissue
structures of liver and kidney compared to normal controls, Sup-
plementary Fig. 1.
New bone was laid down directly onto the implant surface and
grew along the surface without interposed soft tissue layer, imply-
ing direct bonding of bone and implant. No scar tissue or fibrous
429
tissue was presented between bone and implant surface. Enhanced
osseointegration could be observed as directly contacted areas of
bone and implant increased with prolonged implantation time,
as shown in Fig. 9(a) and (c). Bone-implant interfaces were closely
related to the osseointegration and bonding strength. Typical fea-
tures at bone-implant interfaces were presented in Fig. 9(b). In
area A, implant was closely integrated with surrounding bones,
showing well osseointegration. In area B, a transitional zone
existed between the implant and new bone. Signs of new bone
penetration to the implant surface could be observed in this zone,
marked with white arrows. In area C, localized corrosion happened.
Some surface fragmentations of the implant materials could be
found. A white gap without tissue filling presented between
implant and bone tissue, showing delayed osseointegration.
4. Discussion
4.1. Microstructure, mechanical property and corrosion behavior
According to the Mg-Ge binary phase diagram, there is no solu-
bility of Ge in Mg and Mg2Ge phase must be precipitated, as shown
in Fig. 10(a) [34]. Phase diagram also shows a high stability of this
Mg2Ge intermediate phase (melting temperature 1117.4 °C),
substantially higher than Mg17Al12 phase ((melting temperature
455 °C)) in Mg-Al alloys [34]. In our Mg-xGe alloys, Ge content
was below 3.36 wt% (eutectic point). So, they all belonged to the
hypoeutectic group. Mg2Ge phase presented in the form of
a-Mg + Mg2Ge hypoeutectic, surrounding the dendrites of primary
a-Mg phase. Area/volume ratio of those hypoeutectic phases
increased with the increasing addition of Ge. Besides, net-like mor-
phology was formed when Ge content exceeded 2.5 wt%, as shown
in Fig. 1(a) and in Fig. 10(b). The improved strengths of Mg-Ge
430 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
Fig. 8. (a) 3D reconstruction of the implant; (b) quantitative analysis of implant volume; (c) bone-implant contact ratio.
alloys were mainly derived from the second phase strengthening of
Mg2Ge. Effects of solid solution strengthening could be neglected
since there is no solubility of Ge in Mg. Morphology of the eutectic
structures might also have impact on alloy strength as the net-like
second phases could act as a skeleton in the transmission and dis-
tribution of stress, possibly showing similar roles of concrete iron
and stones in reinforced concrete structures in architecture.
The matrix a phase in magnesium alloys is normally anodic to
the second phases and is usually preferentially corroded. Eutectic
phase in our Mg-Ge alloys played two opposite roles during corro-
sion: (1) acted as cathode to the a-Mg matrix which accelerated
the corrosion via galvanic corrosion; (2) acted as a barrier which
provided protection to the Mg matrix and thus slowed down the
corrosion. Those two aspects interacted with each other and they
were closely related to the volume fraction and morphology of
the eutectic phase [35]. Overall corrosion could be enhanced or
suppressed depending on which process was the dominated one.
When the eutectic phase particles were in a low area/volume frac-
tion, they could not form a net-like morphology and could not pro-
vide protection of the matrix, as shown in the cross-section
microstructure of corroded Mg-1.5Ge in Fig. 10(b). On the contrary,
micro-galvanic corrosion between the a phase and eutectic phase
would promote the evolution of severely localized corrosion [47].
However, for a high-volume fraction, the eutectic phase was nearly
continuous like a net over the a matrix. Corrosion of the a phase
was then quite easily obstructed by the continuous eutectic phase,
and so the corrosion was greatly retarded, as demonstrated in the
cross-section microstructure of corroded Mg-3Ge in Fig. 10(b).
Since Ge has no solubility in Mg and Mg-Ge alloys owned the same
constituent phases of a-Mg and Mg2Ge, differences among their
corrosion behaviors should mainly depend on the volume fraction
and morphology of the Mg2Ge phase. The high-volume fraction
and continuously net-like eutectic phase containing Mg2Ge should
be responsible for the high corrosion resistance of Mg-2.5Ge and
Mg-3Ge alloys. At the same time, low volume fraction and inter-
mittent distribution of the eutectic phase (Fig. 10(b)) was believed
to be closely related to the poor corrosion resistance of Mg-1.5Ge.
4.2. Toxicity of Ge and Mg-Ge alloys
The use of germanium compounds in dietary supplements to
promote health or cure diseases has become increasingly popular
in 1970s, especially in Japan. Until 1982, no systemic toxicity of
Ge in man was reported [30]. Thereafter, numerous reports showed
that such supplements present a potential hazard to humans at
high dose [28]. However, till now, Ge-containing health care
products, commonly available in organic forms and in capsules
or tablets, as well as in powdered form, are still available in health
food stores and over the Internet. Cases of Ge induced toxicity in
human were closely related to the high dosage (>100 times higher
than the estimated daily intake) and long-term supplement. Con-
tamination of Ge in the inorganic forms, such as GeO2, was also
suspected to be related to those clinical cases [28].
Ge compounds are relatively less toxic compared to other met-
alloids and metals. However relatively high dose of germanium
dioxide and other inorganic Ge compounds can cause severe
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436 431

Fig. 9. Typical histological staining of hard tissue sections. (a) H&E staining; (b) local magnifications of specific areas in (a); (c) toluidine blue staining.

Fig. 10. (a) Phase equilibrium diagram of binary Mg-Ge system; (b) typical microstructures (upper) of as-cast Mg-1.5Ge and Mg-3Ge alloys, corresponding eutectic phase
morphologies (middle) and corresponding cross-section microstructures (under) after immersion in Hank’s solution for 20 days.

poisoning including death. The median lethal dose (LD50) of GeO2
for mice ranges from 2020 mg/kg to 6300 mg/kg, with correspond-
ing values for rats of 1620 mg/kg and 3700 mg/kg, respectively
[30]. It has been reported that daily intake of Ge from food in
human ranges from 0.4-3.4 mg, of which 96% is absorbed. At this
concentration level, Ge would not cause toxicity [48]. Absorbed Ge
is preferentially excreted in the urine in both humans and animals
[48,49]. Based on human case reports, the lowest observed adverse
effect levels of inorganic Ge, GeO2, organic Ge-132 ((GeCH2CH2
COOH)2O3, a novel organogermanium compound originally
synthesized in Japan in 1967) and Ge-lac-cit (germanium–lac
tate–citrate) are in the range of 0.7–23 mg/kg/d, equivalent to
42–1380 mg/d in an adult weighing 60 kg [28], much higher than
the daily intake.
Except for as-rolled Mg-3Ge, the other experimental Mg-Ge
alloys exhibited no toxicity to MG63 cells in this study. From the
perspective of ion toxicity, the coexistence of 132–160 lg/mL Mg
and 1.8–2.4 lg/mL Ge would not induce toxicity to MG63 cells.
According to the results of Feyerabend et al. [50], 15 mM (360
lg/mL) Mg2+ would not cause cytotoxicity to MG63 cells. So,
 432
Table 2
Some basic properties of typical essential/possibly essential elements in human body which can be used as alloying elements in magnesium.

Element Essential/trace Content Content in bone Blood Recommended Standard Physiological roles Perspective of material science Reference
in humana in human serum daily allowance electrode
level potential
(V) b
Mg Major, 21–28 g 60–65% in bones and teeth 0.75–1.2 320–400 mg 2.37 Activator of many enzymes; maintain muscle Poor mechanical properties of pure [63–65]
essential mM and nerve function; co-regulator of protein Mg hinder its use in load bearing
synthesis and muscle contraction; stabilizer applications; magnesium alloys with
of DNA and RNA sufficient mechanical properties are
needed
Ca Major, 1000 g 99% in bones and teeth 2.1–2.6 1000–1300 mg 2.87 Second messenger in signal transduction Improves strength of Mg (solid [66,67]
essential mM pathways; calcium ions as a cofactor in many solution strengthening, precipitation
enzymes; maintain proper bone formation hardening, grain refinement) and
creep resistance; reduces castability
Zn Trace, essential 2–4 g 29% in bones 12–16 12–15 mg 0.76 Zn has ubiquitous roles; participation in Improves strength, yet reduces [68,69]
mM metabolism of RNA and DNA, signal ductility in high concentrations (solid
transduction, and gene expression, also solution hardening, precipitation);
regulation in apoptosis; appearance in all improves castability

D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436

enzyme classes
Mn Ultra-trace, 10–12 mg 43% in bones 10.9–78.3 1.8–5 mg 1.18 Necessary for development, metabolism, and Improves strength and ductility [70,71]
essential nM the antioxidant system; regulation of cellular (grain refinement); improves creep
energy, bone and connective tissue growth, resistance; improves corrosion
and blood clotting resistance in combination with
aluminium (precipitation that picks
up iron)
Fe Trace, essential 3–5 g 20 mg is transferred daily to 6.3–26.9 0.9 mg 0.44 The most important transition metal in all Fe is the most common impurity in [71–73]
the bone marrow for mM living organisms; iron-containing proteins are Mg; it is detrimental to the corrosion
erythropoiesis and is crucial for the transport and storage of oxygen resistance due to the formation of Fe-
efficiently recycled by containing particles
macrophages
Cu Ultra-trace, 84–126 0.53–5.69 ppm in bones 11–24 0.9–1.2 mg 0.34 Helps with the formation of collagen, Cu is a common impurity in Mg; a [74–76]
essential mg (1.4– mM increases the absorption of iron and plays a small amount of Cu has a beneficial
2.1 mg role in energy production; helps keep blood effect on the creep strength of
per kg of vessels, bones, nerves, and the immune magnesium die castings but strongly
body system healthy accelerates corrosion
weight)
Sr Trace, possibly 0.3 g 99.1% in bones 0.11–2.48 2 mg 2.89 No harmful effects of stable strontium in Improves strength and ductility [5,15,77,78]
essential mM humans; substitution for calcium in a small (grain refinement); improves creep
extent and deposition in bones; showing dose resistance and high temperature
dependent metabolic effect on bone; low strength
doses stimulated new bone formation
Si Trace, possibly 1–2 g No certain value. The highest 7.7–11.1 / 0.86 Involves in bone mineralization and Reduces ductility; improves creep [71,79,80]
essential levels are found in bone and mM prevention of osteoporosis, collagen resistance and high temperature
other connective tissues synthesis, and prevention of the aging of skin, strength (precipitation); reduces
such as, skin, nail, hair, overall condition of hair and nails corrosion resistance and castability
trachea, tendons and aorta
Sn Ultra-trace, 30 mg / 0.067– / 0.14 Tin-deficient diets in rat studies resulted in Improves strength, hardness and [5,21,81,82]
possibly 0.34 mM poor growth, reduced feeding efficiency, creep strength, however too much Sn
essential (in whole hearing loss, and bilateral (male) hair loss impairs strength and elongation; low
blood) concentration positively affects the
corrosion resistance
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436 433

higher Ge concentration (3.8 lg/mL) accompanied with higher

Elements listed below as ‘‘Essential in humans” are those listed by the (US) Food and Drug Administration as essential nutrients. Ultra-trace minerals are those elements with established, estimated, or suspected requirements
Mg concentration (148 lg/mL) in as-rolled Mg-3Ge extract

[27,33,85,86]
Reference

should be responsible for its low cell viability. Even though as-
[5,83,84]
rolled Mg-3Ge induced cytotoxicity to MG63 cells in vitro, the
in vivo tissue response and bone-implant osseointegration were
qualified. New bone could directly lay down onto the implant sur-
lattice structure); reduces corrosion
ductility/formability (change to bcc

face and grew along the surface, implying decent biocompatibility

high temperature strength (grain
Improves room temperature and
of this alloy. Owing to the capability of in vivo circulation system,
Reduces strength yet improves
Perspective of material science

local ion concentration surrounding the implant could be diluted.

refining and second phase

In order to minimize the huge gap between in vitro and in vivo

resistance and density

biocompatibility tests, 6–10 times dilution of extracts for

in vitro cytotoxicity test was recommended for biodegradable
magnesium-based materials [51]. In present study, local Mg and
strengthening)

Ge concentrations in vivo should be much lower than that

in vitro and that’s why as-rolled Mg-3Ge showed good biocom-
patibility in bone while exhibited cytotoxicity in vitro. During
the 3 months’ implantation, the total released Ge was only 0.58
mg, corresponding to 0.0064 mg per day under the assumption
Lithium is used in the treatment of bipolar

of a constant degradation rate. Daily Ge dissolution from the

including anticancer, anti-inflammation,
antioxidation, radioprotection and pro-

implant was far below its daily intake from normal diet and bio-
Organic Ge has been used as a dietary
supplement with therapeutic benefits

safety of this alloy could be guaranteed.

4.3. Mg-Ge alloys compared to other Mg-X (X: essential/possibly

essential elements in human health) alloy systems
Physiological roles

immune functions

Table 2 summarizes the basic properties of essential/possibly

essential elements in human body. As is well-known, elements
Ca and Mg are the major minerals in human body and the other
disorder

elements belong to the group of ‘‘trace elements” [52]. Ca seems

to be a proper alloying element in magnesium, especially for
orthopaedic implants, since it is the most abundant metal in
Standard electrode potential was excerpted from Electronic Science Tutor, G R Delpierre and BT Sewell, 1992–2012.
electrode
potential
Standard

human body and it is crucial to the health of bones and teeth.

3.04

But, poor formability and fast degradation might hinder the appli-
(V) b

0.24

cation of Mg-Ca alloys [7]. Strontium (Sr), zinc (Zn) and silicon (Si)
commonly found in bone tissues are believed to benefit for bone
daily allowance
Recommended

dietary intake

growth and health [53]. Strontium can replace calcium in the

0.4–3.4 mg/d
(estimated

of Ge)
/

(in plasma)
0.07 mM
serum

<1 mM
Blood

level

100 ng/g in bone

Content in bone

/
in human
Content

21–763
ppb

/
Essential/trace

Fig. 11. Comparison of the mechanical properties among various biodegradable

dietary trace
Ultra-trace,

Ultra-trace,

essential?)
in humana

Mg-X alloys (X: essential/possibly essential element in human health). Different

(possibly
essential
possibly

element

symbols represent different alloy systems and symbol color reflects the process
below 1 mg per day.
Table 2 (continued)

condition. Mechanical properties were deduced from the following references: Mg-
Ca alloys from Ref. [61,87,88], Mg-Zn alloys from Ref. [55,62,89,90], Mg-Sr alloys
from Ref. [15,16,18,91], Mg-Sn alloys from Ref. [20–23,82,92], Mg-Si alloys from
Element

Ref. [92,93], Mg-Mn alloys from Ref. [92], Mg-Li alloys from Ref. [24], Mg-Ge alloys
from Ref. [33] and from present study (purple marked). (For interpretation of the
Ge

references to colour in this figure legend, the reader is referred to the web version of
Li

b
a

this article.)
434 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436

Fig. 12. In vitro and in vivo corrosion rates of binary Mg-Ge alloys explored in this study compared to previously reported Mg-X alloys (X: essential/possibly essential element
in human health): (a) corrosion rates in Hank’s solution calculated from electrochemical tests; (b) corrosion rates in Hank’s solution calculated from immersion tests; (c)
corrosion rates in various bone tissues. In vitro corrosion rates: Mg-Ca alloys include as-cast Mg-1Ca [94]; Mg-Zn alloys include as-cast Mg-(1, 5, 7)Zn [55]; Mg-Sr alloys
include as-rolled Mg-(1, 2, 3, 4)Sr [15], and as-extruded Mg-(0.5, 1, 1.5, 2.5)Sr [18]; Mg-Sn alloys include as-cast Mg-(1, 3, 5, 7)Sn [20], as-extruded Mg-(1, 3, 5, 7)Sn [22], and
as-cast and as-rolled Mg-1Sn [92]; Mg-Si alloys include as-cast and as-rolled Mg-1Si [92]; Mg-Mn alloys include as-cast and as-rolled Mg-1Mn [92]; Mg-Li alloys include as-
extruded Mg-3.5Li and Mg-8.5Li [24]; Mg-Cu alloys include as-cast Mg-(0.05, 0.1, 0.25)Cu [13], and Mg-(0.03, 0.19, 0.57)Cu [14]; corrosion data of Mg-Ge alloys were results
in this study. In vivo corrosion rates of as-cast Mg-1Ca, as-extruded Mg-6Zn and as-rolled Mg-2Sr were derived from Ref. [61,62,15], respectively.

mineral of growing bones and thus lead to bone growth problems
[54]. Thus, whether Mg-Sr alloys are applicable to children needs
further research. Zn benefits for the strength and ductility simulta-
neously. It is a proper alloying addition in both orthopaedic and
cardiovascular applications [55,56]. Formability and corrosion
resistance of Mg-Si alloys might be restricted by the brittle Mg2Si
phase since Si has no solubility in Mg and Mg2Si must be precipi-
tated [56,57]. Fe and Cu are normally recognized as impurities in
magnesium since they are inevitably introduced from the raw
materials or during the production process [58]. Mg-Fe and Mg-
Cu based alloys do not seem suitable for load bearing implants as
they possess a fast degradation rate. However, they could be appli-
cable in special situations, such as Mg-Cu alloy in the treatment of
osteomyelitis [13]. Among all the elements in Table 2, lithium (Li)
is the only one who can change the hexagonal close-packed (hcp)
structure of Mg into body-centered cubic (bcc) type, thus improves
ductility/formability. However, Li impairs strength. Mg-Li alloys to
be used as orthopaedic implants should be strengthened by other
alloying elements, such as Ca [59]. Generally, a common problem
faced by Mg-Ca, Mg-Sr, Mg-Si and even Mg-Zn alloys is that they
exhibited fast degradation (>0.5 mm/y) either in vitro or in vivo
[15,60–62]. Information about Mg-Sn and Mg-Mn alloys is quite
limited till now. Possibility of those alloys to be used as orthopae-
dic implants is still uncertain.
Compared to other essential/possibly essential elements, bio-
logical information on Ge is quite limited. But, basically, it is a com-
mon trace element found in human diet and well controlled intake
will not induce safety issues. Blood level of Ge is 3.99 lM (0.29 mg/
L) in human serum, even higher than Li, Sn, Sr and Mn, as shown in
Table 2. Dietary intake of Ge is similar to some other nutrient ele-
ments. Standard electrode potential of Ge (Ge-Ge2+ + 2e, 0.24 V) is
lower than Cu and higher than the remaining elements in Table 2,
for example, group neighbors Sn (Sn-Sn2+ + 2e, 0.14 V) and
Si (Si + 2H2O-SiO2 + 4H++4e, 0.86 V).
An overall comparison of mechanical properties among various
Mg-X alloys (X represents for essential/possibly essential elements
in human health) is displayed in Fig. 11. Under the same processing
condition, at as-cast or as-rolled state, mechanical properties of
Mg-Ge binary alloys are comparable to other alloy systems.
Strengths and elongations of as-cast Mg-Ge alloys in our study
are higher than most of the remaining as-cast binary alloys, as
shown in Fig. 11. Among the limited binary alloys in as-rolled con-
dition, our as-rolled Mg-3Ge exhibits the highest strength and sig-
nificantly improved elongation. Generally, processing history has
 D. Bian et al. / Acta Biomaterialia 64 (2017) 421–436
remarkable impacts on the mechanical properties and corrosion
behaviors. Performances of Mg-Ge alloys can be adjusted through
various heat treatments and by deformation processing. In vitro
corrosion rates of typical Mg-X alloys measured by electrochemical
test and immersion test are displayed in Fig. 12 for comparison.
Corrosion resistance of Mg-Ge alloys is quite outstanding. Among
the Mg-X alloys, as-cast Mg-2.5Ge, as-cast Mg-3Ge and as-rolled
Mg-3Ge exhibited the lowest corrosion rates (electrochemical), as
shown in Fig. 12(a). Superior corrosion resistance of those alloys
was also verified in the immersion corrosion test, as shown in
Fig. 12(b). Besides, as-rolled Mg-3Ge exhibited much better
in vivo corrosion resistance compared to typical Mg-Ca, Mg-Zn
and Mg-Sr alloys of which orthopaedic implantation was
performed, as shown in Fig. 12(c).
5. Conclusions
In this study, biodegradable Mg-based alloys with dietary trace
element germanium were developed and their feasibility to be
used as orthopedic applications was systematically investigated
through in vitro and in vivo evaluations. Mechanical properties of
Mg-Ge alloys were superior to many other biodegradable alloys
strengthened with essential elements in human body. Besides,
they could be further improved by deformation processing, such
as rolling and extrusion. Mg-Ge (>2.5 wt% Ge) alloys exhibited
excellent corrosion resistance since as-rolled Mg-3Ge exhibited a
slow degradation rate in vitro (vcorr = 0.02 mm/y, pH = 0.13 mm/y).
A proper degradation rate of 0.6 mm/y in vivo has also been veri-
fied. Quite limited H2 generated during implant degradation did
not cause bone destruction and it could be completely absorbed
in 3 months. Osseointegration of bone-implant was proved since
new bone could directly lay down onto the implant and grew along
its surface. In conclusion, Mg-Ge alloys show great potential to be
used as orthopaedic implants considering their good combinations
of mechanical property, corrosion resistance, cytocompatibility
and histocompatibility.
Acknowledgements
This work was supported by the National Key Research and
Development Program of China (Grant No. 2016YFC1102402),
National Natural Science Foundation of China (Grant No.
51431002), NSFC/RGC Joint Research Scheme (Grant No.
51361165101 and 5161101031) and NSFC-RFBR Cooperation
Project (Grant No. 51611130054).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.actbio.2017.10.004.
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