Journal of Alloys and Compounds 663 (2016) 156e165

Contents lists available at ScienceDirect

Journal of Alloys and Compounds

journal homepage: http://www.elsevier.com/locate/jalcom

In vitro corrosion behavior and biocompatibility of biodegradable

magnesium-pearl powder metal matrix composite
He Meifeng, Hua Xianyang, Fan Xue, Pan Deng*
School of Materials Science and Engineering University of Shanghai for Science and Technology, Shanghai, 200093, China

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

Article history: Recent studies indicate that there is a high demand to design magnesium alloys with adjustable
Received 27 October 2015 corrosion rates and suitable biocompatibility. An approach to this challenge might be the application of
Received in revised form metal matrix composite (MMC) base on magnesium alloys. Magnesium plays an important role in the
8 December 2015
body, mediating cell-extracellular matrix interactions and bone apatite structure and density. This study
Accepted 15 December 2015
investigated, for the first time the effects of adding pearl powder (PP) as reinforcements to magnesium
Available online 18 December 2015
alloy ZK60 as a matrix for corrosive and cytocompatible properties. Results revealed that PP particles
significantly enhanced osteoblast adhesion and proliferation on MMC-PP composites while stabilizing
Keywords:
Magnesium
the corrosion rate and exhibited more uniform corrosion attack in the simulated body fluid suitable for
Composite biodegradable material applications. Using MTT colorimetric assay, cell proliferation and cytotoxicity
Osteoblasts study of the samples with osteoblasts (or bone-forming cells) indicated that biodegradable MMC-PP
Corrosion composites are cytocompatible biomaterials with adjustable corrosive properties. This research pro-
Cytocompatible vides significant evidence of the potential value of the further study of pearl powder particles for
numerous implanted biomaterials applications.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction implant stability [1].

Biodegradable implant materials in the human body can be
gradually dissolved, absorbed, consumed or excreted, so there is no
need for the secondary surgery to remove implants after the sur-
gery regions have healed [1]. Common metallic biomaterials such
as titanium, stainless steels, and cobaltechromium based alloys
have high mechanical strength and fracture toughness rendering
that more suitable for load bearing applications in comparison with
polymeric or ceramics biomaterials [2,3]. In particular, biodegrad-
able metallic implants can obviate the need for follow-up surgeries
after the tissues have sufficiently healed. Magnesium alloys have
densities between 1.74 and 2.0 g cm
3 similar to that of human
bone (1.74e2.0 g cm-3). Additionally, their Young's moduli of
41e45 GPa are similar to that of human bone compared to synthetic
hydroxyapatites (73e117 GPa), CoeCr alloys (230 GPa), stainless
steels (189e205 GPa), and Ti alloys (110e117 GPa). These favorable
mechanical properties diminish the stress at the implant/bone
interface and can stimulate new bone growth resulting in better
* Corresponding author.
E-mail address: hmf752@163.com (H. Meifeng).
Magnesium based materials are the promising choice due to its
degradability in physiological environment and load bearing ca-
pacity. The mechanical properties of magnesium are also closer to
natural bone which minimizes stress shielding effect that is
generally associated with other-metallic systems [4,5]. However,
the poor corrosion resistance of magnesium alloys limits their ap-
plications [6,7]. High corrosion rate can result in a rapid release of
degradation products, which have a deleterious effect on human
physiology, and rapid corrosion can also cause a reduction in the
mechanical integrity of the implant before the tissue has suffi-
ciently healed [2]. So, controlling the rapid degradation is the
critical issue in developing magnesium based biodegradable im-
plants. The degradation rate of magnesium can be altered by
developing new alloys and composites [8e11], surface coatings and
modifying the microstructure [8e11].
Aragonite and vaterite are two different calcium carbonate
crystals [12,13]. Both, aragonite and vaterite are unstable crystals
and easily transform into calcite crystals in solution [13]. Inorganic
parts of lustrous pearls are aragonite crystals, which are called
aragonite pearls. Aragonite pearl is composed of aragonite crystals
and organic matrixes (＜5 wt. %). Some pearls show no luster,
containing pure vaterite crystals and around 5 wt. % organic

http://dx.doi.org/10.1016/j.jallcom.2015.12.120
0925-8388/© 2015 Elsevier B.V. All rights reserved.
 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165
matrixes, which are known as vaterite pearls [14,15]. It has been
known for a long history in China that pearl powder has great
medicinal value. Pearl powder is rich in calcium carbonate, protein,
water, multiple amino acid and some trace elements, such as iron,
aluminum, magnesium, zinc, selenium, copper, potassium, man-
ganese, strontium, germanium and etc [16]. Dong et al. [17] found
that the highest content of pearl powder is calcium (377.084 mg/g)
as measured by atomic absorption spectrometry (AAS). Since cal-
cium is a major bone-forming mineral, some studies [18e21]
revealed that the calcium of pearl powder can greatly alleviate
the symptoms of osteoporosis and fractures. Pearl powder is
biocompatible and biodegradable when implanted into bone de-
fects [22]. Pearls contain growth factors that are able to increase
osteoblast proliferation [23].
Pearl powder exhibits suitable biodegradability and biocom-
patibility. However, there are only few reports on pearl powder
applications in bone regeneration. Taking advantage of these prior
studies, the objective of the present in vitro study was to charac-
terize the suitability of MMC-PP composite for potential applica-
tions as a bone implant. Various novel techniques are used for
metal/alloy sintering [24e28]. Among these techniques, spark-
plasma-sinter (SPS) technique is widely used to sinter refractory
metals and their carbides, due to its fast heating and short holding
time. In this study, we examined MMC-PP composites with a PP
content of 0~14 wt. % were sintered by SPS method at 400  C. The
corrosion behavior was studied by electrochemical measurement
and the morphological characterization was performed by scanning
electron microscopy (SEM). In addition, cell adhesion and prolif-
eration experiments were performed to assess the osteoblastic
cytocompatibility of this composite.
2. Materials and methods
2.1. Materials
ZK60 magnesium alloy powder was purchased from China
Magnesium and Boron Powder Company. All the reagents were of
analytical grade. Fresh water pearls were obtained from freshwater
assay cultured Hyriopsis cumingii in Zhejiang China. PP was fabri-
cated as described below.
2.1.1. Pearl powder preparation
Pearls were collected from freshwater cultured H. cumingii.
Vaterite and aragonite pearls were acquired from selected lustrous
pearls and lack luster pearls respectively [12]. The two specimens
were put into a beaker filled with ethanol at ambient temperature
for 10 h, then rinsed in ultra-pure water (Milli-Q) and air dried. The
pearls were grounded into powder using a grinder with a
maximum speed of 25,000 rpm for 3 min. Most of the powders in
large size are in the container of the grinder, while a small amount
of powders in small size are sticked to the surface of the lid of the
grinder. Only the powders sticked to the surface of the lid are
scraped and obtained. The mean size of the used powder is 80 nm.
2.1.2. Composite preparation
Commercial ZK60 powder and pearls powder were used as the
starting materials. The ZK60 powder was first put into Al2O3 cru-
cible in glove box filled with high-purity argon (both the H2O and
O2 are limited below 0.1 ppm). The sintering of the mixed powders
with a pearls powder content of 0, 2 wt.%, 6 wt.%, 10 wt.% and
14 wt.% was carried out using the SPS system (Sinter Land Inc.,
Japan, Model LABOX 325). For each sample, 1.0 g composite powder
was fed into a 15 mm graphite die. One scheme was used for the
mixed powder sintering. Samples were heated to target tempera-
ture 400  C with a heating rate of 100  C / min and held for 0 min at
157
sintering temperature. An initial pressure 100 MPa was set from the
very beginning, and it was gradually decreased to the target value
60 MPa in 4 min under program controlling, release the pressure
finally at room temperature. The temperature and pressure pa-
rameters for sintering at 400  C, 120 MPa are shown as an example
in Fig. 1.
2.2. Material characterization
Scanning electron microscopy (SEM) with a FEISION-200 high-
resolution field emission microscope was used to visually charac-
terize the micro-topographical features of the composite surfaces.
The morphologies of the corroded samples and the corrosion
products after electrochemical measurements were observed also
with scanning electron microscope (SEM). Identification of the
phases was verified by X-ray diffraction (XRD, D/max-2550).
2.3. Cell assays
2.3.1. Cell culture
Primary hFOB1.19 osteoblasts (Cell bank of Chinese Academy of
Science, Shanghai, China) were cultured in Dulbecco's Modified
Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS)
and growth factor G418 (300 ml per 50 ml) in a 37  C, humidified, 5%
CO2/95% air environment. G418 (Geneticin) is an aminoglycoside
antibiotic similar in structure to gentamicin B1. Cells at passage
numbers of 4e12 were used in these experiments.
2.3.2. Cell adhesion assay
The composites were cut into 1 cm  1 cm squares, placed
individually into the wells of a 24-well plate, and sterilized under
UV light overnight. Before cell seeding, composites were rinsed
twice with PBS to remove any possible debris. Osteoblasts were
cultured to 90% confluence, rinsed with PBS and trypsinized using
0.25% trypsineEDTA (Sigma). Released cells were centrifuged to
form a pellet and then resuspended in cell growth medium. Sam-
ples were incubated under standard culture conditions for 4 h; then
the medium was aspirated from each well and each sample was
rinsed with PBS. Samples were carefully transferred to a brand new,
clean 24- well plate and 1 ml of cell growth media was added to
each sample along with 200 ml of MTT dye (Cell Tracker™ Green
CMFDA). Samples were placed back into the incubator for 3 h to
allow the MTT to completely react with the metabolic products of
adherent cells, and then 200 ml of solution from each well was
transferred to a 96-well plate in quadruplicate. The 96-well plate
Fig. 1. Schematic heating cycles for sintering process.
158 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165
was placed into iMark Microplate Reader (BioTek instruments) and
the absorbance of the MTT solution in each well was measured at a
wavelength of 490 nm. The absorbance values of blank wells con-
taining composite samples without any seeded cells were sub-
tracted from the absorbance values of the corresponding wells
containing cells to obtain the absorbance of only the metabolic
activity of the cells adherent to each respective composite sample.
The number of cells adherent to each composite sample was
determined by comparing the resulting absorbance values to a
standard curve constructed at the beginning of each trial.
2.3.3. Cell proliferation assay
Cell proliferation assays were conducted in exactly the same
way as the adhesion assay, with the exception that cells were
cultured on samples for 1, 2, 3 and 4 days before the addition of the
MTT dye. The cell growth media was changed after 3 days of
culture.
2.4. Composite degradation
2.4.1. Electrochemical measurements
Electrochemical measurements were performed in simulated
body fluid at 37  C and buffered at pH ¼ 7.2. The electrochemical
tests were carried out in simulated body fluid using a corrosion
measurement system (PARSTAT2273). A three electrode cell was
used for electrochemical measurements, the saturated calomel
electrode (SCE) as a reference, a platinum electrode as the counter,
and the sample as a working electrode. The area of the experi-
mental samples exposed to the solution was 1 cm2. The open circuit
potential (OCP) was measured as a function of time and started
immediately since the sample was immersed in the electrolyte. The
electrochemical impedance spectroscopy (EIS) measurements were
carried out from 100 mHz to 100 MHz at OCP values after the open
circuit potential (OCP) was measured. The amplitude of alternating
current was 10 mV, and the test points were about 150. Potentio-
dynamic polarization tests were carried out at a scanning rate of
1 mV s
1.
2.4.2. Osteoblast proliferation in the composite degradation extracts
To assess the effects of sample degradation products on cell
function, primary hFOB1.19 osteoblasts were cultured in the ex-
tracts of the composites. Extracts were prepared using DMEM with
the surface area of extraction medium ratio 1.25 cm2 mL
1 in a
humidified atmosphere for 72 h, then filtrated through bacterial
filters to remove particulate matter, and then serially diluted to
50e10% concentration. Cells were incubated in 96-well plate at
2  103 cells/100 mL medium in each well and incubated for 1 day to
allow attachment. DMEM containing 10% fetal bovine serum served
as negative controls and those that without cells as blank controls.
After 72 h, the medium was replaced with 100 mL corresponding
extracts, respectively. After incubating the cells in a humidified
atmosphere with 5% CO2 at 37  C for 1, 2, 3 and 4 days, 20 ml MTT
was added to each well and then incubated at 37  C for 4 h. Then
the medium was discarded and replaced by 100 ml DMSO in each
well and kept in a shaker at room temperature for 15 min, and then
the supernatant was measured at a wave length of 490 nm. The
absorbance of blank wells was subtracted from the absorbance of
the corresponding cell-containing wells and then compared to a
standard curve created at the start of each trial to calculate cell
densities in each well.
2.5. Statistical analysis
Cell assay were performed in either triplicate or quadruplicate
and repeated three separate times. The data were analyzed using
single-factor analysis of variance to determine the equality of
population means, and Student's t-tests were performed between
populations of interest, with P < 0.05 considered statistically
significant.
3. Results and discussion
3.1. Material characterization
The morphological change of hybrid composites were observed
using SEM (Fig. 2). The pearl powder particles were dispersive
embedded on the surface of magnesium matrix. No obvious crack
was found on the surface, which proved a good adhesion between
the matrix and the incorporated pearl powder particle. The surface
of hybrid composites was ground to observe the distribution of
pearl powder particles incorporated in magnesium matrix by SEM
(Fig. 2). It was found that the particles uniformly aggregated around
the magnesium matrix with the increase of pearl powder contents.
The aggregation of pearl powder gradually formed an interlinked
framework morphology, which formed a favorable structure for the
subsequent degradation. The phase formation of the hybrid com-
posites with a pearls powder content of 0, 2 wt. %, 6 wt. %, 10 wt. %
and 14 wt. % were characterized by XRD as shown in Fig. 3. The XRD
patterns show that all the samples were composed of
Mg0.97Zn0.03, without X-ray detecting unalloyed Zr intermetallic
phases. The results of XRD indicated the presence of CaCO3 in the
composites of 6 wt. %, 10 wt. % and 14 wt. % in Fig. 3(c, d and e).
Inorganic parts of lustrous pearls are aragonite crystals, which are
called aragonite pearls. Aragonite and vaterite are two different
calcium carbonate crystals. The observed sample weight of PP
decreased about 4%, while the change in the observed sample
weight of the composites was too low for a reliable determination
(Fig. 3b).
3.2. Osteoblast cell adhesion and proliferation
Most significantly, the results of the present study showed that
hFOB1.19 osteoblasts adhered onto samples containing 10%PP
roughly four times better than on plain ZK60 sample (Fig. 4). Pearl
powder-containing samples showed improved osteoblast adhesion
compared to plain ZK60. PP incorporated with Mg seemed to have
an increasing effect on hFOB1.19 osteoblasts cell viability. Pearl
powder-containing samples (98%Mg þ 2%PP, 94%Mg þ 6%PP and
86%Mg þ 14%PP) fell short of sample (90%Mg þ 10%PP), indicating
that the addition of pearl powder particles to ZK60 enhanced the
initial osteoblasteMg interactions. Osteoblasts proliferated to the
highest cell densities on the 90%Mg þ 10%PP samples (Fig. 5A and
B), implying that pearl powder particles enhanced osteoblast
densities through interactions with ZK60 particles similarly to the
benefits Mg ions provide in bone apatite in the body. Pearl powder
in the Mg matrix facilitates heterogeneous nucleation and increases
the rate of apatite crystal formation and growth. Among the five
composites, the PP content of 90%Mg þ 10%PP sample is not the
highest. However, this sample shows improved adhesion and pro-
liferation of cells. The reasons behind the better adhesion of the
cells on the 90%Mg þ 10%PP sample compared to other samples are
embedded PP particles and lower degradation rate.
3.3. Composite degradation
3.3.1. Electrochemical measurements
The open circuit potentials of the composite samples were
recorded for short time. Their electrochemical stabilities as a
function of immersion time in simulated body fluid are shown in
Fig. 6. During the initial 100 s of immersion time, the OCP of the five
 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165 159

Fig. 2. SEM images of each composite. Percentages signify the weight percent of the pearl powder pearl powder (PP) particle and ZK60 magnesium alloy matrix (Mg) in each
composite (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%Mg þ 6%PP, (d) 90%Mg þ 10%PP and (e)86%Mg þ 14%PP.

samples fluctuated with time. After 200 s of immersion, the OCP of
the samples achieve relatively stable values. Fig. 6 shows that, the
composite sample containing 10% pearl powder shows the highest
OCP. In this stage, a relatively stable potential value (
1445 mV/
SCE) is observed; the sample containing 6%PP shows the more
active OCP, the OCP varied rapidly from a low value (
1520 mV/
SCE) to a relatively stable higher value (
1470 mV/SCE); the plain
ZK60 sample shows the OCP is lower, the OCP fluctuation is regular
and the potential is balanced and stable around
1500 mV/SCE, the
sample containing 2%PP shows the OCP is much lower, the OCP
value is closed to linear growth. This relative OCP fluctuation
illustrated that there are some quick and irregular reactions in the
samples, which may not reflect that the samples adsorbed elec-
trolyte, thereby do not to reach stable and saturated state; while the
sample containing 14%PP shows the OCP is lowest. Careful visual
examination of the samples surface after immersion for 900 s re-
veals that the corrosion films are formed on the samples surface,
but no clear pitting occurs and the corrosion is uniform.
Form Fig. 6, the open circuit potentials (OCPs) of the Pearl
powder-containing samples are close and varied in the range
from
1445 to
1650 mV/SCE (fluctuate around that of the plain
ZK60 sample). The pearl powder plays a very important role in the
corrosion behavior of the samples in present study (as shown in
Fig. 6).
160 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165

Fig. 3. XRD patterns of the samples (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%Mg þ 6%PP,
(d) 90%Mg þ 10%PP and (e) 86%Mg þ 14%PP.

Fig. 5. 1, 2, 3, and 4 days osteoblast proliferation determined using an MTT assay (A)
histogram diagram (B) plotting diagram (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%
Mg þ 6%PP, (d) 90%Mg þ 10%PP and (e) 86%Mg þ 14%PP.

Fig. 4. Osteoblast adhesion after 4 h of culture.

Fig. 7 presents the potentiodynamic polarization curves for the

samples after immersion time of 900 s in simulated body fluid.
Generally, the cathodic polarization curves are assumed to repre-
sent the cathodic hydrogen evolution through water reduction,
while the anodic ones represent the dissolution of composite
sample. It is noted that the corrosion potentials (Ecorr) are slightly
different from the OCPs shown in Fig. 6. Since surface conditions of
the electrodes could be modified during the polarization test
because the applied potential is swept from
1800 mV, the po-
tential drifts are reasonable [29]. Such phenomenon is common in
many electrochemical systems. Although the cathodic polarization
behavior according with the Tafel relationship, the corrosion po-
tentials (Ecorr) drifts result in the current densities of the samples at
cathodic region will not have reference value.
The anodic parts of the curves in Fig. 7 reveal clearly the exis-
tence of a different current slope, which indicates the existence of
Fig. 6. Variation of open circuit potentials of the samples after immersed in simulated
some protective coatings on the surface of some samples. Three body fluid. (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%Mg þ 6%PP, (d) 90%Mg þ 10%PP and
samples (98%Mg þ 2%PP, 90%Mg þ 10%PP, 86%Mg þ 14%PP) tested (e) 86%Mg þ 14%PP.
exhibit the same anodic curve-shape: a similar potential-current
density slope with obvious breakdown potential (Eb), after which
high current densities are observed increasing rapidly with little defined as the minimum current density required to maintain
associated increase in potential. The passive current density is passivity. The passive current (corrosion rate with anodic
 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165
Fig. 7. Potentiodynamic curves of the samples after immersed in simulated body fluid.
(a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%Mg þ 6%PP, (d) 90%Mg þ 10%PP and (e) 86%
Mg þ 14%PP.
protection) is evaluated from the anodic potentiodynamic polari-
zation curves such as in Fig. 7. The passive current densities do not
represent the steady-state current densities, which are lower, but
rather a close approximation.
Comparisons with five samples show that containing 10%PP
sample have the lowest corrosion current densities. The sample
containing 10%PP shows the best corrosion resistance in five
samples.
The EIS results of the composite samples with different pearl
Fig. 8. Equivalent circuits for EIS tests in simulated body fluid. (a) 100% Mg, (b) 98%
Mg þ 2%PP, (c) 94%Mg þ 6%PP, (d) 90%Mg þ 10%PP and (e) 86%Mg þ 14%PP.
Fig. 9. Equivalent circuits for EIS tests in simulated body fluid (a) samples except for 98%Mg þ 2%PP and (b) 98%Mg þ 2%PP.
161
powder content are presented in Fig. 8. The Nyquist plots of the
100%Mg 94%Mg þ 6%PP 90%Mg þ 10%PP and 86%Mg þ 14%PP four
samples show two capacitive loops at high frequency and inter-
mediate frequency. In order to further understand the corrosion
mechanisms, the corresponding equivalent circuits for EIS tests are
shown in Fig. 9. It is well known that the high frequency capaci-
tance loop was mainly related to the characteristics of electric
double layer formed at the interface between the metal surface and
the electrolyte, which can be described by Rct and CPE [30]. Rct was
named as charge transfer resistance representing the resistance of
electron transfer during electrochemical reactions at the interface
between the electrode and solution [31]. The larger the distance or
CPE was the constant phase element. Taking account of the rough
metal surface, CPEdl was used to instead of the electric double layer
capacitance. Rs referred to the solution resistance. For the samples,
the impedance loops in Fig. 8(a), (c) (d) and (e) can be modeled
using a parallel combination of a resistance Rct and a capacitance
CPEdl in series with the solution resistance Rs as shown in Fig. 9a.
Obviously, the EIS spectra of the 98%Mg þ 2%PP sample is different.
There are two capacitance loops and one inductance loop, indi-
cating that there were two reactions [30]. The low frequency
inductance loop described with RL (inductance resistance) and L
(inductance) implied the occurrence of corrosion nucleus [32]. The
inductive loop at low frequency is due to the relaxation of adsorbed
species such as Mg(OH) adsþ or Mg(OH)2. That is to say, the
inductive loop can be ascribed to pit formation [33]. The corre-
sponding equivalent circuits for EIS tests are shown in Fig. 9(b). The
high frequency capacitance loop corresponding to charge transfer
reaction can be explained with Rs, Rct and CPEdl. In the present
study, the medium frequency capacitive is observed due to the
short immersion time.
The simulation values obtained by ZSimpWin software for the
equivalent elements are shown in Table 1. As can be observed from
the values of Rct, The sample containing 10%PP has the best corro-
sion resistance, which is in agreement with the results of poten-
tiodynamic polarization test.
Fig. 10 show the SEM morphologies of the corrode samples and
the corrosion products formed on the samples after electro-
chemical test, respectively. The huge corrosion area on the plain
ZK60 is shown in Fig. 10(a), while Pearl powder-containing samples
in Fig. 10(b-e) are corroded from the Mg matrix to the interface of
pearl powder. The interlinked framework morphology prevents the
corrosion spread. Inspecting the pearl powder containing samples,
regions with higher concentrations of pearl powder particles
degraded less extensively than surrounded regions, leaving
distinguishable corrode pits on the sample surface (Fig. 10bee).
Alternatively, the degradation patterns from plain ZK60 sample
(Fig. 10a) appeared less defined, arising from the degradation of
magnesium. Overall, the presence of pearl powder particles
contributed to decreased material degradation, a condition that
could allow the degradation rate of a biomaterial to be tuned by
varying its pearl powder particle content.
The following reactions occur when the samples are immersed
in simulated body fluid, Magnesium hydroxide (Mg(OH)2) is
formed during the degradation of magnesium, and pH of simulated
162 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165

Table 1  Cathodic reactions:

The values of the equivalent elements in the equivalent circuit of different samples.

Samples Rs(U cm2) CPEdl(mF/cm2) Rct(U cm2) O2 þ 2H2O þ 4e / 4OH
(1)

100%Mg 13.05 81 417.1
98%Mg þ 2%PP 9.11 33 206.7 2H2O þ 2e / 2OH
þ H2[ (2)
94%Mg þ 6%PP 10.90 53 466.3
90%Mg þ 10%PP 28.73 65 1087
86%Mg þ 14%PP 12.66 55 447.3

 Anodic reactions:

body fluid increases due to the consumption of hydrogen ions and Mg / Mg2þ þ 2e (3)
release of OH
ions as explained by Wang et al. [5,34].

Fig. 10. The SEM image on corroded morphology of the samples after electrochemical tests in simulated body fluid (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%Mg þ 6%PP, (d) 90%
Mg þ 10%PP and (e) 86%Mg þ 14%PP.
 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165 163

 Consequently the following reaction also occurs:

Mg2þ þ 2OH
/ Mg(OH)2Y (4)

The precipitation of corrosion products led to the formation of a

hydroxide layer, which thickens with time and progressively hin-
ders the corrosion processes. According to literature [31,35,36]
when the corrosion rate decreases, a balance may occur between
the formation and dissolution of the Mg(OH)2 layer, which reaches
a constant thickness. However, this layer is porous and not fully
protective, allowing the diffusion of charged species involved in the
corrosion processes.
Pearl powder easily aggregates to form small or large con-
glomerates in simulated body fluid, possibly affecting the dissolu-
tion of CaCO3. Simulated body fluid containing HPO2
4 and H2PO4
-
2þ
has abilities to stabilize Ca concentration at a nearly constant
value. The electrochemical dissolution of CaCO3 in simulated body
fluid can occur. Ca3 (PO4)2 formed on the corroded samples.
The results of X-ray diffraction (XRD) analysis (Fig. 11) indicate
that the corroded samples (94%Mg þ 6%PP 90%Mg þ 10%PP and 86%
Mg þ 14%PP) surfaces are mainly consisted of Ca3 (PO4)2 and
MgeZn intermetallic compound. Ca3 (PO4)2 (Calcium orthophos-
phate) can be replaced by autologous bone because they participate
in bone remodeling responses similar to natural bones [37]. Also, it
has been proved that the bioceramics enhance osteoblast differ-
entiation as well as osteoblast growth (osteoconductive property)
[38e44].

3.3.2. Cell toxicity

Fig. 12 shows the cell viabilities cultured in all samples extracts
medium for 1, 2, 3 and 4 days. Percentage of cell viability was
calculated using the following formula: Percentage of cell
viability ¼ (OD sample)/ (OD blank). The cell viability for all sam-
ples shows in Table 2.
The cell viability of plain ZK60 extract shows 93.1% of negative
control, indicating slight toxicities (Grade 1) in the first day incu-
bation, while the cell toxicities show Grade 0 after 2 day and 3 day
incubation. After 4 day incubation, the cell viability of plain ZK60
extract shows 88.7% of negative control, also indicating slight tox-
icities (Grade 1). High cell viabilities are observed for 98%Mg þ 2%
PP, 94%Mg þ 6%PP samples extracts (114.9% and 110.6%) and (110.5%
Fig. 12. Cell viabilities cultured in extracts medium for 1, 2, 3, and 4 days determined
using an MTT assay (A) histogram diagram (B) plotting diagram (a) 100% Mg extract,
(b) 98%Mg þ 2%PP extract, (c) 94%Mg þ 6%PP extract, (d) 90%Mg þ 10%PP extract and
(e) 86%Mg þ 14%PP extract.
and 127.4%) after 1 day and 2 day incubation while the cell toxicities
show Grade 1 after 3 day and 4 day incubation. High cell viabilities
are also observed for 90%Mg þ 10%PP sample extract (111.2.9%,
106.2%, 102.9% and 102.0%) after all four days incubation compared
with the negative control, indicating no cell toxicities (Grade 0) of
the extract. The cell viability of 86%Mg þ 14%PP sample extract
shows 105.6%, 100.5% and 110.8% of positive control, indicating no
cell toxicities (Grade 0) after 1 day, 2 day and 3 day incubation,
while the cell toxicities show Grade 1 after 4 day incubation. Thus,
the cytotoxicities of the extracts for all samples are Grade 0e1,
indicating that the results meet the requirement of cellular
applications.
The in vitro cytotoxicities of extracts for all samples are both

Table 2
Percentage of cell viability in different samples extracts medium for 1, 2, 3 and 4
days.

Samples 1day 2day 3day 4day

100%Mg 93.1% 108.6% 110.9% 88.7%

98%Mg þ 2%PP 114.9% 110.6% 99.3% 92.5%
94%Mg þ 6%PP 110.5% 127.4% 99.4% 91.9%
90%Mg þ 10%PP 111.2% 106.2% 102.9% 102.0%
Fig. 11. XRD patterns of the corroded samples (a) 100% Mg, (b) 98%Mg þ 2%PP, (c) 94%
86%Mg þ 14%PP 105.6% 100.5% 110.8% 93.3%
Mg þ 6%PP, (d) 90%Mg þ 10%PP and (e) 86%Mg þ 14%PP.
164 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165
Grades 0e1, indicating that ZK60 and pearl powder composite
material are safe for cells as implant materials. In order to guar-
antee the biosafety of implant materials, the elements of magne-
sium alloys should be toxic free. Zirconium (Zr) is a powerful grain
refiner and usually refines the microstructure to improve the
strength, ductility and corrosion properties of Mg alloys. Further-
more, Zr-based alloys have been intensively investigated as po-
tential implant materials due to their good biocompatibility
[45,46]. Zinc (Zn) is an essential element for humans, and it is
absolutely necessary for the normal functions of a living organism
and non-toxic except at extreme concentration. In conclusion, the
components of magnesium-pearl powder metal matrix composite
suggest good biocompatibility, and the pearl powder can reduce
the release of alloying elements. The cell toxicities show Grade
0 after all four days incubation for the 90%Mg þ 10%PP sample
extract, which is suspected due to the pearl powder no toxicity and
then affects the viability of osteoblast cells.
Osteoblasts grown in the extracts from all samples showed
significant increases in cell density after 2 day of culture when
grown in the extracts from pearl powder containing samples
(Fig. 12), indicating that the pearl powder degradation products
improved osteoblast proliferation. These results together clearly
demonstrated that pearl powder particles are nontoxic materials
that can be used to improve osteoblast functions on biomaterial
surfaces as they degrade.
4. Conclusions
Magnesium-pearl powder metal matrix composites were sin-
tered. It was found that the particles uniformly aggregated around
the magnesium matrix with the increase of pearl powder contents.
The aggregation of pearl powder gradually formed an interlinked
framework morphology. Pearl powder has been researched rarely
as a material for bone tissue engineering. Here, it was shown that
pearl powder particles can be used to enhance the adhesion and
proliferation of bone cells on MMC-PP composites that are suitable
for bone applications. The presence of pearl powder particles
contributed to decreased material degradation, a condition that
could allow the degradation rate of a biomaterial to be tuned by
varying its pearl powder particle content.
Results further showed for the first time that the degradation
products of MMC-PP composites were not toxic to cells, but actually
improved the proliferation of osteoblasts, and further, the slow
degradation kinetics of pearl powder could potentially be used to
tune the corrosion properties of implanted biomaterials with time.
In summary, this study provides significant evidence of the po-
tential value of the further study of pearl powder particles for
numerous implanted biomaterials applications.
Acknowledgments
This study was supported by National Natural Science Founda-
tion, China (Grant No. 51304136), Natural Science Foundation of
Shanghai, China (Grant No. 12ZR1427200 and 13ZR1427700),
Shanghai Education Development Foundation “Selection and
Training the Excellent Young College Teacher”project under Grant
No. Slgl4049 and Key Laboratory of Advanced Metal-based Elec-
trical Power Materials, Shanghai Municipal Commission of
Education.
References
[1] G.L. Song, Control of biodegradation of biocompatible magnesium alloys,
Corros. Sci. 49 (2007) 1696e1701.
[2] M.P. Staiger, A.M. Pietak, J. Huadmai, G. Dias, Magnesium and its alloys as
orthopedic biomaterials: a review, Biomaterials 27 (2006) 1728e1734.
[3] F. Witte, The history of biodegradable magnesium implants: a review,, Acta
Biomater. 6 (2010) 1680e1692.
[4] F. Witte, N. Hort, C. Vogt, S. Cohen, K.U. Kainer, R. Willumeit, F. Feyerabend,
Degradable biomaterials based on magnesium corrosion, Curr. Opin. Solid
State Mater. Sci. 12 (2008) 63e72.
[5] Y. Wang, M. Wei, J. Gao, J. Hu, Y. Zhang, Corrosion process of pure magnesium
in simulated body fluid, Mater. Lett. 62 (2008) 2181e2184.
[6] G.L. Song, A. Atrens, Corrosion mechanisms of magnesium alloys, Adv. Eng.
Mater. 1 (1999) 11e33.
[7] G.L. Song, A. Atrens, Understanding magnesium corrosion e A frame work for
improved alloy performance, Adv. Eng. Mater. 5 (2003) 837e858.
[8] Erkan Koç, M. Bobby Kannan, Mehmet Ünal, Ercan Candan, Influence of zinc
on the microstructure, mechanical properties and in vitro corrosion behavior
of magnesiume zinc binary alloys, J. Alloy Compd. 648 (2015) 291e296.
[9] H.R. Bakhsheshi-Rad, E. Hamzah, A.F. Ismail, Z. Sharer, M.R. Abdul-Kadir,
M. Daroonparvar, Safaa N. Saud, M. Medraj, Synthesis and corrosion behavior
of a hybrid bioceramic-biopolymer coating on biodegradable Mg alloy for
orthopaedic implants, J. Alloy Compd. 648 (2015) 1067e1071.
[10] S. Shaylin, G.J. Dias, Calcium phosphate coatings on magnesium alloys for
biomedical applications: a review, Acta Biomater. 8 (2012) 20e30.
[11] Emee Marina Salleh, Hussain Zuhailawati, Sivakumar Ramakrishnan,
Mohamed Abdel-Hady Gepreel, A statistical prediction of density and hard-
ness of biodegradable mechanically alloyed MgeZn alloy using fractional
factorial design, J. Alloy Compd. 644 (2015) 476e484.
[12] Y.S. Liu, Q.L. Huang, N.M. Hu, Q.L. Feng, O. Albert, Structural feature and
mechanical property of PLLA/pearl powder scaffold, J. Mech. Med. Biol. 13 (1)
(2013) 1350020e1350035.
[13] Y.F. Ma, Y. Gao, Q.L. Feng, Characterization of organic matrix extracted from
fresh water pearls, Mater. Sci. Eng. C 31 (2011) 1338e1342.
[14] L. Qiao, Q.L. Feng, Z. Li, Special vaterite found in freshwater lackluster pearls,
Cryst. Growth Des. 7 (2) (2007) 275e279.
[15] L. Qiao, Q.L. Feng, Y. Liu, A novel bio-vaterite in freshwater pearls with high
thermal stability and low dissolubility, Mate. Lett. 62 (2008) 1793e1796.
[16] O.R. Fennema, Food Chemistry, Marcel DekkR Inc, New York and Basel, 1985,
p. 523.
[17] Dong Na, Liang Jing, Liu Yuqing, Ma Kun, Lv Daoyu, Jin Liming, Hu Wenzhong,
Determination of the trace element in pearl power, J. Anhui. Agri. Sci. 39 (1)
(2011) 315e316.
[18] S.F. Cui, Y. Zhao, W. Sun, P. Cao, Q. Tang, Effect of nano pearl power on the
calcium absorption and utilization in rats, Acta Lab. Anim. Sci. Sin. 13 (4)
(2005) 204e207.
[19] J. Aaseth, G. Boivin, O. Andersen, Osteoporosis and trace elements, J. Trace.
Elem. Med. Bio. 26 (2012) 149e152.
[20] Z.H. Wu, B. Zhou, Y.Z. Tan, A.C. Wu, Experiment study of anti-osteoporotic
action of pearl calcium capsules, Chin. Tradit. Pat. Med. 23 (10) (2001)
766e768.
[21] J.L. Chen, C. Li, T. Li, Experiment study of anti-osteoporotic action of active
protein in pearl, J. Chin. Med. Mater. 27 (5) (2004) 363e365.
[22] J.J. Wang, J.T. Chen, X.R. Zhang, Nacre-induced osteogenesis in the femoral
condyles of New Zealand rabbits, J. South Med. Univ. 29 (2009) 220e223.
[23] Y. Shen, J. Zhu, H. Zhang, F. Zhao, In vitro osteogenetic activity of pearl, Bio-
materials 27 (2006) 281e287.
[24] W. Liu, Y. Ma, J. Zhang, Properties and microstructural evolution of W-Ni-Fe
alloy via microwave sinteringint, J. Refract. Met. Hard Mater. 35 (2012)
138e142.
[25] R. Liu, Y. Zhou, T. Hao, T. Zhang, X.P. Wang, C.S. Liu, Q.F. Fang, Microwave
synthesis and properties of fine-grained oxides dispersion strengthened
tungsten, J. Nucl. Mater. 424 (2012) 171e175.
[26] S.H. Hong, H.J. Ryu, Combination of mechanical alloying and two-stage sin-
tering of a 93We5.6Nie1.4Fe tungsten heavy alloy, Mater. Sci. Eng. A 344
(2003) 253e260.
[27] Z. Zhou, G. Pintsuk, J. Linke, T. Hirai, M. Rodig, Y. Ma, C. Ge, Transient high heat
load tests on pure ultra-fine grained tungsten fabricated by resistance sin-
tering under ultra-high pressure, Fusion Eng. Des. 85 (2010) 115e121.
[28] D.D. Gu, G.Q. Zhang, D.H. Dai, H.Q. Wang, Y.F. Shen, Nanocrystalline tung-
stenenickel heavy alloy reinforced by in-situ tungsten carbide: Mechanical
alloying preparation and microstructural evolution, Int. J. Refract. Met. Hard
Mater. 37 (2013) 45e51.
[29] Jeng-Kuei Chang, Su-Yau Chen, Wen-Ta Tsai, Ming-Jay Deng, I-Wen Sun,
Electrodeposition of aluminum on magnesium alloy in aluminum chloride
(AlCl 3 )-1-ethyl-3-methylimidazolium chloride (EMIC) ionic liquid and its
corrosion behavior, Electrochem. Commun. 9 (2007) 1602e1606.
[30] J.R. Morlidge, P. Skeldon, G.E. Thompson, H. Habazaki, K. Shimizu, G.C. Wood,
Gel formation and the efficiency of anodic film growth on aluminium, Elec-
trochim. Acta 44 (1999) 2423e2435.
[31] G.L. Song, Effect of tin modification on corrosion of AM70 magnesium alloy,
Corros. Sci. 51 (2009) 2063e2070.
[32] A.L.K. Tan, A.M. Soutar, I.F. Annergren, Y.N. Liu, Multilayer solgel coatings for
corrosion protection of magnesium, Surf. Coat. Technol. 198 (2005) 478e482.
[33] C.M.A. Brett, L. Dias, B. Trindabe, R. Fischer, S. Mies, Characterisation by EIS of
ternary Mg alloys synthesised by mechanical alloying, Electrochim. Acta 51
(2006) 1752e1760.
[34] Y. Wang, M. Wei, J. Gao, Improve corrosion resistance of magnesium in
simulated body fluid by dicalcium phosphate dihydrate coating, Mater. Sci.
 H. Meifeng et al. / Journal of Alloys and Compounds 663 (2016) 156e165
Eng. C 29 (2009) 1311e1316.
[35] Mohammed Ibrahim Jamesh, Guosong Wu, Ying Zhao, David R. McKenzie,
Marcela M.M. Bilek, Paul K. Chu, Corros. Sci. 91 (2015) 160e184.
[36] Weihong Jin, Guosong Wu, Hongqing Feng, Wenhao Wang, Xuming Zhang,
Paul K. Chu, Corros. Sci. 94 (2015) 142e155.
[37] P. Habibovic, J. Li, C.M. van der Valk, G. Meijer, P. Layrolle, C.A. van Blitterswijk,
K. de Groot, Biological performance of uncoated and octacalcium phosphate-
coated Ti6Al4V, Biomaterials 26 (2005) 23e36.
[38] N. Verdonschot, C.T. van Hal, B.W. Schreurs, P. Buma, R. Huiskes, T.J. Slooff,
Time-dependent mechanical properties of HA/TCP particles in relation to
morsellized bone grafts for use in impaction grafting, J. Biomed. Mater. Res.
Appl. Biomater. 58 (2001) 599e604.
[39] A.M. Ambrosio, J.S. Sahota, Y. Khan, C.T. Laurencin, A novel amorphous cal-
cium phosphate polymer ceramic for bone repair: I. Synthesis and charac-
terization, J. Biomed. Mater. Res. Appl. Biomater. 58 (2001) 295e301.
[40] K.G. Marra, J.W. Szem, P.N. Kumta, P.A. DiMilla, L.E. Weiss, In vitro analysis of
biodegradable polymer blend/hydroxyapatite composites for bone tissue
engineering, J. Biomed. Mater. Res. 47 (1999) 324e335.
[41] M. Ekholm, J. Hietanen, R.M. Tulamo, J. Muhonen, C. Lindqvist, M. Kellomaki,
R. Suuronen, Tissue reactions of subcutaneously implanted mixture of
caprolactone-lactide copolymer and tricalcium phosphate, an electron
165
microscopic evaluation in sheep, J. Mater. Sci. Mater. Med. 14 (2003)
913e918.
[42] W. Linhart, F. Peters, W. Lehmann, K. Schwarz, A.F. Schilling, M. Amling,
J.M. Rueger, M. Epple, Biologically and chemically optimized composites of
carbonated apatite and polyglycolide as bone substitution materials,
J. Biomed. Mater. Res. 54 (2001) 162e171.
[43] Daniel J. Hickey, Batur Ercan, Linlin Sun, Thomas J. Webster, Adding MgO
nanoparticles to hydroxyapatiteePLLA nanocomposites for improved bone
tissue engineering applications, Acta Biomater. 14 (2015) 175e184.
[44] Sachiko Hiromoto, Motoki Inoue, Tetsushi Taguchi, Misao Yamane,
Naofumi Ohtsu, In vitro and in vivo biocompatibility and corrosion behavior
of a bioabsorbable magnesium alloy coated with octacalcium phosphate and
hydroxyapatite, Acta Biomater. 11 (2015) 520e530.
[45] C.L. Qiu, Q. Chen, L. Liu, K.C. Chan, J.X. Zhou, P.P. Chen, S.M. Zhang, A novel Ni-
free Zr-based bulk metallic glass with enhanced plasticity and good
biocompatibility, Scr. Mater. 55 (2006) 605e608.
[46] L. Saldana, A. Mendez-Vilas, L. Jiang, L. Jiang, M. Multigner, J.L. Gonzalez-
Carrasco, M.T. Perez-Prado, M.L. Gonzalez-Martin, L. Munuera, N. Vilaboa,
In vitro biocompatibility of an ultrafine grained zirconium, Biomaterials 28
(2007) 4343e4354.