Fluoride Conversion Coating on Biodegradable AZ31B Magnesium Alloy

Tingting Yan
1,2)*
, Lili Tan
2)
, Bingchun Zhang
2)
, Ke Yang
2)
1) Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China
2) Institute of Metal Research, Chinese Academic of Sciences, Shenyang 110016, China
[Manuscript received July 29, 2013, in revised form August 27, 2013, Available online xxx]
A fluoride conversion coating was successfully prepared on AZ31B magnesium alloy by chemical reaction in
hydrofluoric acid. Morphologies, composition, bonding strength, corrosion properties, in vitro cytotoxicity and
antibacterial properties of the coating were investigated, respectively. The scanning electron microscopy
observations revealed a dense coating with some irregular pores. The thin-film X-ray diffraction analysis
indicated that the coating was mainly composed of MgO and MgF
2
. The electrochemical impedance
spectroscopy results showed that the fluoride conversion coating significantly improved the corrosion
resistance of AZ31B. The hydroxyapatite formed on the surface of the fluoride coated AZ31B after being
immersed in the simulated blood plasma indicated the good bioactivity of the material. The in vitro
cytotoxicity test showed that the fluoride coated AZ31B alloy was not toxic to BMMSCs (human bone
marrow-derived mesenchymal stem cells). It was also found that the fluoride coated AZ31B alloy had
antibacterial capability.
KEY WORDS: AZ31B magnesium; Fluoride coating; Corrosion resistance; Cytotoxicity; Antibacterial
1. Introduction
Recently, magnesium alloys have attracted much attention as
biodegradable cardiac and orthopaedic implants due to their
unique biological properties
[1e9]
. Biodegradable magnesium
implants would provide a solution for a number of problems
associated with permanent metallic implants such as permanent
physical/mechanical irritation and inability to adapt to growth,
and other ongoing shape changes in the human body. However,
problems, such as alkalization, hydrogen release and high
concentration of magnesium ions
[1,10]
, caused by high corro-
sion rate of magnesium may strongly limit their clinical
application.
Thus, the key issue in the development of biodegradable
magnesium implant is to conne the corrosion attack to a
reasonably low rate. Alloying is one of the effective ways to
control the degradability of magnesium. New magnesium alloys,
such as MgeZneZr
[11]
, MgeMneZn
[5]
, MgeRE
[12]
, MgeZne
MneCa
[13]
, MgeCa
[14]
etc., have been developed. However, the
corrosion rate of those alloys still cannot well meet the practical
demand for implants. Another effective way to reduce the
corrosion rate of magnesium based materials is the surface
treatment. As to biomaterials, surface coating is also a way of
improving their bioactivity. Thus, it is possible to improve both
the surface bioactivity and corrosion resistance of magnesium
alloys by proper surface treatment. Many coating techniques
have been developed to reduce the corrosion of magnesium
alloy, such as calcium phosphate coating
[15]
, hydrogenated
amorphous silicon coating
[16]
, polymer-based coating
[17]
, cal-
cium phosphate conversion coating
[18]
, CaeMgeP containing
coating
[19]
and so on. They could improve the surface bioactivity
and corrosion resistance of magnesium alloys to different extent,
which give the researchers more condences for application of
magnesium implants. Coating techniques, nevertheless, still need
to be developed to meet the more serious requirements to the
implants.
The uoride is essential in the human diet and is thought to be
required for normal dental and skeletal growth
[20]
. Daily uoride
intake is suggested to be 2e5 mg
[21]
. It is also one of the few
known agents that can stimulate the osteoblast proliferation and
increase the new mineral depositions in cancellous bones. The
uoride incorporated into the bone could increase the size and
thus decrease the solubility of the bone apatite crystals
[22]
. The
therapeutic future for sodium uoride in osteoporosis was laid
with early administration and low-dose regimens in which toxic
levels were avoided and mineralization was not impaired
[23]
. It
was revealed that the SR-NaF group signicantly decreased the
risk for vertebral fractures and increased the spinal bone mass
without reduction of bone mass at the femoral neck and total
hip
[24]
. An experimental study in dogs indicated that the uoride
could modify the implant surface to promote osteointegration in
*
Corresponding author. Assoc. Prof., Ph.D.; Tel.: 86 13698721501;
E-mail address: yantting@gmail.com (T. Yan).
1005-0302/$ e see front matter Copyright 2014, The editorial ofce of
Journal of Materials Science & Technology. Published by Elsevier
Limited. All rights reserved.
http://dx.doi.org/10.1016/j.jmst.2013.12.015
Available online at ScienceDirect
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the early phase of healing following the implant installation
[25]
.
Fluoride has been used clinically for prevention and treatment of
osteoporosis although some controversies still existed.
In this work, a magnesium alloy of AZ31B was used as the
substrate material. Though the potential harmful element,
aluminium (Al), exists in the alloy with a content of 3%, the total
amount of aluminium in one set of AZ31B nonbearing bone
implant device (including one X-type bone plate and two bone
screws) is about 12.0 mg. The release of Al for the device with
complete degradation period more than 24 weeks, is much less
than a provisional tolerable weekly intake (PTWI) for Al of
7.0 mg/kg body weight established by World Health Organiza-
tion (WHO)
[26]
. In addition, AZ31B alloy is a commercial ma-
terial with high mechanical property and good deformability, and
is a promising biodegradable material.
In this work, a compact uoride conversion coating was
prepared on the surface of an AZ31B magnesium alloy in order
to control its degradation rate and further improve its biocom-
patibility. The morphology, composition, corrosion behaviour
and biocompatibility of the coating were studied. It can be
concluded that the uoride coating can improve not only the
corrosion resistance, but also the surface biocompatibility of the
magnesium alloy, and the antibacterial property of the coating
was revealed in this study as well.
2. Experimental
2.1. Samples preparation
An extruded AZ31B magnesium alloy was used in this study,
with composition of 1.2%Al, 0.74%Zn, 0.35%Mn, 0.026%Si,
0.003%Fe, 0.0028Cu, 0.0003%Ni (wt%) and balance of Mg.
The alloy was cut into small plates with the size of
11 mm 11 mm 3 mm, then mechanically polished up to
2000 grit with SiC paper, and nally ultrasonically rinsed with
acetone, absolute ethanol and distilled water successively.
2.2. Conversion coating treatment
The samples were immersed in a hydrouoric acid solution
with concentration of 50 wt% at 30

C for 48 h. The treated
samples were then rinsed with distilled water and dried in air.
2.3. Surface characterization
The morphology of the surface layer was examined by scan-
ning electron microscopy (SEM, JEOL JSM-6301F). The
composition of the layer was analysed by thin-lm X-ray
diffraction (TF-XRD, BRUKER AXS D8 ADVANCE).
2.4. Bonding strength test
The bonding strength between uoride coating and the AZ31B
alloy substrate was measured according to ISO 14916
[27]
and the
schematic illustration of the test is shown in Fig. 1, where one
uoride coated sample was vertically adhered to another bare
sample using bisphenol-A epoxy resin (HTE-51, Shenyang
Southeast Institute of Chemical Industry) with an adhesive
strength of about 45 MPa. The test was performed on a universal
tensile tester (AG-I 500 kN, Shimadzu) with loading rate of 1 mm/
min. Five experimental values and standard deviations were
calculated from fractures of the specimens in the test.
2.5. Electrochemical impedance spectroscopy test
The corrosion resistance of bare substrate and uoride treated
AZ31B samples was evaluated by means of electrochemical
impedance spectroscopy (EIS) in the simulated blood plasma,
containing NaCl 6.8 g/l, MgSO
4
0.1 g/l, NaHCO
3
2.2 g/l, Na
2
HPO
4
0.216 g/l, NaH
2
PO
4
0.026 g/l, CaCl
2
0.2 g/l and KCl 0.4 g/l, at
37

C. The tests were carried out with a lock-in amplier (model
5210, EG&G PAR) coupled with a potentiostategalvanostal
(model 273A, EG&G PAR), by using a typical three-electrode
conguration. The cell consisted of a saturated calomel electrode
as the reference, a platinum sheet as the counter electrode and the
sample as the workingelectrode. The workingelectrode includedan
electrical connection wire, which was initially attached to the sur-
face of samples with conductive glue, 1 cm
2
surface of the sample
exposed in the electrolyte and the rest of the sample sealed by epoxy
resin. The samples were immersedinthe electrolyte for 30 minprior
to each test, allowing the system to become equilibrated with the
electrolyte. The EIS measurements were carried out in the fre-
quency range of 100 kHz to 10 mHz. The signal amplitude was
10 mV. The EIS data were presented as the Nyquist and Bode plots.
2.6. Immersion test
Fluoride treated AZ31B samples were immersed in a simu-
lated blood plasma. The ratio of the specimen area to the solution
volume was 3 cm
2
/ml, according to ISO 10993-12
[28]
. All the
immersion containers were kept at 37

C in a 5% CO
2
incubator.
The immersion solution was changed every day. At different
immersion time points, samples were removed from the simu-
lated blood plasma, gently rinsed with distilled water and dried
in air. The surface and cross section morphologies and micro-
structures of the samples before and after immersions were
characterized by scanning electron microscopy (SEM, JEOL
JSM-6301F), equipped with energy-disperse spectrometer (EDS,
Oxford INCA Energy 300) attachment, and an X-ray diffrac-
tometer (XRD, D/max 2500 PC).
2.7. Cytotoxicity test
The cytotoxicity in vitro was measured by MTT assay ac-
cording to ISO 10993-5
[29]
. The extract liquid was prepared by
immersing samples in RPMI-1640 medium. The volume of
Fig. 1 Schematic illustration of the bonding strength test.
2 T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9
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solution was calculated based on a volume-to-sample area ratio
of 1 ml/3 cm
2
, which was in accord with ISO10993-12
[28]
.
Monolayer cultures of the BMMSCs (human bone marrow-
derived mesenchymal stem cells) were initiated at a density of
1 10
3
cells per 96 well and incubated in 5% CO
2
incubator at
37

C for 24 h. After the cell attachment, 50 ml extract liquid was
added to each well that was further incubated in 5% CO
2
incu-
bator at 37

C for 24 h. After then, the culture uid was taken out
of the wells and 200 ml MTT was added to each well. After
incubation at 37

C for 4 h, MTT uid was taken out of the wells
and 150 ml per well dimethyl sulfoxide (DMSO) was added into
the wells. The cell contents of the individual cultures were
measured colorimetrically. The optical density (O.D.) was
recorded at 492 nm by using a micro plate reader (GF M-2000,
China), and the cell proliferation rate (P) was dened
P
O:D:
testmaterial
O:D:
negative
100% (1)
The P was used to evaluate the cytotoxicity grade of each
group according to the standard of GB/T 16175
[30]
(see the
notation under Table 1). Each experiment was repeated for at
least 5 times.
2.8. Antibacterial test
The lm attachment method
[31]
was adopted for the antibac-
terial test, which is one of the most commonly used testing
methods for antibacterial properties evaluation of solid materials,
quantitatively measuring the antibacterial rate of the material.
Antibacterial tests were conducted against a standard Gram-
negative bacterium, Escherichia coli (E. coli) ATCC25922. The
culturing broth was prepared by dissolving 5 g esh extract, 5 g
NaCl and 10 g peptone into 1000 ml of distilled water and its pH
was adjusted to 7.0e7.2. The culturing solution containing the
bacteria was diluted to 10
6
cfu/ml (colony forming units/ml).
0.3 ml of this solution was homogeneously added dropwise by a
dispenser onto the surface of samples, which had been hori-
zontally placed into a sterilized Petri-dish, and then covered with
sterile plastic lm. The bacteria on samples were incubated at
37

C for 24 h in an incubator. After the incubation, the bacterial
solution on the sample surface was diluted with 15 ml of
phosphate buffer solution, and 0.1 ml of it was added into a
Petri-dish. The antibacterial effect was recognized by the anti-
bacterial rate, which was calculated as the following:
Antibacterial rate (%) 100 (A B)/A (2)
where A is the number of bacteria colonies in the Petri-
dish for the contrast stainless steel (316L stainless
steel) acting with E. coli, and B is the number of bacteria
colonies for the uoride treated AZ31B acting with
E. coli.
3. Results
3.1. Coating characteristics
Both the surface morphology and chemical composition of the
uoride treated samples were examined in this study. The surface
morphology of the conversion coating on AZ31B presented in
Fig. 2 shows that a black, dense and smooth coating with some
small irregular pores scattering was formed on the surface of
AZ31B sample. The irregular pores in the coating should be
generated by the evolution of hydrogen and could be reduced or
lled by precipitations of MgF
2
and MgO particles
[32]
during
uoride conversion treatment. The SEM image of cross section
of the uoride treated sample shown in Fig. 3 indicates that the
coating layer adhered well to the AZ31B substrate, with thick-
ness of about 1.9 mm.
The result of TF-XRD, as shown in Fig. 4, clearly indicates
that the conversion coating is composed of MgF
2
and MgO.
Since the coating was very thin, the magnesium alloy substrate
was also detected and showed high resolution in the spectrum.
The result of bonding strength test indicates that the average
bond strength between the uoride coating and substrate is
43.2 5.1 MPa. Fig. 5 shows one of the test curves. However,
as one can see from Fig. 6, the uoride coating on the sample
after the test still keeps the original bond with the substrate and
the fracture occurs in the epoxy resin layer because the strength
of epoxy resin is less than the bonding strength between the
uoride coating and the AZ31B substrate. So it can be deduced
that the strength of 43.2 5.1 MPa tested in this study should be
the strength of epoxy resin and the bonding strength of the
uoride coating with AZ31 substrate should be more than
43.2 MPa.
Bonding strength is, quite naturally, an important property of a
coating on materials. It is especially an important property of the
coating used for biomaterials. Geng et al.
[33]
reported that the
bonding strength between b-TCP coating and Mg substrate was
16.1 MPa. Lin et al.
[34]
studied the bonding strength between
hydroxyapatite (HA) coating and 316L stainless steel substrate
Table 1 Fitted data of the elements in the model of Fig. 8
Materials R
p
(U cm
2
) R
c
(U cm
2
) R
s
(U cm
2
) CPE
p
(F cm
2
) CPE
c
(F cm
2
)
Bare AZ31B 500 1000 24 7 10
6
0.009094
Fluoride coated AZ31B 429640 e 86.2 1.2794 10
5
e
Fig. 2 Surface morphology of uoride coating on AZ31B alloy.
T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9 3
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and the bonding strength was in a range of 22e37 MPa. Xiao
et al.
[35]
reported that the bonding strength between HA coating
and Ti substrate was between 11e22 MPa. According to ASTM
1147-F
[36]
, the minimum bonding strength of 22 MPa is required
for a coating on the medical implants. In the present study, all the
tested coatings showed high bonding strength and the average
bonding strength between the uoride coating and AZ31B sub-
strate was over 43.2 MPa and meets the requirement of the
medical implants although the precise bonding strength value
was not measured. The strong bonding between the uoride
coating and AZ31B substrate should come from two aspects, the
mechanical interlocking and the chemical bonding, which were
developed on the coating-substrate during the coating generation
process.
3.2. Corrosion behaviour
The EIS spectra of the samples measured in the simulated
blood plasma are shown in forms of Nyquist and Bode plots, as
shown in Fig. 7. It can be seen from the Nyquist plots
(Fig. 7(a)), the capacitance loop diameter of the samples with
uoride conversion coating is much larger than that of the bare
sample. It can also be observed that the spectra of the bare
AZ31B sample have two loops and those of the uoride coated
sample have only one, the former representing the characteristics
of electric double layer. The impedance modulus at different
frequencies can be directly read from the Bode spectra, and the
capacitive behaviour is indicated by the increase of the imped-
ance with decreasing frequency. In the high frequency region,
the impedance is independent of frequency, which is the resis-
tance of the electrolyte between the sample and the reference
electrode. At the low frequency limit, the impedance is attrib-
uted to the polarization resistance (R
p
) of the sample in the
electrolyte. As shown in the Bode plots (Fig. 7(b)), the imped-
ance modulus of the uoride coated AZ31B sample is much
higher than that of the bare one.
In accordance with the impedance spectra, the equivalent
circuits in Fig. 8 are proposed to t the data. The tted data are
also presented in Fig. 7 and the parameters of the equivalent
circuit are given in Table 1. There is a good agreement between
the experimental and the tted data. For the bare AZ31B sample,
the circuit was based on the three contributions, the resistance of
the electrolyte (R
s
), the impedance of the electrolyteeoxide
interface (the resistance of oxide layer R
p
in parallel with the
electrolyteeoxide interface capacitance CPE
p
), and at the highest
frequencies a contribution associated to the oxide-substrate
interface (resistance and capacitance of the interface R
c
and
CPE
c
). This equivalent circuit is consistent with the presence of
two loops found in the experimental Nyquist plots. For the
uoride coated AZ31B samples, the equivalent circuit was
composed of the resistance of the electrolyte (R
s
) and the
Fig. 3 SEM micrograph of cross section of uoride coated AZ31B
sample.
Fig. 4 TF-XRD spectrum of uoride coating on AZ31B alloy.
Fig. 5 Diagram bonding strength test result.
Fig. 6 Photo of uoride coated AZ31B sample after bonding strength
test.
4 T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9
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impedance of the uoride layer (resistance and capacitance of the
uoride layer R
p
and CPE
p
). As shown in Table 1, the R
p
of the
uoride coated sample is 429640 U cm
2
, much larger than that of
the bare AZ31B sample (500 U cm
2
). This difference can also be
seen from the Bode plots. The R
c
of the substrate is about
1000 U cm
2
. The circuit capacitance of the uoride coated
AZ31B samples (1.2794 10
5
F cm
2
) is much lower than
that of the bare AZ31B sample (sum of CPE
p
and CPE
c
). The
higher R
p
and lower CPE clearly demonstrate a lower corrosion
rate of the uoride coated samples than that of the bare sample.
The above EIS result conrms that the uoride coating can offer
an effective protection to delay the degradation process of
AZ31B magnesium alloy.
Fig. 9 shows the surface and cross section morphologies of the
AZ31B samples with uoride conversion coating immersed in
the simulated blood plasma for different time intervals. After 5
days immersion, white particles deposit on the surface. After 20
days, a thin deposition coating is formed on the surface and there
is no severe pitting corrosion. On the 45th day, an obvious
deposition coating with some cracks is found. The deposition is
increased and the coating gradually grows up with increasing
immersion time. On the 120th day, a thick coating with some
cracks composed of spherical crystals, with thickness about
4.3 mm, is formed on the surface. The cracks in the deposition
coating are possibly attributed to desorption of water from the
coating during the drying process. From the cross section mor-
phologies, it can be seen that the cracks occur in the uoride
conversion coating on the 90th day, indicating the breaking of
the uoride coating. Once the conversion coating broke, the
AZ31B substrate would start to degrade. On the 120th day, the
uoride conversion coating cannot be found. The EDS analysis
on the sample immersed for 90 days, as shown in Fig. 10, reveals
that the deposition contains Mg, O, F, Ca, P and C, and the Ca/P
ratio is about 1.39. In order to identify the structure of the
deposition, XRD was conducted on the surface of the uoride
coated sample after immersion in the simulated blood plasma for
90 days, as shown in Fig. 11 with XRD standard JCPDS patterns
of Mg, HA, MgF
2
and MgO for comparison. It can be found that
HA phase was detected from the coating. The formation of HA
illustrated the bioactivity of the coating.
3.3. Cytotoxicity test
Table 2 shows the result of cytotoxicity test. The relative
proliferation rates of BMMSCs on the groups of AZ31B samples
with and without uoride conversion coating were 89.7% and
36.8%, respectively, the corresponding toxicity grade being 1
and 3. The grade 1 means that the tested samples have no
cytotoxicity, while the grade 3 represents moderately cytotoxic.
Thus it can be concluded that the uoride coating can eliminate
the cytotoxicity of AZ31B magnesium alloy and the uoride
coating has no toxicity to the BMMSCs.
3.4. Antibacterial test
Table 3 shows the antibacterial rate of the AZ31B samples with
uoride conversion coating. The antibacterial rate of the contrast
316L stainless steel is 0, while the antibacterial rate of uoride
coated AZ31B sample is over 99.99%, indicating that uoride
conversion coating can provide AZ31B an excellent antibacterial
surface. Fig. 12 presents the breeding and agglomeration status of
E. coli after actions with the AZ31B with uoride conversion
coating and the contrast stainless steel. It can be clearly seen that
after 24 h cultivation of E. coli contacting with the samples, there is
almost no bacterium in the dish where the bacteria act with the
uoride coated AZ31B (Fig. 12(a)), but large amount of bacteria in
the dish where bacteria act with the contrast stainless steel
(Fig. 12(b)), indicating that the bacteria cultivated on the surface of
the uoride coated AZ31Bsamples have almost been extinguished.
4. Discussion
The rapid corrosion rate of magnesium in the electrolytic
physiological environment is one of the greatest limitations for
Fig. 7 EIS spectra of bare and uoride coated AZ31B samples in the simulated blood plasma: (a) Nyquist plots, (b) Bode plots.
Fig. 8 Equivalent electrical circuits for bare AZ31B (a) and uoride
coated AZ31B (b) samples.
T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9 5
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its biomedical applications. The unprotected magnesium
exposed to a typical atmosphere will produce a gray oxide lm
of magnesium hydroxide (Mg(OH)
2
) which slows the corro-
sion
[37]
, but the chloride ions (Cl

) in physiological environment
are aggressive to magnesium, since the absorption of Cl

in the
oxide lms of the magnesium surface can transform Mg(OH)
2
to
the easily soluble MgCl
2
[37]
. The following reactions summarize
the corrosion reactions to magnesium:
Fig. 9 Surface (1) and cross section (2) morphologies of uoride coated AZ31B immersed in the simulated blood plasma for different time intervals: (a)
5 days, (b) 20 days, (c) 45 days, (d) 90 days, (e) 120 days.
6 T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9
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Mgs 2H
2
O/MgOH
2
H
2
g (3)
Mgs 2Cl

aq/MgCl
2
(4)
MgOH
2
s 2Cl

/MgCl
2
(5)
One of the effective ways to reduce the corrosion of magne-
sium alloys is surface modication. As to biomaterials, a surface
modication is also an effective way to improve its bioactivity.
Therefore, it is possible to reduce the corrosion of magnesium
alloy and improve the surface bioactivity by selecting a proper
surface treatment to the alloy.
For the above purpose, the present uoride conversion coating is
potentially applied for biomedical magnesium alloy. Li et al.
[38]
found that the uoride lm deposited on magnesium alloy did
not improve its corrosion resistance in the 3.5% NaCl solution and
they considered that the uoride on magnesiumalloy surface could
only improve its reactivity at the active site. Chiu et al.
[39]
studied
the corrosion property of MgF
2
treated pure Mg in Hanks solution
and found an increase in corrosion resistance resulting from the
uoride conversion coating on Mg. In the present work, a
remarkably improved corrosion resistance has been obtained for
the uoride coated uoride AZ31B in the simulated blood plasma,
shown by the electrochemical impedance spectroscopy.
TF-XRD result indicates that the coating is mainly composed
of MgF
2
and MgO. It is well known that Mg can react with
hydrouoric acid via a replacement reaction as the following:
Mg 2HF/MgF
2
H
2
[ (6)
Simultaneously, an oxidation reaction occurs as the following:
Mg 2H
2
O/MgOH
2
H
2
[ (7)
Since Mg(OH)
2
is not stable in the acidic solution
[40]
, re-
actions should occur as the following:
MgOH
2
2HF/MgF
2
2H
2
O (8)
MgOH
2
/MgO H
2
O (9)
These reactions should take place at the lm/solution inter-
face
[32]
. The insoluble MgF
2
layer which acts as the passive lms
can form on the magnesium alloy surface by a chemical reaction.
The MgF
2
lm will grow up with the immersion time, but the
growth rate should slow down as the immersion continues
[39]
.
Slow growth of the reaction layer is an indication of the barrier
effect of MgF
2
to the reaction.
The toxicity of uoride has been investigated. Matsui et al.
[41]
found that NaF could increase the intracellular Ca
2
concen-
tration, which might be one of common features of the uoride
toxicity. Khalil et al.
[42]
reported that the uoride had a sort of
cytotoxicity/cytostatic effect on the cultured rat bone marrow
cells. It was also found that toxicity of the uoride was related
with its concentration. Kleinsasser et al.
[43]
investigated the
cytotoxicity extent of the uoride using the trypan blue exclusion
test. They found that non vital cells of less than 10% could be
shown for the uoride concentrations of 2 10
6
to 35 10
6
and there were 15% and 43% damaged cells after incubation
with 71 10
6
and 213 10
6
uoride, respectively.
Fig. 10 EDS analysis result of uoride coated AZ31B incubated in the
simulated blood plasma for 90 days.
Fig. 11 XRD patterns on surface of the uoride coated AZ31B after
immersion in the simulated blood plasma for 90 days: (a)
uoride coated AZ31B, (b) Mg (JCPDS89-5003), (c) HA
(JCPDS09-0432), (d) MgF2 (JCPDS 38-0882), (e) MgO
(JCPDS87-0652).
Table 2 Results of in vitro cytotoxicity test
O.D. (sd) P (%) Toxicity grade
*
Control 0.5476 0.0162 100 /
Fluoride coated AZ31B 0.4914 0.0517 89.7 1
AZ31B 0.2015 0.0400 36.8 3
Notes: * Toxicity grade 0 means P 100, grade 1 means
P75%; 99%, grade 2 means P50%; 74%, grade 3 means
P25%; 49%, grade 4 means P1%; 24%, grade 5 means
P 0
[30]
.
Table 3 Antibacterial rate of uoride coated AZ31B (E. coli, 106 cfu/
ml)
Materials Antibacterial rate
316L stainless
steel as a contrast
0
Fluoride coated
AZ31B
> 99.99%
T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9 7
Please cite this article in press as: T. Yan, et al., Journal of Materials Science & Technology (2014), http://dx.doi.org/10.1016/j.jmst.2013.12.015
Meanwhile, the interaction between uoride and magnesium
has also been investigated. Colly et al.
[44]
found that uoride ion
affected the enamel hardening and prevented its annealing, but
this effect diminished after administration of Mg. ODell et al.
[45]
studied the interaction of dietary uoride and magnesium in
guinea pigs, and they found that the uoride in the food and
water consumed by man would be particularly valuable if
magnesium was limiting in the diet. One the other hand, a high
intake of magnesium should be highly benecial in areas where
uorosis prevails and the toxicity of uoride decreased with
increasing amount of magnesium in the diet of the guinea
pigs
[45]
.
In the present study, the in vitro cytotoxicity evaluation results
demonstrated that AZ31B alloy samples with uoride conver-
sion coating were not toxic to BMMSCs, indicating the stability
of compound MgF
2
formed in the conversion coating with safe
concentration of uoride released into the cell culture medium.
By comparing with that on uoride conversion coating, the
relative proliferation rate of BMMSCs on the groups of bare
AZ31B samples was 35.8%, indicating moderately cytotoxic. It
is mainly because the corrosion rate of bare AZ31B is too fast
that results in the higher pH values in MTT. Groos et al.
[46]
indicated that colony-forming ability of RT112 cells was
reduced signicantly at pH values greater than 8.4 after 24 h
exposure. Meanwhile, the hydrogen released during the bare
AZ31B samples degrading in the solution also had some effects
on cell growth
[47]
. All the above results proved that the uoride
conversion coating can provide AZ31B with lower biodegrada-
tion rate and decreased cytotoxicity.
Surface morphologies of the samples with uoride conversion
coating after immersion in simulated blood plasma illustrated that
the bioactive HAwere formed on the surface of samples. For bone
implants, the prerequisite to bond to living bone is the formation of
biologically active apatites on their surface in the body. The apatite
layer bridges chemically the bone and the implant. Thus, the
formation of HA illustrates the bioactivity of the uoride con-
version coating and that is benecial to the application of AZ31B
with uoride conversion coating as bone implants. It was reported
that a bonelike apatite layer was observed after only 3 days of bare
magnesium immersing in SBF, indicating the bioactive of mag-
nesium
[48]
. Thus, we can deduce that the release of magnesium
ions fromuoride conversion coating and AZ31Bsubstrate is one
of the contributions to the bioactivity of AZ31B with uoride
conversion coating. Meanwhile, uoride can enhance osteoblastic
differentiation and interfacial bone formation
[49]
. Bernstein
et al.
[50]
also reported that the uoride could prevent the osteo-
porosis and provoke the metastatic calcication. So the uoride in
the conversion coating is a possible contribution to the bioactivity
of the sample in this study.
Already in 1940, it was demonstrated that the carbohydrate
metabolism in pure cultures of oral streptococci and lactobacilli
was inhibited by the uoride
[51]
. Since then, many reports have
been published about the effect of uoride on bacteria
[52e54]
.
Yoshinari et al.
[55]
demonstrated that the bacteria were injured by
the pharmacological effect of the metal-uoride complexes with
their inhibition of enzymatic activity. In this work, the uoride
coated AZ31Bsamples well showed good antibacterial capability,
which might be due to the pharmacological effect of MgeF
complex (MgF
2
) in the conversion coating. As for biomedical
implants and devices, infection caused by bacteria from per-
operative contamination during the surgery is the primary cause
for failure. Therefore, biomaterials with anti-microbial properties
are highly desirable because the reducing risk of infection. Thus,
the antibacterial property of the uoride coated AZ31Bsamples is
benecial to their application as the medical implants.
5. Conclusion
A compact uoride conversion coating was successfully pre-
pared on an AZ31B magnesium alloy. The coating was mainly
composed of MgF
2
and MgO. The bonding strength between the
uoride coating and AZ31B substrate was over 43.2 5.1 MPa.
Electrochemical tests showed an improved corrosion resistance
for the uoride coated AZ31B alloy in the simulated blood
plasma compared with that of the bare AZ31B alloy. The in vitro
cytotoxicity test showed that the uoride coated AZ31B was not
toxic to BMMSCs. The uoride coated AZ31B had antibacterial
capability. To conclude, the uoride conversion treatment is an
effective way for application by using AZ31B magnesium alloy
as a biomaterial.
Acknowledgements
The authors thank the nancial support of the National Basic
Research Program of China (973 Program, No. 2012CB619101)
and the Basic Application Research of Yunnan Province (No.
KKSA201151053).
Fig. 12 Photos of breeding status of E. coli after actions with uoride coated AZ31B (a) and 316L stainless steel (b).
8 T. Yan et al.: J. Mater. Sci. Technol., 2014, -(-), 1e9
Please cite this article in press as: T. Yan, et al., Journal of Materials Science & Technology (2014), http://dx.doi.org/10.1016/j.jmst.2013.12.015
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Please cite this article in press as: T. Yan, et al., Journal of Materials Science & Technology (2014), http://dx.doi.org/10.1016/j.jmst.2013.12.015