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Thin Solid Films xxx (2009) xxx–xxx
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Thin Solid Films
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t s f
Improvement of bioactivity with magnesium and fluorine ions incorporated
hydroxyapatite coatings via sol–gel deposition on Ti6Al4V alloys
Yanli Cai a, Sam Zhang a,⁎, Xianting Zeng b, Yongsheng Wang a, Min Qian b, Wenjian Weng c
a
b
c
School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore
Singapore Institute of Manufacturing Technology, 71 Nanyang Drive, 638075, Singapore
Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang, 310027, PR China
a r t i c l e
i n f o
Available online xxxx
Keywords:
Magnesium
Fluoridated hydroxyapatite
Simulated body fluid
Bioactivity
a b s t r a c t
Magnesium (Mg) is a trace element in natural bone, its existence plays an important role in cell adhesion and
bone formation. To improve the biological properties, Mg and fluorine (F) are simultaneously incorporated in
hydroxyapatite (HA) to form MgxFHA coating on titanium alloy via sol–gel process. In vitro bioactivity of the
coating is evaluated by examination of apatite precipitation on surface of the coatings during immersion in
simulated body fluid. The chemical states of Mg and F in the coating are examined by X-ray photoelectron
spectroscopy. Grazing incidence X-ray diffraction and scanning electron microscopy are employed for phase
identification and surface morphology changes are compared after soaking in the SBF solutions for 7 to
28 days. The results show that both Mg and F ions are indeed incorporated into the HA crystal structure. The
presence of F promotes Mg incorporation into the HA crystal structure. The presence of Mg makes the
coatings more bioactive in promoting bone formation. However, at high Mg concentration, formation of βTCMP (Mg substituted β-TCP) takes place.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
In orthopaedic field, hydroxyapatite (HA, Ca10(PO4)6(OH)2) coated
metal implants have been studied extensively due to their outstanding
biological responses in the physiological environment [1–11]. However, pure HA suffers from high dissolution in physiological environment that poses long term stability threat leading to implant failures
[12]. It has been proven that the bioactivity of pure HA is inferior to
real bone mineral [13]. Important trace elements exist in the biological
apatite: Na+ (ca. 0.7 wt.%), K+ (ca. 0.03 wt.%), Mg2+ (ca. 0.55 wt.%), Cl−
(ca. 0.10 wt.%), and F− (ca. 0.02 wt.%) [14]. This may hold the key to
improving the biological performance: incorporation of trace element
in the otherwise pure HA coating. Fluorine has been used to substitute
some of the hydroxyl groups to form fluoridated hydroxyapatite
(FHA). FHA has a more compact structure than HA, decreased
dissolution rate as well as enhanced adhesion strength between the
coating and substrate [12], thus possesses improved stability in the
physiological environment. Mg is the fourth most abundant cation in
living organisms. Mg is not only essential for cellular and enzymatic
reactions [15], but also directly affects the process of mineralization
and mechanical properties of bones. The in vitro and in vivo studies of
FGMgCO3Ap-collagen composite showed that Mg ions incorporated
into the apatite crystals may contribute to the acceleration of
osteoblast adhesion to apatite and promote bone formation [16].
⁎ Corresponding author. Tel.: +65 6790 4400; fax: +65 6791 1859.
E-mail address: [email protected] (S. Zhang).
Therefore, it is logical to incorporate Mg and F ions into HA coating
simultaneously in a bid to improve both the short term bioactivity and
long term stability of the coating.
Deposition of HA coating on metal substrate can take place in many
ways: plasma spraying [1–3], chemical treatment [4,17], pulsed laser
deposition / ablation [5,6], sol–gel deposition [8–10,18], etc. So–gel
deposition is preferred due to its capability of precision control of
chemical composition. In this study, sol–gel deposition is employed.
The in vitro bioactivity is assessed by examination of growth of bonelike apatite on the coating surface after soaking in Kokubo's SBF
solution.
2. Experimental procedures
2.1. Coating preparation
Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, Merck, AR), phosphorous pentoxide (P2O5, Merck, GR), magnesium nitrate hexhydrate
(Mg(NO3)2·6H2O, Merck, AR) and hexafluorophosphoric acid (HPF6,
Sigma-Aldrich, GR) were selected to prepare the Ca-precursor, Pprecursor, Mg-precursor and F-precursor, respectively. Ca(NO3)2·4H2O
and Mg(NO3)2·6H2O were dissolved in the ethanol to prepare 2M
Ca-precursor and Mg-precursor, and then mixed together to form the
Ca–Mg mixture. 2M P-precursor was prepared by dissolving P2O5 in
the ethanol followed by a refluxing process for 24 h, then a fixed
amount of F-precursor was added to P-precursor to obtain the P–F
mixture. The Ca–Mg mixture was added drop-wise into the P–F
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doi:10.1016/j.tsf.2009.03.071
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Table 1
Inorganic ion concentrations of human blood plasma and Kokubo's SBF.
Inorganic ion concentration (mM)
Human blood plasma
Kokubo's SBF
Na+
K+
Mg2+
Ca2+
Cl−
HCO−
3
HPO42−
SO42−
142.0
142.0
5.0
5.0
1.5
1.5
2.5
2.5
103.0
148.8
27.0
4.2
1.0
1.0
0.5
0.5
mixture to form a solution with (Ca, Mg)/P ratio of 1.67. This mixed
solution was refluxed for 24 h to obtain the sol. The designed degree
of substitution of Ca2+ by Mg2+ was indicated by the x value in the
general formula of Ca(10 − x)Mgx(PO4)6F(OH), where x = 0/2, 1/2, 2/2,
3/2 and 4/2, and the corresponding coatings were labeled as FM0, FM1,
FM2, FM3 and FM4, respectively.
Fine polished Ti6Al4V substrate slab of 20 × 30 × 1.2 mm3 in size
was dipped vertically into the sol and withdrawn at a speed of 3 cm/
min for the first layer. Then the as-dipped coating was dried at 150 °C
for 15 min followed by firing at 600 °C for 15 min. This dippingdrawing-drying-firing process was repeated 4 times at a withdrawal
speed of 4.5 cm/min for a thicker coating each time. The final coating
thickness was 1.5–2 μm.
2.2. In vitro bioactivity in simulated body fluid
Simulated Body Fluid (SBF) solution was prepared according to
Kokubo's recipe [19], in which the inorganic ion concentrations in the
SBF solution were designed almost the same with that in human blood
plasma, as shown in Table 1. The samples were immersed in the SBF
solution in sterilized bottles for various periods up to 28 days at 37 ±
0.1 °C in a water bath. After immersion, the samples were taken out,
gently washed with deionized water and dried at room temperature
before surface morphology/apatite precipitation and other
examination.
2.3. Coating characterization
The chemical states of fluorine and magnesium were determined by
X-ray photoelectron spectroscopy (XPS, Kratos-Axis Ultra System) using
monochromatic Al Kα X-ray source (1486.7 eV). Grazing incidence X-ray
diffraction (GIXRD, Rigaku Ultima 2000) was employed to analyze the
phases of the coatings before and after soaking in the SBF solution, using
CuKα radiation (λ = 0.15406 nm) at 40 kV and 40 mA with a step size of
0.02°. Scanning electron microscopy (SEM, JEOL JSM-5600LV) was used
Fig. 2. XPS profiles of MgxFHA coatings on Ti6Al4V substrate ((a): survey scan; (b): F 1s
narrow scan; (c): Mg 2p narrow scan).
to observe the surface morphology of the coatings before and after
immersion in SBF.
3. Results and discussion
Fig. 1. GIXRD profiles of the as-deposited MgxFHA coatings on Ti6Al4V substrate.
The gracing incident X-ray diffraction (GIXRD) profile of the asdeposited MgxFHA coatings is shown in Fig. 1. The diffraction peaks of
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Fig. 3. GIXRD patterns of MgxFHA coatings on Ti6Al4V substrate after soaking in the SBF
solutions for 7 days (a) and 28 days (b).
all the samples are assigned to apatite phase and to that of the
Ti6Al4 V substrate. With reference to pure HA (PDF#09-0432), no any
other calcium phosphate is found in the coating. All the main
diffraction peaks in FM0 sample have a slight shift towards higher
degree, especially the (300) peak shifting from 32.902° of pure HA to
33.040°. That is because some of the hydroxyl groups (OH−) are
substituted by the fluorine ions (F−) to form fluoridated hydroxyapatite (FHA) [20,21]. There is not much difference in peak
positions in low concentration MgxFHA (see FM1 and FM2) except
slight peak shift towards higher degree. This is expected the
substitution takes place of smaller Mg2+ (0.069 nm) for larger Ca2+
(0.099 nm) ion. As Mg concentration increases further, i.e. FM3 and
FM4, a new phase of β-TCMP (Mg substituted β-TCP) with weak
intensity is detected, signaling it is beyond the maximum “solubility”
of Mg in HA crystal structure. In our previous study [22], β-TCMP was
formed as the main phase when Mg was incorporated into HA
coatings with the same concentration of FM2 in the sol, because
the substitution of Mg2+ into Ca2+ positions made the apatite
structure resemble β-TCP. Moreover, Mg favoured the conversion of
HA into β-TCMP during the heat treatment [23,24]. However,
when Mg and F ions are incorporated in HA coating at the same
time, only a small amount of β-TCMP was found in FM3 and FM4,
meaning F-incorporation in HA increases the “solubility” of Mg in the
HA structure. Similar results were also reported by other researchers
who prepared apatite powders in the presence of Mg and F [25]. The
shrinkage along c-axis caused by the substitution of hydroxyl groups
by fluorine ions would lead to larger “rattling” space thus the lattice
3
structure becomes less stable. As a result, Mg ions have more chances
to enter into the HA crystal structure to occupy the Ca position.
The chemical states of MgxFHA coatings are confirmed by XPS, as
shown in Fig. 2. The survey scans in Fig. 2(a) demonstrate that all the
elements in the coatings are detected (the presence of C in all the
samples is due to inevitable surface contamination even from air). The
narrow scans of F 1s is shown in Fig. 2(b), the binding energy of F 1s at
684.3 ± 0.1 eV, revealing that F ions have been incorporated into the
HA crystal structure (F 1s at 684.3 ± 0.1 eV is the fingerprint of F in
FHA or FA, while the peak of F 1s in CaF2 is at 686.7 eV [12,26]). This is
also supported by GIXRD analysis. Fig. 2(c) is the narrow scans of Mg
2p. With increasing Mg in the sols, Mg 2p peak gradually intensifies,
indicating more Mg ions are included in the HA coating. Moreover,
only one symmetrical peak is located at 50.3 eV belonging to Mg 2p,
that means one chemical state of Mg existing in the coating, which is
the fingerprint of Mg in apatite structure [22]. Therefore, this result
confirms that Mg ions have been incorporated into the HA lattice
structure in all the samples. In both Mg-containing HA and Mgsubstituted β-TCP, Mg bonds with phosphate group, so the binding
energy of Mg 2p between them does not have difference. As a result,
only one symmetrical peak of Mg 2p is detected for FM3 and FM4
samples, though a second phase of β-TCMP is discovered by GIXRD.
Fig. 3 exhibits the GIXRD profiles of MgxFHA coatings after
soaking in the SBF solutions for 7 and 28 days. After 7 days, as
shown in Fig. 3(a), an obvious difference is observed around 32° for
FM3 and FM4, while not obvious for FM0, FM1 and FM2 as compared
with that in Fig. 1. For FM3 and FM4, the peaks of (211), (112) and
(300) are combined into one broad peak, which implies the poorly
crystallized apatite grown in the solution – evidence of precipitation
of a new apatite layer on the primary coating. The diffraction peaks of
the substrate and calcium phosphate are found in all the samples. Fig.
3(b) shows the GIXRD patterns after 28 days. By now, broad peaks
around 32° are also obvious for FM2, which are assigned to the newly
formed apatite layer. Some other calcium phosphates as intermediate
products like CaH2P2O7 and CaP4O11 during the formation of apatite
are also detected.
Fig. 4 shows SEM images of MgxFHA coatings before and after
soaking in the SBF solutions for 7 and 28 days. After soaking in the SBF
solution for 7 days, the surface morphology of FM3 shows a newly
formed porous layer with cracks (Figure FM3b). Similar observations
are found for FM4 (Fig. 4 FM4b). Other surfaces exhibit some grooves
due to dissolution of the coatings. After 28 days (the c-series images),
FM2c, FM3c and FM4c exhibit similar porous layer as FM3b and FM4b
(precipitation of apatite), but FM0c and FM1c still show the
dissolution characteristics. The dissolution and precipitation behavior
of HA coatings are two of the main factors governing the bioactivity of
HA coatings [27]. When samples are immersed in SBF, both dissolution
of the coatings and precipitation of the new apatite occur at the same
time. When precipitation is dominant, the new apatite layer forms.
The higher the Mg concentration in the coatings (i.e. FM3 and FM4),
the faster the dissolution is. This is attributed to two possible reasons:
the incorporation of Mg into HA speeds up the dissolution of HA
[25,28]; the small amount of β-TCMP favors the dissolution of the
coating due to its higher solubility than Mg-containing HA [29]. As a
result, the concentrations of calcium ions and phosphate groups
increase sharply, leading to the increase of local supersaturation,
which is beneficial to nucleation and growth of the new apatite
crystals. As the dissolution of FM3 and FM4 proceeds much faster than
that of FM0, FM1 and FM2, the precipitation process predominates.
This is evident in Fig. 4 where new apatite layer is observed only after
7 days in SBF. The new apatite layer is also observed on FM2 after
28 days, while not found on FM0 and FM1 even after 28 days. This
again confirms that high Mg concentration favors the formation of the
new apatite layer. Both surface morphology observation and phase
analysis show that the existence of Mg in the coating significantly
affects the bone growth (formation of new apatite layer) in SBF, i.e. the
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Fig. 4. SEM images of MgxFHA coatings on Ti6Al4V substrate before (a) and after soaking in the SBF solutions for 7 days (b) and 28 days (c).
in vitro bioactivity of the coating. Higher concentration of Mg has
better in vitro bioactivity.
4. Conclusions
Mg and F ions are incorporated into HA crystal structure at
the same time via sol–gel deposition on Ti6Al4V substrate. The
presence of F promotes Mg incorporation into the HA crystal structure.
In Ca(10−x)Mgx(PO4)6F(OH), x = 1 seems to be the maximum Mg the
HA structure can take. Over and above this amount, β-TCMP phase
forms. Mg in the coating results in better in vitro bioactivity as
revealed by faster formation of a new apatite layer on the surface of
the coating as submerged in simulated body fluid.
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